Improving the PCR protocol to amplify a repetitive DNA sequence

• Published in: Genetics and molecular research 2017-09, Vol.16 (3), p.1
• Main Authors: Riet, J, Ramos, L R V, Lewis, R V, Marins, L F
• Format: Article
• Language: English
• Subjects: Alanine; Amino acid sequence; Base Composition; Conserved sequence; Denaturation; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA structure; DNA-directed DNA polymerase; Fibroins - genetics; Glycine; Inverted Repeat Sequences; MASP-1 protein; Nucleic Acid Denaturation; Nucleotide sequence; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; Repetitive Sequences, Amino Acid
• ISSN: 1676-5680
• DOI: 10.4238/gmr16039796

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 is the key determinant in the amplification of the MaSp1 partial gene sequence.