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Full-length cDNA nucleic acid linear amplification method and kit

A kit and nucleic acid technology, applied in the field of molecular biology, can solve problems such as low reaction sensitivity, short-cut products, insufficient kinetics of enzyme activity, etc.

Active Publication Date: 2013-12-25
SHANGHAI OFFO BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have a long test cycle, high cost, low PCR amplification efficiency, and short-cutting of the product. Therefore, it is not easy to obtain a complete full-length gene, especially the 5' of the gene, by using these methods. end
[0004] In addition, the existing cDNA amplification technology still has the problem of insufficient kinetics of reverse transcriptase activity under low mRNA template amount, and the reaction sensitivity is low when reverse transcription of very small amount (such as fg-level mRNA template) of RNA. Therefore, it is urgently needed A cDNA amplification method to improve the reaction sensitivity of cDNA amplification and obtain full-length cDNA

Method used

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  • Full-length cDNA nucleic acid linear amplification method and kit
  • Full-length cDNA nucleic acid linear amplification method and kit
  • Full-length cDNA nucleic acid linear amplification method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Synthesis and linear amplification reaction of embodiment 1 trace cDNA

[0076] 1. Preparation of total RNA

[0077] The leaves of rice were collected, put into liquid nitrogen and ground into powder, and 100 mg of powder was used to extract the total RNA of rice leaves using the Trizol method (refer to the product manual of Invitation Company).

[0078] 2. How to use

[0079] 2.1 The inventive method

[0080] 2.1.1 Primer synthesis

[0081] 1) Oligo dT linker primer: 5'-Biotin-GCCCAGCCAATCACCTAAAGTCAATTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO: 3).

[0082] 2) Cofactor: 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaaa-3' (SEQ ID NO: 1).

[0083] 3) Oligo modified primer: 5'-GCCCCAGCCAATCACCTAAAGTCANNNNNNNNN'-3 (SEQ ID NO: 4), wherein NNNNNNNNNN' is a random primer, and the outermost base at the 3' end of the Oligo modified primer is modified by amination.

[0084] 4) Anchor primer: 5'-GCCCAGCCAATCACCTAA-3' (SEQ ID NO:5)

[0085] 2.1.2 Full-length cDNA nucleic acid linear am...

Embodiment 2

[0103] Synthesis and linear amplification reaction of embodiment 2 trace cDNA

[0104] 1. Preparation of total RNA

[0105] Human placental tissue was collected, put into liquid nitrogen and ground into powder, and 100 mg was extracted using the Trizol method (refer to the product manual of Invitation Company) to extract human total RNA.

[0106] 2. Method

[0107] 2.1 Primer synthesis

[0108] 1) Oligo dT adapter primer: 5’-Biotin-GCCCAGCCAATCACCTAAAGTCAA ggccGAGGCggccAGCAGTgcag TTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO: 6), where the underlines are the restriction enzyme cutting site sequences of sfi, EcoP151 and BsgI.

[0109] 2) Cofactor: 5'-gacguacguagcuaggccuauucggccaaaaaaaaaaaaaaaaaaa-3' (SEQ ID NO: 2)

[0110] 3) Oligo modified primer: 5'-GCCCAGCCAAATCACCTAAAGTCA ggccgaggcggcc NNNNNNNN'N'N'-3 (SEQ ID NO:7), wherein NNNNNNNN'N'N' is a random primer, and the three outermost consecutive bases at the 3' end of the Oligo modified primer are treated with dideoxy modificatio...

Embodiment 3

[0122] Synthesis and linear amplification reaction of embodiment 3 trace cDNA

[0123] 1. Preparation of total RNA

[0124]Collect planarian samples, vannamei samples, clam samples, aloe vera samples, and cypress seed samples, and grind them into powders with liquid nitrogen, and take 100 mg of powder from each of the five samples and use the Trizol method (see Invitation company product manual) to extract each sample total RNA.

[0125] 2. Experimental method

[0126] 2.1 Primer synthesis

[0127] 1) Oligo dT linker primer: 5'-Biotin-GCCCAGCCAATCACCTAAAGTCAA ggccGAGGCggccAGCAGTgcag TTTTTTTTTTTTTTTTTTTVN-3' (SEQ ID NO: 6), where the underlines are the restriction enzyme cutting site sequences of sfi, EcoP151 and BsgI.

[0128] 2) Cofactor: 5'-cgacguacguagcuagacuuaucguaaaaaaaaaaaaaaaaaa-3' (SEQ ID NO: 1), where the underline is the restriction enzyme cutting site of RsaI.

[0129] 3) Oligo-modified primer: 5'-GCCCCAGCCAATCACCTAAAGTCANNNNNNNNN'-3 (SEQ ID NO: 4), wherein NN...

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Abstract

The present invention relates to the field of molecular biology, and particularly discloses a full-length cDNA nucleic acid linear amplification method. The method comprises the following steps: separating and purifying mRNA, carrying out reverse transcription synthesis of first strand cDNA, carrying out first strand cDNA 3' terminal extension to obtain full-length first strand cDNA, enriching and purifying the full-length strand cDNA, synthesizing double-stranded cDNA, and the like, such that the full-length cDNA with both known terminal sequences is synthesized. The full-length cDNA nucleic acid linear amplification method has characteristics of high sensitivity and simpleness, and is suitable for efficient synthesis of the full-length cDNA.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a full-length cDNA nucleic acid linear amplification method. Background technique [0002] Changes in gene expression levels and patterns drive major biological processes in organisms. Isolating and cloning genes is the basis for studying gene structure, function and expression. In the past, the classic methods of establishing and screening cDNA and DNA libraries to isolate and clone target genes were cumbersome and heavy workload. [0003] The emergence of PCR technology has greatly improved the efficiency of isolating and cloning target genes. Technologies such as RT-PCR, pot handle PCR, and junction-mediated PCR provide shortcuts for amplifying unknown DNA fragments next to known sequences. Sequence information Design primers for gene transformation, use the reverse transcribed first-strand cDNA as a template to amplify the 3' end and 5' end sequences by PCR respectively and...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 唐荣苏丽唐冬梅
Owner SHANGHAI OFFO BIOPHARM CO LTD
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