DNA sequencing techniques were first described in the 1970’s by Gilbert & Maxam using a chemical sequencing approach and by Sanger using a chain termination method. Both lead investigators were awarded the Nobel Prize for chemistry for their work. The Sanger sequencing method is the simpler of the two and is widely used for Sequence Based Typing.
The sequencing step in sequence based typing is preceded by locus specific PCR amplification to generate templates for the sequencing step. The sequencing step requires single stranded DNA templates, DNA primers, a DNA polymerising enzyme, deoxynucleotidephosphates (dNTPs) and fluorescently labelled di-deoxynucleotidephosphates (ddNTPs). At a minimum, HLA class I requires sequencing at exons 2 and 3 and HLA class II requires sequencing at exon 2. Other exons are often also sequenced to help increase the resolution. The amplified PCR products are divided into four separate sequencing reactions (eight if sequencing in the forward and reverse directions), each containing all four dNTPs (dATP, dCTP, dGTP & dTTP) and the DNA polymerase. To each reaction, one of the ddNTPs is added. The ddNPT will terminate chain elongation once incorporated into the growing sequence as they lack the 3’-OH group required for the formation of a phosphodiester bond between themselves and an incoming nucleotide. The products of the sequencing reaction are analysed using automated high-throughput DNA sequence analyzers. Modern DNA sequencers use capillary electrophoresis for size separation, detection of dye fluorescence and data output as peak traces on a chromatogram.
Sequence based typing is critical to Haematopoietic Stem Cell transplantation where HLA typing at high resolution may be needed in the related setting and is critical in the unrelated setting. In stem cell transplantation, Petersdorf et. al., and others have shown that the risk of GvHD increases with increasing numbers of mismatches in the Graft versus Host direction (i.e. HLA genes present in the patient but absent in the donor). Similar studies have also shown that the risk of graft failure increases with increasing numbers of mismatches in the Host versus Graft direction (i.e. HLA genes present in the donor but absent in the patient). Sequence based typing is generally used to obtain high resolution 4 digit allele level for HLA-A, B, C, DR and DQ and in some cases for HLA DP.
Sequence based typing is also an option in some disease association and drug sensitivity cases where a high resolution HLA type is required. Examples include HLA-B*57:01 typing for Abacavir drug hypersensitivity in HIV positive patients and HLA-DRB1*06:02 typing in Narcolepsy cases.
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