[Establishment of an assay for cloning and sequencing the full-length HLA-Cw gene].
OBJECTIVE
To establish a reliable assay for cloning and sequencing the full-length HLA-Cw gene.


METHODS
In this study, a fragment of 4.5 kb full-length HLA-Cw gene was amplified using the self-designed PCR primer pair by long template PCR, purified PCR products was cloned into the pGEM-Teasy plasmid vector and the plasmid DNA isolated from positive clones was subjected to haplotype sequencing by both directions. A total of 12 samples having been previously-genotyped by PCR sequence-based-typing (PCR-SBT) were amplified by using the TaKaRa LA Taq and Stratagene Pfu polymerase, respectively. PCR products of full length HLA-Cw gene were subjected to cloning and sequencing and the obtained haplotype sequence were compared with the PCR-SBT results.


RESULTS
The specific target fragment of HLA-Cw gene could be amplified and the full-length HLA-Cw allele sequence covering from nucleotide position -962 in 5'untranslated region (5'-UTR) to nucleotide position 3576 in downstream area of 3'-UTR region could be obtained using our method. The results of cloning and sequencing analysis indicated that the Stratagene Pfu polymerase had better fidelity than the TaKaRa LA Taq polymerase in this experiment. By comparing the sequences of Cw*07020101 with Cw*010201, 11 SNPs as well as 2 insertions/deletions in nt-962--284 of 5'-UTR, and 11 SNPs as well as 1 insertion/deletion in nt3067-3576 downstream of 3'-UTR were identified.


CONCLUSIONS
Our results indicate that the technique for cloning and sequencing full-length HLA-Cw gene has been established, it has a broad application in full-length HLA-Cw gene polymorphism study and the regulation and expression of HLA-Cw gene.
Deng
Zhi-hui
ZH
Shenzhen Blood Center, Shenzhen Key Laboratory of Histocompatibility and Immunogenetics, Shenzhen, Guangdong, 518035 PR China. zhihui_deng@yahoo.com.cn
Xu
Yun-ping
YP
Gao
Su-qing
SQ
Li
Da-cheng
DC
Yu
Qiong
Q
Su
Yu-qing
YQ
Zeng
Jian-qiang
JQ
Yang
Bao-cheng
BC
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002681:China / Q000208:ethnology; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D006680:HLA Antigens