ABI PE9700 PCR Cycler

Genome Diagnostics BV:  Investigation of the Suitability of Kyratec’s SuperCycler™ for HLA Typing

Introduction

Human Leucocyte Antigens (HLA) are encoded by highly polymorphic genes on chromosome 6. These genes play a major role in the immune response but it has also been found that they have a role in transplantation rejection. The high level of polymorphisms translate into the many alleles that each gene has eg. the HLA-B gene currently encompasses 1605 alleles. Amplification and sequencing of HLA genes to determine these allelic variants (typing) has been challenging. This is due to a combination of the high number of polymorphisms and homology found in these genes. Genome Diagnostics BV has developed amplification and sequencing primers (SBTexcellerator®) and software (SBTengine®) to facilitate the process of Sequencing Based Typing (SBT), the gold standard in high resolution HLA typing. This study aimed to compare the performance of our SBTexcellerator® products on the ABI PE9700 thermocycler and Kyratec’s SuperCycler™, thereby determining the suitability of Kyratec’s SuperCycler™ for use in HLA Typing.

Materials and Methods

The HLA-B gene from 16 samples was amplified and sequenced using SBTexcellerator® primers in both the ABI PE9700 thermocycler and Kyratec’s SuperCycler™. This was carried out according to the protocol described in the manual.

Results

Amplification of samples 1 – 16 (HLA-B) in ABI (above) versus Kyratec (below). Amplicons (3.5kb) were run on a 1.5% agarose gel.

Sequencing

Below is a representative example of the results from one of the samples tested: .

Conclusions

Comparison of amplification and sequencing reactions with HLA-B, using the ABI PE9700 and Kyratec SuperCycler, showed minor variance of <10% in the representation of 2 alleles, which is of no functional difference when it comes to typing HLA loci.