A MutS-based protein chip for detection of DNA mutations.
This paper describes a new protein chip method for detection of single-base mismatches and unpaired bases of DNA, using a genetic fusion molecular system Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutS (THLSLM). The THLSLM coding sequence was constructed by attaching Strep-tag II and mutS gene to pET32a (+) sequentially with insertion of a linker peptide coding sequence before and behind Strep-tagII gene, respectively. THLSLM was expressed in E. coli AD494 (DE3) and purified using Ni(2+)-chelation affinity resin. THLSLM retained both mismatch recognition activity and streptavidin binding affinity. THLSLM was then immobilized on the chip matrix coated with streptavidin through the Strep-tag II-streptavidin binding reaction. The resulting protein chip was used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides, as well as a single-base mutation in rpoB gene from Mycobacterium tuberculosis, with high specificity. The method could potentially serve as a platform to develop the high-throughput technology for screening and analysis of genetic mutations.
Bi
Li-Jun
LJ
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Zhou
Ya-Feng
YF
Zhang
Xian-En
XE
Deng
Jiao-Yu
JY
Zhang
Zhi-Ping
ZP
Xie
Bin
B
Zhang
Cheng-Gang
CG
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D020137:Base Pair Mismatch; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004361:Drug Tolerance; D015870:Gene Expression; D051719:MutS DNA Mismatch-Binding Protein; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; D012293:Rifampin