During chain elongation by DNA polymerase there is an attack on the alpha phospate of the ATP molecule but during DNA sequencing for preparation of radiolabelled primer there is a radiolabelling on the gamma phosphate....why so?

Question

During chain elongation by DNA polymerase  there is an attack on the alpha phospate of the ATP molecule but during DNA sequencing for preparation of radiolabelled primer there is a radiolabelling on the gamma phosphate....why so?

anonymous · Answer

In DNA sequencing,doesn't the Alpha Phosphate of all ddNTPs are radio labelled?so that when they form 5'-phosphoester bond,beta & gamma phospates are knocked out,but radio labelled alpha phosphate remains in the phosphoester bond....I am also asking this Question,Does primer is needed to be radiolabelled in dna sequencing??If anuone know the cause,please share??From what i know,primer doesnot need to be labelled....

munish · Answer

Actually in dna sequencing either ddntps are radiolabelled or the primers are radiolabelled at 5' end in this kinase is used but to radiolabel at 5'end gamma phosphate of atp is radiolabelled so this is the question why gamma phosphate as we know it is the nucleophilic attack of the alpha phospate on the 3'hydroxyl group and beta and gamma phosphate are taken out as pyrophosphate ions.

anonymous · Answer

yeah...thats right....but,donot know about primer labelled dna sequencing.....can u give me a protocol of it?so,i can study that.

anonymous · Answer

are u referring Maxam–Gilbert sequencing?

anonymous · Answer

http://www.youtube.com/watch?v=lqWZ-duHfu8 This is Maxam–Gilbert sequencing which uses 5' gamma phosphate.///from my opinion,the labelling is done on a primer is this manner: 5'-(32p)ATTACG-3' > This is a primer...It has already labelled at the very 5' end. The dna synthesis is not possible in that end...so,that labelling is unharmed in whole dna sequencing process.Moreover,it is necessary because,at the end,when the dna fragments are run under gel electrophoresis and further autoradiographed, this 5' gamma phosphate gives the sign of how far the strand has run. Hope that answers the question...

anonymous · Answer

n.b. The primer i gave example of is not an actual primer...just used as an example...

munish · Answer

so first question is how is this labeling done.........secondly why only gama phospate why not beta or alpha?