Suppose one has extracted RNA from a plant, converted it to the corresponding cDNA & amplified it but now wants to cut out a particular, already-sequenced gene out from it, how does one proceed?

i.e. How does one cut out that particular segment of the whole DNA which one wants? Does one always find restriction sites (or rather cleavage sites) at both ends of the desired gene segment & then one treats the cDNA sample with those specific restriction enzymes?

To get the gene shown below I'd find an enzyme that cuts at the GGCCAC site and another to cut at AAAAAA?

enter image description here

  • 2
    $\begingroup$ You could also PCR amplify the gene using primers with fitting restriction enzyme recognition sites (+ some extra bases) on the ends. $\endgroup$ – Chris Feb 17 '16 at 9:18
  • 1
    $\begingroup$ PCR, as Chris suggested. Also, when you made the cDNA library, you don't really have "whole DNA" as you suggested, but many pieces of DNA corresponding to the mRNAs you extracted. Some of those should be the gene of interest and can be amplified. $\endgroup$ – user137 Feb 17 '16 at 10:30
  • $\begingroup$ @user137 Thanks! Must one go through the mRNA & cDNA stages? Or is there an easier way to get the gene? e.g. Can't one extract the DNA itself from the plant cell nucleus. Why start from mRNA? Sorry if some of my questions are naive. $\endgroup$ – curious_cat Feb 17 '16 at 10:47
  • $\begingroup$ mRNAs are generally used because they tend to have a much higher copy number than genomic DNA and because the introns are already removed, so you are left with the coding sequence. You can isolate genomic DNA and do PCR directly off that, but the introns will be there, potentially making for a much much much larger gene. If you just want the exons, use the mRNA -> cDNA approach. It should be pointed out that if you want to express protein in bacteria, the introns will probably not be removed properly on transcription, so the protein won't be right. $\endgroup$ – user137 Feb 17 '16 at 15:10
  • $\begingroup$ @user137 Makes a lot of sense now the way you described it! Thank you. $\endgroup$ – curious_cat Feb 17 '16 at 15:11

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.