Good day house.
Please what is the function of the universal primer, Inosine, in a primer? I need to use the primer for PCR. If I don't include the Inosine in the primer as published what is the consequence?
While using primer blast to confirm my primers, I discovered that replacing Inosine with any of the four nucleotides, the primer blast gave me the set of organisms I am interested in. So, I am wondering rather than embed Inosine in the primer, why not replace it with any of the four nucleotides.
Inosine is used because it binds to all the 4 bases of dna. It is the equivalent of N ( any base) at its position so can be used when there is a polymorphism at that position and the primer will still anneal. You can make the primer with N at the inosine position and the company will add all 4 bases so you will end up with 4 slightly different primers in your tube and pcr. This may make a difference to whether the primer will anneal at some temperatures depending on whether the primer has an A or a c/g and the amount of any specific primer will be less than you normally use for pcr as for any sequence 3 out of 4 primers will not be annealing but the pcr primers are in large excess so that will not make much difference. If you want to amplify specific organisms then using specific sequenced primers would be better. If you want to amplify all organisms to see what is in your sample then N or I will amplify more organisms and give you a better idea what is present
Inosine is used because it binds to all the 4 bases of dna. It is the equivalent of N ( any base) at its position so can be used when there is a polymorphism at that position and the primer will still anneal. You can make the primer with N at the inosine position and the company will add all 4 bases so you will end up with 4 slightly different primers in your tube and pcr. This may make a difference to whether the primer will anneal at some temperatures depending on whether the primer has an A or a c/g and the amount of any specific primer will be less than you normally use for pcr as for any sequence 3 out of 4 primers will not be annealing but the pcr primers are in large excess so that will not make much difference. If you want to amplify specific organisms then using specific sequenced primers would be better. If you want to amplify all organisms to see what is in your sample then N or I will amplify more organisms and give you a better idea what is present
inosine does not pair with G .... A Adenine G Guanine C Cytosine T Thymine U Uracil R Purine (A or G) Y Pyrimidine (C or T) N Any nucleotide W Weak (A or T) S Strong (G or C) M Amino (A or C) K Keto (G or T) B Not A (G or C or T) H Not G (A or C or T) D Not C (A or G or T) V Not T (A or G or C)
you can replace it with D ...
you have to replace it , do not skip it . if you do you will get completely different primer especially if it is on 3' end - and it will not bind to your template, do not get your pcr product ....
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