GTest Contradiction in primer_task definition Contradiction in primer_task definition Unrecognized PRIMER_TASK Unrecognized PRIMER_TASK Illegal TARGET value: %1 Illegal TARGET value: %1 Illegal PRIMER_DEFAULT_PRODUCT value: %1 Illegal PRIMER_DEFAULT_PRODUCT value: %1 Illegal PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION value: %1 Illegal PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION value: %1 Illegal INCLUDED_REGION value: %1 Illegal INCLUDED_REGION value: %1 Illegal EXCLUDED_REGION value: %1 Illegal EXCLUDED_REGION value: %1 Missing SEQUENCE tag Missing SEQUENCE tag Error in sequence quality data Error in sequence quality data Sequence quality data missing Sequence quality data missing PRIMER_PAIRS_NUMBER is incorrect. Expected:%2, but Actual:%3 PRIMER_PAIRS_NUMBER is incorrect. Expected:%2, but Actual:%3 %1 is incorrect. Expected:%2,%3, but Actual:NULL %1 is incorrect. Expected:%2,%3, but Actual:NULL %1 is incorrect. Expected:NULL, but Actual:%2,%3 %1 is incorrect. Expected:NULL, but Actual:%2,%3 %1 is incorrect. Expected:%2,%3, but Actual:%4,%5 %1 is incorrect. Expected:%2,%3, but Actual:%4,%5 %1 is incorrect. Expected:%2, but Actual:%3 %1 is incorrect. Expected:%2, but Actual:%3 Primer3Dialog Main Main Excluded regions Excluded regions Targets Targets <html><head/><body>Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list ofstart,lengthpairs where start is the index of the first base of the excluded region, and length is its length. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs.</body></html> <html><head/><body>Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list ofstart,lengthpairs where start is the index of the first base of the excluded region, and length is its length. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs.</body></html> <html><head/><body>If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-separated list of start,lengthpairs where start is the index of the first base of a Target, and length is its length.</body></html> <html><head/><body>If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-separated list of start,lengthpairs where start is the index of the first base of a Target, and length is its length.</body></html> Product size ranges Product size ranges <html><head/><body>A list of product size ranges, for example: 150-250 100-300 301-400 Primer3 first tries to pick primers in the first range. If that is not possible, it goes to the next range and tries again. It continues in this way until it has either picked all necessary primers or until there are no more ranges. For technical reasons this option makes much lighter computational demands than the Product Size option.</body></html> <html><head/><body>A list of product size ranges, for example: 150-250 100-300 301-400 Primer3 first tries to pick primers in the first range. If that is not possible, it goes to the next range and tries again. It continues in this way until it has either picked all necessary primers or until there are no more ranges. For technical reasons this option makes much lighter computational demands than the Product Size option.</body></html> Mispriming/Repeat library Mispriming/Repeat library <html><head/><body>This selection indicates what mispriming library (if any) Primer3 should use to screen for interspersed repeats or for other sequence to avoid as a location for primers.</body></html> <html><head/><body>This selection indicates what mispriming library (if any) Primer3 should use to screen for interspersed repeats or for other sequence to avoid as a location for primers.</body></html> Number to return Number to return Max repeat mispriming Max repeat mispriming Max template mispriming Max template mispriming Max 3' stability Max 3' stability Pair max repeat mispriming Pair max repeat mispriming Pair max template mispriming Pair max template mispriming <html><head/><body>The maximum number of primer pairs to return.Primer pairs returned are sorted by their "quality", in other words by the value of the objective function (where a lower number indicates a better primer pair).Caution: setting this parameter to a large value will increase running time.</body></html> <html><head/><body>The maximum number of primer pairs to return.Primer pairs returned are sorted by their "quality", in other words by the value of the objective function (where a lower number indicates a better primer pair).Caution: setting this parameter to a large value will increase running time.</body></html> <html><head/><body>The maximum stability for the last five 3' bases of a left or right primer.Bigger numbers mean more stable 3' ends. The value is the maximum delta G (kcal/mol) for duplex disruption for the five 3' bases as calculated using the Nearest-Neighbor parameter values specified by the option of 'Table of thermodynamic parameters'.</body></html> <html><head/><body>The maximum stability for the last five 3' bases of a left or right primer.Bigger numbers mean more stable 3' ends. The value is the maximum delta G (kcal/mol) for duplex disruption for the five 3' bases as calculated using the Nearest-Neighbor parameter values specified by the option of 'Table of thermodynamic parameters'.</body></html> <html><head/><body>The maximum allowed weighted similarity with any sequence in Mispriming Library.</body></html> <html><head/><body>The maximum allowed weighted similarity with any sequence in Mispriming Library.</body></html> <html><head/><body>The maximum allowed sum of similarities of a primer pair (one similarity for each primer) with any single sequence in Mispriming Library. Library sequence weights are not used in computing the sum of similarities.</body></html> <html><head/><body>The maximum allowed sum of similarities of a primer pair (one similarity for each primer) with any single sequence in Mispriming Library. Library sequence weights are not used in computing the sum of similarities.</body></html> <html><head/><body>The maximum allowed similarity to ectopic sites in the sequence from which you are designing the primers.The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> <html><head/><body>The maximum allowed similarity to ectopic sites in the sequence from which you are designing the primers.The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> <html><head/><body>The maximum allowed summed similarity of both primers to ectopic sites in the sequence from which you are designing the primers.The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> <html><head/><body>The maximum allowed summed similarity of both primers to ectopic sites in the sequence from which you are designing the primers.The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> Start codon position Start codon position <html><head/><body>Index of the first base of a start codon.This parameter allows Primer3 to select primer pairs to create in-frame amplicons e.g. to create a template for a fusion protein. Primer3 will attempt to select an in-frame left primer, ideally starting at or to the left of the start codon, or to the right if necessary. Negative values of this parameter are legal if the actual start codon is to the left of available sequence. If this parameter is non-negative Primer3 signals an error if the codon at the position specified by this parameter is not an ATG. A value less than or equal to -10^6 indicates that Primer3 should ignore this parameter. Primer3 selects the position of the right primer by scanning right from the left primer for a stop codon. Ideally the right primer will end at or after the stop codon.This parameter should be considered EXPERIMENTAL. Please check the output carefully; some erroneous inputs might cause an error in Primer3.