Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination

A primer combination and primer set technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problem of poor specificity of target sequence amplification, insufficient tissue sample acquisition, and disadvantageous large-scale promotion and other issues, to achieve the effect of low price, good practical application value and good fidelity

Active Publication Date: 2016-10-26
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of the last base mismatch in ARMS technology cannot completely block the amplification of wild-type DNA, there is a risk of false positives, and it can only be detected for specific sites
In addition, such as high performance liquid chromatography, capillary electrophoresis, etc., require special equipment, and the operation is complicated, which is not conducive to large-scale clinical promotion
Nucleic acid sequence amplification (NASBA), self-sequencing sequence replication (3SR) and strand displacement replication (SDA) are all isothermal amplification methods, but their specificity for the amplification of target sequences is not strong
[0005] despite this, P53 The detection method of mutation is still based on the detection of tumor tissue samples as the gold standard, but in clinical practice, the detection is often limited due to insufficient tissue samples

Method used

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  • Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination
  • Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination
  • Primer combination for detecting mutation of genes P53 in micro-tissues and application of primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: In the DNA template of 1000pg P53 Gene Mutation Detection

[0032] 1. Experimental materials

[0033] phi29 DNA polymerase, 10×reaction buffer, dNTP (10nM), 100×BSA, 1000pg DNA template, deionized water, primer1-prime18 (ie Primer Mix), 2×Pfu Mix, primer19-primer26, PCR machine, super Thin product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd. DP203), 1% agarose gel, electrophoresis apparatus.

[0034] 2. Experimental procedures and results

[0035] 2.1 Pre-amplification

[0036] (1) Primer dilution

[0037] Primer1-prime18 was diluted and mixed to form a Primer Mix (Table 1), wherein the final concentration of primer1-primer16 primers was 0.1 μM, and the final concentration of primer17-primer18 primers was 2 μM. Configure the following system:

[0038] ;

[0039] (2) After mixing the above reagents, pre-denature at 98°C for 10 minutes, and then quickly put them on ice for 20 minutes;

[0040] (3) Then add the following system:...

Embodiment 2

[0057] Embodiment 2: In the DNA template of 500pg P53 Gene Mutation Detection

[0058] 1. Experimental materials

[0059] phi29 DNA polymerase, 10×reaction buffer, dNTP (10nM), 100×BSA, 1000pg DNA template, deionized water, primer1-prime18 (ie Primer Mix), 2×Pfu Mix, primer19-primer26, PCR machine, super Thin product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd. DP203), 1% agarose gel, electrophoresis apparatus.

[0060] 2. Experimental steps

[0061] 2.1 Pre-amplification

[0062] (1) Primer dilution. Primer1-prime18 was diluted and mixed to form a Primer Mix (Table 1), wherein the final concentration of primer1-primer16 primers was 0.1 μM, and the final concentration of primer17-primer18 primers was 2 μM. Configure the following system:

[0063] ;

[0064] (2) After mixing the above reagents, pre-denature at 98°C for 5 minutes, and then quickly put it on ice for 20 minutes.

[0065] (3) Then add the following system:

[0066] ;

[0067] A...

Embodiment 3

[0082] Example 3: In the DNA template of 100pg P53 Gene Mutation Detection

[0083] 1. Experimental materials

[0084] phi29 DNA polymerase, 10×reaction buffer, dNTP (10nM), 100×BSA, 1000pg DNA template, deionized water, primer1-prime18 (ie Primer Mix), 2×Pfu Mix, primer19-primer26, PCR machine, super Thin product purification kit (Tiangen Biochemical Technology (Beijing) Co., Ltd. DP203), 1% agarose gel, electrophoresis apparatus.

[0085] 2. Experimental steps

[0086] 2.1 Pre-amplification

[0087] (1) Primer dilution. Primer1-prime18 was diluted and mixed to form a Primer Mix (Table 1), wherein the final concentration of primer1-primer16 primers was 0.1 μM, and the final concentration of primer17-primer18 primers was 2 μM.

[0088] Configure the following system:

[0089] ;

[0090] (2) After mixing the above reagents, pre-denature at 98°C for 8 minutes, and then quickly put it on ice for 20 minutes.

[0091] (3) Then add the following system:

[0092]

[009...

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Abstract

The invention discloses a primer combination for detecting mutation of genes P53 in micro-tissues. The primer combination comprises pre-amplification primer groups, primer groups for detecting mutation of NO.5 exons, primer groups for detecting mutation of NO.6 exons, primer groups for detecting mutation of NO.7 exons and primer groups for detecting mutation of NO.8 exons. The primer combination has the advantages that a method aims to pre-amplifying micro-DNA (deoxyribonucleic acid) to obtain high-concentration DNA templates, and the DNA templates and direct sequencing are combined with one another to detect mutation conditions of the genes P53 in samples; the primer combination is applied to preparing detection reagents for detecting mutation of the genes P53, the concentration of DNA in the samples can be reduced and reaches 100 pg, all mutation of the NO.5, NO.6, NO.7 and NO.8 exons of the genes P53 can be simultaneously detected, and the detection method is high in sensitivity and specificity, low in cost and applicable to detecting mutation of genes P53 of clinical tumor patients and has excellent clinical application value.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a P53 Primer combination for gene mutation detection and its application in tumor drug selection and disease diagnosis. Background technique [0002] P53 The gene is the gene most closely related to human tumors found so far, and it has been reported that there are even more than 50% of tumor patients. p53 genetic changes. The human p53 gene is located in band 3.1 (17q13.1) of the short arm of chromosome 17, with a full length of 16-20kb, including 11 exons and 10 introns, and is divided into wild type (wt-p53) and mutant (mt-p53) (Reich and Levine, 1982; Lane, 1984). wt-p53 is a tumor suppressor gene, and its function change or loss is closely related to a large number of different types of human tumor cells. Wt-p53 can integrate signals of various cellular emergencies, respond to these signals through transcriptional or non-transcriptional pathways, including ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2531/113
Inventor 盛苗苗罗瑛唐文如李珊珊王芳赵月光张继虹贾舒婷吴晓明刘静周若宇旦菊花
Owner KUNMING UNIV OF SCI & TECH
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