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A mutS-based protein chip for detection of DNA mutations

Bi, Li-Jun ; Zhou, Ya-Feng ; Zhang, Xian-En ; Deng, Jiao-Yu ; Zhang, Zhi-Ping ; Xie, Bin LU and Zhang, Cheng-Gang (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.

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author
; ; ; ; ; and
publishing date
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}},
}