Riet J, Ramos L R V, Lewis R V, Marins L F
Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, , , Brasil.
Departamento de Produção Animal, Instituto de Zootecnia, Universidade Federal Rural do Rio de Janeiro, Seropédica, RJ, Brasil.
Genet Mol Res. 2017 Sep 21;16(3):gmr-16-03-gmr.16039796. 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.
尽管基于聚合酶链式反应（PCR）的技术已成为分子和基因研究领域的重要工具，但重复DNA序列的扩增受到限制。这是由于扩增序列的截短性质，其在基于DNA聚合酶的扩增过程中也容易出错。重复DNA的复杂结构可形成发夹环，这会促进聚合酶与模板解离，损害完全扩增，并导致形成作为大引物的不完全片段。这些大引物与其他序列退火，在每个PCR循环中产生意外片段。我们的基因模型MaSp1长1037个碱基对，GC含量为68%，其氨基酸序列的特征是聚丙氨酸-甘氨酸基序，代表重复密码子共有序列。我们描述了通过对PCR程序进行微小改变来扩增MaSp1基因。结果表明，98°C的变性温度是扩增MaSp1部分基因序列的关键决定因素。
