Short fragment nucleic acid chain detection method and pre-amplification method

Abstract

The invention relates to a short fragment nucleic acid chain detection method and a pre-amplification method. The detection method is characterized in that during reverse transcription, the 5' end ofa target sequence is lengthened through TSO sequence template conversion, the target sequence is identified through a reverse transcription prime, and the 3' end of the target sequence is lengthened;during nucleic acid amplification, 5' and 3' specific primes are designed for the reverse transcription prime, the 5' specific prime spans the connecting part of the TSO sequence and the target sequence, and the 3' specific prime spans the connecting part of the target sequence and the reverse transcription prime, so that a short fragment nucleic acid sequence can be accurately amplified and detected; during nucleic acid amplification, 5' and 3' universal primes are designed for the reverse transcription prime, the 5' universal prime is designed inside the TSO sequence, and the 3' universal prime is designed inside the reverse transcription prime, so that a plurality of short fragment nucleic acid sequence pre-amplification products can be obtained.

Description

technical field [0001] The invention relates to the field of nucleic acid detection in molecular biology, in particular to a short-segment nucleic acid chain detection method and a pre-amplification method. Background technique [0002] In recent years, hot life science research often requires the amplification and detection of short-segment nucleic acid chains, such as the detection of short-segment nucleic acid chains that regulate biological growth and development, or the detection of highly degraded genetic material. General nucleic acid fragments such as DNA and mRNA can be detected and quantified by nucleic acid amplification such as PCR. It is necessary to design primers with a length of about 20 bases at both ends of the target fragment to amplify the target fragment; however, due to some short fragments The length of the nucleic acid chain sequence is not enough to design upstream and downstream primers, or although the length is sufficient, its sequence characteris...

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Problems solved by technology

[0013] The advantage of this method is that the use of dual-specific primers improves the specificity, but there is a fatal weakness - the 5' primer is only a dozen bases complementary to the target miRNA, and it is difficult to combine with the cDNA of the target miRNA in the first step of PCR , leading to great limitations in primer design: First, the Tm value of a dozen bases complementary to the template should not be too low, otherwise no matter how many bases are artificially introduced at the 5' end, the Tm value cannot be raised to a suitable temperature , the primers will not work; the second is that in the first cycle of PCR, the artificially introduced bases are not complementary to the template, which will reduce the efficiency of nucleic acid amplification

[0014] Due to this defect, the method in background document 2 has not been accepted by the public despite its unique advantages, and the current mainstream method is still using the polyA tailing method or the general stem-loop primer method in background document 1

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