Coronary artery disease (CAD) and acute myocardial infarction (AMI) susceptibility diagnosis kit and use of single nucleotide polymorphism (SNP) in preparation thereof
A kit and susceptibility technology, applied in the field of SNP in the preparation of coronary heart disease and acute myocardial infarction susceptibility detection kits, can solve the problems of individual differences in treatment effects, difficult diagnosis and discovery, and variable clinical manifestations
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Embodiment 1
[0055] Example 1: Detection of rs12050757 in samples by direct sequencing
[0056] DNA extraction:
[0057] A 5ml venous EDTA anticoagulated blood sample was obtained from the subject. Then follow the common salting-out method or use a special DNA extraction kit, such as the DNA extraction kit purchased from Omega, USA, to extract the genomic DNA of the blood sample to be tested.
[0058] PCR amplification:
[0059] The sample DNA was amplified by PCR using the upstream primer 5'GGAAGTCCCCAAGTAGAAA 3' and the downstream primer 5'CCCACAGTAACACCCAAG 3' (such as a Takara PCR Thermal Cycler from TaKaRa, Japan). Use 50μl PCR reaction system for gene amplification of rs12050757, containing 1×PCR buffer, 1.5mM MgCl 2 , 100-150ng of genomic DNA, both upstream and downstream primers are 0.5μM, dNTP is 0.2mM, and ExTaq DNA polymerase from TaKaRa Company is 1.5U. The PCR amplification cycle parameters are as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30...
Embodiment 2
[0065] Example 2: Detection of rs12050757 by Taqman probe SNP detection method
[0066] Primers were designed to specifically amplify the PCR product containing the rs12050757 site, and two Taqman-MGB probes were designed for the C and T alleles of the rs12050757 site, respectively.
[0067] Primer design principles are:
[0068] 1. Sequence selection should be in the conserved segment of the gene
[0069] 2. Avoid the formation of 4 or more consecutive pairings between the primers themselves or with the primers, and avoid the formation of a circular hairpin structure by the primers themselves
[0070] 3. The length of the primer is 18 to 24 nucleotides.
[0071] 4. Tm value is 55-65℃, GC content is 40%-60%
[0072] 5. The Tm value difference between the primers should not exceed 2°C
[0073] 6. Avoid using base A at the 3' end of the primer, and avoid 3 or more consecutive identical bases at the 3' end of the primer.
[0074] 7. The length of the PCR amplification fragme...
Embodiment 3
[0086] Example 3: Detection of rs12050757 by PCR-single-strand conformational polymorphism (SSCP) method
[0087] The rationale for this method is as follows:
[0088] In the neutral polyacrylamide gel without denaturant, the mobility of single-stranded DNA is not only related to the length of DNA, but also mainly depends on the spatial conformation formed by single-stranded DNA. The single-stranded DNA of the same length will have different conformations due to the difference in sequence or single base difference. When PCR products are subjected to single-strand DNA gel electrophoresis after denaturation, each single strand is in a certain position. When base deletions, insertions or single base substitutions occur in the target DNA, migratory displacement will occur, which suggests that there is a group variation in the fragment. For example, in SNP detection, C allele homozygous, T allele homozygous and C / T heterozygous will show different band positions after electrophor...
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