Q1. Katsuyama et al. (2009) had designed the primers for the full-length cDNA of CURS2 using a pair of primers, 5′-GCTAATCAGTCAATCCAGATGG-3′ and 5′-CGTCTATCGATTGATCGATCGTG-3′. These primers were designed on the basis of the previously reported expressed sequence tag (EST) sequences (NCBI accession no. DY393763 and DY387045). A 1.3-kb cDNA fragment encoding CURS2 was obtained. I want to know how can I do that on my own? Is it possible to know the actual sequence in between those primers in full length CURS encoding cDNA?
Q2. Katsuyama et al. (2009) had also used gene-specific forward and reverse primers (5′-TGTTGCCGAACTCGGAGAAGAC-3′ and 5′-TCGGGATCAAGGACTGGAACAAC-3′ for CURS2 in qRT-PCR for gene expression study. Is it possible to find out which region is actually amplified in CURS cDNA using those primers? Please suggest me how to do that on my own?
DOI: 10.1016/j.febslet.2009.07.029
I am also using those same primers for qRT-PCR, and I have to sequence full length cDNAs and have to order primers. Is there any better way to do so? Please suggest me.
Hi Dipendra,
it's very specific....if you're looking for informations on curcuma with CURS2, try the uniprot site (http://www.uniprot.org/uniprot/C6L7V8) and you'll get some details that may help you (end of this page, sequence database/AB506762/view or download....
fred
Dear Fred,
Thank you for your suggestion. I already have downloaded all those database sequences for all CURS genes and I also have some idea about how to design the specific primers for qRT-PCR using those sequence information using Primer3 (NCBI) but I have to amplify the full length CURS encoding cDNA from the total cDNA synthesized from mRNA. Author has designed those primers from EST sequence tags but I don't get it, how they did it? Because when I searched for ESTs they used it was just only 756 base pairs length and one of the forward primer matched the sequence somewhere in EST but another didn't show any match. The point is they were able to amplify 1176 bps using those primers. I also have to design primers for amplifying the same, how?
Hi Dipendra,
if you have the right sequence, there are lot of softwares (I use OLigo7) and tools, just try this one (https://www.eurofinsgenomics.eu/en/dna-rna-oligonucleotides/oligo-design-more/primer-design-tools.aspx).
fred
We also order the primers from Eurofins, thank you for the link. I will try this out. But the thing is I want to know how to design primers using ESTs? The ESTs are smaller fragments but the gene encoding region is longer than that.
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