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SNP (Single Nucleotide Polymorphism) marker composition for detecting lung cancer susceptibility, primer composition and kit

A primer combination and susceptibility technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of high cost, long detection cycle and expensive direct sequencing method

Inactive Publication Date: 2018-05-15
SINOGENOMAX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the direct sequencing method has high labor costs and a long detection cycle. The main reagent BigDye used in sequencing is imported, and the cost of consumables is high. The cost of kits for PCR product purification is also high, resulting in high cost and complicated detection steps for direct sequencing. , The detection cycle is long, it is difficult to realize the detection of high-throughput samples
However, the TaqMan fluorescent probe method requires expensive and non-versatile two-color probes. At the same time, it has high requirements for sample quality. In addition to no sample degradation, the concentration must be as consistent as possible.

Method used

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  • SNP (Single Nucleotide Polymorphism) marker composition for detecting lung cancer susceptibility, primer composition and kit
  • SNP (Single Nucleotide Polymorphism) marker composition for detecting lung cancer susceptibility, primer composition and kit
  • SNP (Single Nucleotide Polymorphism) marker composition for detecting lung cancer susceptibility, primer composition and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 sequence search

[0054] Log in to NCBI, and query the sequence corresponding to the SNP site in GenBank according to the SNP site. The specific sequence is shown in Table 1.

[0055] Table 1 The allelic variation sequence corresponding to the SNP site

[0056]

Embodiment 2

[0057] Embodiment 2 primer design and synthesis

[0058] For the SNP site described in Example 1, a primer combination and a probe combination dedicated to KASP technology were designed, wherein the primers included a primer combination dedicated to the detection of the aforementioned 5 SNP sites, and the probe combination included a fluorescent probe. Needles 1-2 and quenching probes 1-2, wherein the nucleotide sequences of fluorescent probes 1 and 2 are the same as tag sequences 1 and 2 respectively, and the nucleotide sequences of quenching probes 1-2 are respectively tag sequences 1, 2 Reverse complementary nucleotide sequences (see Table 2 for specific sequences).

[0059] The aforementioned primers and probes were synthesized, and the 5' end of fluorescent probe 1 was labeled with FAM, the 5' end of fluorescent probe 2 was labeled with HEX, and the 3' ends of quencher probes 1 and 2 were labeled with BHQ.

[0060] Table 2 KASP-specific primers, labels and probe sequence...

Embodiment 3

[0063] The establishment of embodiment 3KASP detection method

[0064] Using the extracted DNA of the individual to be detected as a template, use the KASP-labeled special primers and probes developed in Example 2 for PCR amplification and detection. The specific method is as follows:

[0065] 1) Prepare Primer Mix according to the ratio shown in Table 3, and store it in the refrigerator at 4 degrees after preparation.

[0066] Table 3 The ratio of each component in Primer Mix

[0067] Primer

volume

Upstream primer 1 (100mM)

12μL

Upstream primer 2 (100mM)

12μL

Downstream primer (100mM)

30μL

h 2 o

46μL

total

100μL

[0068] 2) Add human DNA template to be tested (sample comes from buccal swab or blood) (20-30 ng / μL) into 384-well plate, 2.5 μL per well.

[0069] 3) Configure the Assay Mix according to the ratio shown in Table 4 (calculated according to the number of samples), wherein the Master Mix inc...

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Abstract

The invention relates to an SNP (Single Nucleotide Polymorphism) marker composition for detecting lung cancer susceptibility, a primer composition and a kit. The SNP marker composition comprises rs1048943, rs1051730, rs13181, rs1799793 and rs25487. The SNP marker composition provided by the invention can systemically and comprehensively reflect lung cancer susceptibility, is particularly suitableto be used by Chinese populations, and is conducive to early discovering lung cancer populations, so that a living environment and a living habit are specifically improved, and the lung caner incidence rate is reduced.

Description

technical field [0001] The invention relates to the field of molecular markers, in particular to a SNP marker combination, a primer combination and a kit for detecting lung cancer susceptibility. Background technique [0002] Lung cancer has become the most common malignant tumor and the leading cause of cancer death in the world in recent years. In my country, lung cancer is the leading cause of cancer death. Genes, environment, and lifestyle are the three causes of lung cancer, among which genetics play the most important role. According to the age statistics of lung cancer incidence, 27% of lung cancer patients around 50 years old are caused by genetic genes, 42% are caused by the joint influence of genes and smoking, and 27% are caused by smoking and environmental factors. 4% were caused by other reasons. So far, the key to improving the curative effect of lung cancer lies in early detection, early diagnosis, and early treatment. However, limited by the current diagnos...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 娄海娟高文娟王柏婧侯全民张晓柠
Owner SINOGENOMAX
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