Protein chip for detection of DNA mutations

Area of Science:

• Biotechnology
• Molecular Biology
• Genetics

Background:

• Gene mutations are a primary cause of human genetic diseases and bacterial drug resistance.
• Accurate and rapid detection of these mutations is crucial for diagnostics and research.
• Existing methods may lack the speed or throughput required for large-scale screening.

Purpose of the Study:

• To develop a novel protein chip-based method for high-throughput DNA mutation detection.
• To create and characterize a specific fusion protein capable of binding mismatched DNA.
• To validate the utility of the developed protein chip for identifying various DNA mutations.

Main Methods:

• Construction and expression of a Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM) fusion protein in E. coli.
• Purification of the THLSLM protein using Ni(2+)-chelation affinity chromatography.
• Immobilization of THLSM on a streptavidin-coated chip via Strep tagII-streptavidin interaction.
• Detection of mismatched and unpaired DNA bases using the immobilized protein chip.

Main Results:

• The THLSLM fusion protein demonstrated bioactivity, binding to mismatched DNA and streptavidin.
• The protein chip successfully detected single-base mismatches and unpaired bases in synthesized oligonucleotides.
• Specific detection of a single-base mutation in the rpoB gene of Mycobacterium tuberculosis was achieved with high specificity.
• The developed method exhibits potential for high-throughput genetic mutation screening.

Conclusions:

• A novel protein chip method based on the THLSLM fusion protein has been successfully developed.
• This method offers high specificity and sensitivity for detecting DNA mutations, including single-base changes.
• The developed protein chip serves as a potential platform for high-throughput screening and analysis of genetic mutations, aiding in disease diagnostics and research.