I want to isolate a gene from the potato which has 20 gene that are 90% identical to the one i want to use ,How will i design my primers and make sure that i have only extracted my desired gene of interest? Or will i just suppress all of them and randomly discuss the one i want. Any input on this matter will be very much appreciated.
Thank you
Thank you Sarit Charuchaibovorn any recommendation on where I can find information about Primers design for Nested PCR?
Marcus' answer is great. It is not too detailed, but gives a complete overview.
BTW: it depends, if you want to amplify it from DNA or mRNA ... be aware that the upstream and downstream regions of the genes might be quite variable. Which is good in this case, as it gives you the possibility to search for unique sequence features (SNP states, Indels). If you use mRNA/cDNA be aware that the 3'UTR is quite variable also in gene families but can be short. to make primers for a region 100bp downstream the STOP might not result in an amplicon.
I worked with gene families of 25 and now work with one of ca 100 members, but god bless not in a recent polyploid species, where identity of homologues is definitelly higher.
There are some dirty tricks in making primers more specified (e.g. insert a mismatch base at the second or third last position at the 3'end of your primer.) You have to find out for your system what works be--... (I will omit the word "best", as this is quite difficult issue and you will be happy about a working solution, even it is not the best.)
I think, To enhance the primer's specificity, designed DPO (dual priming oligonucleotide) primers. These are the primers for Multiplex PCR that also used for conventional PCR but have high specificity. These primers reduce the risk of undesirable extension of gene.
good luck.
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