Multiplex PCR with Syber Green I

[miss-pcr]

Hi...
I'm using Multiplex PCR with Syber Green I, do I have to add all the reagents with the same volume in one capillary except the volume of water which is going to be reduced?

[amberpdidit]

Sorry, since SYBR is a generic DNA dye it cannot be multiplexed since it lacks specificity.

[admin]

Hmm i didnt know that SYBR cant be used in multiplex pcrs.

Which dyes can?

Thanks amberpdidit.

[amberpdidit]

well, someone can correct me if I'm wrong but I believe that you can only multiplex with probe based chemistries.

Since any of the DNA dyes can bind any nonspecific DNA sequence it makes it so you wouldn't be able to tell which sequence a specific dye is binding. Probe based chemistries (i.e. TaqMan) are sequence specific so you can multiplex. Multiplexing is a big chore as far as I've heard because it becomes more critical to have perfect primer probe sets and amazing efficiencies, etc.

We do SYBR and just run genes seperately, unless you have a ton of assay development/optimization time upfront it is fastest and simplest to do 2-step SYBR running your genes of interest seperately.

That is just my opinion and experience. Best of luck

[chovek69]

Originally Posted by amberpdidit

well, someone can correct me if I'm wrong but I believe that you can only multiplex with probe based chemistries.

This is not quite true. A lot of papers already proved that if two or more PCR products have enough differencies in there Tm (say >3C) it is no problem at all to multiplex the primer pairs with SYBR detection and melting-curve analysis. Recently I read a paper where 8 different tetracycline genes (namely tetA,B,C...) were effectively detected in a octaplex SYBR green real-time PCR. Besides there are already many instruments capable of doing high-resolution melting-curve (HRM) down to < 0.1C which further facilitate the multiplexing/detection.

So google a little and you'll find out

Regards

[jpinzon]

Hi
Very interesting. I knew that sybr green can use for detection of mutation, but no in multiplex pcr assays

[Aga]

I use SYBR Green I for multiplex assay and it's possible on one condition - the Tm temperatures of the products must differ of approximately 3-4 degrees to obtain good resolution of the peaks and the primers must be constructed in such a way that they don't interfere with one another and not produce by-products.
It is possible though, but any sort of quantification is impossible, as SYBR Green in non-specific dye.

I haven't heard of any works utilizing octaplex SYBR real time PCR and - chovek - if you can give me the reference I will appreciate it.

[chovek69]

Check it out ....! If you cannot access it ... provide your e-mail and I will send it.
Fan W, Hamilton T, Webster-Sesay S, Nikolich MP, Lindler LE.
Multiplex real-time SYBR Green I PCR assay for detection of tetracycline efflux genes of Gram-negative bacteria. Mol Cell Probes. 2007 Aug;21(4):245-56.

[Aga]

Thanks. I've just read this publication. I would say that out of 8 analysed genes the differentiation of 3 of them (J, D &A in Fig. 1) is ambiguous on the basis of melting curves. You can only differentiate 8 products using both melting curve analysis and gel electrophoresis, but I'd rather do two separate tetraplex reactions than electrophoresis, which takes longer (ethidium bromide!).

[chovek69]

Yeah, I agree with you... For differentiation of all genes one must have machine capable of doing high-resolution melting curve analysis. I saw one recently and I must say I was impressed.