We want to detected the base deletion of plasmid in our experiment,however,there are only less than 5bases deletion of sequence. For now, we can only have one method: sequencing.
I want to ask if anyone know other methods, can detected 1-5bases deletion of DNA sequence instead of sequencing?
You can use 20-25% polyacrylamide electrophoresis. But there is no guatantee that it could resolve 1 bp difference.
Yes, if we run the gel, 1bp difference cannot be detectable, That's the case.
Yes, you can use an EasyBeacon combined with asymmetric PCR. This allow you to detect it easily using only one probe. We have developed a workflow for that, which is extremely straight forward. It is a real-time PCR as you now it, with an end-point affinity measurement (melt). I am not unbiased as this is our own products, but please feel free to browse our webpage though that it is not very up to date (a new is being developed) but prices can be found.
Try T7 endonuclease assay, if you know in which region mutation has occured then design primers for that region and amplify it followed by denaturation and re-annealing to create hteroduplexes. Then digest the mixture with T7 endonuclease ,if two three bands occur that means mutation has occured.
You can use an ARMS PCR.
The best variant is Tetra primer ARMS PCR (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55900/)
You probably have to play around the way to design internal primers with a 5bp indel
here the tool for primer design: http://primer1.soton.ac.uk/primer1.html
You can find an option also in batch primer 3 (http://probes.pw.usda.gov/batchprimer3/)
Bioanalyzer can do this job, especially high sensitivity DNA kit.
But you need cut your plasmid into two fragments: one fragment with the same length for both original plasmid and 1-5 base deleted plasmid; the other fragment is much shorter ideal ~50bp which contains potential deletion.
When you run bioanalyzer, it will clearly show the difference.
if you know the position of the deletion and it affects a restriction site te use restriction digest. If it is not a restriction site then use a mismatched oligo such that it creates a restriction site with the normal sequence then if the pcr amplimer of that region does not cut then you have a base change or small deletion at that site . Method is ACRS also known as dcaps which googles to a site that designs the mismatched oligo
I quote Paul!
Dcaps Finder is good!
http://helix.wustl.edu/dcaps/dcaps.html
if the size of deletion is not important to you, after PCR, you can run your PCR products on a 20-25% acrylamid gel. this concentration is able to distinguish templates with difference even about 1bp!
first, try then decide. i think it will work and worth trying.
good luck
did you check for loss or gain of a restriction endonuclease site?
You can label your forward primer with fluorochrome (e.g. 6-FAM ) and than run your PCR samples on genetic analyzer e.g. ABI 3130, in case of 3bp difference in my products it worked perfectly and the results are easy to analyze
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