Primer, kit and method related to determination of full-length sequence of human leukocyte antigen HLA-E, -F and-G genes
A leukocyte antigen, HLA-E technology, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve the problems of expensive and limited large-scale application of HLA gene sequencing and typing
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Embodiment 1
[0084] This example provides a complete set of full-length sequences of HLA-E 3.8kb, HLA-F 3.5kb and HLA-G 3.1kb alleles.
[0085] Take HLA-E gene full-length amplification primers E-Genome-F and E-Genome-R in the present invention, HLA-F gene full-length amplification primers F-Genome-F and F-Genome-R, HLA-G Gene full-length amplification primers G-Genome-F and G-Genome-R were used to amplify the full-length HLA-E, -F, and -G genes of 8 random Asian DNA samples respectively, and carried out in ABI 9700 PCR instrument. The amplification reaction system consists of:
[0086]
[0087] In the HLA-E full-length sequence amplification reaction system, the cycle parameters are:
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[0089] In the HLA-F full-length sequence amplification reaction system, the cycle parameters are:
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[0091]
[0092] In the HLA-G full-length sequence amplification reaction system, the cycle parameters are:
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[0094] Take 2 μL of PCR products, stain with cyber-gre...
Embodiment 2
[0128] This example presents the experimental results related to the whole gene sequencing of HLA-E in Example 1.
[0129] 2.1 Identification of HLA-E whole gene PCR amplification products
[0130] Using the PCR amplification primers designed in this paper, the entire HLA-E gene fragments of 16 samples were successfully amplified, and the length of the PCR product was about 3.6Kb. Take 5 μL and electrophoresis in 1.2% agarose gel, and a clear amplification band can be seen. The Marker is DL10000 DNA Marker (Takara, Dalian Baosheng Company). Amplification results see figure 1 . figure 1 The upper and middle figures show the electropherograms of the amplification products of Marker and samples C1-C8 respectively, and the lower figures show the electrophoresis figures of the amplification products of Marker and samples C9-C16 respectively.
[0131] 2.2 Sequencing Peak Map and Splicing Effect
[0132] Using the step-by-step sequencing primers ESF1-ESF7 designed in this paper, ...
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