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Cont..... How to do neutrophil and platelet genotyping GENOTYPING
OF HLA-1 TO HPA-13 BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM
Genomic DNA (200-400 ng) was amplified by the use of
gene-specific primer pairs as describe in Table 1. Amplification was
performed on a DNA thermal cycler (9600, Applied Biosystem, Langen,
Germany) under polymerase chain reaction (PCR) condition as shown in
Table @. PCR products were analyzed by 1.8-percent agarose gel
electrophoresis using the Tris borate EDTA buffer system (TBE, Biozyme,
Hameln, Germany); the molecular weight standard was MW5 (Boehringer
Mannheim, Mannheim, Germany). Table
1. Primers used for amplification of Gpla, GPlba,
GPlbb,
and GPIIIa encompassing the polymorphic bases responsible for HPA-1 to
-13
*
F: forward primer; R: reverse primer. The position of the first 5
nucleotide of primers is given. Mismatched bases are underlined.
Intronic bases (italic) located downstream or upstream of the
polymorphic exons are indicated with (+) or (-), respectively. The
numbering of primers is based on publishedcDNA sequences 17-21 and
genomic DNA sequences. 22-26 Table
2. PCR conditions for HPA genotyping*
*
Genotyping was performed with the use of DNA polymerase(AmpliTaq Gold [5
U/mL],
Perkin Elmer) on a thermal cycler. After denaturation for 10 minutes at
96°C,
DNA samples (100 ng/mL)
were amplified by using gene-specific primers (5mM)
in the presence of dNTP (1.25 mM). Samples were heated for 5
minutes at 72°C
for the final extension. +
PRC was performed in the presence of 5 mL
of dimethyl sulfoxide.
Table 3. RFLP analysis of HPA-1 to 13
* RFLP was modified in this study. Allele-specific fragments are
italicized. Aliquot (8 mL) of PCR products were digested in a thermoblock (Trio Biometra, Gottingen, Germany) with a specific endonucleases for restriction fragment length polymorphism (RFLP) as described in Table 3. Restriction fragments were analyzed on 3 % agarose gel using TBE buffer. GENOTYPING
OF NA1, NA2, AND SH BY ALLELE-SPECIFIC PCR
Genotyping of NA1, NA2 and SH was performed by using PCR with
sequence-specific primers (SSP). Allele-specific PCR products were
analyzed on 2% agarose gel using TBE buffer.
DNA derived from these B-LCLs was typed by polymerase chain
reaction-restriction fragment length polymorphism and
sequence-specific primers. The results were in accordance with the
genotyping from peripheral blood cells.
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