Improving sequencing quality from PCR products containing long mononucleotide repeats

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Stutter products are common artifacts in PCR amplification of genetic markers with mononucleotide simple sequence repeats.
• Accurate determination of nucleotide sequence and allele size is crucial, yet progress in reducing PCR stutter products has been limited.

Purpose of the Study:

• To investigate methods for decreasing stutter product formation during PCR amplification.
• To evaluate the impact of various PCR conditions and polymerases on sequence quality for mononucleotide repeats.

Main Methods:

• Tested effects of lowered extension temperatures, co-solutes, PCR cycle number, and different DNA polymerases.
• Analyzed sequence quality for DNA sequences containing mononucleotide A/T repeats (10-17 bp).

Main Results:

• Sequence quality of mononucleotide repeats up to 15 bp was greatly improved using proofreading DNA polymerases fused to nonspecific dsDNA binding domains.
• The number of nucleotides interacting with DNA polymerase appears critical in reducing slipped-strand mispairings.

Conclusions:

• Proofreading DNA polymerases fused to dsDNA binding domains are effective in improving PCR sequence quality for mononucleotide repeats.
• Optimizing DNA polymerase interaction with nucleotides can minimize stutter product formation.