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Optical sequence probing with the homologous recombination protein RecA

Paper ID Volume ID Publish Year Pages File Format Full-Text
23305 43431 2013 6 PDF Available
Title
Optical sequence probing with the homologous recombination protein RecA
Abstract

In this study, we used the homologous recombination protein RecA to locate a specific sequence on DNA. Single-stranded (ss) DNA (80-mer, 5′-biotinylated), complementary to the sequence of interest, was labeled with quantum dots (Qdots®) via biotin–avidin binding. The DNA was then mixed with RecA to form a fluorescent-labeled ssDNA–RecA complex. λ DNA, which was used as the target DNA, was stretch-and-positioned onto microelectrodes by using the electrostatic method. When the ssDNA–RecA complex was fed to the suspended target DNA, clear fluorescence spots were observed on individual target DNA molecules. The histogram of the probe-binding position along the target DNA was measured, and the peak was found to correspond to the location complementary to the probe ssDNA. This result shows a potential use for recombination proteins in facilitating the optical detection of DNA sequences.

► A method to visualize a specific DNA sequence by using RecA was developed. ► The probe consisted of the ssDNA–RecA complex fluorescence-labeled with Qdots. ► A target strand of DNA was stretch-and-positioned using the electrostatic method. ► Clear fluorescent spots were observed on the stretched DNA molecules. ► The peak-binding frequency of the probe was at the site of sequence complementarity.

Keywords
ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; FISH, fluorescence in situ hybridization; DTT, dithiothreitol; BSA, bovine serum albuminRecA; Optical mapping; Homologous recombination; Electrostatic manipulation
First Page Preview
Optical sequence probing with the homologous recombination protein RecA
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 164, Issue 2, March 2013, Pages 254–259
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering