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Anchor primer for efficient and rapid amplification of cDNA ends and amplification method

An anchoring and rapid technology, applied in DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of low amplification efficiency, low gene abundance, poor specificity, etc., and achieve good specificity and amplification. High efficiency and easy operation effect

Active Publication Date: 2017-06-20
SUN YAT SEN UNIV CANCER CENT
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Problems solved by technology

[0003] In many biological studies, the abundance of the target gene studied is often very low. In this case, the traditional RACE technology highlights its shortcomings, such as poor specificity and low amplification efficiency. Long cDNA becomes incredibly difficult

Method used

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  • Anchor primer for efficient and rapid amplification of cDNA ends and amplification method
  • Anchor primer for efficient and rapid amplification of cDNA ends and amplification method
  • Anchor primer for efficient and rapid amplification of cDNA ends and amplification method

Examples

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Embodiment 1

[0022] A method for efficiently and rapidly amplifying cDNA 5' and 3' ends, comprising the steps of:

[0023] a. Design a specific locking primer connected to the 5' end of the cDNA, that is, the anchor primer, denoted as LT. There are 3 guanines at the 3' end of the sequence, the first two guanines are riboguanosine, and the last guanine base is locked nucleic acid modified guanine (LNA). The primer can form 3 'Stem-loop structure protruding from the terminal portion. The special modification at the end of the adapter sequence and its own structure can significantly improve the anchoring efficiency of the locked primer;

[0024] b. Design an anchor sequence combined with the poly(A) tail at the 3' end, denoted as MT. The end of the sequence contains 30 thymines and does not form a hairpin structure by itself;

[0025] c. Design a primer that is partially complementary to the MT sequence, denoted as B;

[0026] d. Design two specific primers for the target gene, wherein th...

Embodiment 227227

[0029] Example 2 Rapid amplification of 27227 lncRNA 5' and 3' ends

[0030] 27227lncRNA is a long non-coding RNA, and the function and gene name of this long non-coding RNA are still uncertain. The accession number of its UCSC database is known as TCONS_00027227, named 27227 by itself, and its chromosome location position: chr19: 22025306-22035178.

[0031] According to the method described in Example 1, the 5' and 3' ends of 27227lncRNA were amplified, and its key parts were:

[0032] 1. Nucleic acid sequence design

[0033] The specific nucleic acid sequence used in this embodiment, the specific sequence is as follows:

[0034] The specific nucleic acid sequence used in the present embodiment of table 1

[0035]

[0036] Wherein, rG in the above table is monodeoxyguanine (riboguanosine), +G is locked nucleic acid modified guanine (LNA), and then the reverse transcription reaction system is prepared according to Table 2:

[0037] Table 2 Reverse transcription reaction s...

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Abstract

The invention discloses an anchor primer for efficient and rapid amplification of cDNA 5' and 3' ends. The two anchor primers can be specifically bound to a target nucleic acid sequence and trigger reverse transcription elongation reaction, thereby efficiently and rapidly amplifying cDNA 5' and 3' ends, and especially can be applied to rapid amplification of the 5' and 3' ends of cDNA in low abundance transcripts. In addition, the invention also provides a kit and an amplification method for efficient and rapid amplification of cDNA 5' and 3' ends.

Description

technical field [0001] The invention relates to a method for rapidly amplifying cDNA ends, in particular to an anchor primer and an amplification method for efficiently and rapidly amplifying cDNA ends. Background technique [0002] Obtaining full-length genes is a focus of bioengineering and molecular biology research. The classic RACE (rapid-amplification of cDNA ends) technology is a technology invented by Frohman et al. in 1988, which can effectively obtain the full-length sequence of the gene. RACE is based on RT-PCR technology, adding locking primers at the end to obtain cDNA with complementary or identical sequences to the locking primers at the end, and then performing PCR amplification with specific primers to obtain complete cDNA 5' and 3' end sequences. The principle of RACE technology is simple, and it has the advantages of quickness, high efficiency and low cost. [0003] In many biological studies, the abundance of the target gene studied is often very low. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
CPCC12N15/1096C12N15/11C12Q2531/113C12Q2521/107
Inventor 蔡木炎项志成谢丹王凤伟陈杰伟凌逸虹李鹏
Owner SUN YAT SEN UNIV CANCER CENT
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