Histocompatibility screening by molecular techniques: Use of polymerase chain reaciton products and heteroduplex formation

Abstract

Novel molecular approaches have recently become available allowing improved major histocompatibility complex (MHC) matching of potential allogeneic bone marrow donors and recipients. Current cellular and serological assays are hindered by aberrant cell populations and limited reagents which only detect an individuals' phenotype. Therefore, a molecular screening protocol which discriminates at the genotypic level would be advantageous. Here we describe a two-step DNA-based approach that can be applied to large-scale screening of potential donors. A primary screen, utilizing polymerase chain reaction (PCR), reduces the potential donor population, whereas a secondary or fine resolution screen uses DNA heteroduplex analysis to determine identity or non-identity at specific loci. Heteroduplex analysis generates a DNA migration pattern that is unique for alleles at a given locus, and is more sensitive than serology in discriminating among individuals. Here we demonstrate the potential feasibility of this approach by analyzing results at one MHC locus, HLA-DQ. Since this method does not rely on typing sera or viable lymphocytes, it is not subject to the variability found in the traditional methods. In contrast to traditional methods, these molecular techniques can provide the critical information needed to select a potential bone marrow donor.

PAGE analysis of the heteroduplexes formed between PCR-amplified 16S rRNA genes: estimation of sequence similarity and rDNA complexity

“…Formation of heteroduplexes also occurs during PCR amplification of partially homologous genes and the pattern obtained from a single species enables allelic polymorphism and mutations in heterozygous individuals to be detected (Bowen et al, 1997;White et al, 1992), and has also been applied to the genotyping of different species (Jensen & Hubner, 1996;Rubocki et al, 1992;Tang et al, 1995Tang et al, , 1996Zimmerman et al, 1995).…”

High‐resolution DNA melting curve analysis to establish HLA genotypic identity

“…Heteroduplex analysis of polymerase chain reaction (PCR) products was used in the past in order to establish identity at HLA loci (5, 6). However, these subjective gel techniques have lost favor to high‐resolution probe techniques and sequencing methods that are more robust.…”