[High through-put genomic DNA isolation technique and its application in HLA genotyping for samples from bone marrow donor program].
This study was aimed to develop and establish an efficient method for high through-put automatically extracting genomic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 microl blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrity of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambda rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity (A260/A280) of genomic DNA extracted from 400 microl whole blood samples were 3.217+/-0.715 microg and 1.710+/-0.103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was >600 RFU, however, the fluorescent intensity for negative bead for each sample was <50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.
Wang
Da-Ming
DM
Shenzhen Blood Center, Shenzhen 518035, Guangdong Province, China.
Tang
Si
S
Li
Zhen
Z
Cheng
Xi
X
Gao
Su-Qing
SQ
Deng
Zhi-Hui
ZH
D018070:Biological Specimen Banks; D001853:Bone Marrow; D004247:DNA / Q000302:isolation & purification*; D017931:DNA Primers; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D019520:Living Donors; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods
D017931:DNA Primers; D006680:HLA Antigens; D004247:DNA