Homogeneous fluorescence detection method for human leukocyte antigen-DR typing following polymerase chain reaction amplification with sequence-specific primer.
A fluorescent homogeneous method for the detection of sequence-specific amplification of human leukocyte antigen (HLA) alleles has been developed. In this approach, polymerase chain reaction sequence-specific primers (PCR-SSP) are used to amplify DRB1, DRB3, and DRB4 alleles. Lambda exonuclease and Exonuclease I are added to reduce background by digesting template DNA, partial primer dimer, and primer. PCR amplicons are then detected following the addition of a fluorescent dye (thiazole yellow dimer) which binds to double-stranded DNA. No transfer or wash steps are required. Thus, the risk of sample contamination, which is a major source of inaccuracy for DNA amplification methods, is greatly reduced. This approach is also faster and more easily automated than the standard approach using gel electrophoresis and ethidium bromide staining. Speed and automation are important considerations for HLA typing since the number of possible alleles for each HLA type is substantial. A homogeneous HLA-SSP typing method may be especially useful for clinical labs doing large numbers of samples and for the eventual automation of HLA DNA typing.
Okamoto
N
N
Biomedical Research Center, Olympus America Inc., East Setauket, New York 11733.
Lee
A
A
Kano
T
T
Lee
T D
TD
D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000097:blood / Q000302:isolation & purification; D017931:DNA Primers; D045643:DNA Repair Enzymes; D005090:Exodeoxyribonucleases; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens / Q000097:blood* / Q000235:genetics; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013698:Templates, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens; D014764:Viral Proteins; D004247:DNA; C114331:EXO1 protein, human; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; C043586:exo protein, Bacteriophage lambda; D045643:DNA Repair Enzymes