Mononucleotide polymorphic site of CDC6 gene correlated to primary liver cancer and application thereof

A single nucleotide polymorphism and nucleic acid technology, which is applied in the direction of recombinant DNA technology, microbial measurement/testing, DNA/RNA fragments, etc., to achieve the effect of promoting discovery

Inactive Publication Date: 2007-09-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching domestic and foreign literature and patents of the prior art, there has been no report on the relationship between CDC6 gene polymorphism and susceptibility to primary liver cancer

Method used

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  • Mononucleotide polymorphic site of CDC6 gene correlated to primary liver cancer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Collection of blood samples and extraction of genomic DNA

[0037]A total of 514 sporadic primary hepatocellular carcinoma specimens were collected from the Cancer Hospital Affiliated to Sun Yat-sen University in Guangzhou, with an average age of 48.6±11.8 years (standard deviation), and 44 of them were female. All cases were patients who were treated in the cancer hospital, and were diagnosed as primary hepatocellular carcinoma according to the World Health Organization criteria. The control samples were 509 unrelated healthy individuals from Guangdong Province, with an average age of 46.2±14.0 years (standard deviation), including 45 female samples.

[0038] Leukocytes were separated from the blood samples collected above by conventional methods, and genomic DNA of leukocytes was extracted by the phenol-chloroform method, and the sample DNA was uniformly diluted to 10 ng / ul as a DNA template.

Embodiment 2

[0039] Embodiment 2: design nucleotide primer sequence, carry out polymerase chain reaction (PCR) amplification

[0040] The PCR reaction was carried out on MJ PTC-200PCR instrument.

[0041] The PCR reaction system and procedures are as follows:

[0042] Perform an amplification reaction with a total volume of 20ul for each sample, which contains 20ng of the prepared genomic DNA dilution, 2ul of 10×Taq Buffer, 0.8ul of 5mM dNTP, and 25mM MgCl 2 1.6ul, 0.25 units of Taq DNA polymerase and 0.5ul each of amplification primers (diluted to 10uM), and finally add ddH 2 O make the total volume 20ul. After the reaction was pre-denatured at 94°C for 2 minutes, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, and finally a fill-in at 72°C for 6 minutes were performed.

[0043] Amplification primers are as follows:

[0044] Forward primer: 5'-CTAGGCTTTTCACTGTCACCAA-3' (SEQ ID NO: 2)

[0045] Reverse primer: 5'-GA...

Embodiment 3

[0047] Example 3: SNP typing on the amplified DNA sequence

[0048] For the product obtained in Example 2, single-strand conformational polymorphism (SSCP) was used for gene polymorphism typing. The specific method is: add 1 μl PCR product to 4 μl loading buffer (98% deionized formamide, 10 mM EDTA (pH8.0), 0.1% xylene cyanol, 0.1% bromophenol blue), denature at 95°C for 10 minutes, place Place on ice for 2 minutes, quickly spot 4 μl on a 10% non-denaturing polyacrylamide gel (Arc:Bis=37.5:1), and place in a refrigerator at 4°C for electrophoresis. 1.5W constant power electrophoresis for 10 hours, silver staining. The results of SSCP were sequenced and verified, and three genotypes of GG, AG, and AA were found. Some of the sequencing results are shown in Figure 1.

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Abstract

The invention discloses the polymorphic site (rs4134994) in CDC6 gene associated with primary liver cell cancer and its application. The invention also provides a method for detecting the polymorphic site and susceptibility of the primary liver cell cancer. By use of the nucleic acid sequence of the polymorphic loci and its detection method, it can produce kit for preventing primary liver cell cancer and early diagnosis and treatment.

Description

technical field [0001] The present invention relates to a single nucleotide polymorphic site of the CDC6 gene associated with primary hepatocellular carcinoma, the invention also relates to a method for detecting the polymorphic site, and the polymorphic site in prevention and auxiliary diagnosis and its use in the treatment of primary hepatocellular carcinoma. The invention belongs to the field of biotechnology. technical background [0002] Primary hepatocellular carcinoma is one of the most common malignant tumors in the world. my country is the main place where liver cancer occurs, and the incidence of liver cancer is as high as 35 / 100,000. In recent years, the incidence of liver cancer has a rising trend, seriously endangering human health and life safety. [0003] Single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. SNPs can appear in the gene regulatory region and coding reg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/113
Inventor 庄诗美熊兴东程家森杨金娥
Owner SUN YAT SEN UNIV
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