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Kit for in-vitro single base damage repair detection through real-time quantitative PCR (Polymerase Chain Reaction)

A damage repair and in vitro detection technology, applied in the field of DNA single base damage repair detection, can solve the problems of restricted environment, human injury, pollution, etc., and achieve the effect of ensuring specificity

Inactive Publication Date: 2011-09-14
BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most previous in vitro studies of BER have used 32 P radioisotope-labeled oligonucleotides are used as substrates, but this method can only give semi-quantitative results, and the radiolabeling method limits its use in general laboratory research where radioisotopes are prohibited (to avoid environmental pollution and human injury)

Method used

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  • Kit for in-vitro single base damage repair detection through real-time quantitative PCR (Polymerase Chain Reaction)
  • Kit for in-vitro single base damage repair detection through real-time quantitative PCR (Polymerase Chain Reaction)
  • Kit for in-vitro single base damage repair detection through real-time quantitative PCR (Polymerase Chain Reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] First: Template Annealing:

[0047]Take 200 pmol of oligonucleotides (Primer1-7) shown in SEQ ID No.1 to SEQ ID No.7 respectively, combine them according to Table 1 and add them to four EP tubes respectively. Table 1 is templates A, B, C, D. Template A consists of Primer1 (SEQ ID No.1), Primer2 (SEQ ID No.2) and Primer5 (SEQ ID No.5); Template B consists of Primer3 (SEQ ID No.3), Primer2 (SEQ ID No.2) ) and Primer5 (SEQ ID No.5); Template C is composed of Primer4 (SEQ ID No.4) and Primer5 (SEQ ID No.5); Template D is composed of Primer6 (SEQ ID No.6) and Primer7 (SEQ ID No.5) No.7) composition. Add 20ul of 10x annealing buffer (250mM NaCl; 20 mM Tris-Cl, pH=8.0; 10mM EDTA) to four EP tubes, make up to 200ul with water, anneal at 72°C for 10 minutes, then gradually drop to room temperature. Store at -20°C for later use.

[0048] Table 1 Template A, B, C, D combination

[0049]

[0050] Second: single base deletion damage repair ability detection and sensitivity a...

Embodiment 2

[0065] Example 2 Validation of the kit and detection method of the present invention

[0066] In the usual in vitro single-base damage repair experiments, people use isotopes 32 P-labeled damaged oligonucleotide substrates and purified enzyme and nucleotide extracts to determine DNA polymerase, DNA ligase and glycosidase activities. However, due to the application of isotopes, the study of single-base damage repair in vitro is greatly limited. In this research mode, the templates include: single-strand break template A, single-nucleotide removal template B, single-base modified oligonucleotide template C and control oligonucleotide template D, specifically as follows figure 1 shown. In this novel qPCR method, we designed two different target sequence probes for modified and control oligonucleotides. The modified oligonucleotide target probes were labeled with 5'-FAM, and the control oligonucleotide target probes were labeled with 5'-Tet. All oligonucleotides (Primer 1 to 7...

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Abstract

The invention discloses a kit for in-vitro single base damage repair detection through real-time quantitative PCR (Polymerase Chain Reaction). The kit comprises a 10X detection buffer solution, a 5X low-salt cell lysis solution, a 2X cell nucleoprotein or mitochondrion protein extract, a 20X detection substrate and a 2XqPCR damage repair buffer solution, wherein the repair buffer solution comprises taq enzyme, dNTP (Deoxyribonucleotide Triphosphate), probes and primers. In the kit, primers of an amplification internal reference and damage base qPCR (quantitative PCR) templates are the same, the amount of damage base repair can be calculated according to the amount of the internal reference. Since a complementary template only has 14 bases which are complementary with the primer 2, annealing of the complementary template and the primer 2 is not produced in a qPCR process, and specificity of damage repair detection is guaranteed. A 3' tail end of the complementary template is dideoxyoligonucleotide, so that extension of the template per se is avoided. The probe for detecting the damage template and the probe for detecting the internal reference template are marked with different fluorescent groups, so that absolute quantification of repair of the damage template is realized through detection in the same qPCR system.

Description

technical field [0001] The invention relates to a DNA single base damage repair detection technology, in particular to a real-time quantitative PCR in vitro detection single base damage repair technology. Background technique [0002] The repair of DNA damage in mammalian cells induced by reactive oxygen species, alkylation and ionizing radiation is usually through the single base excision repair (BER) pathway, so BER plays an important role in maintaining genome integrity. The ability to study cellular BER in vitro is of great significance for our understanding of the mechanisms of aging, tumors, and degenerative diseases. BER involves the process of DNA single-strand break repair (SSBR), AP site excision after base loss, replacement of damaged nucleotides and religation. The activities of different stages of DNA BER repair enzymes such as APE1, OGG1, UNG, DNA polymerase and ligase should be accurately detected. [0003] Most previous in vitro studies of BER have used 32...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈德喜李宁张洪海金荣华张玉林孙玉魏飞力石英
Owner BEIJING YOUAN HOSPITAL CAPITAL MEDICAL UNIV
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