HLA class II typing of whole genome amplified mouth swab DNA.
Postal collection of mouth swabs provides a cheap and convenient means of DNA sampling but hitherto has not provided sufficient genetic material for HLA typing by polymerase chain reaction using sequence-specific primers (PCR-SSP). This study examined the feasibility of collecting mouth swabs from a test population by post, amplifying the DNA by whole genome amplification and genotyping for selected HLA class II alleles. We optimised a strategy for whole genome amplification or primer extension preamplification using a random 15 base pair primer which resulted in a 1,000-fold increase in DNA template. The amplified DNA was of sufficient quality for analysis of selected HLA Class II alleles by PCR-SSP and PCR using sequence-specific oligonucleotide probes. To test the reliability of our data, blood DNA from 30 individuals in 10 families, previously tested for all DRB1 alleles in a routine diagnostic laboratory, was then tested in our laboratory for DRB1 *03 and *04 following whole genome amplification. Further whole genome amplified product from another 10 families was tested for DRB1 *03, *04 in our laboratory and then tested for all DRB1 alleles in a routine diagnostic laboratory. One repeat typing was required to achieve 100% concordance between laboratories. Amplification of whole genome amplified DNA by PCR-SSP was then extended successfully to low-resolution HLA DRB1, DQA1, DQB1 and DPB1 typing. Mouth swab collection by post, followed by whole genome amplification of DNA provides an effective strategy for genetic analysis of large cohorts. We have optimised conditions for HLA class II typing on whole genome amplified DNA collected by mouth swab, but this method could potentially be applied to low concentrations of DNA from other sources.
Gillespie
K M
KM
Diabetes and Metabolism, Division of Medicine, University of Bristol, UK. K.M.Gillespie@bristol.ac.uk
Valovin
S J
SJ
Saunby
J
J
Hunter
K M
KM
Savage
D A
DA
Middleton
D
D
Todd
J A
JA
Bingley
P J
PJ
Gale
E A
EA
D000328:Adult; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics / Q000276:immunology; D005192:Family Health; D015894:Genome, Human; D006682:HLA-DP Antigens / Q000235:genetics; D059868:HLA-DP beta-Chains; D006683:HLA-DQ Antigens / Q000235:genetics; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; D006684:HLA-DR Antigens / Q000235:genetics; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D009055:Mouth; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D013048:Specimen Handling
D006682:HLA-DP Antigens; D059868:HLA-DP beta-Chains; C071170:HLA-DPB1 antigen; D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; C069050:HLA-DQA1 antigen; C069051:HLA-DQB1 antigen; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II; D015345:Oligonucleotide Probes