I am working on a dna sequence which can fold into hairpin or quadruplex.I am confused about how to proceed this problem and invitro/invivo techniques used to establish the role of that sequence invivo. I haven`t find any good paper regarding this
Schaffitzel, C., Berger, I., Postberg, J., Hanes, J., Lipps, H. J., and Plückthun, A. (2001). In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. U.S.A. 98, 8572-8577.
http://www.bioc.uzh.ch/plueckthun/pdf/APpub0202.pdf
If you want to measure then experimentally, you can probably use X-ray crystallography, circular dichroism or various other methods (really not my area, I'm afraid!).
There are also several algorithms on line that you can use to predict the free energies of hairpin and/or G-quadruplex formation for a given sequence; this may give you an idea of which is more favourable?
See, for example:
http://bioinformatics.ramapo.edu/QGRS/index.php
Hope that helps!
Cheers,
Dan
Dear varun,
For invitro assays,
1)you run the quadruplexes and unfolded Same sequence of DNA in Native polyacrylamide gel. By UV shadowing, you will see a shifted migration pattern for the same. Alternatively, radiolabel both the DNA (unfolded or folded), and see by EMSA on Native polyacrylamide gel.
2)there is one more way of confirming the formation of quadruplexes which is DMS footprinting or Bromine Footprinting.
3) DNA POLYMERASE STOP Assay.
FOr invivo studies, clone your sequence after promoter of GFP gene in a plasmid.
Transform it and quantify GFP signal. Formation of Quadraplexes will reduce the signal of GFP.
How i made it clear here.
hi Varun,
It also depends on the structures that will arise from your sequence. E.g in Quadraplexes, Intermolecular species will migrate slower than the intramolecular species. Intramolecular species will run even faster than the linear DNA form which will be your control structure for comparison in EMSA.
hi manoj. r u working with quadruplex.can you share proteocol for dms footprinting and other quadruplex related techniquw.
Discovery and Structural Characterization of G-quadruplex DNA in Human Acetyl-CoA Carboxylase Gene Promoters: Its Role in Transcriptional Regulation and as a Therapeutic Target for Human Disease
Mangesh Kaulage†‡, Basudeb Maji‡, Jyotsna Bhat§, Yasumasa Iwasaki∥, Subhrangsu Chatterjee§, Santanu Bhattacharya‡, and K. Muniyappa*†
Please follow this link. Its one of the journal from our lab. Am not working on Quadraplxes.
can we generate G-quadruplex structures for ssDNA sequences online if we know there is a probability that those seq might generate one?
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