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Aim: Dried Blood Spots (DBS) on FTA cards serve as a good resource for
HLA and other SNP genotyping studies. Isolation of gDNA from small punches
of DBS for genetic testing, such as HLA typing, is cumbersome and
inefficient. Herein, we describe a quick and inexpensive protocol for the
preparation of DNA extract from DBS that shows promise for HLA typing based
on results from LIFECODES HLA-A SSO typing kit. Methods: Small 3 mm discs
of DBS were washed with FTA washing buffer to remove possible PCR
inhibitors while gDNA and cells remain attached to the discs. One washed
disc was treated with 10 @ml of alkaline lysis solution for 10 minutes on
ice followed by the addition of 10 @ml of neutralizing solution. 1@ml of
this crude DNA extract was used for PCR amplification for HLA-A typing
using a Hot Start Taq DNA polymerase. The rest of the HLA typing assay
protocol remained essentially as described in the kit product insert.
Results: Successful genotyping of several DBS was achieved for HLA-A
alleles using only 1@ml of the 20 @ml crude DNA extract obtained from one 3
mm disc of DBS, while leaving enough DNA extract for the genotyping of
other HLA loci. The genotyping results from this procedure were concordant
to the reference genotyping results obtained from the gDNA isolated from
the corresponding blood samples using LIFECODES HLA-A SSO typing kit.
Conclusions: This procedure has potential to eliminate the need for gDNA
isolation from DBS for HLA typing assays, thus reducing the requirements of
large amounts of biological samples, assay time and costs. Validation of
these procedures could find enormous use in HLA typing of archived blood
samples. Such validation studies using larger number of DBS, and using
LIFECODES SSO Typing Kits for other HLA loci are pending.
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