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Research Article Improving the PCR protocol to amplify a repetitive DNA sequence.

2017; Research Foundation of Ribeirão Preto; Volume: 16; Issue: 3 Linguagem: Inglês

10.4238/gmr16039796

1676-5680

Job van Riet, Leonardo Rocha Vidal Ramos, Randolph V. Lewis, Luís Fernando Marins,

Antimicrobial Peptides and Activities

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited.This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification.The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers.These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle.Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus.We describe the amplification of the MaSp1 gene through minor changes in the PCR program.The results show that a denaturation temperature of 98°C