primer design

[adele13]

I am new to molecular biology and unfortunately I don't understand the primer design process.

I need to carry out PCR prior direct sequencing, for example. I know there are several programs that design primers like Primer3, Primer express. I have to paste the DNA sequence, right? But I don't need the whole DNA, I only need one exon to be genotyped later, so what should I do? Should I paste the sequence of this exon only? or should I copy a part of gene DNA that is a little bit longer (both sides) than my exon? however, an intron goes after the exon I need, so should I copy the mRNA sequence in this case?

My second question is how can I design a sequencing primer? can I use a forward primer already designed for PCR?

I know the questions are very simple and may even be stupid, but I just don't get it I would be very grateful for your answers

[obama]

How big is the exon.

If you designing primer on exon then you have design primer based on cDNA of the gene/DNA.

For your second question, Theoretically I don't the answer, but once i used my pcr primer for sequencing it worked.

[adele13]

Thank you for your reply!

The exon is about 200bp long. Can you please tell me where I can find the cDNA sequence?
Will the primers designed for the cDNA sequence work for the genome DNA? I thought the fact that there is an intron between my exon and the one next to it won't let the primer designed for a flanking exon-exon cDNA region attach to my DNA. I would appreciate if you could explain me if I am wrong.

[obama]

Sorry am wrong in my last answer. If your purpose of pcr is to genotype then you have to design primer from DNA sequence. (am confused , i thought you doing it for gene expression only in that case you have to design primer from cDNA).

If your exon is 200 base pairs then you can manually design primers with in the 200bp. with product size of 180 to 150. it is good size of product size. Best to avoid intron sequence in pcr design, intron not conserved like entron so you may get false negative result in some cases.

Try to get good primer region within your exon.

[adele13]

Thank you for your explanation, I appreciate it!