Most of the informations show that both PCR and cloning can be used before Sanger Sequencing to amplify the DNA template. But how is it possible for us to design the primers if we don't know the DNA sequence yet? So, in my opinion, only cloning is possible to amplify the sequences. Can anyone tell me how PCR can work in this way? Thank you!
$\begingroup$
$\endgroup$
2
-
$\begingroup$ Can you link these informations? $\endgroup$– Chris ♦May 31, 2018 at 7:07
-
$\begingroup$ Some comments in this question. There are different aspects which confuses me. biology.stackexchange.com/questions/40988/… $\endgroup$– IvyMay 31, 2018 at 7:11
Add a comment
|
2 Answers
$\begingroup$
$\endgroup$
PCR is used to amplify a sequence included inside two known regions. So, if you're able to clone (for example with recombination) two sequence forward and backward to your sequence, by using PCR you'll amplify also the sequence included.
This image in example will clarify my statement.
$\begingroup$
$\endgroup$
There are several tricks that can be used to PCR amplify an "unknown" sequence.
1) If the protein sequence is known, than it is possible to guess the entire range of DNA coding possibilities for a small region of that protein. Making the complementary primers for two regions is thus possible.
2) If you know several homologous of that gene several species, you can attempt to identify highly conserved regions within the gene. ie parts of that genes that does not change between species.
3) If the genome is small and your lab is well to do, you can just sequence the whole genome. Then you will have the DNA sequence and can thus design primers at your leisure.
4) If you have isolated the gene (via BAC cloning) but do not know the sequence, you may still PCR amplify it using primer that bind to the vector containing the gene of interest.
5) You may ligate dsDNA oligos to the unknown DNA sequence. Then use primers complementary to the oligos
