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Biochemistry

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Nanyang Technological University

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

9.6K Views

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11:08 min

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June 19th, 2018

DOI :

10.3791/57862-v

June 19th, 2018

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Kang-Yi Su*¹^,², Steven D. Goodman*³, Hung-Ming Lai¹, Rong-Syuan Yen¹, Wei-Yao Hu¹, Wern-Cherng Cheng², Liang-In Lin¹^,², Ya-Chien Yang¹^,², Woei-Horng Fang¹^,²

¹Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, ²Department of Laboratory Medicine, National Taiwan University Hospital, ³Center for Microbial Pathogenesis, Nationwide Children's Hospital and the Department of Pediatrics, The Ohio State University

Summary

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

Explore More Videos

MALDI TOF Mass Spectrometry

Single Nucleotide Extension

Primer template Preparation

Mismatch Substrate

Reaction Buffer

DNA Polymerase Dilution

Reaction Temperature

Reaction Termination

Chapters in this video

0:05

Title

1:17

Proofreading Reactions

3:16

Terminate the Reaction and Transfer Reaction Products to a Matrix Chip

4:54

Setup the Assay Information on the Mass Spectrometry

6:18

MALDI-TOF Mass Spectrometry (MS) Operation and Data Analysis

8:00

Results: DNA Proofreading and Repair Analyzed by MALDI-TOF MS

9:49

Conclusion

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