Q-PCR problem - melt curve analysis

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Amtekoth
Amtekoth's picture
Q-PCR problem - melt curve analysis

Any experts around here that can tell me how to get rid of a major 2nd peak? I don't think it's from template contamination, since it's come up in all my DNA samples but not my +/- controls. I'm thinking that I may need a different primer pair, but any other possibilities?

Thanks,

Ed

Tony Rook
Tony Rook's picture
Hi Ed:

Hi Ed:

Take a look at these papers to see if they help you at all. However, the best thing for you might be to simply redesign some new primers.

Jo Vandesompele, Anne De Paepe, and
Frank Speleman. Elimination of Primer±Dimer Artifacts andGenomic Coamplification Using a Two-Step SYBR Green I Real-Time RT-PCR. Analytical Biochemistry. 2002. doi:10.1006/abio.2001.5564

Abstract:
Gene expression analysis plays an increasingly important role in many fields of biological research. The recently developed real-time PCR quantification method has many advantages over the conventional quantifications in terms of accuracy, sensitivity, dynamic range, high-throughput capacity, and absence of
post-PCR manipulations. Sequence-specific fluorescence- labeled probes (e.g., TaqMan) have been considered as a standard detection format in many diagnostic and research applications, but are not very well suited for quantification of a large number of different sequences, because a new and relatively expensive probe is generally required for each amplicon under investigation. We have therefore optimized and
validated a reverse transcriptase PCR (RT-PCR) assay for accurate expression profiling using the double stranded DNA-binding dye SYBR green I, which is a much more economical alternative to quantify any given transcript in a reaction. Using such a generic dye, different PCR amplicons and/or nonspecific amplification products could accurately be distinguished by the generation of so-called DNA melting curves and first-derivative melting peaks.

Jannine Brownie, Susan Shawcross, Jane Theaker, David Whitcombe, Richard Ferrie, Clive Newton and Stephen Little. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Research. 1997. Volume 25, Number 16, p. 3235-3241.

Abstract:
We attempted to produce primer-dimers (PDs) from a variety of primers with differing types and extents of complementarity. Where PDs were produced they were cloned and sequenced. We were unable to produce detectable PDs either with individual primers alone or with similar sequence primers even if they had 3' complementarity. These observations led to the hypothesis that a system could be developed whereby the accumulation of PDs in a PCR may be eliminated. We demonstrate a method for the general suppression of PD formation that uses a sequence of additional nucleotides (a Tail) at the 5' ends of amplimers. Tailed amplimers are present at low concentration and only participate during early cycles of PCR. In subsequent PCR cycles, amplification is achieved using a single primer that has the same sequence as that of the Tail portion of the early cycle primers, here we refer to this as a Tag. When products are small, as with PDs, there is a high local concentration of complementary sequences derived from the Tail. This favours the annealing of the complementary ends of a single strand produced by tailed primer interactions and gives rise to `pan-handle' structures. The formation of these outcompetes the annealing of further Tag primers thereby preventing the accumulation of non-specific PD products. This aids the design of large multiplex reactions and provides a means of detecting specific amplicons directly in the reaction vessel by using an intercalating dye.

Good luck!

EvoRoadster
EvoRoadster's picture
Is this a SYBR-Green assay?

Is this a SYBR-Green assay? Did you optimize your primer pairs via normal pcr to obtain only specific product before running realtime? What is the melt temp for your specific product vs you non-specific product? Much more info is needed to help.

Amtekoth
Amtekoth's picture
The primer pairs were

The primer pairs were generated in silico by somebody else before I joined the project. The Tms were 60 and 62 degrees and I chose to start with an annealing gradient of 54-60 and saw no real differences.

What I found out yesterday is that the original designer wasn't going to use SYBR Green but rather a 6FAM-TAMRA probe specific for the predicted product, and thus didn't care if there were non-specific products generated.

So now I've ordered a 6FAM-TAMRA probe and I'll give that a go. This will be new for me...

Thanks,

Ed