Method for externally amplifying specific ring type or concatemer nucleic acid

Abstract

The externally amplifying process of specific ring form or concatemer nucleic acid includes the pre-denaturation of ring type or concatemer nucleic acid containing target sequence as the template, one pair of 5'-phosphorylated oligonucleotide primers, four kinds of triphosphodeoxyribonucleotides and thermonucleic acid synthetase in proper buffing system at 90-99 deg.c for 1-30; and subsequent 1-40 thermal circulation treatment. The said thermal circulation includes three steps of: denaturation via maintaining at 90-99 deg.c for 1 sec to 5 min; renaturation and connection at 30-85 deg.c for 10 sec to 15 min; and extension at 65-85 deg.c for 1-30 min. The present invention can produce and amplify ring form or serial DNA externally and optionally.

Description

technical field [0001] The present invention relates to a method for in vitro amplification of circular or tandem nucleic acids, ie DNA. In particular, the invention relates to methods for amplifying circular or tandem DNA in a cell-free system. Background technique [0002] Polymerase chain reaction (PCR) is a powerful and rapid method for exponentially amplifying target nucleic acids, as disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR has become the core technology of molecular biology. PCR has greatly developed gene isolation and identification and molecular cloning techniques, including gene sequencing, identification of alleles, and detection of infectious and genetic diseases. PCR is amplified by heat-denaturing the DNA template and primers through thermal cycling, annealing the primers with the complementary DNA strand, and extending the primers with a heat-resistant DNA polymerase. Nucleic acid sequences located between the two primers can be exponentially...

AI Technical Summary

Problems solved by technology

It is very tedious, time-consuming, labor-intensive and costly work to construct a whole genome library or a full-length cDNA library and screen the target sequence, and has a high false positive rate

In comparison, RACE and reverse PCR are more labor-saving and cost-effective, but they also have many problems: not only need to overcome difficulties and take time to design, synthesize and detect suitable RACE primers and reverse primers, but also need to carry out a series of PCR amplification

In addition, the amplification of large circular or linear DNA with sequence-specific primers is time-consuming and inefficient, so random primers are usually used instead of sequence-specific primers for multibranch amplification (hRCA), which makes the RCA reaction more sensitive, but also loss of specificity and selectivity

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