I'm working on a Rice mutant (spotted-leaf mutant) in which there is a SNP in the coding region of a gene. We are considering that the mutant allele is semidominant. We have T1 complementation plants (WT allele transferred into mutant) which shows different intensity of lesion phenotype on leaves. We wish to detect abundance of WT and mutant allele by semiquantitative-RT PCR. Can anyone tell how to design the primers and do the semi-RT PCR?
Thanks in advance!
you can use forward and reverse primers flanking the SNP in addition to use two probes FAM and VIC that detect both homozygous and heterozygous SNPs by using RT-PCR,
I. A. Abdulhassan
Thank you for ur suggestion, but we have already done homozygous and heterozygous screening by dCAPS analysis. Now we want to check the abundance of WT and mutant allele (in heterozygous T1 transgenic plants) so as to see if we can correlate this abundance with phenotype (lesion intensity). Due to unavailability of special probes like FAM and VIC in our lab, I need to rely on simple PAGE analysis.
You can design primer by using BLAST program available on web site of NCBI but you can depending on previous published papers if you don’t have experiment in primer design. And about Semi-quantitative RT-PCR you must be extracted RNA from your sample and transfer to cDNA by Reverse Transcription and then amplified your targeted gene by use PCR after select specific primer. afterthat PCR products loading on polyacrylamide gel with presence DNA ladder for analysis
You can use simple HRM (High Resolution Melting) designing probe covering your SNPs between forward and reverse primers through qRT-PCR.
- Badges
- Science method