High resolution, high throughput hla genotyping by clonal sequencing
A genotype, HLA-A technology, applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of phase ambiguity retention, time-consuming, etc.
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[0073] multiplex pyrosequencing
[0074] Analysis of multiple HLA loci across multiple samples in a single 454 run is facilitated by the incorporation of molecular ID (MID) tags into the PCR primers. Table 1 shows the sequence of the 454 HLA-specific fusion primer with adapter sequence (for bead capture) and 4-base MID tag. Additional sequences giving 5-base MID tags are provided.
[0075] In preliminary experiments, 24 cell lines with known HLA genotypes (Table 2) were analyzed. In subsequent experiments, 48 samples were analyzed.
[0076] Fifteen primer pairs were designed for exons 2, 3 and 4 of HLA-A, B and C loci, exons 2 of DRB1, DPB1, DPA1, DQA1, and exons 2 and 3 of DQB1 . Primers with twelve different MID tags were designed for each target sequence, a total of 180 (15 x 12). Primers for exon 2 of DRB1 also amplified the DRB3, DRB4 and DRB5 loci, which are genes present on specific DRB1 haplotypes. After amplification of various samples, PCR products were analy...
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