Search research articles

Search research articles

Related Experiment Videos

Solution-based scanning for single-base alterations using a double-stranded DNA binding dye and fluorescence-melting

K S Elenitoba-Johnson¹, S D Bohling

¹Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA. kojo.elenitobaj@path.utah.edu

The American Journal of Pathology

|September 11, 2001

Summary

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

MMSET stimulates myeloma cell growth through microRNA-mediated modulation of c-MYC.

Leukemia·2012

Same author

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

Molecular pathology : MP·2003

Same author

Real-time multiplex PCR assays.

Methods (San Diego, Calif.)·2002

Same author

Intrinsic deoxyguanosine quenching of fluorescein-labeled hybridization probes: a simple method for real-time PCR detection and genotyping.

Laboratory investigation; a journal of technical methods and pathology·2001

Same author

Complex regulation of telomerase activity: implications for cancer therapy.

The American journal of pathology·2001

Same author

Loss of p53 tumor suppressor function is required for in vivo progression of Friend erythroleukemia.

Oncogene·2001

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

This study introduces a novel solution-based fluorescence method to detect single-base DNA mutations. The technique uses melting temperature analysis to distinguish wild-type DNA from mutant sequences across large regions, improving mutation detection sensitivity.

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Traditional methods for distinguishing DNA sequences, like gel electrophoresis, are limited by fragment size and probe length.
Existing solution-based fluorescence methods are restricted to detecting mutations in very short DNA segments (approx. 20 bp).

Purpose of the Study:

To develop a novel solution-based fluorescence method for detecting single-base DNA substitutions.
To expand the detectable region for fluorescence-based mutation detection in solution.

Main Methods:

Utilized melting theory and fluorescence monitoring of melting temperatures.
Analyzed GC-clamped amplicons undergoing thermal denaturation with urea.
Differentiated wild-type and mutant DNA by analyzing melting profiles of heteroduplex mismatches.

Related Experiment Videos

Main Results:

Successfully distinguished wild-type from mutant DNA sequences across regions larger than 200 nucleotides.
Identified all four possible sets of mismatches in 17 heterozygous mutations across 20 amplicons.
Demonstrated that heterozygous samples show lower melting temperatures due to less stable heteroduplexes.

Conclusions:

The developed method obviates the need for gel electrophoresis or labeled oligonucleotide probes.
Significantly expands the interrogation region for single-base change detection using fluorescence in solution.
The methodology is suitable for automation and high-throughput systems.