Polymerase preference index
Disclosed is a method for calculating a Polymerase Preference Index (PPI) for potential primers for DNA sequencing and/or amplification, and thereby increasing the efficiency of DNA sequencing and/or amplification performed using primers selected according to their PPI number.
1. A method for producing primers to improve the efficiency of amplification by a polymerase, the method comprising:
a) performing multiple amplifications using randomly-generated primers to amplify target sequences using a polymerase, each randomly-generated primer comprising a sequence of about 20 nucleotides, the 3′ end of each primer binding to a 6-mer of a 10-nucleotide sequence in a target sequence, wherein the remaining 4-mer of the 10-nucleotide sequence comprises a runway sequence, thereby producing amplicons;
b) selecting at least one primer with the greatest polymerase preference index, the polymerase preference index determined by the multiple of a first percentage, representing the number of times a 6-mer of the 10-nucleotide sequence is found at the 5-prime ends of amplicons from the multiple amplifications divided by the number of times the 6-mer is found elsewhere in the amplicons, and a second percentage representing the number of times that a 4-mer of the 10-nucleotide sequence is found at the 3-prime end of a the 6-mer in the amplicons divided by the number of times that the 4-mer is found elsewhere in the amplicon sequences; and
c) performing amplification of at least one target sequence using at least one primer selected with the greatest polymerase preference index.
2. The method of claim 1 , wherein at least two primers with the greatest polymerase preference index are selected.
3. The method of claim 2 , wherein the lengths of the at least two primers with the greatest polymerase preference index are increased or decreased to produce primers with approximately the same melting temperatures.