RESUMO
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.
Assuntos
DNA/química , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Aminoácidos , Composição de Bases , Fibroínas/genética , Sequências Repetidas Invertidas , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase/normasRESUMO
The aim of this study was to determine the isotopic-turnover rate (RIT ) and trophic-discrimination factor (FTD ) in muscle tissues of Lebranche mullet Mugil liza fed an experimental diet (δ13 C = -27·1; δ15 N = 1·0). Juvenile M. liza exhibited a relatively fast RIT , with a half-life (t50 ) of only 16 and 14 days for δ13 C and δ15 N respectively and a nearly complete isotopic turnover (t95 ) of 68 and 60 days for δ13 C and δ15 N.