real-time PCR - Multiple melting curve peaks (Aug/23/2005 )
I have greater than 2 peaks in my melting curve for samples that should only contain 2 differently coded CPR products.....
It was my belief that one PCR product:probe complex should equal one melting peak... Am I hurrendously wrong?
I have proved that my samples are contaminated - ie have sample DNA and a contamination which is proved by RFLP to be consistent with one of the expected PCR products (ie is a wildtype DNA contamination)...but according to the theory I understand - this should only produce 2 peaks maximum - ie the wild type and the mutant???.....
If anyone has any suggestions or would like to set me right on my knowledge I wold extremely grateful!
(a very tired postgrad desperately writing up a MSc diss)
Hi, may I ask the length of your PCR product please?
Hi! Can you except mutations (SNPs)? You using probes for melting curve analysis, what kind of?
I know the mutations in the area - they are very well documented and I am deliberately using the probes to genotype the DNA - hence I know that should only be two types of DNA - a wild type and a mutated code.
I have considered tha there maybe more mutations in the PCR product - this would change the melting temp - and hence give me more peaks - but because this is such a well studied gene area I would think it was unlikely.
I am using two adjacent hybridisation probes - one with a 5' FAM and one with 3'CY5 - hence the FAM is quenched when the probes are bound.
Can you give a picture of the melting curve? Perhaps your probes are of no good qualitity and hences there is a population of nucleotides (G and C e.g.) at a special position?
Hi, Ive attempted ot attache the melting peak (s) of my homozygote samples - ie should contain 1 type of DNA.
The probe codes are;
5’– G A G C A G A G A T A T A C G T A C C A G G T G G A G C –JOE– 3’
5’CY5– C C A G G C C T G G A T C A G C C C C T C A T T G T G –Phosphate–3’
Could you explain further what you mean by population of nucleotides?
Apparently my file attachment didnt work, I wil try one more time
hi, I am not an expert on PCR, but I just checked the sequence of your pcr primers on a software, it turn out that your second primer will form dimers and hairpin. It might indicate that you have some unspecific products. However, my experience is limited and thus I am unable to comment.
Thanks for the check - but those are my probes - not my primers. The codes on the previous reply have a 3' end phosphate that stops them from being able to function as a primer.
But still useful info about the dimers and hairpin - as maybe the probe is forming a dimer itself and then as the temperature rises it denatures itself and this changes the fluorescence.... hmmm
Can I ask what software you used? was it on a website or a private thing?
Any chance you would mind checking my primers??? please please please? The codes are below - you would be really helping me out!
5’ – C C T A C C A G G G C T G G A T A A C C – 3’
5’- C A A A G A G C A G A T C C T C A T C T C A C -3’
5’ – T C A G A G C A G G A C C T T G G T C T T T C – 3’
5’ – G G C T T G A A A T T C T A C T G G A A A C C C – 3’
Thankyou thankyou thankyou!