DNA Combing Technology: A Simple But Useful Technique
DNA combing is a simple technique which can be used for visualization of single DNA molecules by following simple steps. A single DNA molecule is 2 nm wide but when intercalated by a fluorescent dye, it can be seen with the help of charge coupled device camera.
To perform DNA combing pure genomic DNA is extracted from tissues and is mixed in combing buffer solution carrying TE, MgCl2 ions, a fluorescent dye such as SybrGold or SybrGreen and a surfactant such as Triton-X. A drop of combing buffer solution is taken and placed over an APTES or PEI surface coated glass slide. Surface coating of the glass slide is necessary for imparting positive surface such that the DNA molecules can bind to it.
DNA combing allows us to perform single molecule analysis. Single molecule analysis of DNA molecules provides better resolution as compared to bulk techniques such as agarose gel electrophoresis. Fluorescent probes can be used to perform fluorescent in situ hybridization for detection of desired genomic sequences. Multiple probes intercalated by different dyes can be used to detect genic rearrangements and deletions in the genomic regions in a simple and easy manner as compared to DNA sequencing. Gene deletions and rearrangements can be studied by DNA combing technology much easily than Next Generation Sequencing methods as it requires only multiple probes to perform Fiber-FISH on single combed DNA molecules whereas a large data set of DNA molecules is required for Next Generation Sequencing methods.
Combed DNA molecules can be denatured to treat them with single-stranded binding proteins for studying DNA-protein interactions. Other proteins such as RecA can also be used to study DNA-protein interaction. Restriction digestion of combed DNA by restriction enzymes will lead to fragments of different sizes which can be analyzed by software to generate physical maps by using fluorescent intensity of the molecules through CCD cameras. Such sort of physical mapping is known as optical mapping and different optical maps of various bacterial genomes can be generated by this technology. Optical mapping is very fast as compared to other similar technologies.
Recently there has been a trend to study drug-DNA interactions since some anticancerous drugs bind to DNA molecules and form DNA adducts. Such adducts can be easily studied by performing drug-DNA interactions on combed DNA molecules. Other potential uses of DNA combing include the study of DNA replication on combed DNA molecules. It thus provides a unique robust platform not only for visualization of single DNA molecules by simple fluorescent microscope but this simple technology can be used for carrying out genomic studies on single DNA molecules which is far better than techniques involving DNA molecules in bulk.
Thus this technology has several unique advantages over other technologies and can be useful for the development of future rapid molecular diagnostic tools. It can be applied in multiple fields such as to diagnose genetic diseases and for detection of nitrogen-fixing genes and genes responsible for phosphate solubilization on the combed bacterial genomic DNA.
These findings are described in the article entitled Enhanced post wash retention of combed DNA molecules by varying multiple combing parameters, recently published in the journal Analytical Biochemistry. This work was conducted by Hemendra Yadav from the University of Rajasthan and Pulkit Sharma from Bioinventree Pvt Ltd.