Reagent for improving replication efficiency of single-stranded nucleic acid molecule with secondary structure, nucleic acid fragment replication method and application

Abstract

The invention provides a reagent for improving the replication efficiency of a single-stranded nucleic acid molecule having a secondary structure, wherein the reagent is an oligonucleotide chain, andsequentially comprises a specific sequence for complementarily pairing with a first stem arm sequence of the secondary structure from the side where the 5' end of a single-stranded nucleic acid molecule template is located, and a terminal modification structure for preventing the 3' end of the specific sequence from extending through nucleic acid polymerase, wherein the first stem arm sequence isall or at least one end sequence segment of a stem arm, close to one side of the 5' end of a template, of the secondary structure. The invention also provides a nucleic acid fragment replication method and an application. According to the reagent disclosed by the invention, in a single-stranded nucleic acid replication process, a specific sequence competes with a complementary paired secondary structure in the template to form a hybridization product of the specific sequence and a partial region of the secondary structure of the template, and meanwhile, the secondary structure in the templateis unlinked; furthermore, the hindering effect of the secondary structure in the template on the nucleic acid polymerase is eliminated in the primer extension process.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a reagent for improving the replication efficiency of a single-stranded nucleic acid molecule with an intramolecular secondary structure, a nucleic acid fragment replication method, and an application. Background technique [0002] When a single-stranded nucleic acid has a complementary sequence inside, it can form an intramolecular secondary structure according to the principle of complementary base pairing. First of all, the intramolecular secondary structure of nucleic acid plays an important biological role in organisms. For example, the clover-like intramolecular secondary structure formed by transfer RNA is crucial for its amino acid translocation during translation, while the highly complex intramolecular secondary structure formed by ribosomal RNA is to ensure the structure of ribosomes and realize It is necessary for the function of synthesizing peptid...

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Problems solved by technology

[0006] In summary, for molecular biology methods that use nucleic acid molecules as templates and synthesize new nucleic acid molecules by nucleic acid polymerases based on the principle of complementary base pairing, such as PCR, sequencing, transcription reactions, and reverse transcription reactions, etc., in the template single strand Potentially stable intramolecular secondary structures often have an adverse effect on the technical performance of the corresponding method, and so far there is no widely applicable, clear mechanism and no side effect method to eliminate such adverse effects

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