</body></html> <html><head/><body>Index of the first base of a start codon.This parameter allows Primer3 to select primer pairs to create in-frame amplicons e.g. to create a template for a fusion protein. Primer3 will attempt to select an in-frame left primer, ideally starting at or to the left of the start codon, or to the right if necessary. Negative values of this parameter are legal if the actual start codon is to the left of available sequence. If this parameter is non-negative Primer3 signals an error if the codon at the position specified by this parameter is not an ATG. A value less than or equal to -10^6 indicates that Primer3 should ignore this parameter. Primer3 selects the position of the right primer by scanning right from the left primer for a stop codon. Ideally the right primer will end at or after the stop codon.This parameter should be considered EXPERIMENTAL. Please check the output carefully; some erroneous inputs might cause an error in Primer3.</body></html> Pick left primer Pick left primer or use left primer below or use left primer below Pick hybridization probe (internal oligo) Pick hybridization probe (internal oligo) or use oligo below or use oligo below Pick right primer Pick right primer or use right primer below (5' to 3' on opposite strand) or use right primer below (5' to 3' on opposite strand) General Settings General Settings <html><head/><body>This section covers such settings as primer size, temperature, GC and other general settings.</body></html> <html><head/><body>This section covers such settings as primer size, temperature, GC and other general settings.</body></html> Primer size Primer size Min Min Opt Opt Max Max Primer Tm Primer Tm <html><head/><body>Minimum, Optimum, and Maximum lengths (in bases) of a primer oligo.Primer3 will not pick primers shorter than Min or longer than Max, and with default arguments will attempt to pick primers close with size close to Opt.Min cannot be smaller than 1. Max cannot be larger than 36. (This limit is governed by maximum oligo size for which melting-temperature calculations are valid.)Min cannot be greater than Max.</body></html> <html><head/><body>Minimum, Optimum, and Maximum lengths (in bases) of a primer oligo.Primer3 will not pick primers shorter than Min or longer than Max, and with default arguments will attempt to pick primers close with size close to Opt.Min cannot be smaller than 1. Max cannot be larger than 36. (This limit is governed by maximum oligo size for which melting-temperature calculations are valid.)Min cannot be greater than Max.</body></html> <html><head/><body>Minimum, Optimum, and Maximum melting temperatures (Celsius) for a primer oligo.Primer3 will not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions will try to pick primers with melting temperatures close to Opt.</body></html> <html><head/><body>Minimum, Optimum, and Maximum melting temperatures (Celsius) for a primer oligo.Primer3 will not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions will try to pick primers with melting temperatures close to Opt.</body></html> <html><head/><body>Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo.</body></html> <html><head/><body>Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo.</body></html> <html><head/><body>Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo.</body></html> <html><head/><body>Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo.</body></html> <html><head/><body>The minimum, optimum, and maximum melting temperature of the amplicon.Primer3 will not pick a product with melting temperature less than min or greater than max. If Opt is supplied and the Penalty Weights for Product Size are non-0 Primer3 will attempt to pick an amplicon with melting temperature close to Opt.</body></html> <html><head/><body>The minimum, optimum, and maximum melting temperature of the amplicon.Primer3 will not pick a product with melting temperature less than min or greater than max. If Opt is supplied and the Penalty Weights for Product Size are non-0 Primer3 will attempt to pick an amplicon with melting temperature close to Opt.</body></html> Max Tm difference Max Tm difference <html><head/><body>Maximum acceptable (unsigned) difference between the melting temperatures of the left and right primers.</body></html> <html><head/><body>Maximum acceptable (unsigned) difference between the melting temperatures of the left and right primers.</body></html> Table of thermodynamic parameters Table of thermodynamic parameters <html><head/><body>Specifies details of melting temperature calculation.First method uses the table of thermodynamic parameters from the paper [Breslauer KJ, et. al (1986), Proc Natl Acad Sci 83:4746-50 http://dx.doi.org/10.1073/pnas.83.11.3746]Second method (recommended) was suggested in the paper [SantaLucia JR (1998), Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460].</body></html> <html><head/><body>Specifies details of melting temperature calculation.First method uses the table of thermodynamic parameters from the paper [Breslauer KJ, et. al (1986), Proc Natl Acad Sci 83:4746-50 http://dx.doi.org/10.1073/pnas.83.11.3746]Second method (recommended) was suggested in the paper [SantaLucia JR (1998), Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460].</body></html> Breslauer et. al 1986 Breslauer et. al 1986 SantaLucia 1998 SantaLucia 1998 Primer Designer Primer Designer <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list of start,length pairs where start is the index of the first base of the excluded region, and length is its length. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Primer oligos may not overlap any region specified in this tag. The associated value must be a space-separated list of start,length pairs where start is the index of the first base of the excluded region, and length is its length. This tag is useful for tasks such as excluding regions of low sequence quality or for excluding regions containing repetitive elements such as ALUs or LINEs.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-separated list of start,length pairs where start is the index of the first base of a Target, and length is its length.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> If one or more Targets is specified then a legal primer pair must flank at least one of them. A Target might be a simple sequence repeat site (for example a CA repeat) or a single-base-pair polymorphism. The value should be a space-separated list of start,length pairs where start is the index of the first base of a Target, and length is its length.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> A list of product size ranges, for example: 150-250 100-300 301-400 Primer3 first tries to pick primers in the first range. If that is not possible, it goes to the next range and tries again. It continues in this way until it has either picked all necessary primers or until there are no more ranges. For technical reasons this option makes much lighter computational demands than the Product Size option.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> A list of product size ranges, for example: 150-250 100-300 301-400 Primer3 first tries to pick primers in the first range. If that is not possible, it goes to the next range and tries again. It continues in this way until it has either picked all necessary primers or until there are no more ranges. For technical reasons this option makes much lighter computational demands than the Product Size option.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> This selection indicates what mispriming library (if any) Primer3 should use to screen for interspersed repeats or for other sequence to avoid as a location for primers.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> This selection indicates what mispriming library (if any) Primer3 should use to screen for interspersed repeats or for other sequence to avoid as a location for primers.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum number of primer pairs to return. Primer pairs returned are sorted by their "quality", in other words by the value of the objective function (where a lower number indicates a better primer pair). Caution: setting this parameter to a large value will increase running time.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum number of primer pairs to return. Primer pairs returned are sorted by their "quality", in other words by the value of the objective function (where a lower number indicates a better primer pair). Caution: setting this parameter to a large value will increase running time.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum stability for the last five 3' bases of a left or right primer. Bigger numbers mean more stable 3' ends. The value is the maximum delta G (kcal/mol) for duplex disruption for the five 3' bases as calculated using the Nearest-Neighbor parameter values specified by the option of 'Table of thermodynamic parameters'. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum stability for the last five 3' bases of a left or right primer. Bigger numbers mean more stable 3' ends. The value is the maximum delta G (kcal/mol) for duplex disruption for the five 3' bases as calculated using the Nearest-Neighbor parameter values specified by the option of 'Table of thermodynamic parameters'. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed weighted similarity with any sequence in Mispriming Library.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed weighted similarity with any sequence in Mispriming Library.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed sum of similarities of a primer pair (one similarity for each primer) with any single sequence in Mispriming Library. Library sequence weights are not used in computing the sum of similarities.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed sum of similarities of a primer pair (one similarity for each primer) with any single sequence in Mispriming Library. Library sequence weights are not used in computing the sum of similarities.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed similarity to ectopic sites in the sequence from which you are designing the primers. The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed similarity to ectopic sites in the sequence from which you are designing the primers. The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed summed similarity of both primers to ectopic sites in the sequence from which you are designing the primers. The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowed summed similarity of both primers to ectopic sites in the sequence from which you are designing the primers. The scoring system is the same as used for Max Mispriming, except that an ambiguity code is never treated as a consensus.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Index of the first base of a start codon. This parameter allows Primer3 to select primer pairs to create in-frame amplicons e.g. to create a template for a fusion protein. Primer3 will attempt to select an in-frame left primer, ideally starting at or to the left of the start codon, or to the right if necessary. Negative values of this parameter are legal if the actual start codon is to the left of available sequence. If this parameter is non-negative Primer3 signals an error if the codon at the position specified by this parameter is not an ATG. A value less than or equal to -10^6 indicates that Primer3 should ignore this parameter. Primer3 selects the position of the right primer by scanning right from the left primer for a stop codon. Ideally the right primer will end at or after the stop codon. This parameter should be considered EXPERIMENTAL. Please check the output carefully; some erroneous inputs might cause an error in Primer3. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Index of the first base of a start codon. This parameter allows Primer3 to select primer pairs to create in-frame amplicons e.g. to create a template for a fusion protein. Primer3 will attempt to select an in-frame left primer, ideally starting at or to the left of the start codon, or to the right if necessary. Negative values of this parameter are legal if the actual start codon is to the left of available sequence. If this parameter is non-negative Primer3 signals an error if the codon at the position specified by this parameter is not an ATG. A value less than or equal to -10^6 indicates that Primer3 should ignore this parameter. Primer3 selects the position of the right primer by scanning right from the left primer for a stop codon. Ideally the right primer will end at or after the stop codon. This parameter should be considered EXPERIMENTAL. Please check the output carefully; some erroneous inputs might cause an error in Primer3. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> This section covers such settings as primer size, temperature, GC and other general settings.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> This section covers such settings as primer size, temperature, GC and other general settings.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> </body></html> Product Tm Product Tm Primer GC% Primer GC% <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Minimum, Optimum, and Maximum lengths (in bases) of a primer oligo. Primer3 will not pick primers shorter than Min or longer than Max, and with default arguments will attempt to pick primers close with size close to Opt. Min cannot be smaller than 1. Max cannot be larger than 36. (This limit is governed by maximum oligo size for which melting-temperature calculations are valid.) Min cannot be greater than Max.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Minimum, Optimum, and Maximum lengths (in bases) of a primer oligo. Primer3 will not pick primers shorter than Min or longer than Max, and with default arguments will attempt to pick primers close with size close to Opt. Min cannot be smaller than 1. Max cannot be larger than 36. (This limit is governed by maximum oligo size for which melting-temperature calculations are valid.) Min cannot be greater than Max.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Minimum, Optimum, and Maximum melting temperatures (Celsius) for a primer oligo. Primer3 will not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions will try to pick primers with melting temperatures close to Opt.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Minimum, Optimum, and Maximum melting temperatures (Celsius) for a primer oligo. Primer3 will not pick oligos with temperatures smaller than Min or larger than Max, and with default conditions will try to pick primers with melting temperatures close to Opt.</body></html> Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo. Minimum, Optimum, and Maximum percentage of Gs and Cs in any primer or oligo. <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The minimum, optimum, and maximum melting temperature of the amplicon. Primer3 will not pick a product with melting temperature less than min or greater than max. If Opt is supplied and the Penalty Weights for Product Size are non-0 Primer3 will attempt to pick an amplicon with melting temperature close to Opt.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The minimum, optimum, and maximum melting temperature of the amplicon. Primer3 will not pick a product with melting temperature less than min or greater than max. If Opt is supplied and the Penalty Weights for Product Size are non-0 Primer3 will attempt to pick an amplicon with melting temperature close to Opt.</body></html> Maximum acceptable (unsigned) difference between the melting temperatures of the left and right primers. Maximum acceptable (unsigned) difference between the melting temperatures of the left and right primers. <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Specifies details of melting temperature calculation. First method uses the table of thermodynamic parameters from the paper [Breslauer KJ, et. al (1986), Proc Natl Acad Sci 83:4746-50 http://dx.doi.org/10.1073/pnas.83.11.3746] Second method (recommended) was suggested in the paper [SantaLucia JR (1998) , Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460].</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Specifies details of melting temperature calculation. First method uses the table of thermodynamic parameters from the paper [Breslauer KJ, et. al (1986), Proc Natl Acad Sci 83:4746-50 http://dx.doi.org/10.1073/pnas.83.11.3746] Second method (recommended) was suggested in the paper [SantaLucia JR (1998) , Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460].