A mutS-based protein chip for detection of DNA mutations
(2003) In Analytical Chemistry 75(16). p.4113-4119- Abstract
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... (More)
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.
(Less)
- author
- Bi, Li-Jun ; Zhou, Ya-Feng ; Zhang, Xian-En ; Deng, Jiao-Yu ; Zhang, Zhi-Ping ; Xie, Bin LU and Zhang, Cheng-Gang
- publishing date
- 2003-08-15
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Analytical Chemistry
- volume
- 75
- issue
- 16
- pages
- 7 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- scopus:0041975869
- pmid:14632124
- ISSN
- 0003-2700
- DOI
- 10.1021/ac020719k
- language
- English
- LU publication?
- no
- id
- 15748687-d65d-45d8-8b3d-0fecdc281074
- date added to LUP
- 2021-10-26 23:01:44
- date last changed
- 2024-01-05 19:00:14
@article{15748687-d65d-45d8-8b3d-0fecdc281074, abstract = {{<p>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-His<sub>6</sub>-Linker peptide-Strep-tagII-Linker peptide-MutS (THLSLM). The THLSLM coding sequence was constructed by attaching <i>Strep-tag II</i> and <i>mutS </i>gene to pET32a (+) sequentially with insertion of a linker peptide coding sequence before and behind <i>Strep-tagII</i> gene, respectively. THLSLM was expressed in <i>E. coli</i> AD494 (DE3) and purified using Ni <sup>2+</sup>-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 <i>rpoB </i>gene from <i>Mycobacterium tuberculosis</i>, with high specificity. The method could potentially serve as a platform to develop the high-throughput technology for screening and analysis of genetic mutations.</p>}}, author = {{Bi, Li-Jun and Zhou, Ya-Feng and Zhang, Xian-En and Deng, Jiao-Yu and Zhang, Zhi-Ping and Xie, Bin and Zhang, Cheng-Gang}}, issn = {{0003-2700}}, language = {{eng}}, month = {{08}}, number = {{16}}, pages = {{4113--4119}}, publisher = {{The American Chemical Society (ACS)}}, series = {{Analytical Chemistry}}, title = {{A mutS-based protein chip for detection of DNA mutations}}, url = {{http://dx.doi.org/10.1021/ac020719k}}, doi = {{10.1021/ac020719k}}, volume = {{75}}, year = {{2003}}, }