Never Break the Chain

Q: 'If PCR amplifies DNA how do you know if the sequence you're seeing is original number or amplified? For example is AAGGT the sequence n u got AAGGTAAGGTAAGGT how do I know what's the original?'



A:  The three essential steps in a PCR (Polymerase Chain Reaction) are:

 1) Denaturation: 'Melting' of DNA into single strands a higher temperatures
2) Annealing: Addition of Complementary DNA Primers at lower temperatures
3) Elongation: Replication of DNA by DNA polymerase


 The processes of denaturation, annealing and elongation constitute a single cycle.

 Multiple cycles are required to amplify the DNA target to millions of copies. 

 Now while we're calculating the amount of DNA present in a sample, we retrace to the original number of copies using certain formulae.

 The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n, where n is the number of cycles. 

 Thus, a reaction set for 50 cycles results in 2^50, or 1,125,899,906,842,624 copies of the original double-stranded DNA target region.

 The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies.

 References:

 1. Saiki, R.; Scharf, S.; Faloona, F.; Mullis, K.; Horn, G.; Erlich, H.; Arnheim, N. (1985). "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia". Science. 230 (4732): 1350–1354. doi:10.1126/science.2999980. PMID 2999980.

 2.Saiki, R.; Gelfand, D.; Stoffel, S.; Scharf, S.; Higuchi, R.; Horn, G.; Mullis, K.; Erlich, H. (1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–491. Bibcode:1988Sci...239..487S. doi:10.1126/science.2448875. PMID 2448875.

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