</body></html> Max self complementarity Max self complementarity Max 3' self complementarity Max 3' self complementarity Inside target penalty Inside target penalty Outside target penalty Outside target penalty Concentration of monovalent cations Concentration of monovalent cations Max #N's Max #N's Max poly-X Max poly-X First base index First base index CG clamp CG clamp Concentration of divalent cations Concentration of divalent cations Salt correction formula Salt correction formula <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Specifies the salt correction formula for the melting temperature calculation. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Specifies the salt correction formula for the melting temperature calculation. </body></html> Schildkraut and Lifson 1965 Schildkraut and Lifson 1965 Owczarzy et. 2004 Owczarzy et. 2004 Concentration of dNTPs Concentration of dNTPs Annealing oligo concentration Annealing oligo concentration <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between left and right primers.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between left and right primers.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers. Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers. Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed.</body></html> Maximum number of unknown bases (N) allowable in any primer. Maximum number of unknown bases (N) allowable in any primer. The maximum allowable length of a mononucleotide repeat, for example AAAAAA. The maximum allowable length of a mononucleotide repeat, for example AAAAAA. <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Non-default values valid only for sequences with 0 or 1 target regions. If the primer is part of a pair that spans a target and does not overlap the target, then multiply this value times the number of nucleotide positions from the 3' end to the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include nearness to the target as a term in the objective function.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Non-default values valid only for sequences with 0 or 1 target regions. If the primer is part of a pair that spans a target and does not overlap the target, then multiply this value times the number of nucleotide positions from the 3' end to the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include nearness to the target as a term in the objective function.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> This parameter is the index of the first base in the input sequence. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> This parameter is the index of the first base in the input sequence. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Require the specified number of consecutive Gs and Cs at the 3' end of both the left and right primer. This parameter has no effect on the internal oligo if one is requested.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Require the specified number of consecutive Gs and Cs at the 3' end of both the left and right primer. This parameter has no effect on the internal oligo if one is requested.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> The millimolar (mM) concentration of monovalent salt cations (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo and primer melting temperatures. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> The millimolar (mM) concentration of monovalent salt cations (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo and primer melting temperatures. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> The millimolar concentration of divalent salt cations (usually MgCl^(2+)) in the PCR. Primer3 converts concentration of divalent cations to concentration of monovalent cations using following formula: [Monovalent cations] = [Monovalent cations] + 120*(([divalent cations] - [dNTP])^0.5) In addition, if the specified concentration of dNTPs is larger than the concentration of divalent cations then the effect of the divalent cations is not considered. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> The millimolar concentration of divalent salt cations (usually MgCl^(2+)) in the PCR. Primer3 converts concentration of divalent cations to concentration of monovalent cations using following formula: [Monovalent cations] = [Monovalent cations] + 120*(([divalent cations] - [dNTP])^0.5) In addition, if the specified concentration of dNTPs is larger than the concentration of divalent cations then the effect of the divalent cations is not considered. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> The millimolar concentration of the sum of all deoxyribonucleotide triphosphates. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> The millimolar concentration of the sum of all deoxyribonucleotide triphosphates. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> A value to use as nanomolar (nM) concentration of each annealing oligo over the course the PCR. Primer3 uses this argument to esimate oligo melting temperatures. The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research --0.5 microliters of 20 micromolar concentration for each primer in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius. The value of this parameter is less than the actual concentration of oligos in the initial reaction mix because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> A value to use as nanomolar (nM) concentration of each annealing oligo over the course the PCR. Primer3 uses this argument to esimate oligo melting temperatures. The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research --0.5 microliters of 20 micromolar concentration for each primer in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius. The value of this parameter is less than the actual concentration of oligos in the initial reaction mix because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Non-default values valid only for sequences with 0 or 1 target regions. If the primer is part of a pair that spans a target and overlaps the target, then multiply this value times the number of nucleotide positions by which the primer overlaps the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include overlap with the target as a term in the objective function.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Non-default values valid only for sequences with 0 or 1 target regions. If the primer is part of a pair that spans a target and overlaps the target, then multiply this value times the number of nucleotide positions by which the primer overlaps the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include overlap with the target as a term in the objective function.</body></html> Liberal base Liberal base Show debuging info Show debuging info Do not treat ambiguity codes in libraries as consensus Do not treat ambiguity codes in libraries as consensus Lowercase masking Lowercase masking Internal Oligo Internal Oligo <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Parameters governing choice of internal oligos are analogous to the parameters governing choice of primer pairs. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> Parameters governing choice of internal oligos are analogous to the parameters governing choice of primer pairs. </body></html> Hyb oligo excluded region Hyb oligo excluded region <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Middle oligos may not overlap any region specified by this tag. The associated value must be a space-separated list of <start>,<length> pairs, where <start> is the index of the first base of an excluded region, and <length> is its length. Often one would make Target regions excluded regions for internal oligos.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Middle oligos may not overlap any region specified by this tag. The associated value must be a space-separated list of <start>,<length> pairs, where <start> is the index of the first base of an excluded region, and <length> is its length. Often one would make Target regions excluded regions for internal oligos.</body></html> Hyb oligo size Hyb oligo size Hyb oligo Tm Hyb oligo Tm Hyb oligo GC% Hyb oligo GC% Min: Min: <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of Primer Size for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of Primer Size for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of Primer Tm for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of Primer Tm for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer GC% for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer GC% for the internal oligo. </body></html> Opt: Opt: <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of Primer GC% for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of Primer GC% for the internal oligo. </body></html> Max: Max: Hyb oligo self complementarity Hyb oligo self complementarity Max #Ns Max #Ns Hyb oligo mishyb library Hyb oligo mishyb library Hyb oligo min sequence quality Hyb oligo min sequence quality Hyb oligo conc of monovalent cations Hyb oligo conc of monovalent cations Hyb oligo conc of divalent cations Hyb oligo conc of divalent cations <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max self complemntarity for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max self complemntarity for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max #Ns for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max #Ns for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Similar to primer mispriming library, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather than priming from them.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Similar to primer mispriming library, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather than priming from them.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer minimum quality for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer minimum quality for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer concentration of monovalent cations for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer concentration of monovalent cations for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer concentration of divalent cations for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer concentration of divalent cations for the internal oligo. </body></html> Hyb oligo max 3' self complementarity Hyb oligo max 3' self complementarity Hyb oligo max poly-X Hyb oligo max poly-X Hyb oligo max mishyb Hyb oligo max mishyb <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max 3' self complementarity for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max 3' self complementarity for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max poly-X for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer max poly-X for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Similar to primer max library mispriming. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Similar to primer max library mispriming. </body></html> Hyb oligo [dNTP] Hyb oligo [dNTP] Hyb oligo DNA concentration Hyb oligo DNA concentration <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer concentration of the dNTPs for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Гибридизация олиго [dNTP]. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Equivalent parameter of primer DNA concentration for the internal oligo. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> ДНК концентрация гибридизации олиго. </body></html> Penalty Weights Penalty Weights <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> This section describes "penalty weights", which allow the user to modify the criteria that Primer3 uses to select the "best" primers. There are two classes of weights: for some parameters there is a 'Lt' (less than) and a 'Gt' (greater than) weight. These are the weights that Primer3 uses when the value is less or greater than (respectively) the specified optimum.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> This section describes "penalty weights", which allow the user to modify the criteria that Primer3 uses to select the "best" primers. There are two classes of weights: for some parameters there is a 'Lt' (less than) and a 'Gt' (greater than) weight. These are the weights that Primer3 uses when the value is less or greater than (respectively) the specified optimum.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:10pt; font-weight:400; font-style:normal;"> For Primers</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:10pt; font-weight:400; font-style:normal;"> For Primers</body></html> Tm Tm Size Size GC% GC% Lt: Lt: <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for corressponding parameter of a primer less than (Lt) or greater than (Gt) the optimal value. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for corressponding parameter of a primer less than (Lt) or greater than (Gt) the optimal value. </body></html> Gt: Gt: Self complementarity Self complementarity 3' self complementarity 3' self complementarity #N's N Mispriming Mispriming Sequence quality Sequence quality End sequence quality End sequence quality Position penalty Position penalty End stability End stability Template mispriming Template mispriming <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for a primer parameter different from predefined optimum value. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for a primer parameter different from predefined optimum value. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Determines the overall weight of the position penalty in calculating the penalty for a primer.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Determines the overall weight of the position penalty in calculating the penalty for a primer.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:10pt; font-weight:400; font-style:normal;"> For Primer Pairs</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:10pt; font-weight:400; font-style:normal;"> For Primer Pairs</body></html> Product size Product size <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for corressponding parameter of a primer pair less than (Lt) or greater than (Gt) the optimal value. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for corressponding parameter of a primer pair less than (Lt) or greater than (Gt) the optimal value. </body></html> Tm difference Tm difference Any complementarity Any complementarity 3' complementarity 3' complementarity Pair mispriming Pair mispriming Primer penalty weight Primer penalty weight Hyb oligo penalty weight Hyb oligo penalty weight Primer pair template mispriming weight Primer pair template mispriming weight <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for a primer pair parameter different from predefined optimum value.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for a primer pair parameter different from predefined optimum value.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:10pt; font-weight:400; font-style:normal;"> For Hyb Oligos</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:10pt; font-weight:400; font-style:normal;"> For Hyb Oligos</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for corressponding parameter of a internal oligo less than (Lt) or greater than (Gt) the optimal value. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for corressponding parameter of a internal oligo less than (Lt) or greater than (Gt) the optimal value. </body></html> Hyb oligo #N's Hyb oligo #N's Hyb oligo mishybing Hyb oligo mishybing Hyb oligo sequence quality Hyb oligo sequence quality <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for an internal oligo parameter different from predefined optimum value.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> Penalty weight for an internal oligo parameter different from predefined optimum value.</body></html> RT-PCR RT-PCR <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:9pt; font-weight:400; font-style:normal;"> In this section user can specify if primer product must overlap exon-exon junction or span intron. This only applies when designing primers for a cDNA (mRNA) sequence with annotated exons. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:9pt; font-weight:400; font-style:normal;"> RT-PCR. </body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:9pt; font-weight:400; font-style:normal;"> This feature allows to specify if primer product must span intron-exon boundaries. Checking this option will result in ignoring Excluded and Target Regions from Main section.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'Sans Serif'; font-size:9pt; font-weight:400; font-style:normal;"> This feature allows to specify if primer product must span intron-exon boundaries. Checking this option will result in ignoring Excluded and Target Regions from Main section.</body></html> Design primers for RT-PCR analysis Design primers for RT-PCR analysis Exon annotation name: Exon annotation name: <html><head/><body>The name of the annotation which defines the exons in the mRNA sequence.</body></html> <html><head/><body>The name of the annotation which defines the exons in the mRNA sequence.</body></html> exon exon Minimum exon junction overlap size Minimum exon junction overlap size <html><head/><body>If the range is set, primer search will be restricted to selected exons. For example:1-5If the range is larger than actual exon range or the starting exon number exceeds number of exons, error message is shown. </body></html> <html><head/><body>If the range is set, primer search will be restricted to selected exons. For example:1-5If the range is larger than actual exon range or the starting exon number exceeds number of exons, error message is shown. </body></html> <html><head/><body>This number of found primer pairs wil bel queried to check if they fullfill the requirements for RTPCR.</body></html> <html><head/><body>This number of found primer pairs wil bel queried to check if they fullfill the requirements for RTPCR.</body></html> <html><head/><body>In this section user can specify sequence quality of target sequence and related parameters.</body></html> <html><head/><body>In this section user can specify sequence quality of target sequence and related parameters.</body></html> <html><head/><body>A list of space separated integers. There must be exactly one integer for each base in the Source Sequence if this argument is non-empty. High numbers indicate high confidence in the base call at that position and low numbers indicate low confidence in the base call at that position.</body></html> <html><head/><body>A list of space separated integers. There must be exactly one integer for each base in the Source Sequence if this argument is non-empty. High numbers indicate high confidence in the base call at that position and low numbers indicate low confidence in the base call at that position.</body></html> <html><head/><body>The minimum sequence quality allowed within a primer.</body></html> <html><head/><body>The minimum sequence quality allowed within a primer.</body></html> <html><head/><body>The minimum sequence quality allowed within the 3' pentamer of a primer.</body></html> <html><head/><body>The minimum sequence quality allowed within the 3' pentamer of a primer.</body></html> <html><head/><body>The minimum legal sequence quality.</body></html> <html><head/><body>The minimum legal sequence quality.</body></html> <html><head/><body>The maximum legal sequence quality.</body></html> <html><head/><body>The maximum legal sequence quality.</body></html> <html><head/><body>This section allows to set output annotations' settings.</body></html> <html><head/><body>This section allows to set output annotations' settings.</body></html> Help Help Save settings Save settings Load settings Load settings Reset form Reset form Pick primers Pick primers mRNA sequence mRNA sequence <html><head/><body>Specifies the salt correction formula for the melting temperature calculation.</body></html> <html><head/><body>Specifies the salt correction formula for the melting temperature calculation.</body></html> <html><head/><body>The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between left and right primers.</body></html> <html><head/><body>The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between left and right primers.</body></html> <html><head/><body>The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers.Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed.</body></html> <html><head/><body>The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers.Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed.</body></html> <html><head/><body>Maximum number of unknown bases (N) allowable in any primer.</body></html> <html><head/><body>Maximum number of unknown bases (N) allowable in any primer.</body></html> <html><head/><body>The maximum allowable length of a mononucleotide repeat, for example AAAAAA.</body></html> <html><head/><body>The maximum allowable length of a mononucleotide repeat, for example AAAAAA.</body></html> <html><head/><body>Non-default values valid only for sequences with 0 or 1 target regions.If the primer is part of a pair that spans a target and does not overlap the target, then multiply this value times the number of nucleotide positions from the 3' end to the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include nearness to the target as a term in the objective function.</body></html> <html><head/><body>Non-default values valid only for sequences with 0 or 1 target regions.If the primer is part of a pair that spans a target and does not overlap the target, then multiply this value times the number of nucleotide positions from the 3' end to the (unique) target to get the 'position penalty'. The effect of this parameter is to allow Primer3 to include nearness to the target as a term in the objective function.</body></html> <html><head/><body>This parameter is the index of the first base in the input sequence.</body></html> <html><head/><body>This parameter is the index of the first base in the input sequence.</body></html> <html><head/><body>Require the specified number of consecutive Gs and Cs at the 3' end of both the left and right primer.This parameter has no effect on the internal oligo if one is requested.</body></html> <html><head/><body>Require the specified number of consecutive Gs and Cs at the 3' end of both the left and right primer.This parameter has no effect on the internal oligo if one is requested.</body></html> <html><head/><body>The millimolar (mM) concentration of monovalent salt cations (usually KCl) in the PCR.Primer3 uses this argument to calculate oligo and primer melting temperatures.</body></html> <html><head/><body>The millimolar (mM) concentration of monovalent salt cations (usually KCl) in the PCR.Primer3 uses this argument to calculate oligo and primer melting temperatures.</body></html> <html><head/><body>The millimolar concentration of divalent salt cations (usually MgCl^(2+)) in the PCR.Primer3 converts concentration of divalent cations to concentration of monovalent cations using following formula:[Monovalent cations] = [Monovalent cations] + 120*(([divalent cations] - [dNTP])^0.5)In addition, if the specified concentration of dNTPs is larger than the concentration of divalent cations then the effect of the divalent cations is not considered.</body></html> <html><head/><body>The millimolar concentration of divalent salt cations (usually MgCl^(2+)) in the PCR.Primer3 converts concentration of divalent cations to concentration of monovalent cations using following formula:[Monovalent cations] = [Monovalent cations] + 120*(([divalent cations] - [dNTP])^0.5)In addition, if the specified concentration of dNTPs is larger than the concentration of divalent cations then the effect of the divalent cations is not considered.</body></html> <html><head/><body>The millimolar concentration of the sum of all deoxyribonucleotide triphosphates.</body></html> <html><head/><body>The millimolar concentration of the sum of all deoxyribonucleotide triphosphates.</body></html> <html><head/><body>A value to use as nanomolar (nM) concentration of each annealing oligo over the course the PCR.Primer3 uses this argument to esimate oligo melting temperatures.The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research --0.5 microliters of 20 micromolar concentration for each primer in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius.The value of this parameter is less than the actual concentration of oligos in the initial reaction mix because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.