Rapid genotyping of human platelet antigen 1 (HPA‐1) with fluorophore‐labelled hybridization probes on the LightCycler^TM

Abstract

Summary.Genotyping of human platelet alloantigens (HPA) has become an important procedure in the diagnosis and prevention of disorders such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and refractoriness to platelet transfusion therapy. We present a single-tube method for HPA-1 genotyping that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. A fragment covering the polymorphic site is amplified in the presence of two fluorescentlylabelled hybridization probes. During the annealing step of the thermal cycling, both probes bind to their complementary sequences in the amplicon resulting in resonance energy transfer, thus providing real-time fluorescence monitoring of PCR. Continuous aquisition of fluorescence data during a melting curve analysis at the completion of PCR revealed that loss of fluorescence occurred in an allele-specific manner as the detection probe, which was fully complementary to the HPA-1b allele, melted off the template. By determining the temperature at which maximum melting of the hybrids occurred, the two alleles were readily distinguishable. Using this method, genotyping of 32 samples was completed within 30 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The genotypes determined with the LightCycler TM were identical when compared with a conventional PCR and restriction fragment length polymorphism technique. The genotyping of HPA-1 on the LightCycler is a rapid and reliable method that is suitable for typing both small and large numbers of samples.

Association study on the DUSP6 gene, an affective disorder candidate gene on 12q23, performed by using fluorescence resonance energy transfer-based melting curve analysis on the LightCycler

“…13 There were two reasons why we tested a two-step protocol, that is, performing PCR amplification and fluorescence analysis in separate tubes. First, the quality of our sample DNAs was variable, and PCR yield of amplicons did not reach a satisfactory level for some of the samples.…”

“…Continuous fluorescence monitoring of the reaction as the temperature is raised from annealing to denaturation results in a sharp decrease in fluorescence at the temperature at which the 'detection probe' dissociates from the template. 12,13 The single base change caused by the DUSP6 polymorphism results in a decrease in the fluorescence of the 'detection probe' that can easily be distinguished with the LightCycler™ (Roche).…”

The Factor V Leiden, Prothrombin Gene 20210GA, Methylenetetrahydrofolate Reductase 677CT and Platelet Glycoprotein IIIa 1565TC Mutations in Patients With Acute Ischemic Stroke and Atrial Fibrillation

“…2,8 The MTHFR and GPIIIa mutations were detected using hybridization-based polymerase-chain reaction in a LightCycler real-time system. 9,10 We used stratification to control for the presence of other potentially confounding variables. Relative risks were calculated as the odds of having the gene mutation for cases, compared with the odds for controls, with 95% CI for the odds ratios.…”

Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing

“…Except for those directly focused on polymerase chain reaction (PCR) and ligation methods (Landegren et al 1988;Bottema et al 1993;Livak 1995), most such approaches can be roughly divided into techniques based on primer extension or on the recognition of heteroduplex DNA. The former category includes several assay types, such as Solid Phase Minisequencing (SPM) and detection of dissimilarly sized extension fragments by MALDI-TOF mass spectroscopy (Little et al 1997;Syvänen 1999), whereas the latter involves a wide number of applications, including mismatch cleavage detection, oligoarray hybridization, molecular beacon signaling, fluorescence monitoring of PCR, and electronic dot blot assay (Pease et al 1994;Mashal et al 1995;Southern 1996;Tyagi and Kramer 1996;Gilles et al 1999;Nauck et al 1999; for review, see Graber et al 1998).…”