A nested primer for amplification of mitochondrial control region of white-crowned brook robin and method for amplification, cloning and sequencing

Abstract

The invention discloses a nested primer for amplification, cloning and sequencing of the mitochondrial control region of the white-crowned brook robin and a method for amplification, cloning and sequencing, and belongs to the field of biotechnology. 2 times of PCR amplification, the product of the first round of PCR can be directly sequenced, and the fragments amplified by the second round of PCR combined with agarose gel recovery and purification rapid cloning technology can greatly improve the purity and concentration of the sequencing template to eliminate the sequencing process The heterogeneity in the polynucleotide sequence effectively overcomes the problem of sequencing failure caused by the polynucleotide sequence. The combination of direct sequencing and cloning sequencing greatly reduces the workload of the operator. The present invention uses amplified nested primers with less secondary structure and stable conformation. , the 3' ends of the six nested primers avoid the introduction of A bases, thus greatly reducing the risk of non-specific amplification.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to the technical field of amplification and cloning, and in particular relates to a nested primer for the amplification, cloning and sequencing methods of the white-crowned brook robin mitochondrial control region. Background technique [0002] As an important wild bird, the white-crowned brook (Chaimarrornis leucocephalus) has a wide distribution range. The study of its biodiversity by molecular means is helpful to guide the protection of the wild resources of the white-crowned brook. [0003] White-crowned Brookbird belongs to the Passeriformes Flycataceae taxonomically, with various species, diverse forms, and wide distribution; there is a big controversy about the phylogenetic relationship among the birds of this family or within the genera. Compared with the nuclear genome, the mitochondrial genome of birds has the following advantages in phylogenetic analysis: small molecular we...

AI Technical Summary

Problems solved by technology

[0004] There are a large number of polynucleotide tandem repeat sequences in the mitochondrial control region of the white-crowned brook robin. During the DNA sequencing process, the effective read length of the sequencing reaction is 600-800bp, and this repeated polynucleotide sequence can Causes changes in the secondary conformation of DNA, resulting in the failure of normal sequencing, premature signal failure, and interruption of sequencing reactions leading to sequencing failures

At the same time, due to the high degree of methylation in mitochondrial DNA, PCR primers may have poor specificity and are not suitable for sequencing, resulting in sequencing failure and the inability to obtain molecular data for research

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