Detection The Location Of DNA Damage With RDPCR And Apply It To Detect DNA Damage In K-ras Gene Induced By Environmental Cancerogen

Randomized terminal linker-dependent PCR(RDPCR), which was established on the base of LMPCR by making up some deficient of LMPCR, is a kind of molecular biology method that can detect the loci of all kinds of DNA damage. The method was founded in success in 2002 with the support of the National Nature Fund. The method has been applied to detect the damage of the exon 7 in p53 induced by a varity enviomental chemicals and concentrated water, also applied to detect the damage of exon 1 and exon 2 in N-ras, H-ras and so on. While either LMPCR or upgraded PDPCR has some deficient, for example, many steps, the experiment optimized condition can be gain success or not only can be observed after a lot of steps, while the optimized condition is critical for the experiment. Furthermore, for different chemicals, the damage hot points induced by them may be different, if the damage point can be located on the exact nucleotides, which should be very meaningful for the research of carcinogenesis mechanism. The objective of the study is to choose a facility method to optimized RDPCR, and to apply the method in the research of DNA damage of exon 1 and exon 2 in the K-ras induced by a variety of environmental cancerogens, and to study the carcinogenesis mechanism of the chemicals, also provide more data of RDPCR.The subject is divided into 3 parts.Part 1: Short-fragment optimized the RDPCR condition. The TK6 cell DNA was abstracted after conventional cultured and used to amplify some target gene fragments, and used to prepare single probe of exon 1 and exon 2 in K-ras.The target fragments of exon 1 and exon 2 in K-ras (called short fragment followed) were amplified and purified, and used to built the damage mold casused by HinfI, then were colorated with single probe after RDPCR amplification.Every products of RDPCR were carried on agarose gel electrophoresis (AGE) to definite the optimize condition of every experiment steps. The optimize experiment condition were applied to detect the damage induced by HinfI, and to verify whether the optimized condition is suitable or not.Result: the sequencing result of amplified exon 2 in K-ras is concord to the sequence in Gene Bank, AGE and hybtidization of exon 1 and exon 2 showed the preparation of probes is successful.Southern blot showed that short fragment method optimized the RDPCR detecting condition successful, the experiment condition were applied to detect the damage of exon 1 and exon2 in K-ras caused by HinfI, both the short fragment and genome DNA showed hybrided fragment on the predicted position. That means short fragment method to optimize the RDPCR experiment condition is convenient in saving labour, money and time, is suitable in detecting the DNA damage caused by environmental chemicals.Part 2: Establishing the method which can detect the DNA lesion at the Level of nucleic acid. The RDPCR product of exon2 in k-ras cut by hinf I was amplified, and whose products were compared with the blotting products, and were purified, then were sequenced. The sequencing result was compared with that of exon2 in K-ras in Gene Bank, and to analyse the damage points. The correctness and profession of the method can be difinited by comparing the damage point with the enzyme cut point.The sequencing result showed that the damage point was located in the enzyme cut point. So, combining RDPCR with sequencing can locate the damage on the exact single nucleotide, is meaningful to go on researching and spreading.Part 3: Detection the DNA damage induced by environmental cancerogens by RDPCR method. The TK6 cell were treated by DCB, BaP, MMS, SA, DCA, TCA, TNF and 4NQO,then the cell DNA were abstracted used to RDPCR. Some RDPCR products were used to southern blot, and some used to nested primers amplified, and puried, coloned, and sequenced.Result: the DNA damage of exon 2 in K-ras induced by DCB was dectected by RDPCR, and the southern blot showed that there was a blot fragment a little lag from the control position; and the sequencing result showed that DNA damge position is G. on the 65th in exon2(ie second base G, in 59th code, in K-ras).In the study of the DNA damage induced by BaP (S9⁺) detected by RDPCR, southern blot result showed that there was a blot fragment a little lag from the control position; and the sequencing result showed that DNA damage position is G, on the 66th in exon 2 (ie third base T, in 59th code, in K-ras); no damage induced by BaP (S₉^-) was detected.In the study of the DNA damage induced by MMS detected by RDPCR,southern blot result showed that there was a blot fragment a little lag from the control position; and the sequencing result showed that DNA damage position is G, on the 13th upstream the exon2 (ie intronl in K-ras).In the study of the DNA damage induced by SA detected by RDPCR,southern blot result showed that there was a blot fragment a little lag from the control position; and the sequencing result showed that the DNA damage position is T, on the 127th in exon2(ie third base T, in 80th code, in K-ras).In the study of the DNA damage induced by DCA and TCA detected by RDPCR, southern blot result showed that there was a single blot fragment lag from the control position induced by the lower concentration, and two blot fragments were seen induced by lowest concerntration, one lag from the control position and the other advanced it. No damage induced by TCA was detected.In the study of the DNA damage induced by 4NQO and TNF detected by RDPCR, a single blot fragment lag from the control position can be seen after either of the southern blot.Based on the results of the study, DCB and other environmental chemicals can cause the DNA damage of exon2 in K-ras, while their sequencing results showed their damage location were different, which means that different chemicals assaults different position on DNA. Both DCB and BaP(S₉⁺) assaulted the 59th code of exon2 in k-ras, which means the code is easily to be assaulted. The RDPCR method hasn't detected the DNA damage caused by TCA, which means the TCA target assult position maybe is not in exon 2 in K-ras.