Author: Narrandes, Shavira; Xu, Wayne
Title: Gene Expression Detection Assay for Cancer Clinical Use Document date: 2018_6_5
ID: wheblwm3_5
Snippet: Primers are required in qRT-PCR analysis to anneal to a specific portion of the mRNA so that the DNA polymerase can extend from the primer and amplify the gene of interest. The factors to consider in primer design are the melting temperatures (T m ) and GC content of the forward and reverse primers. A range of T m s have been reported, with 58Ëš~60ËšC considered as stringent. Tight annealing of the primer to the mRNA template will reduce the chan.....
Document: Primers are required in qRT-PCR analysis to anneal to a specific portion of the mRNA so that the DNA polymerase can extend from the primer and amplify the gene of interest. The factors to consider in primer design are the melting temperatures (T m ) and GC content of the forward and reverse primers. A range of T m s have been reported, with 58Ëš~60ËšC considered as stringent. Tight annealing of the primer to the mRNA template will reduce the chances of primer-dimers or hairpins. 11 , 12 Lastly, primers spanning exon-exon junctions allow for the strict amplification of mRNA samples and not any contaminating DNA as DNA contains alternating regions of exons and introns while mRNA lacks introns. 11 There are a few freeware to help the primer/probe design, for example Primer3, the primer designing tool from NCBI.
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