PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment

A sulfite and methylation technology, applied in the field of molecular biology, can solve problems such as inability to amplify, difficult PCR amplification, cumbersome and complicated, etc., and achieve the goal of expanding the combination range, improving amplification efficiency, and high amplification efficiency Effect

Inactive Publication Date: 2013-01-23
LIAONING UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] (1) Since the DNA sequence will change after sulfite treatment, and the formed thymine T is easy to form a hairpin structure, the general annealing temperature cannot completely open the double strand, which brings difficulties to PCR amplification, and many alternative primers need to be designed , relying on the method of multiple attempts to amplify;
[0005] (2) If the primer annealing temperature is increased, the genomic DNA has been broken into fragments of about 800 bp after sulfite treatment, so the primers designed by conventional methods are not easy to amplify or cannot be amplified;
[0006] (3) Using high-fidelity Taq enzymes (gold medal rTaq, LATaq, ExTaq, etc.) does not improve the amplification effect, and the success rate is low and the cost is high;
[0007] (4) Need to design primers for methylated and non-methylated sites separately, which is cumbersome and complicated

Method used

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  • PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment
  • PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment
  • PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment

Examples

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Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 PCR method for detecting the DNA methylation modification of the left LTR of the retrotransposon Tos17 located on chromosome 10 in the rice genome after sulfite treatment

[0046] a) Design internal and external primers

[0047] 1) According to the clone number AC087545 of Tos17 on Genebank, download the Tos17 sequence on the NCBI website, and the left LTR sequence is 138bp, and then download the 600bp sequences of the upstream and downstream of the left LTR sequence through the NCBI website, and use the primer design software pimer5 Primers were designed according to the following method;

[0048] 2) The primer size is 28-35nt, and A and T should be avoided for more than 3 consecutive repetitions;

[0049] 3) The end of the forward primer cannot be C;

[0050] 4) The forward primer is less than 5 C, and the reverse primer is less than 5 G;

[0051] 5) C in the forward primer replaces C / T with the merged base Y; G in the reverse primer replaces G / A with t...

Embodiment 2

[0071] Example 2 PCR method for detecting the DNA methylation modification of the left LTR of the retrotransposon Tos17 located on chromosome 7 in the rice genome after sulfite treatment

[0072] a) Design internal and external primers

[0073] 1) According to its clone number AP005292 on Genebank, this sequence was downloaded from the NCBI website, which is located at the position of 186486-190599 on chromosome 7, and then the sequence of 600 bp upstream and downstream of the left LTR sequence was downloaded through the NCBI website, which is located at At 185886-187086 of chromosome 7, use the primer design software pimer5 to design primers according to the following method;

[0074] 2) The primer size is 28-35nt, and A and T should be avoided for more than 3 consecutive repetitions;

[0075] 3) The end of the forward primer cannot be C;

[0076] 4) The forward primer is less than 5 C, and the reverse primer is less than 5 G;

[0077] 5) C in the forward primer replaces...

Embodiment 3

[0097] Example 3 PCR method for detecting DNA methylation modification of sequence AP001551 in GENE BANK after sulfite treatment

[0098] a) Design internal and external primers

[0099] 1) According to its clone number AP001551 on Genebank, download this sequence on the NCBI website, which is located at the position 59722-64876 of chromosome 1, and then download the sequence of 300 bp upstream and downstream of the left sequence through the NCBI website, which is located at a At 59422-60022 of chromosome number, use the primer design software pimer5 to design primers according to the following method:

[0100] 2) The primer size is 28-35nt, and A and T should be avoided for more than 3 consecutive repetitions;

[0101] 3) The end of the forward primer cannot be C;

[0102] 4) The forward primer is less than 5 C, and the reverse primer is less than 5 G;

[0103] 5) Using the merger method, the C in the forward primer is replaced by the merged base Y for C / T; the G in the ...

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Abstract

The invention relates to a PCR (polymerase chain reaction) method for quickly and efficiently detecting DNA (deoxyribonucleic acid) methylation modification after sulfite treatment. The technical scheme is as follows: 1) a pair of inner primers A1 and A2 and a pair of outer primers B1 and B2 are designed; 2) PCR amplification: the PCR amplification comprises two PCR processes: the first PCR process is touchdown PCR, and uses the primers A1 and A2, or A1 and B2, or B1 and A2; the second PCR amplification process is common PCR, and uses the primers B1 and B2, the template is the first PCR process product 1-2ul, and the enzyme is Extaq or common rTaq; and 3) the amplification product is less than 700bp. The invention has the characteristics of high amplification efficiency, low experimental cost, short experimental period, quick and manageable detection and high experimental result stability, can be easily completed in the laboratory, and can be widely used in methylation modification detection of genome specific genes of humans, mammals and plants.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a detection method for DNA methylation modification in epigenetic modification. Background technique [0002] DNA methylation is the most common form of DNA covalent modification in eukaryotic genomes and is an important aspect of epigenetics. DNA methylation mainly forms 5-methylcytosine (5-mC) and a small amount of N6-methyladenine (N6-mA) and 7-methylguanine (7-mG). In eukaryotes, DNA methylation mainly exists in the form of 5-methylcytosine. Since DNA methylation, especially cytosine methylation, is very common in eukaryotic genomes, it has attracted great attention from the life science community. Numerous studies have shown that DNA methylation is involved in various cellular activities of higher organisms. These mainly include tissue-specific gene expression, heterochromatin formation, developmental mechanisms, transposition activation of transposons, X chr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王红艳王秋雨韩阳周婵王铮
Owner LIAONING UNIVERSITY
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