Protein chip for detection of DNA mutations.
A large number of human genetic diseases, bacterial drug resistances, and single-nucleotide polymorphisms are caused by gene mutations. Rapid and high-throughput mutation detection methods are urgently demanded. A protein chip method for detection of single-base mismatches and unpaired bases of DNA was developed using a genetic fusion molecular system Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM). The THLSLM coding sequence was constructed by attaching Strep tag II and mutS gene to the vector pET32a (+) sequentially with insertion of a (Ser-Gly)6 coding sequence before and behind Strep tagII gene, respectively. The fusion protein THLSLM was expressed in Escherichia coli AD494 (DE3) and purified using Ni(2+)-chelation affinity resin. The results of bioactivity assay showed that THLSLM both binds to mismatched DNA and interacts with streptavidin. THLSLM was immobilized on the chip matrix coated with the streptavidin through Strep tagII-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.
Zhang
Xian-En
XE
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Bi
Li-Jun
LJ
D001426:Bacterial Proteins / Q000235:genetics*; D020137:Base Pair Mismatch; D004252:DNA Mutational Analysis / Q000379:methods*; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics* / Q000378:metabolism; 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; D019809:Streptavidin / Q000378:metabolism
D001426:Bacterial Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D019809:Streptavidin; D012321:DNA-Directed RNA Polymerases; D051719:MutS DNA Mismatch-Binding Protein; D012293:Rifampin