Primer Design Question

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Kelly M.
Kelly M.'s picture
Primer Design Question

I'm new at designing primers and am looking for some help.  I'm trying to amplify a gene that is 13.3KB.  I realize this is too large to accurately do a PCR to amplify the whole gene.  There are multiple papers that cite primers they designed/used for this receptor.  I've been able to locate the forward/sense primer sequence in the sequence I obtained off of NCBI, however i have NOT been able to locate any of the reverse sequences.  Does anyone have suggestions as to what I am doing wrong that I can't find the reverse/antisense primer sequences that are published in the published NCBI sequence?

Ivan Delgado
Ivan Delgado's picture
You may want to consider

You may want to consider using a primer design program. One that works well is NCBI's primer BLAST: 

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

Biju
Biju's picture
Hi Kelly

Hi Kelly
1.You need to check which strand(sense/anti-sense) you are using to locate the primer sequence.
If the gene is very recently discovered the published sequence need not necessarily contain the complete sequence and hence you might  not be able to locate your forward or reverse primer.
Hope this  answers your query atleast partially.
Biju

Kelly M.
Kelly M.'s picture
I guess let me rephrase it. 

I guess let me rephrase it.  In 4 separate references, there are listed primers for my receptor of interest.  They are either listed as forward/reverse or positive/negative or sense/antisense.  None of the primers listed in any of the paper are the same. I am only able to find the forward/positive/sense primers in the NCBI sequence (I don't believe this to be a new sequence) when I use the "Find" feature under "Edit". The sequences published at reverse/negative/antisense I am unable to find. 

I'm not opposed to just using a primer design program , I just never have before and am unsure of what I must enter and what I am getting as far as results.  The only time that I have previously designed primers the gene of interest was <3KB... not 13 KB... will this huge size difference affect anything? 

The person who taught (and I use that term loosely) me to design primers didn't do a great job and had zero patients for teaching.  I'm unsure of where to even start!

Biju
Biju's picture
Hi Kelly

Hi Kelly
My question now  is whether the gene is on a single stranded or double stranded DNA
Biju

wilsonj826
wilsonj826's picture
Hi,

Hi,
A gene of 13.3KB is certainly too large to be amplified using a single PCR run.
As per my suggestions:
1) You should either break down the target or ROI into smaller fragments and then amplify the gene in multiple pieces. Finally you can clone them which is a tedious task. Amplifying a smaller fragment in this way would require knowledge of the actual sequence of the gene (which I believe you already know).
2) Else, try to design reverse/antisense primer with the help of the forward/sense primer and the template sequence you have using any available automated software program. I rely on Beacon Designer wherein you can specify a forward primer and it designs a compatible antisense primer. Here is the link to Beacon Designer:
http://www.premierbiosoft.com/molecular_beacons/index.html

Regarding “not finding a reverse/antisense primer sequences that are published in the published NCBI sequence”, did you try the reverse complement sequence. It is possible that the reverse primers reported are in a different orientation due to which you are unable to find them on the sequence.
Cheers!!
John