RAPID ALKALINE EXTRACTION OF GENOMIC DNA SUITABLE FOR HLA TYPING WITH SEQUENCE SPECIFIC PRIMERS AND PCR.
RW Allen, RD Zarrabi, and SA Ward, HLA Laboratory, Chapman Institute of Medical Genetics, Tulsa, OK.

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Extraction and purification of genomic DNA is fundamental to PCR-SSP testing. Several techniques for the extraction and purification of genomic DNA involve hydrolysis with proteinases or detergents followed by purification with organic solvents (Phenol/chloroform) or high salt. Alkaline extraction is simpler than these techniques, requiring less time and fewer pipetting steps. Alkaline extraction of genomic DNA from whole blood and buccal epithelial cells can be accomplished in less than 10 minutes at room temperature.

DNA was extracted from the whole blood (50 m L) of 16 individuals using phenol/chloroform (organic), high salt (inorganic), and alkaline extraction techniques. Yields of DNA from the various techniques were analyzed on the FluorImager 595 (Molecular Dynamics, Sunnyvale, CA) using PicoGreenä (Molecular Probes, Eugene, OR). Since PicoGreenä intercalates double stranded DNA with greater affinity than single stranded DNA, DNA extracted using organic or inorganic techniques was quantified both as double stranded and heat denatured DNA. Comparison of yields between methods were studied, with the greatest yield coming from alkaline extraction.

Genomic DNA for use in PCR-SSP testing was extracted with one of two protocols, depending on the amount of genomic DNA required. EDTA-anticoagulated whole blood or frozen buffy coat was used (10 m L for DR/DQ; 40 m L for AB/DR). Whole blood or frozen buffy coat was lysed with 1 mL of 4° C molecular biology grade water (MBGW). The lysate was centrifuged at 12000 x G for 1 minute, and the supernatant discarded. 0.2 M NaOH was added to the pellet (10 m L for DR/DQ; 40 m L for AB/DR), mixed, and incubated at room temperature for 5 minutes. 0.16M Tris-HCl (pH 7.5) was added to stop the reaction (45 m L for DR/DQ; 180 m L for AB/DR). This extract was then used directly in PCR-SSP testing. SSP kits from Genysysä , PelFreezâ , and One Lambda have all been successfully used with this extraction protocol. We now are able to perform PCR-SSP typing from extraction to gel image analysis in less than 3 hours, or very nearly the same time required for serological typing.

Extraction of genomic DNA from buccal epithelial cells using alkaline extraction has been performed in this laboratory with successful subsequent amplification of non-MHC loci. To date we have not tried this technique for HLA typing, but believe it will also be successful.