Simultaneous Genotyping of Human Platelet Antigens by Hot Start Sequence- Specific Polymerase Chain Reaction with DNA Polymerase AmpliTaq Gold

Abstract

Objectives: 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-la and lb, 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. Conclusion: 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.

Genotyping of the Human Platelet Antigen-1 by ELISA Detection of Allele - Specific Amplicons

“…Until recently, only al-ternative strategies such as ligase chain reactions [9] offered adequate specificity. Two groups [7,8] have recently demonstrated that a hot-start technique may enhance both the specificity and the efficiency of sequence-specific PCR for HPA typing. The present study demonstrates that the specificity of PCR catalyzed by AmpliTaq Gold TM suffices for reliable HPA-1 genotyping by ELISA.…”

“…This amino acid dimorphism is due to a single base pair substitution in the coding gene (C^T sub stitution at nucleotide position 196 of the coding DNA) [1]. Polymerase chain reactions (PCR) with allele-specific primers have been established as a valid method for HPA genotyping [2][3][4][5][6][7][8], This method still requires identification of the amplificates by gel-electrophoretic analysis of the products from two reactions per DNA sample essentially limiting the sample throughput.…”

Human Platelet Antigens

“…Each data entry (bar) represents the mean and standard deviation for the number of studies indicated in parentheses. References: White, 36,[188][189][190][191][192][193][194][195][196][197][198][199][200][201][202][203]220 Southeast Asian, 36 Asian, 36,189,190,204,205,[207][208][209][210][211][212][213]215 Central African, 36 North African, 36,216,217 black American, [188][189][190]203,220 Amerindian, 188,189,218 Native American, 220 and Aboriginal. 202,219…”

“…Each data entry (bar) represents the mean and standard deviation for the number of studies indicated in parentheses. References: White,36,[188][189][190][191][192][193][194][195][196][197][198][199][200][201][202][203] Southeast Asian,…”

Improvement of HLA class I and class II PCR‐SSP typing by using timed‐release activity of DNA polymerase

“…To obtain a higher level of specificity while simultaneously genotyping for human platelet antigens (HPA) (3) and for HLA class I and class 11, we employed a DNA polymerase AmpliTaq Gold (Perkin Elmer). As described by the manufacturer, this polymerase is provided in an inactive state and becomes activated when heated.…”