two DNA fragment having same length but different sequence (i.e high GC and low GC-content). what about their migration on agarose gel?
Thanks.
Best Wihses.
according to Fermentas information para 3.6 (attached) AT rich dna runs slower than GC rich dna of the same length. Possibly due to more triple hydrogen bond carrying a partial charge but that is my guess
Hi
since migration is related to sizes and/or charges, I would say have a look at (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643323/).
fred
Migration in chemically inert gels depends nearly exclusively on mass : charge ratio. Normally, there is no influence of the DNA sequence on migration velocity. There are, however, influences of DNA bending on migration. In this case, deviations from "linear migration" may be seen. As this refers to shorter molecules, the effect is seen only on polyacrylamide gels. A bent conformation of a fragment will slow down migration a little bit. Consequently, a fragment appears a little larger than it is in reality.
Agarose is not informative for these purposes. You should use SSCP or CGSE, and migration in PAGE in denaturating conditions with urea.
melting temperature is dependent upon GC content of sequence. gel run is independent of GC content proportion.
GC content is an important factor in Denaturing Gradient Gel Electrophoresis (DGGE):
1. DGGE analyses are employed for the separation of double-stranded DNA fragments that are identical in length, but differ in sequence (DNA bases). In practice, this refers to the separation of DNA fragments produced via PCR amplification. The technique exploits (among other factors) the difference in stability of G-C pairing (3 hydrogen bonds per pairing) as opposed to A-T pairing (2 hydrogen bonds). A mixture of DNA fragments of different sequence are electrophoresed in an acrylamide gel containing a gradient of increasing DNA denaturants. In general, DNA fragments richer in GC will be more stable and remain double-stranded until reaching higher denaturant concentrations. Double-stranded DNA fragments migrate better in the acrylamide gel, while denatured DNA molecules become effectively larger and slow down or stop in the gel. In this manner, DNA fragments of differing sequence can be separated in an acrylamide gel.
[ http://ddgehelp.blogspot.com/2005/11/dgge-guide.html ]
2. Denaturing Gradient Gel Electrophoresis (DGGE) Procedure: Denaturing Gradient Gel Electrophoresis (DGGE) is a microbial fingerprinting technique that separates amplicons of roughly the same size based on sequence properties. These properties dictate the threshold at which DNA denatures. The DGGE gel uses a gradient DNA denaturant (a mixture of urea and formamide), or a linear temperature gradient. When the fragment reaches its melting point (threshold of enough denaturant), it stops moving. This is due to the fact that a partially melted double-stranded DNA can no longer migrate through the gel. A GC clamp (about 40 bases with high GC content) is used as a special primer to anchor the PCR fragments together once they have denatured.
https://en.wikipedia.org/wiki/Community_Fingerprinting
3. See attached Figures for DGGE
If the buffer inside the migration machine is old and mainly durty, your DNA will migrate faster and also further, and the second point the solution will warm up more than before.
So if you don't watch for a clean buffer (I'm using TBE buffer) in machine, you'll get some troubles. You'll migrate your gel during the time and at the voltage, and maybe lost your DNA.
I also read (but never saw it), the solution can warm up a lot and melt the gel inside the migration machine.
After bp (A,T,C,G) will not interfere during migration.
according to Fermentas information para 3.6 (attached) AT rich dna runs slower than GC rich dna of the same length. Possibly due to more triple hydrogen bond carrying a partial charge but that is my guess
Attached is a paper describing factors affecting the mobility of DNA in a gel.
Title: Effect of the matrix on DNA electrophoretic mobility (2009)
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology