Method for separating long terminal repeats of retrotransposons

Abstract

The invention discloses a method for separating long terminal repeats of retrotransposons. Genome DNA is taken as a template, an RNaseH1 primer is respectively combined with different annealing control primers for first polymerase chain reaction (PCR) reaction, the first PCR product is diluted to serve as a template, an RNaseH2 primer and a universal primer UP are subjected to secondary PCR reaction amplification, conversion and extraction of plasma DNA are performed, and a sequence is subjected to sequencing to obtain an amino acid sequence of RNaseH enzyme, and proteins are encoded. Compared with the common PCR technology, the method has the advantages that: the operation is simple, long terminal repeat regions of the retrotransposons are easily aligned, and the pertinence is high; compared with a flow type magnetic microbead inverse hybrid method, the method has the advantages of saving hybrid, eluting, screening and other processes, simplifying the flow and having high efficiency;and compared with other separation methods, the method has the advantages of reducing time consumption, quickly obtaining target segments, cloning the long terminal repeats in short time, and reducing false positive rate.

Description

technical field [0001] The present invention relates to a method for isolating retrotransposons, specifically Ty1 -copia A method for retrotransposon-like long terminal repeats. Background technique [0002] Retrotransposons have the characteristics of widespread existence, high copy number, high heterogeneity, and irreversible insertion sites, and play an important role in the size, structure, function, and evolution of the genome. It is an important tool for the study of its function, biodiversity and phylogenetic evolution. However, its wide application is limited by the isolation of full-length sequences, especially the acquisition of the long terminal repeat sequence LTR; retrotransposons are the most abundant and widely distributed transposons in eukaryotes, including long terminal repeats Repeated sequences, LTR and non-long terminal repeats, non-LTR retrotransposons (Grandbastien, 1992; Bennetzen, 1996; Kunze, 1997), LTR retrotransposons are the most widely distrib...

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Problems solved by technology

[0004]The LTR sequence is relatively conservative but has a certain degree of variation. It is currently an ideal target site for primer development in retrotransposon molecular markers, but the reverse The acquisition of primers in the analysis of transcriptional transposon molecular markers needs to know the nucleic acid sequence in advance, and it is difficult and expensive to isolate the full-length or partial fragments of retrotransposons from specific species, which limits its wide application. At present, magnetic bead enrichment method, inhibition PCR method, SiteFinding PCR method and other methods are commonly used to isolate LTR sequences. However, these methods have the disadvantages of complicated operation and high false positive rate. At the same time, high false positive rate is one of the main problems of nested PCR. Due to the short random primers, between 10-13bp, an annealing temperature of 40-45°C is usually required for PCR. Low-temperature annealing can easily lead to non-specific binding of primers and templates, resulting in high false positives and poor reproducibility of results.

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