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Enhancing PCR* Efficiency with MasterTaq

ially. One way of achieving this aim is by designing oligonucleotide primers to confirm the expression of the target sequence via RT-PCR in the cells under examination. If the RT-PCR is positive, the amplicon can be used as probe for detecting the corresponding mRNA on Northern blots or for semi-quantitative RT-PCR. As many sequences are low-copy genes and thus difficult to detect on Northern blots, RT-PCR is preferred in these cases. The results from subtractive hybridization in many cases consist of very short sequences (80250 bp), so the amount of possible PCR primers is limited. Nevertheless, to obtain any results from RT-PCR, a reliable amplification system is required.

Experimental setup

In the experiment described, the influence of an inhibitor of histone deacetylases on the gene expression of human lung adenocarcinoma cells was analyzed using subtractive hybridization4 of treated cells versus control cells. Of 36 oligonucleotide primer pairs designed to amplify candidate sequences, three failed to yield any PCR product with the standard Taq DNA Polymerase used for routine amplifications. PCR reaction conditions were modified using temperature gradient PCR (Eppendorf Mastercycler gradient)** for determining the optimal annealing temperature of the primers (50C70C), variation of MgCl2 concentration (1.5 mM to 5 mM) and touchdown PCR. None of these yielded any PCR product. Five different polymerases from other suppliers were then tested according to the manufacturers instructions, including two different proofreading polymerases and two hot start polymerases. Finally, the MasterTaq kit, containing a PCR enhancer, was used and resulted in positive RT-PCRs following the standard
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