Hi everyone, i need some idea immediately. What I meant above is that the resistant gene which caused by single mutation. Cos there are few types of single mutations in the same codon which gives the resistance for a drug. What I planned is to design a primer that is universal for the codon...but differentiate the mutation points by specific probes...However, there will be a problem cos the probes will be only differ in terms of a single nucleotide. So, can hybridization differentiate the single nucleotide changes in the probe? or any other methods or technology which I can refer 2...except RT- PCR
Hi Thanes!
Have a look at this: http://seq.mc.vanderbilt.edu/DNA/html/ola.html
Hi everyone, thanks for the kind reply..will look into OLA or SNP analysis...btw can anyone give a details or elaboration on this method...thanks.
You can use different methods:
I think the problem is the cost.
Use RFLP, ASO-PCR, MLPA (modifying the protocol in accordance to your needs).
Good Luck.
Micro or macro- arrays really could differentiate SNP.
I think it's also possible to try two primers with different nucleotides at 3' end in standart reaction.
Thanks to all replies first of all..btw for your information I am planning to use microarray method in combination with the OLA in order 2 differentiate the SNPs. However, i m still a bit confused with the OLA probe preparation for the immobilization on the membrane of a a chip? Can anyone give some idea on this..thanks.
Hi Jean, thanks for the suggestion but my company wants me 2 use the company's platform which is detection based on Chip method.
simplest method is perform RFLP... search for the restriction enzymes which cuts at the specific point and perform RFLP with both normal and mutated gene.. because of the mutation the restriction site is altered in the mutated gene so the enzyme didnt cut at that position..
other method is design allele specific primers such as CA primers... and perform PCR if there is mutation u will get product otherwise u didnt get the product.
Thanks for the suggestions. for now i m going to some prove of concept on OLA method. Can anyone suggest me any denaturing agent for DNA? I am planning to use chemical denaturation method. Are there any other chemicals which I can use other than acid, alkali, UREA and formamide? any enzymatic method which i can use 2 get a single stranded DNA?
The better enzymatic method to get ssDNA is so called asymmetric PCR. The idea is to amplify a short amplicon (about 100 bp or better less if it is practically convenient) when one of the primers is in a great excess compared to the other. The size of amplicon and right ratio of primers could be experimentally determined, ssDNA has altered mobility in agarose gel compared to ds form, the less fragment the less quantity of undesired short products will be seen. If I right remember it is about 1:100 (20 pm: 0.2pm). Predominant primer should produce a strand that supposed to be hybridized with the allelle specific primer.
We are looking for beta testers for our temperature gradient microarray hybridizing equipment. It has the ability to distinguish such mismatches with proper probe design.
http://www.arraymelter.com/
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