[Study on the location on the nucleotide of DNA lesion detected by RDPCR].
OBJECTIVE
To simplify the establishing procedure of RDPCR and detect the DNA lesion on the precise position at the level of nucleic acid.


METHODS
The TK6 cell was cultured and its genomic DNA was extracted and prepared to the single-stranded probes of the exon 2 of k-ras gene. The exon 2 of k-ras gene was amplified,called short fragment. The digestion of the genomic DNA and the short fragment were treated by the endonuclease HinfI. To the former, the products come from the every step of RDPCR were imaged after gel electrophoresis (GE) and the end products was taken coloration after southern hybridization with the single-stranded probes of the exon 2 of k-ras gene as well as were sequenced after re-amplified. To the latter, the genomic DNA were treated as the former except the GE.


RESULTS
From the image of GE, the straps could be seen clearly that come from every step of amplifying of short fragments which were cleaved by the endonuclease HinfI in RDPCR. The clear hybridized bands caused by the products of RDPCR were seen at the expectant position. The sequencing analysis showed that the position was linked by the linker was just the restriction site of HinfI.


CONCLUSIONS
Results indicated that RDPCR could be easily established by the short fragment method. This may be the first time detect the precise position of DNA lesion at the level of nucleic acid by RDPCR.
Gao
Xufang
X
Department of Environmental Health, School of Public Health, Sichuan University, Chengdu 610041, China. liligxf@163.com
Rao
Chaolong
C
Wen
Weihua
W
Shuai
Peiqiang
P
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004249:DNA Damage; D005091:Exons / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D008214:Lymphocytes / Q000166:cytology; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D009696:Nucleic Acids; D004247:DNA