Abstract
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 Ni2+-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.
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References
Stewart, P. R., el-Adhami, W., Inglis, B., and Franklin, J. C. (1993) Analysis of an outbreak of variably methicillin-resistant Staphylococcus aureus with chromosomal RFLPs and mec region probes. J. Med. Microbiol. 38, 270–277.
Meggouh, F., Benomar, A., Rouger, H., et al. (1998) The first de novo mutation of the connexin 32 gene associated with X linked Charcot-Marie-Tooth disease. J. Med. Genet. 35, 251–252.
Zoller, B. and Dahlback, B. (1995) Resistance to activated protein C caused by a factor V gene mutation. Curr. Opin. 2, 358–364.
Everett, L. A., Glaser, B., Beck, J. C., et al. (1997) Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat. Genet. 17, 411–422.
Wong, C., Dowling, C. E., Saiki, R. K., Higuchi, R. G., Erlich, H. A., and Kazazian, H. H., Jr. (1987) Characterization of β-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature 330, 384–386.
Saiki, R. K., Sharf, S., Faloona, F., et al. (1985) Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Sugano, K., Fukayama, N., Ohkura, H., et al. (1995) Single-strand conformation polymorphism analysis by perpendicular temperature-gradient gel electrophoresis. Electrophoresis 16, 8–10.
Guldberg, P. and Guttler, F. (1993) A simple method for identification of point mutations using denaturing gradient gel electrophoresis. Nucleic Acids Res. 21, 2261–2262.
Deeble, V. J., Roberts, E., Robinson, M. D., Woods, C. G., Bishop, D. T., and Taylor, G. R. (1999) Comparison of enzyme mismatch cleavage and chemical cleavage of mismatch on a defined set of heteroduplexes. Genetic Testing 1, 253–259.
Hacia, J. G. (1999) Resequencing and mutational analysis using oligonucleotide microarrays. Nat. Genet. 21, 42–47.
Lu, A. L., Clark, S., and Modrich, P. (1983) Methyl-directed repair of DNA basepair mismatches in vitro. Proc. Natl. Acad. Sci. USA 80, 4639–4643.
Lieb, M. (1987) Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites. J. Bacteriol. 169, 5241–5246.
Jiricny, J., Su, S. S., Wood, S. G., and Modrich, P. (1988) Mismatch-containing oligonucleotide duplexes bound by the E. coli mutS-encoded protein. Nucleic Acids Res. 25, 7843–7853.
Wagner, R., Debbie, P., and Radman, M. (1995) Mutation detection using immobilized mismatch binding protein (MutS). Nucleic Acids Res. 11, 3944–3948.
Ellis, L. A., Taylor, G. R., Banks, R., and Baumberg, S. (1994) MutS binding protects heteroduplex DNA from exonuclease digestion in vitro: a simple method for detecting mutations. Nucleic Acids Res. 11, 2710–2711.
Gotoh, M., Hasebe, M., Ohira, T., et al. (1997) Genetic Analysis. Bimolecular Eng. 14, 47–50.
Geschwind, D. H., Rhee, R., and Nelson, S. F. (1996) A biotinylated MutS fusion protein and its use in a rapid mutation screening technique. Genet. Anal. 13, 105–111.
Nelson, S. F. (1995) Genomic mismatch scanning: current progress and potential applications. Electrophoresis 16, 279–285.
Behrensdorf, H. A., Pignot, M., Windhab, N., and Kappel, A. (2002) Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format. Nucleic Acids Res. 15, E64.
Shao, W. H., Zhang, X. E., Liu, H., Zhang, Z. P., and Cass, A. E. (2000) Anchorchain molecular system for orientation control in enzyme immobilization. Bioconjug. Chem. 11, 822–826.
Shi, J. X., Zhang, X. E., Xie, W. H., et al. (2004) Improvement of homogeneity of analytical biodevices by gene manipulation. Anal. Chem. 76, 632–638.
Feng, G. and Winkler, M. E. (1995) Single-step purifications of His6-MutH, His6-MutL and His6-MutS repair proteins of Escherichia coli K-12. Biotechniques 19, 956–965.
Laurent, G., Celine, B., and Peter, B. (1999) ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL. Nucleic Acids Res. 27, 2325–2331.
Modrich, P. (1991) Mechanisms and biological effects of mismatch repair. Annu. Rev. Genet. 25, 229–253.
Gutierrez, M. C., Galan, J. C., Blazquez, J., Bouvet, E., and Vincent, V. (1999) Molecular markers demonstrate that the first described multidrug-resistant Mycobacterium bovis outbreak was due to Mycobacterium tuberculosis. J. Clin. Microbiol. 37, 971–975.
Miller, L. P., Crawford, J. T., and Shinnick, T. M. (1994) The rpoB gene of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 38, 805–811.
Biswas, I., Ban, C., Fleming, K. G., et al. (1999) Oligomerization of a MutS mismatch repair protein from Thermus aquaticus. J. Biol. Chem. 274, 23,673–23,678.
Zhou, Y. F., Zhang, X. E., Liu, H., Zhang, Z. P., Zhang, C. G., and Cass, A. E. (2001) Construction of a fusion enzyme system by gene splicing as a new molecular recognition element for a sequence biosensor. Bioconjug. Chem. 12, 924–931.
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© 2007 Humana Press Inc., Totowa, NJ
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Zhang, XE., Bi, LJ. (2007). Protein Chip for Detection of DNA Mutations. In: Rampal, J.B. (eds) Microarrays. Methods in Molecular Biology, vol 382. Humana Press. https://doi.org/10.1007/978-1-59745-304-2_11
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DOI: https://doi.org/10.1007/978-1-59745-304-2_11
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