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SNP (single nucleotide polymorphism) typing detecting method for utilizing locking-type probe for rolling ring amplification

A padlock probe and rolling circle amplification technology, applied in the biological field, can solve the problems of increasing the probability of operator infection, high cost, cumbersome operation, etc., shorten the operation steps and detection time, reduce the risk of cross-infection, and detect accurate results

Active Publication Date: 2016-04-06
华桥生物工程科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The composition of peripheral blood is complex, and the influence of various enzymes, proteins, heme, etc., results in incomplete exposure of the genome or inhibition of the ring formation reaction, poor amplification efficiency, etc., especially the padlock probe-RCA technology system. The purified genome is used as a template, so the current detection based on rolling circle amplification is not only cumbersome to operate, heavy workload, high cost, easy to cause cross-contamination between samples, but also increases the probability of operator infection; A report on the direct detection of peripheral blood samples by the probe-rolling circle amplification system

Method used

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  • SNP (single nucleotide polymorphism) typing detecting method for utilizing locking-type probe for rolling ring amplification
  • SNP (single nucleotide polymorphism) typing detecting method for utilizing locking-type probe for rolling ring amplification
  • SNP (single nucleotide polymorphism) typing detecting method for utilizing locking-type probe for rolling ring amplification

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Effect test

Embodiment 1

[0026] 1. Design of padlock probes and amplification primers

[0027] Obtain all reference genotypes of the SNP site (rs27044) on the ERAP1 gene from the genebank database, and obtain the gene sequence of 500 bp upstream and downstream of the single nucleotide polymorphism site, and use the primer5.0 software to design two allele-specific A sex padlock probe (to simplify the description, it will be referred to simply as a padlock probe hereinafter) and two amplification primers. in:

[0028] Detection of rs27044 site: two padlock probes (as shown in SEQ ID NO: 1, 2): Pro-15'-TGAGGCGTCGTAAGCCATAAGTAGGATAGGACAGGAAAGAAACGCTCCTCATCATCAAG-3', Pro-25'-TGAGGCGTCGTAAGCCATAAGTAGGATAGGACAGGAAAGAAACGCTCCTCATCATCAAC-3'; two amplification primers (such as SEQ ID NO: 3 , 4): the first primer 5'-CTTTTCCTGTCCTATCCTACTTATG-3', the second primer 5'-CTCCTCATCATCAACTG-3';

[0029] 2. Padlock probe phosphorylation

[0030] Phosphorylate the 5′ end of the designed allele-specific padlock probe, ...

Embodiment 2

[0041] In order to verify the detection effect of a SNP typing detection method using padlock probe rolling circle amplification of the present invention, the applicant carried out the operation of Example 2, specifically:

[0042] Take sample 1, purify the DNA genome, and use the SNP typing detection method of the present invention to detect, and the detection results are as follows: figure 2 Shown; And by adopting existing recognized Sanger method to carry out SNP typing detection to peripheral blood sample I to carry out contrast, then test result shows, the detection result of the present invention's detection method is consistent with the detection method of existing recognized Sanger method;

[0043] Take sample II, purify the DNA genome, and use the SNP typing detection method of the present invention to detect, and the detection results are as follows: image 3 shown; and by adopting the existing recognized Sanger method to carry out SNP typing detection on the periph...

Embodiment 3

[0045] In order to verify the detection effect of a SNP typing detection method using padlock probe rolling circle amplification of the present invention, the applicant carried out the operation of Example 2, specifically:

[0046] Get the peripheral blood sample I and use the SNP typing detection method of the present invention to detect, and the detection results are as follows: Figure 4 Shown; The result corresponding to this embodiment is figure 2 ;

[0047] Take peripheral blood sample II and use the SNP typing detection method of the present invention to detect, and the detection results are as follows: Figure 4 Shown;; The result corresponding to this embodiment is Figure 4 ;

[0048] Therefore, the detection result of the detection method of the present invention is accurate and feasible.

[0049] In addition, it should be noted that the percentage of each component in the present invention: the solid-liquid system is the mass percentage, and the liquid-liquid ...

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Abstract

The invention provides an SNP (single nucleotide polymorphism) typing detecting method for utilizing a locking-type probe for rolling ring amplification. At first, the locking-type probe and an amplification primer are designed, then the designed locking-type probe is phosphorylated, then a locking-type probe cyclization reaction is carried out, an enzyme digestion reaction is adopted for digesting an uncyclized locking-type probe and genomic DNA to obtain a cyclized locking-type probe, the cyclized locking-type probe is subjected to a rolling ring amplification reaction, a reactant obtained through the rolling ring amplification reaction is taken and subjected to 1% agarose gel electrophoresis detection, and the gene type of SNP can be judged through an electrophoresis detecting result. According to the method, SNP typing detection can be directly carried out on biological samples like peripheral blood, the genomic extraction process is omitted, the operation steps are greatly reduced, detecting time is greatly shortened, cost is saved, the risk of cross infection of operators is reduced, the detecting method has great popularization prospects, and the detecting result is accurate and sensitive.

Description

【Technical field】 [0001] The invention belongs to a detection method in the field of biology, and in particular relates to a SNP typing detection method using padlock probe rolling circle amplification. 【Background technique】 [0002] As the third generation of DNA genetic markers, single nucleotide polymorphism (SNP) has the characteristics of wide distribution, large number, easy detection and relatively stable in the genome. Its different genotypes are related to many important physiological phenomena and Its development process has great relevance. Detection of SNP genotype is the key development direction of modern molecular biology and one of the research directions of modern molecular pathology, and has become a hot spot in related research fields. [0003] At present, the commonly used SNP detection methods are mostly to extract the relevant genome first, then perform gene amplification based on PCR technology, and then combine with other detection methods such as D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2521/501C12Q2531/125C12Q2533/107
Inventor 杨会勇饶华春刁勇王清瑶郑晨娜许超尘
Owner 华桥生物工程科技有限公司
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