Amplification method for 3'-RACE adaptor primer and 3'-end unknown gene sequences

Abstract

The invention discloses an amplification method for 3'-RACE adaptor primer and 3'-end unknown gene sequences. The 3'-RACE adaptor primer is of a stem-loop structure composed of a stem handle region, astem-loop region and a PolyA binding region; the stem handle region and the stem-loop region have a nucleotide sequence shown in SEQ ID NO.1; and 15, 16, 17, 18, 19 or 20 continuous thymine are designed in the PolyA binding region. Two fragments of the stem handle region of the 3'-RACE adaptor primer have complementary sequence base so as to form a handle of the stem loop. Due to base stacking atthe handle, the stability of the primer is increased, and the reverse transcription efficiency is improved. A stem-loop configuration is formed due to a double-chain structure and self-complementaryproperty, so that hybridization between a reverse transcription primer and a target RNA template can be avoided, and background interference is reduced. More G / C bases are particularly inserted into the stem-loop region of the 3'-RACE adaptor primer, so that the annealing temperature is increased, and the non-specific PCR amplification is reduced.

Description

technical field [0001] The invention relates to a 3'-RACE adapter primer and a method for amplifying unknown gene sequences at the 3' end. Background technique [0002] Rapid Amplification of cDNA Ends (RACE) is a method to quickly amplify cDNA from complex and low-abundance transcripts through the organic combination of reverse transcription and PCR based on the known partial sequence of the target gene An efficient method for the 5' and 3' ends of the Using the known sequence at the 5' end of the gene coding region to obtain the unknown sequence at the 3' end is called 3'-RACE, which is currently the most commonly used technique for amplifying the unknown 3' end sequence. [0003] The basic principle of conventional 3′-RACE: Use 3′-RACE linker primers to reverse transcribe the RNA sample containing the mRNA of the target gene to obtain an eDNA library containing the target gene; then use the known sequence of the target gene to design gene-specific primers, and anchor Th...

AI Technical Summary

Problems solved by technology

Using a general template as a nested PCR material, although high-throughput gene cloning can be performed, but there is a problem of high complexity and no selectivity for genes

In complex templates, the cDNA content of the target gene is relatively low, and for genes with low expression abundance, cloning is more difficult

[0006] When preparing a universal template, the reverse transcription reaction extends from the 3' end to the 5' end of the mRNA, which increases the workload of 3'-RACE, increases the complexity of the template, and may affect the 3'-RACE results

Moreover, the more complex the template, the easier non-specific amplification will occur in the PCR reaction, which will affect the amplification efficiency of the target fragment in the PCR reaction, and the non-specific amplification products will also interfere with the selection of the target band.

[0007] Conventional 3′-RACE adapter primers are linear sequences, and there is likely to be non-specific binding to the RNA template in the reverse transcription reaction, which further increases the complexity of the cDNA product, reduces the efficiency of reverse transcription, and ultimately leads to inaccurate amplification results. ideal

For less abundant mRNAs, it is even difficult to obtain amplification products

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