Real Time PCR suggestions

4 posts / 0 new
Last post
Firoz Ahmad
Firoz Ahmad's picture
Real Time PCR suggestions

Hi!
I have one query with respect to real time primer and probes. Can i multiplex a target (132bp) and housekeeping gene(123bp) in the same tube. The size of the PCR products are mentioned in the bracket. The probes are labelled with FAM and HEX respectively with TAMRA as a quenchor.
Please suggest me at your earliest.
Regards,
Firoz Ahmad

Tony Rook
Tony Rook's picture
Firoz Ahmad:

Firoz Ahmad:

The design and optimization of multiplex PCR assays consist of a careful and systematic approach to primer selection. Here is a reference to a very thorough discussion of the design, testing, and optimization of multiplex assays.

Reference:

Richard Schoske, Pete M. Vallone, Christian M. Ruitberg, John M. Butler. Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Anal Bioanal Chem (2003) 375 : 333343

Link:

http://www.cstl.nist.gov/div831/strbase/pub_pres/Schoske2003.pdf

Abstract:

The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures (Tm) and primerdimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixture constructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a wellbalanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used.

Tony Rook
Tony Rook's picture
Firoz Ahmad:

Firoz Ahmad:

Additionally, please refer to http://genomics14.bu.edu:8080/MuPlex/MuPlex.html

This site hosts the Multi-Objective Multiplex PCR Design software from Boston University. This may be helpful with you're multiplex designs.

Firoz Ahmad
Firoz Ahmad's picture
Hello!!

Hello!!
Thanks for your valuable suggestions!! I will go through the paper which you sent to me !!
With regards,
Firoz