DNA Gel Electrophoresis
DNA Gel Electrophoresis
Last Updated on Wednesday, 11 May 2011 05:53 Written by Administrator Wednesday, 11 May 2011 05:46
DNA Gel Electrophoresis. Information on how to separate DNA deoxyribonucleic acid on an agarose or polyacrilamide gel.
Separation of DNA by Gel Electrophoresis
DNA is usually separated by agarose gel electrophoresis although polyacrilamide gel electrophoresis for DNA is also often done. Large DNA or RNA is usually done by agarose gel electrophoresis whereas smaller fragments of DNA or RNA, or sequencing gels are run on polyacrylamide.
In the case of DNA and other nucleic acids the direction of migration on a gel matrix is from the negative electrode to the positive electrode. This is due to the inherent positive electrical charge present on the DNA backbone which is composed of deoxyribose sugars and phosphates (PO4 3-).
Double-stranded DNA fragments or dsDNA behave differently from single stranded DNA as they are coiled in a helix, and migrate as long rods. The migration of dsDNA in a gel matrix is therefore directly related to their radius of gyration. Interesingly, super-coiled DNA migrates even faster than non-supercoiled DNA on a gel due to its smaller radius.
Nicked DNA (non-supercoiled or partially single stranded) double-stranded plasmids for example, migrate even slower than their supercoiled DNA counterparts.
For non-cyclic DNA fragments (non-plasmid DNA such as PCR fragments), DNA migrates according to size.
Single-stranded DNA (ssDNA) and RNA tend to create secondary structures and thus fold upon themselves to create hairpins and other structures.
Thereofore, these ssDNA and RNA molecules will migrate through the gel in a complicated manner based on their secondary structure unless formamide, high heat, or other denaturing methods (agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide) are used to run them on a gel.