</body></html> <html><head/><body>A value to use as nanomolar (nM) concentration of each annealing oligo over the course the PCR.Primer3 uses this argument to esimate oligo melting temperatures.The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research --0.5 microliters of 20 micromolar concentration for each primer in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius.The value of this parameter is less than the actual concentration of oligos in the initial reaction mix because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.</body></html> <html><head/><body>Non-default values valid only for sequences with 0 or 1 target regions.If the primer is part of a pair that spans a target and overlaps the target, then multiply this value times the number of nucleotide positions by which the primer overlaps the (unique) target to get the 'position penalty'.The effect of this parameter is to allow Primer3 to include overlap with the target as a term in the objective function.</body></html> <html><head/><body>Non-default values valid only for sequences with 0 or 1 target regions.If the primer is part of a pair that spans a target and overlaps the target, then multiply this value times the number of nucleotide positions by which the primer overlaps the (unique) target to get the 'position penalty'.The effect of this parameter is to allow Primer3 to include overlap with the target as a term in the objective function.</body></html> <html><head/><body>Parameters governing choice of internal oligos are analogous to the parameters governing choice of primer pairs.</body></html> <html><head/><body>Parameters governing choice of internal oligos are analogous to the parameters governing choice of primer pairs.</body></html> <html><head/><body>Middle oligos may not overlap any region specified by this tag. The associated value must be a space-separated list of<start>,<length>pairs, where <start> is the index of the first base of an excluded region, and <length> is its length. Often one would make Target regions excluded regions for internal oligos.</body></html> <html><head/><body>Middle oligos may not overlap any region specified by this tag. The associated value must be a space-separated list of<start>,<length>pairs, where <start> is the index of the first base of an excluded region, and <length> is its length. Often one would make Target regions excluded regions for internal oligos.</body></html> <html><head/><body>Equivalent parameter of Primer Size for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of Primer Size for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of Primer Tm for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of Primer Tm for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer GC% for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer GC% for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of Primer GC% for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of Primer GC% for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max self complemntarity for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max self complemntarity for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max #Ns for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max #Ns for the internal oligo.</body></html> <html><head/><body>Similar to primer mispriming library, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather than priming from them.</body></html> <html><head/><body>Similar to primer mispriming library, except that the event we seek to avoid is hybridization of the internal oligo to sequences in this library rather than priming from them.</body></html> <html><head/><body>Equivalent parameter of primer minimum quality for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer minimum quality for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer concentration of monovalent cations for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer concentration of monovalent cations for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer concentration of divalent cations for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer concentration of divalent cations for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max 3' self complementarity for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max 3' self complementarity for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max poly-X for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer max poly-X for the internal oligo.</body></html> <html><head/><body>Similar to primer max library mispriming.</body></html> <html><head/><body>Similar to primer max library mispriming.</body></html> <html><head/><body>Equivalent parameter of primer concentration of the dNTPs for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer concentration of the dNTPs for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer DNA concentration for the internal oligo.</body></html> <html><head/><body>Equivalent parameter of primer DNA concentration for the internal oligo.</body></html> <html><head/><body>This section describes "penalty weights", which allow the user to modify the criteria that Primer3 uses to select the "best" primers.There are two classes of weights: for some parameters there is a 'Lt' (less than) and a 'Gt' (greater than) weight. These are the weights that Primer3 uses when the value is less or greater than (respectively) the specified optimum.</body></html> <html><head/><body>This section describes "penalty weights", which allow the user to modify the criteria that Primer3 uses to select the "best" primers.There are two classes of weights: for some parameters there is a 'Lt' (less than) and a 'Gt' (greater than) weight. These are the weights that Primer3 uses when the value is less or greater than (respectively) the specified optimum.</body></html> Primers <html><head/><body>Penalty weight for corressponding parameter of a primer less than (Lt) or greater than (Gt) the optimal value.</body></html> <html><head/><body>Penalty weight for corressponding parameter of a primer less than (Lt) or greater than (Gt) the optimal value.</body></html> <html><head/><body>Penalty weight for a primer parameter different from predefined optimum value.</body></html> <html><head/><body>Penalty weight for a primer parameter different from predefined optimum value.</body></html> <html><head/><body>Determines the overall weight of the position penalty in calculating the penalty for a primer.</body></html> <html><head/><body>Determines the overall weight of the position penalty in calculating the penalty for a primer.</body></html> Primer Pairs <html><head/><body>Penalty weight for corressponding parameter of a primer pair less than (Lt) or greater than (Gt) the optimal value.</body></html> <html><head/><body>Penalty weight for corressponding parameter of a primer pair less than (Lt) or greater than (Gt) the optimal value.</body></html> <html><head/><body>Penalty weight for a primer pair parameter different from predefined optimum value.</body></html> <html><head/><body>Penalty weight for a primer pair parameter different from predefined optimum value.</body></html> Hyb Oligos <html><head/><body>Penalty weight for corressponding parameter of a internal oligo less than (Lt) or greater than (Gt) the optimal value.</body></html> <html><head/><body>Penalty weight for corressponding parameter of a internal oligo less than (Lt) or greater than (Gt) the optimal value.</body></html> <html><head/><body>Penalty weight for an internal oligo parameter different from predefined optimum value.</body></html> <html><head/><body>Penalty weight for an internal oligo parameter different from predefined optimum value.</body></html> <html><head/><body>In this section user can specify if primer product must overlap exon-exon junction or span intron. This only applies when designing primers for a cDNA (mRNA) sequence with annotated exons.</body></html> <html><head/><body>In this section user can specify if primer product must overlap exon-exon junction or span intron. This only applies when designing primers for a cDNA (mRNA) sequence with annotated exons.</body></html> <html><head/><body>This feature allows to specify if primer product must span intron-exon boundaries.Checking this option will result in ignoring Excluded and Target Regions from Main section.