Detection method of human proto-oncogene KRAS and kit

A detection method and kit technology, applied in the biological field, can solve problems such as insufficient accuracy, high requirements for experimental conditions, and inability to screen unknown mutations, and achieve the effect of improving accuracy and standardization

Inactive Publication Date: 2010-06-16
GENESKY BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent number is 200480043145 (Korean patent), which requires high experimental conditions, unstable results, and insufficient accuracy, so it is not conducive to clinical standardized application
Moreover, the chip hybridization method can only detect known mutations, and cannot screen unknown mutations.

Method used

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  • Detection method of human proto-oncogene KRAS and kit
  • Detection method of human proto-oncogene KRAS and kit
  • Detection method of human proto-oncogene KRAS and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 KRAS gene mutation detection

[0065] Specimen source: This method can be applied to the detection of Kras gene mutations in DNA from blood, tissue, paraffin specimens, pathological sections and other samples. We used DNA extraction from blood, tissue, paraffin and other specimens from Qiagen, Germany. Extraction kit (for specific extraction method, please refer to the kit instruction manual). The samples we selected in this example were tumor tissue samples from 64 cases of colorectal cancer (Fujian Provincial Cancer Hospital, No. 1-64), and DNA was extracted from each tissue.

[0066] PCR amplification:

[0067] Prepare the reaction mixture in a microcentrifuge tube, the reaction system is as follows:

[0068] components

1×

8.5×

wxya 2 o

6.52μl

55.42μl

10×Buffer

1μl

8.5μl

dNTP (10mM)

0.2μl

1.7μl

MgCl 2 (25mM)

0.2μl

1.7μl

Primer Mix (2μM)

1μl

8.5μl

Ta...

Embodiment 2

[0107] Embodiment 2 KRAS gene mutation detection

[0108] The treatment and experimental steps of samples 1-5 are the same as in Example 1. In the PCR amplification step, Primer Mix is ​​a mixture of two pairs of primers, and the concentration of each primer is 2 μM; primer SEQ ID NO: 5 / SEQ ID NO : 6 is a primer for amplifying Kras exon 1; primer SEQ ID NO: 9 / SEQ ID NO: 10 is a primer for amplifying Kras exon 4.

[0109] The sequencing primers in the reaction mixture in the sequencing reaction are KrasE02F and KrasE04F, respectively.

[0110] Sequencing results show that samples 2 and 3 have missense mutations, see image 3 , Figure 4 .

Embodiment 3

[0111] The preparation of embodiment 3KRAS gene mutation detection kit

[0112] Prepare a kit with the following components, each in a tube:

[0113] 1. PCR master mix: Contains 3 pairs of primers (each 2uM), MgCl 2 (25mM), dNTP(10mM), 10×Buffer

[0114] name sequence

Sequence (5'→3')

SEQ ID NO:

forward primer

KrasE02F: gagtttgtattaaaaggtactggtgga

KrasE03F: cgtcatctttggagcaggaac

KrasE04F: ggaaggaaaatttggtgtagtgg

SEQ ID NO: 5

SEQ ID NO: 7

SEQ ID NO: 9

[0115] name sequence

Sequence (5'→3')

SEQ ID NO:

reverse primer

KrasE02R: gtgcaggacccattctttgataca

KrasE03R: cactgctctaatcccccaaga

KrasE04R: aagaaaccaaagccaaaagca

SEQ ID NO: 6

SEQ ID NO: 8

SEQ ID NO: 10

[0116] 2. Taq DNA polymerase: 1Uhotstar taq enzyme (Germany QIAGEN company)

[0117] 3. PCR product purification enzyme: Contains 0.5U SAP (Promega) and...

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Abstract

The invention provides a detection method of KRAS gene mutation and a kit. The detection method comprises the following steps of: (a) extracting DNA in the sample, and carrying out PCR amplification by using specific primers under specified conditions; and (b) and sequencing the amplified products, comparing with normal KRAS gene, and determining whether gene mutation exists. The specific primers are 2-4 pairs of primers aiming at different exons of the KRAS gene. By adopting the detection method and the kit of the invention, the detection speed can be improved and the reagent and time are saved. By utilizing the detection result, the administration for clinical chemotherapy of alimentary tract tumors can be guided.

Description

technical field [0001] The invention relates to clinical molecular diagnosis technology in the field of biotechnology, in particular to a detection method and kit for human proto-oncogene KRAS. Background technique [0002] Existing technologies for detecting the human proto-oncogene KRAS are divided into two categories. One is to use enzyme-linked immunosorbent assays to detect the expression level of the kras gene rather than the gene mutation level. The expression level can only provide reference data at the mRNA level, not DNA level, and the expression level is only related to tumorigenesis, and has nothing to do with the efficacy of chemotherapy drugs. [0003] The second type also uses the detection of gene mutations at the DNA level, but uses chip hybridization technology (oligonucleotide hybridization detection technology, that is, the use of oligonucleotides with relevant mutations to hybridize with the DNA of the subject to determine the type of mutation). The pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 姜正文丁达
Owner GENESKY BIOTECH INC
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