start,lengthpairs where start is the index of the first base of a Target, and length is its length.
start,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.
Primer3 uses the oligo melting temperature formula given in Rychlik, Spencer and Rhoads, Nucleic Acids Research, vol 18, num 12, pp 6409-6412 and Breslauer, Frank, Bloeker and Marky, Proc. Natl. Acad. Sci. USA, vol 83, pp 3746-3750. Please refer to the former paper for background discussion.
5' ATCGNA 3' || | | 3' TA-CGT 5'is allowed (and yields a score of 1.75), but the alignment
5' ATCCGNA 3' || | | 3' TA--CGT 5'is not considered. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable local alignment between two oligos.
5' ATGCCCTAGCTTCCGGATG 3' ||| ||||| 3' AAGTCCTACATTTAGCCTAGT 5'or
5` AGGCTATGGGCCTCGCGA 3' |||||| 3' AGCGCTCCGGGTATCGGA 5'The scoring system is as for the Max Complementarity argument. In the examples above the scores are 7.00 and 6.00 respectively. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable 3'-anchored global alignment between two oligos. In order to estimate 3'-anchored global alignments for candidate primers and primer pairs, Primer assumes that the sequence from which to choose primers is presented 5'->3'. It is nonsensical to provide a larger value for this parameter than for the Maximum (local) Complementarity parameter because the score of a local alignment will always be at least as great as the score of a global alignment.