High-throughput identification of mutations using a combination of CEL I fragmentation and SAGE technology.
A new method to detect mutations based on the serial analysis of gene expression (SAGE) technique, ligation-mediated (LM) PCR, and recombinant nuclease CEL I named LM-SAGE assay is reported in the present study. Mismatched DNA heteroduplexes formed from wild-type and mutant DNA are fragmented with CEL I nuclease at the mutant site to produce a double-strand fragment with an overhanging base at the 3'-end. The fragment is ligated to a linker, and digested with MmeI and then ligated to another linker. PCR is performed to amplify the ligation products, and NlaIII is used to release 17-bp tags containing mutation sites followed by purification, concatemerization, cloning, and sequencing. The locations of mutations can be identified from the homology analysis of tags. This new LM-SAGE assay can detect both known and unknown mutations with a sensitivity of 1:50 (mutant:wild-type DNA ratio) in 2.4 x 10(6) copies starting DNA sample. Our results show that this method could be used as a potentially high-throughput assay for mutation detection, particularly for the discovery of unknown mutations in genomic DNA.
Zhang
Chi
C
MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Li
Yuan-Yuan
YY
Wang
Xian-Liang
XL
Zhang
Li-Jun
LJ
Li
Xiao-Bo
XB
Wang
You-Jie
YJ
Xu
Shun-Qing
SQ
D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000379:methods* / Q000706:statistics & numerical data; D017931:DNA Primers / Q000235:genetics; D004720:Endonucleases; D005189:False Positive Reactions; D015870:Gene Expression; D020180:Heteroduplex Analysis / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D004247:DNA; D004720:Endonucleases; C406922:CEL I nuclease