I'm working on Alpha thalassemia both of multiplex-PCR and single PCR for sequences of Alpha-alpha, alpha 4.2, alpha 3.7 and SEA by using Takara taq Hot Start version. Amplification result for DNA samples with various alpha-globin genotypes by Single PCR were positive but the multiplex-PCR weren't . I don't know what is the problem ????.
Thank for reading and helping!!
Hi Pham,
test first your primers by in-silico PCR just to verify if you're amplifying the right positions and see the differences between respective Tm. they must be very closed to ensure multiplex success. if it's OK, you have to divide the amount of each primers by the number of simplex to add in PCR mix without changing other parameters.
the UCSC site gives you some tools as in silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr).
fred
Hi Pham,
First, verify the amplicon with appropriate positive control specimen. there is a higher chance of non-specific amplification at expected size. The primary step in optimization of conventional or real time PCR is generation of known positive and negative control. If you don't have try to get it. Before that no body can make a comment on the sensitivity and specificity of your experiment.
Hi Pham,
Alpha deletion and multiplication PCRs are quite problematic because of the product sizes, high GC content and few other reasons. Our alpha deletion multiplex PCRs targeting two or three deletions work well when used with Q solutions, but multiplexing above that is usually difficult with high failure rates.. If you are in diagnostic side I would suggest MLPA as it is more informative and covers more deletions that deletion PCR.
- Badges
- Science method