I got genomic dna samples and I wonder if need to break the circular strand in order to sequence it. How can I break it? And how can i know if I have circular or linear dna in my samples?
Thanks a lot!
Are sure that your DNA sample still circular?. I think you need to give some more information about your experiments to be able to help you. I have just attached a paper about making new plasmids as an example of circular DNA constrcution
Article The pJan25 vector series: An enhancement of the Gateway-comp...
You should first identify a single restriction site (sequence) in the circular DNA that a single restriction endonuclease enzyme could cut, just once on the circle, then run a 1% agarose gel of the product (using Gel Red as the stain; ethidium bromide has been conventionally used - but safety concerns abound on that) to visualize a single band at a MW that matches the linearized form (comparing to a lane loaded with pre-sized MW DNA standards). Uncut circular supercoiled DNA will appear at a smaller MW on the gel (since supercoiled DNA appears smaller to the gel's ability to impede travel, and if you get two bands, you know you haven't entirely linearized your circular DNA yet). Finding a restriction endonuclease enzyme that only cuts the circle at exactly one site is your first goal... e.g., EcoRI, HindIII etc. Then, learn the process of running a 1% agarose gel (easy to find on Google)...
However... rolling circle sequencing may be another option for you:
see: http://www.pnas.org/content/110/49/19872.abstract
sanger will work even on circular genomes as long as specific primers are provided ... is your dna of mitochondrial origin or of chloroplast origin or is it bacterial? these are known to be circular
If you are looking towards NGS then any of the sequencers with long reads can give you that answer. Sonication can be used to randomly shear the DNA (circular and linear both). If your genome is small less than 10kb then you can try rolling circle sequencing as recommended above by Jack
Also maybe digestion with "RecBCD Nuclease, E. coli" can tell you whether its circular or linear. If it is able to digest than its linear if the dna remains intact than its circular ... please refer this link for details http://www.epibio.com/enzymes/nucleases-glycosylases-dna-binding-proteins/quick-guide-to-enzyme-properties
I am getting problems because I have dna strands inmobilised onto nanoparticles and this probe recognises its complementary strand but I am not getting results with the bacterial genomic dna. This is why I was wondering if I should use a restriction enzyme to break the genomic dna. Genomic dna has been isolated by CTAB procedure. Sonication is enough for breaking DNA? Thanks for your answers.
in that case yes sonication should be enough ... assuming ofcourse that the dna strand immobilized on nanoparticles contains sequences of your bacterial dna
- Badges
- Science method