Clone method of SGAE label 3' end cDNA segment

Abstract

The invention relates to a clone method of SGAE label 3' end cDNA segment, belonging to the field of biotechnology. The method comprises the following steps: reverse transcription is carried out on all mRMAs by utilizing the decorated oligo(dT16), 3-5 cytimidine basic groups are added the 3' end of a first cDNA which is synthesized by murine reverse transcriptase, complementary strand of the first cDNA is synthesized by primers, a pair of primers are synthesized according to the known sequence at the two ends of the cDNA for PCR augmentation, and all the cDNAs are concentrated; the cDNA concentrated in the step one is taken as a template, specific segments containing tag are cloned by adopting semi-nested PCR. By adopting the invention, the specific segments containing tag label can be more easily obtained, the process is simpler and the cost is lower.

Description

technical field [0001] The invention relates to a method for cloning cDNA fragments in the field of biotechnology, in particular to a method for cloning cDNA fragments at the 3' end of SGAE (serial analysis of gene expression) tags. Background technique [0002] Serial analysis of gene expression (SAGE, serial analysis of gene expression) technology was first proposed by Velculesce et al. in 1995. The basic principle of gene expression series is: in theory, every nucleotide fragment of 9 bases can represent a specific sequence of a transcription product. SAGE is a high-throughput method for qualitative and quantitative research on transcript mRNA at the genome-wide level. Specifically, a 14bp-length fragment, namely the SAGE tag, is intercepted at a specific position of each transcript mRNA to represent the transcript. cDNA sequence, and then all SAGE tags are sequenced in tandem with each other to analyze their type and abundance, and then qualitatively and quantitatively ...

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Problems solved by technology

[0003] Found through document retrieval to prior art, Chen, J.J. etc. published a paper titled "Generation of longer cDNA fragments from serial" in "Proceedings of the National Academy of Sciences" (Proceedings of the National Academy of Sciences) 2000 97 phase 349-353 pages Analysis of gene expression tags for gene identification" (generating long cDNA fragments from SAGE tags for gene identification method) papers, which stated that an anchored oligo (dT) (anchored oligodeoxyribonucleotide thymine) was used as the The antisense primer uses the sequence containing the SAGE tag tag as the sense primer at the same time, and under the action of high-fidelity DNA polymerase (Pfu Taq), the specific band is amplified by PCR reaction, but this method faces the following problems: A large amount of initial RNA is required, the efficiency of amplifying low-abundance expression tags is low, and there are many non-specific bands in amplification, and the process is cumbersome

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