What are the merits and demerits of primer binding inside and outside the gene of interest?
I don't understand why it would create any confusion, even if someone choose to design a primer out side a gene, he would definitely knowing the amplicon size. So no confusion in gel. Again, I believe whether it would be within, or outside of gene is absolutely dependent on the purpose of your experiment. . But yes, if you think I am wrong, please correct me.
Very simple question.
Well, a primer should be specific to your gene of interest only. It should not bind outside the gene otherwise a false result will obtained on agarose gel electrophoresis run after the PCR step.. This can be avoided by designing a primer using a software. You can go for Primer-BLAST of NCBI.
If primer binds outside the gene of interest it will give wrong/false or rather confusive result during electrophoresis. In other words if your GOI is 1 kb and primer binds about 10 bases away from gene sequences than the size will be that many bases more.
On the controry if primer attaches inside gene of interest in such case also the result will be false and if you are doing SNP study or want to sequence the gene there are more possibilities that you may get wrong answer.
A properly designed primer must anneal inside the gene of interest
I don't understand why it would create any confusion, even if someone choose to design a primer out side a gene, he would definitely knowing the amplicon size. So no confusion in gel. Again, I believe whether it would be within, or outside of gene is absolutely dependent on the purpose of your experiment. . But yes, if you think I am wrong, please correct me.
I agree with my previous writer that in principal it doesn't matter where the primer is located. Specificity does not depend on the location, but on the complementary (primer) sequence within the targeted DNA. If someone needs the full DNA sequence of a gene a primer has to be located outside the gene of interest. If this is not necessary and someone wants to amplify the gene of interest for distantly related organisms, it is better to design a primer inside, since coding sequences are more conserved.
Anyway you need sequence information (if available in e.g. NCBI) for designing primers either inside or outside a gene.
Hey Kashif. Primers depending on what kind they are can bind both inside and outside the gene of interest. The primers that bind inside are called gene specific primers and the primers that bind outside are called vector specific primers. Depending upon the purpose of your experiments, one can select the primers; say for example you amplify your gene of interest for cloning using gene specific primers. While sequencing a cloned gene, you will be using vector specific primers.
The question is not clear to me. I believe it depends upon how you define outside the gene of interest. A few bases away may not affect the result but if it is several thousands bases away then it may become an issue. Also, if the gene has several alternate splicing sites with different transcripts then your amplicon may be different. Yes, if the gene gives you only one product then it makes no difference using primers insdie or outside of the target (if the primer is not really too far away from your target). But, if the primers "outside" of you target gene give you a product that is too big it will decrease the efficiency of the amplification which should also be considered.
Thanks alot every one for yours valuable answer......
actually i was confuse because when i have ask this Question in my group then sum said outside and sum said inside so i was confused ......... but now i think depending upon the type of experiment the primer are to be binds either inside or outside .....
I think that if you want to express a protein from a protein coding gene, you need it to include the start and stop codons, so the primers may be outside (without including another start codon) or inside if you are including the start and stop codon. However, if you want to see if the gene is present, it should be inside the gene because that primer will give you the specificity to that gene.
In denaturation step, two stands of a DNA are seperated and primers come inside to be placed in special position, not outside. By the way, hydrogen bonds are sahped exactly in front of complementary pyrimidine and purines (A=T and G=C) as a result they are attached each other exactly inside at least in linear form.
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