Simultaneous genotyping of human platelet antigens by hot start sequence-specific polymerase chain reaction with DNA polymerase AmpliTaq Gold.
OBJECTIVE
Human platelet alloantigen (HPA) typing has potential clinical relevance in a variety of contexts. We can improve methods for HPA genotyping by complementing our knowledge of the DNA sequence polymorphisms of HPA genes and experience with various DNA-based HPA typing techniques.


METHODS
A newly available DNA polymerase, AmpliTaq Gold (Perkin Elmer), provided in an inactive state and activated by heat, makes it possible to perform a hot start polymerase chain reaction (PCR) in order to prevent nonspecific amplification during the setup of PCR. To establish a practical procedure for HPA-1, 2, 3 and 5 genotyping, we applied the AmpliTaq Gold for a hot start PCR and employed 8 pairs of published sequence-specific primers (SSP). A simple simultaneous genotyping of these 4 HPA systems could be rapidly achieved with high specificity.


RESULTS
The HPA gene frequencies observed in 126 randomly selected German blood donors were 0.82 and 0.18 for HPA-1a and 1b, 0.92 and 0.08 for HPA-2a and 2b, 0.63 and 0.37 for HPA-3a and 3b and 0.90 and 0.10 for HPA-5a and Sb, respectively.


CONCLUSIONS
Using our hot start PCR-SSP procedure with AmpliTaq Gold a simple, rapid and reproducible genotyping for HPA-1, 2, 3 and S systems could be achieved.
Chen
D F
DF
Blutbank-Immunhämatologie-Transfusionsmedizin, Medizinische Hochschule Hannover, Deutschland.
Pastucha
L T
LT
Chen
H Y
HY
Kadar
J G
JG
Stangel
W
W
D000483:Alleles; D016824:Antigens, Human Platelet / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D005787:Gene Frequency; D005838:Genotype; D005858:Germany; D006358:Hot Temperature; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
D016824:Antigens, Human Platelet; D004259:DNA-Directed DNA Polymerase