[Screening the differentially methylated DNA sequences of colorectal cancer by methylated CpG islands amplification coupled with representational difference analysis].
OBJECTIVE
To screen the differentially methylated DNA sequences between mucosa adjacent to colorectal cancer (MACC) and normal colonic mucosa.


METHODS
The methylated DNA sequences were enriched by methylation CpG islands amplification (MCA), and the differentially methylated DNA sequences between MACC and normal colonic mucosa were isolated by representational difference analysis (RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with BLAST program system in GenBank. With the separated fragment 1A12 as probe, dot blot was used to study its distribution between RDA products (No. 1-4 rounds), MACC (tester) and normal colonic mucosa(driver).


RESULTS
Twenty-five differentially methylated DNA sequences were obtained. Preliminary studies indicated that 1A01 fragment was concerned with two different genes (LOC256866 and CECR7), it was located in the first exon of CECR7. 1A12 fragment was located in 5  flanking region of GR6 gene. By dot blot with 1A12 probe, hybridized signals were detected in MCA product of MACC and RDA products of No. 1-4 rounds, respectively. No signal was detected in MCA product of normal colonic mucosa.


CONCLUSIONS
The differentially methylated DNA sequences can be isolated effectively between two different tissues with MCA coupled with RDA. Different methylated DNA fragments exist between MACC and normal colonic mucosa and these fragments may be concerned with colorectal cancer.
Zhu
Yi-min
YM
School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 310031 PR China.
Lin
Jie
J
Huang
Qiong
Q
Lai
Mao-de
MD
D000328:Adult; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005820:Genetic Testing; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D004273:DNA, Neoplasm