As you will be able to tell, I am a bit of a novice in this field. I have the cDNA sequence of the glutathione reductase gene from a transcriptome of a tubificine worm I am studying. I was able to find exon 1 and intron 1 by choosing a forward primer at the the 5' end of the CDS and trying several reverse primers paired with it. The one successful reverse primer was at the beginning of exon 2. Exon 1 is 126 nt long and intron 1 is 321 nt long. The entire CDS is 1368 nt long. To continue this sequencing, I can choose a new forward primer from the 3' end of intron 1, but do I just keep choosing various reverse primers from the cDNA sequence, hoping that the primer is not split by an exon/intron boundary or that the amplicon too long to amplify easily? Is there a way to determine where the exon/intron boundaries are from my cDNA sequence? I cannot find any related annelid sequences, except one that is also just from mRNA. Thanks for any help!
coding sequences are often conserved between species (while the introns are not conserved) so you could get some ideas from NCBI BLASTing the cdna against the genomic dna for some similar species and you should get regions of good homology (exons) and regions of no homology where the introns are
To facilitate the strategy mentioned by Paul Rutland ,you can try using the BLAT tool at the UCSC Genome Browser. At https://genome.ucsc.edu/cgi-bin/hgBlat, you can try entering your cDNA sequence and running it against any or all of the genomes listed there. What BLAT does is to align a cDNA against a genomic sequence, revealing the intron/exon structure. Hopefully, there is some species there with enough similarity to the one of your interest to provide some useful guidance for your primer designs.
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Infectious Diseases