Genotype-related fingerprints from HLA-DPB1 exon 2 low-stringency PCR.
Techniques based on the formation of heteroduplexes between relevant heterologous amplified HLA loci have proved to be simple and cost-effective screening methods for the detection of DNA sequence diversity. However, the banding patterns produced may not be as complex as required. We used the original procedure of Pena et al. (Proceedings of the National Academy of Sciences USA 91, 1946-1949, 1994) to generate fingerprints from a specific, polymorphic PCR product. HLA-DPB1 exon 2 was amplified, recovered from agarose gel, and used as a template for subsequent low-stringency (30 degrees C) amplification cycles (LS-PCR) in the presence of a single primer. The LS-PCR products were run on 8% PAGE and silver-stained. In total, 22 subjects were characterized by this method. The issues of the reproducibility and specificity of the patterns obtained were addressed by comparing fingerprints from individuals with the same genotype. The results showed that LS-PCR was robust. A further step was the evaluation of the diversity that can be generated, i.e. the sensitivity of the method. Genotype-related fingerprints were produced, and differences as small as a single nucleotide in heterozygous samples could be detected. We then demonstrated the usefulness of LS-PCR in the evaluation of donor/recipient pairs. We believe that LS-PCR may be a valuable adjunct to the battery of tests aimed at the verification of HLA matching before unrelated bone marrow transplantation. We suggest that it could be used to speed up the search process when several candidate donors are retrieved from registries before embarking on SSOP typing or sequencing.
Porfirio
B
B
Department of Clinical Physiopathology, University of Florence, Italy.
Chiarelli
I
I
Rombolà
G
G
Mattiuz
P L
PL
D016026:Bone Marrow Transplantation; D016172:DNA Fingerprinting / Q000379:methods*; D005091:Exons; D005838:Genotype; D006682:HLA-DP Antigens / Q000145:classification* / Q000235:genetics; D059868:HLA-DP beta-Chains; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D006682:HLA-DP Antigens; D059868:HLA-DP beta-Chains; C071170:HLA-DPB1 antigen