Method for acquiring flanking sequence from genome DNA

A flanking sequence and gene sequence technology, applied in the field of obtaining flanking sequences, can solve the problems that traditional TAIL-PCR application potential cannot be brought into full play, high-efficiency TAIL-PCR is time-consuming and labor-intensive, and primer design and synthesis are expensive, so as to reduce contamination. Odds, time-saving, short-time effects

Inactive Publication Date: 2015-03-25
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the improved high-efficiency TAIL-PCR is still time-consuming and labor-intensive, and the cost of primer design and synthesis is expensive
At the same time, hi TAIL-PCR and FPNI-PCR technologies have also encountered huge challenges, such as: DNA polymerase activity problems and non-specific amplification, etc., so it is often accompanied by low-efficiency amplification results
In summary, these improved methods are still time-consuming and labor-intensive, and cannot exert the broad application potential of traditional TAIL-PCR

Method used

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  • Method for acquiring flanking sequence from genome DNA
  • Method for acquiring flanking sequence from genome DNA
  • Method for acquiring flanking sequence from genome DNA

Examples

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Embodiment 1

[0054] Example 1. Preparation of kit for obtaining flanking sequences from genomic DNA and method of use thereof

[0055] 1. Preparation of kits for obtaining flanking sequences from genomic DNA

[0056] The kit for obtaining flanking sequences from genomic DNA provided by the present invention comprises 15 random degenerate primers SAP, nested primers SEP and primers SNP.

[0057] According to the principles of TAIL-PCR, suppression PCR and nested PCR, a batch of random degenerate primers (SAP1-15) with adapter sequences were designed for the first round of PCR reaction (as shown in Table 1); according to the principle of suppression PCR, Mixed nested primers (SEP) were used to extend the adapter sequence and minimize non-specific amplification; according to the principle of nested amplification, the primer SNP was designed for the third round of PCR reaction. For specific principles and procedures, see figure 1 .

[0058] Random degenerate primer SAP, nested primer SEP an...

Embodiment 2

[0102] Example 2, using the kit prepared in Example 1 to obtain the 5' end sequence of LaTCTP gene from larch genomic DNA

[0103] 1. Design of LaTCTP gene-specific primers

[0104] According to the principle of specific primer design in Example 1, using primer design software primer5, three gene-specific primers 5′LaTCTP1, 5′ were designed according to the 5′ partial genome sequence (sequence 19) of larch translation regulation tumor protein LaTCTP LaTCTP2 and 5' LaTCTP3 (Table 3) were used for amplification of their 5' flanking sequences.

[0105] Table 3 LaTCTP gene-specific primers

[0106]

[0107] 2. Extraction of larch genomic DNA

[0108] Take 0.1-0.5 g of embryogenic tissue of Japanese larch (Larix leptolepis), put it in liquid nitrogen and freeze it rapidly, and use the CTAB method described in Example 1 to extract genomic DNA. Finally, 50 μL of sterilized water was used to dissolve the DNA, and the DNA concentration was determined by NanoDrop1000 to be 471.9 n...

Embodiment 3

[0127] Example 3, using the kit prepared in Example 1 to obtain the 5' end sequence of LaNFYA1 gene from larch genomic DNA

[0128] 1. Design of LaNFYA1 gene-specific primers

[0129] According to the principle of specific primer design in Example 1, using the primer design software primer5, three gene-specific primers 5'LaNFYA1-1 and 5'LaNFYA1 were designed according to the 5'UTR sequence (sequence 26) of the larch LaNFYA1 gene -2 and 5' LaNFYA1-3 (Table 4) for its 5' flanking sequence walk.

[0130] The specific primer of table 4LaNFYA1 gene

[0131]

[0132]

[0133] 2. Extraction of larch genomic DNA

[0134] With embodiment 2.

[0135] 3. The first round of PCR amplification

[0136] About 400ng of genomic DNA (about 1 μL) was used as a template, SAP1 and SAP2 (see Table 1) were selected as upstream primers, and 5′LaNFYA1-1 was used as a downstream primer, and the first round of PCR was performed.

[0137] The preparation of the first-round PCR reaction system ...

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Abstract

The invention discloses a method for acquiring a flanking sequence from genome DNA. The method comprises the following steps: 1) designing three specific primers having consistent directions and used for amplifying the flanking sequence according to a known sequence on the genome DNA, and sequentially marking the primers as GSP1, GSP2 and GSP3 according to the tapered distance between the primers and the flanking sequence; 2) performing first PCR (polymerase chain reaction) amplification by using the genome DNA as a template through a primer SAP (any of sequences 1-15) and the GSP1 to obtain a PCR product 1; 3) performing second PCR amplification by using the PCR product 1 as a template through primers SEP (sequences 16 and 17) and the GSP2 to obtain a PCR product 2; and 4) performing third PCR amplification by using the PCR product 2 as a template through a primer SNP (sequence 18) and the GSP3 to obtain a PCR product 3, thus acquiring the flanking sequence. The method has the following advantages of simplicity in operation, high specific amplification, high success rate, short time and the like; and large fragments are easily obtained.

Description

technical field [0001] The invention relates to a method for obtaining flanking sequences from genomic DNA, in particular to a method for obtaining flanking sequences from genomic DNA through SNM-PCR. Background technique [0002] The advent of the genome era has had a huge impact on all fields of biology. For a few species whose genomes have been sequenced (such as human, Arabidopsis, rice, etc.), flanking sequences of known sequences can be found directly from the database. However, the genome DNA sequence of most organisms in nature is still unknown, and chromosome walking technology is a very effective method to know the DNA sequence on both sides of a known region. Therefore, chromosome walking technology (Genome Walking) is an important modern molecular biology research technology, which can effectively obtain unknown sequences adjacent to known sequences. [0003] Chromosome walking technology mainly has the following applications: 1. According to the known fragment...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 张立峰韩素英齐力旺李水根张俊红
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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