</body></html> <html><head/><body>This feature allows to specify if primer product must span intron-exon boundaries.Checking this option will result in ignoring Excluded and Target Regions from Main section.</body></html> <html><head/><body>The name of the annotation which defines the exons in the mRNA sequence.</body></html> <html><head/><body>The name of the annotation which defines the exons in the mRNA sequence.</body></html> At 5' side (bp): At 5' side (bp): At 3'side (bp) At 3'side (bp) Exon range: Exon range: <html><head/><body>If the range is set, primer search will be restricted to selected exons. For example:1-5If the range is larger than actual exon range or the starting exon number exceeds number of exons, error message is shown. </body></html> <html><head/><body>If the range is set, primer search will be restricted to selected exons. For example:1-5If the range is larger than actual exon range or the starting exon number exceeds number of exons, error message is shown. </body></html> Primer product must span at least one intron on the corresponding genomic DNA Primer product must span at least one intron on the corresponding genomic DNA Max number of pairs to query: Max number of pairs to query: <html><head/><body>This number of found primer pairs wil bel queried to check if they fullfill the requirements for RTPCR</body></html> <html><head/><body>This number of found primer pairs wil bel queried to check if they fullfill the requirements for RTPCR</body></html> Sequence Quality Sequence Quality <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> In this section user can specify sequence quality of target sequence and related parameters.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> In this section user can specify sequence quality of target sequence and related parameters.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> A list of space separated integers. There must be exactly one integer for each base in the Source Sequence if this argument is non-empty. High numbers indicate high confidence in the base call at that position and low numbers indicate low confidence in the base call at that position.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:8.25pt; font-weight:400; font-style:normal;"> A list of space separated integers. There must be exactly one integer for each base in the Source Sequence if this argument is non-empty. High numbers indicate high confidence in the base call at that position and low numbers indicate low confidence in the base call at that position.</body></html> Min sequence quality: Min sequence quality: The minimum sequence quality allowed within a primer. The minimum sequence quality allowed within a primer. Min 3' sequence quality: Min 3' sequence quality: The minimum sequence quality allowed within the 3' pentamer of a primer. The minimum sequence quality allowed within the 3' pentamer of a primer. <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> This section allows to set output annotations' settings.</body></html> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0//EN" "http://www.w3.org/TR/REC-html40/strict.dtd"> <html><head><meta name="qrichtext" content="1" /><style type="text/css"> p, li { white-space: pre-wrap; } </style></head><body style=" font-family:'MS Shell Dlg 2'; font-size:7.8pt; font-weight:400; font-style:normal;"> This section allows to set output annotations' settings.</body></html> Sequence quality range min: Sequence quality range min: The minimum legal sequence quality. The minimum legal sequence quality. Sequence quality range max: Sequence quality range max: The maximum legal sequence quality. The maximum legal sequence quality. Result Settings Result Settings QObject Primer Primer PCR primer design PCR primer design Excluded regions Excluded regions Targets Targets Product size ranges Product size ranges Number to return Number to return Max repeat mispriming Max repeat mispriming Max template mispriming Max template mispriming Max 3' stability Max 3' stability Pair max repeat mispriming Pair max repeat mispriming Pair max template mispriming Pair max template mispriming U2::FindExonRegionsTask Failed to search for exon annotations. The sequence %1 doesn't have any related annotations. Failed to search for exon annotations. The sequence %1 doesn't have any related annotations. U2::GTest Illegal TARGET value: %1 Illegal TARGET value: %1 Illegal PRIMER_DEFAULT_PRODUCT value: %1 Illegal PRIMER_DEFAULT_PRODUCT value: %1 Illegal PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION value: %1 Illegal PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION value: %1 Illegal INCLUDED_REGION value: %1 Illegal INCLUDED_REGION value: %1 Illegal EXCLUDED_REGION value: %1 Illegal EXCLUDED_REGION value: %1 Unrecognized PRIMER_TASK Unrecognized PRIMER_TASK Contradiction in primer_task definition Contradiction in primer_task definition Missing SEQUENCE tag Missing SEQUENCE tag Error in sequence quality data Error in sequence quality data Sequence quality data missing Sequence quality data missing PRIMER_PAIRS_NUMBER is incorrect. Expected:%2, but Actual:%3 PRIMER_PAIRS_NUMBER is incorrect. Expected:%2, but Actual:%3 %1 is incorrect. Expected:%2,%3, but Actual:NULL %1 is incorrect. Expected:%2,%3, but Actual:NULL %1 is incorrect. Expected:NULL, but Actual:%2,%3 %1 is incorrect. Expected:NULL, but Actual:%2,%3 %1 is incorrect. Expected:%2,%3, but Actual:%4,%5 %1 is incorrect. Expected:%2,%3, but Actual:%4,%5 %1 is incorrect. Expected:%2, but Actual:%3 %1 is incorrect. Expected:%2, but Actual:%3 U2::Primer3ADVContext Primer3... Primer3... Error Error Cannot create an annotation object. Please check settings Cannot create an annotation object. Please check settings U2::Primer3Dialog NONE NONE HUMAN HUMAN RODENT_AND_SIMPLE RODENT_AND_SIMPLE RODENT RODENT DROSOPHILA DROSOPHILA Start Codon Position Start Codon Position Product Min Tm Product Min Tm Product Opt Tm Product Opt Tm Product Max Tm Product Max Tm Inside Penalty Inside Penalty Internal Oligo Opt Tm Internal Oligo Opt Tm Excluded Regions Excluded Regions Internal Oligo Excluded Regions Internal Oligo Excluded Regions Included region is too small for current product size ranges Included region is too small for current product size ranges Product Size Ranges Product Size Ranges Sequence Quality Sequence Quality Cannot create an annotation object. Please check settings. Save primer settings Save primer settings Load settings Load settings Can not load settings file: invalid format. Can not load settings file: invalid format. Opt GC% Opt GC% Targets Targets Sequence quality list length must be equal to the sequence length Sequence quality list length must be equal to the sequence length Can't pick hyb oligo and only one primer Can't pick hyb oligo and only one primer Nothing to pick Nothing to pick The field '%1' has invalid value The field '%1' has invalid value U2::Primer3Plugin Primer3 Primer3 Integrated tool for PCR primers design. Integrated tool for PCR primers design. U2::Primer3Task Pick primers task Pick primers U2::Primer3ToAnnotationsTask Search primers to annotations Search primers to annotations Failed to find any exon annotations associated with the sequence %1.Make sure the provided sequence is cDNA and has exonic structure annotated Failed to find any exon annotations associated with the sequence %1.Make sure the provided sequence is cDNA and has exonic structure annotated The first exon from the selected range [%1,%2] is larger the number of exons (%2). Please set correct exon range. The first exon from the selected range [%1,%2] is larger the number of exons (%2). Please set correct exon range. The the selected exon range [%1,%2] is larger the number of exons (%2). Please set correct exon range. The the selected exon range [%1,%2] is larger the number of exons (%2). Please set correct exon range. U2::QDPrimerActor %1 invalid input. Excluded regions. %1 invalid input. Excluded regions. %1 invalid input. Targets. %1 invalid input. Targets. %1 invalid input. Product size ranges. %1 invalid input. Product size ranges.