The purified RNA can be used in a number of downstream applications including real time PCR, RT-PCR, Northern blotting, RNase protection, primer extension
, expression array assays and NGS.
This company has to call a tender for the supply of a system of pharmacogenomics with mass spectrometry technology primer extension
in cancer diagnostics.
First, primer extension
is performed using site-specific primers and a combination of dNTPs and ddNTPs, selected so that different alleles will result in products of different and predicted sizes.
During this work, we found that this measuring system was not able to detect changes in methylation levels during patient treatment, and so 2 different primer extension
schemes were investigated.
observed that the single mismatches present at the last five positions from the primer 3 end could totally block the primer extension
reactions were conducted using the SNaPshot ddNTP Primer Extension
Kit (Applied Biosystems, Foster City, CA, USA).
The PCR cycle involves three steps: denaturation, primer annealing, and primer extension
The PCR program consisted of an initial denaturation step at 95[degrees]C for 5 min; followed by 30 cycles of DNA denaturation at 95[degrees]C for 1 min, primer annealing at 52[degrees]C for 1 min, and primer extension
at 72[degrees]C for 1 min; followed by a final extension at 72[degrees]C for 5 min.
Ramping time can also be reduced by converting from a three-step to a two-step protocol in which the annealing and extension steps are combined at a temperature that's optimal for primer annealing yet sufficient for primer extension
CYP2D6 genotyping by a multiplex primer extension
We tested single-base primer extension
using the species-specific SNPs identified in oysters (results not shown here).
in an aqueous buffer at alkaline pH results in at least tow-fold increase in enzyme activity in less than 20 minutes which allow formation of primer extension