Histocompatibility screening by molecular techniques: use of polymerase chain reaction products and heteroduplex formation.
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.
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-3135.
R J
RJ
Rubocki
J L
JL
Wisecarver
D D
DD
Hook
S M
SM
Cox
K W
KW
Beisel
D001483:Base Sequence; D016026:Bone Marrow Transplantation / Q000276:immunology; D004247:DNA / Q000235:genetics; D015343:DNA Probes, HLA; D005069:Evaluation Studies as Topic; D006680:HLA Antigens / Q000235:genetics; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D014019:Tissue Donors
D015343:DNA Probes, HLA; D006680:HLA Antigens; D009692:Nucleic Acid Heteroduplexes; D004247:DNA