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Sequencing primer for qualitative detection of KRAS genetic typing and kit thereof

A technology of genotyping and sequencing primers, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effect of simple sample processing, high sensitivity and high sensitivity

Active Publication Date: 2013-01-30
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there is no product on the market that uses pyrophosphate technology for KRAS gene polymorphism detection

Method used

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  • Sequencing primer for qualitative detection of KRAS genetic typing and kit thereof
  • Sequencing primer for qualitative detection of KRAS genetic typing and kit thereof
  • Sequencing primer for qualitative detection of KRAS genetic typing and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: the preparation of kit

[0049] 1. Design and synthesis of primers and probes

[0050] Studies have shown that the current KRAS gene mutations are mainly concentrated in codons 12 and 13 of exon 2 and codon 61 of exon 3, select specific mutation sites, and use PyroMark Assay Design2.0 software to design primers ; Wherein the amplification primer and the sequencing primer are first purified by PAGE, and then purified by HPLC, wherein the 5' of SEQ ID NO:1 and SEQ ID NO.5 is biotin-labeled.

[0051] Mutation site and type Table 1:

[0052] Mutation

[0053] The amplified sequence is shown in Table 2:

[0054] Table 2. Sequences of specific amplification primers and sequencing primers

[0055]

[0056] 2. Control substance selection

[0057] A synthetic oligonucleotide chain TAYGGTTTGCA control oligo was used as a quality control product; DNAase RNA-free water was used as a blank control.

[0058] 3. Composition of PCR reaction solution

...

Embodiment 2

[0065] Embodiment 2: the use of kit

[0066] 1. sample testing

[0067] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, UNG enzyme, and Taq DNA polymerase, aliquot the system, add sample DNA, blank control or positive control as templates, and form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0068] The main components of the KRAS1213 system are as follows:

[0069]

[0070] The main components of the KRAS61 system are as follows:

[0071]

[0072] The system reaction procedure is as follows:

[0073] Table 5. PCR reaction program

[0074]

[0075] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.

[0076] 2. Pyrosequencing

[0077] The sequencing operation was carried out according to the sta...

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Abstract

The invention provides a sequencing primer for detecting mutation of codons 12 and 13 in a second exon and a codon 61 in a third exon of a KRAS gene, and a kit thereof, and belongs to the field of in vitro nucleic acid detection. The kit comprises uracil DNA glycosylase, Taq polymerase, PCR amplification primers and sequencing primers. The kit provided by the invention has high sensitivity and good specificity; the PCR products can be simply treated for sequencing on a phosphate sequenator; and the kit has advantages of simple operation, short reaction time, and higher sensitivity than a gold standard-capillary electrophoresis sequencing, and is more suitable for mutation analysis.

Description

technical field [0001] The invention relates to the field of in vitro nucleic acid detection, and uses PCR combined with pyrosequencing technology to detect the KRAS genotype in patient tissue DNA, in particular to a pair of sequencing primers and a kit for qualitatively detecting the KRAS genotype. Background technique [0002] There are three genes in the RAS gene family related to human tumors—HRAS, KRAS and NRAS. Among them, KRAS has the greatest impact on human cancer. It is like a molecular switch: when it is normal, it can control the pathway of regulating cell growth; when it is abnormal, the gene is permanently activated. Causes cells to continue to grow and prevents cells from self-destruction, allowing cells to proliferate uncontrollably and become cancerous (see figure 1 ). According to statistics, KRAS mutation colorectal cancer patients account for about 30-40%, and the mutation rate of lung cancer patients accounts for about 20-30%. [0003] The NCCN expert ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 肖芳
Owner CHANGSHA 3G BIOTECH
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