PMID: 9174137May 1, 1997Paper

Sequencing-based typing of HLA-A locus using mRNA and a single locus-specific PCR followed by cycle-sequencing with AmpliTaq DNA polymerase, FS

Tissue Antigens
L NorgaardA Svejgaard

Abstract

The large number (59) of alleles now known at the HLA-A locus is a serious challenge to the existing methods for HLA typing, including many of the DNA based methods. Here, we describe a sequencing-based typing (SBT) protocol for typing of HLA-A alleles using a single A-locus-specific PCR. This reaction amplifies an 824 base pair product from cDNA, prepared from mRNA, covering exons 1-3 and most of exon 4. This product allows identification of all possible combinations of two alleles from this locus. The sequencing strategy used for allele assignment contains several improvements compared to those previously published. The enzyme AmpliTaq DNA Polymerase, FS, used, combines high-quality sequencing, i.e. long reads, low background, and uniform peak heights making the identification of heterozygous positions very reliable in a fast and easy protocol developed by determining the optima for a number of variables. Thus, this strategy meets most of the requirements for the use of sequencing in HLA typing. Furthermore, this method is very flexible. The use of a PCR primer-pair tailed with the recognition sites for two different sequencing primers allows the application of the same sets of fluorescent-labelled sequencing primers regardle...Continue Reading

References

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Citations

Jun 7, 2005·Human Immunology·Wendy T N SwelsenElla M van den Berg-Loonen
Nov 7, 1999·Tissue Antigens·L NorgaardA Svejgaard
Jan 1, 2010·BMC Molecular Biology·Elena Hidalgo AshrafiNatasha Paul
Jun 14, 2012·PloS One·Benjamin StevensYale E Goldman
Dec 3, 2014·Forensic Science International. Genetics·Tao Geng, Richard A Mathies

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