HLA related SNP marker and detection primer pair and determination method thereof
A technique for determining methods and labeling primers, applied in the medical field
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Embodiment 1
[0027] Embodiment 1 Obtaining a group of HLA-associated SNP markers
[0028] 1.1 Obtaining of blood samples in Example 1
[0029] Example 1 The blood samples were 6 blood samples randomly drawn from the pre-transplant whole blood samples of kidney transplant patients collected by the Organ Transplantation Department of the First Affiliated Hospital of Sun Yat-sen University for genomic DNA extraction.
[0030] 1.2 Example 1 Extraction of Genomic DNA from Blood Sample
[0031] In this experiment, TIANamp Blood DNA Kit (catalogue number: DP348) was used to extract genomic DNA from the blood sample of Example 1. The specific steps are as follows:
[0032] (1) Gently invert and mix the whole blood samples of the six patients in Example 1, and draw 200ul from it into a new 1.5ml EP tube (if the DNA extraction concentration is too low, you can also centrifuge the blood sample at 500g for 5min to separate the layers, and take The buffy coat cells in the middle are 200ul for subsequ...
Embodiment 2
[0061] The difference between this embodiment and embodiment 2 is that the primers used during amplification are different:
[0062] Upstream primer: 5'CTTTGAATTTAGGCAGAACG 3' (SEQ ID NO.3)
[0063] Downstream primer: 5'GCAGCATCACTTGTCTCC 3' (SEQ ID NO.4).
[0064] The nucleotide fragment where the SNP to be tested is located is amplified.
[0065] The PCR amplification product can be sequenced in the forward direction or in the reverse direction, preferably forward sequencing.
[0066] Forward sequencing primer 5'CTTTGAATTTAGGCAGAACG 3' (SEQ ID NO.3);
[0067] Reverse sequencing primer: 5'GCAGCATCACTTGTCTCC 3' (SEQ ID NO.4).
[0068] Since base 32610275 is one of the most important SNPs affecting the expression of DQ, the expression level of DQ is higher when the base at position 32610275 is T, and the expression level of DQ is lower when the base at position 32610275 is C. Based on the above principles, by measuring the SNP at the site above the DQ promoter of the patien...
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