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I understand that in PCR we're able to amplify only selected portions of the DNA... however despite reading it from multiple sources, I cannot figure out how this selection actually takes place.

I know that by using proper primers, one each for coding and template strands, we can direct the DNA polymerases to specific sites to start with. I also understand that these primers make the replication proceed in opposite directions, towards each other. Like follows:

My understanding of so far

However, I wonder what makes the replication terminate when the polymerase reaches the primer at the other end? Because as far as I understand, replication should continue indefinitely until the physical end of the sample strand is reached... such that we may decide the starting points but never the ending points.

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Termination of replication on the original template is dependent on the processivity of the enzyme or the length of the DNA, but think about what happens if a newly synthesized strand is used as the template. –  canadianer Aug 6 at 11:10

2 Answers 2

up vote 7 down vote accepted

Note: In your PCR program you always set extension time.

Case:

  • Product length = 500bp
  • PCR extension time = 50sec
  • Assuming that polymerase adds 1000 nt/min

Cycle 1:

  • Strand that binds FP: extends ~800nt to the right (as per the polymerization rate): 300 bp ahead of RP complementary site. This product is lets say P1
  • Strand that binds RP: extends ~800nt to the left: 300bp ahead of FP complementary site. This is P2

enter image description here

Cycle 2:

The products of cycle-1 become templates for next round. FP binds to P2 -300nt from its 3' end. Similarly RP binds to P1 -300nt from its 3' end. The resultant products will be 500nt which give rise to a 500bp perfect dsDNA.

enter image description here

Some P1 and P2 will keep forming at a linear rate; since your template is low in amount compared to the amplified cycle1 and cycle-2 products, your expected PCR product will be much higher (which gets produced at an exponential rate).

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The long products are amplified linear, while the short product (with the primer sequences on the ends) are amplified exponential. –  Chris Aug 6 at 11:42
    
yep exactly... :) –  WYSIWYG Aug 6 at 11:42
    
Wow, beautifully explained. –  Zuhaib Ali Aug 6 at 14:06
    
Only now I understand why there are highly controlled timings and temperature control in PCR. –  Zuhaib Ali Aug 6 at 14:13
    
I think that the DNA produced with overhanging ends (i.e that made of P1 and P2) must grow as exponentially as the one of perfect size... only that it will lag one order of magnitude behind. –  Zuhaib Ali Aug 6 at 14:41

I'm not completely clear when you say "what makes the replication terminate when the polymerase reaches the primer at the other end" since when you perform a PCR you go through three phases. The denaturation, whereby the two DNA strands become single stranded, then the annealing, which is when primers attach to their appropriate matching site (but the original DNA template is still single stranded) and extension, which is when DNA polymerase use the primers to extend make the matching strand the primer is attached to (http://en.wikipedia.org/wiki/Polymerase_chain_reaction). DNA polymerase continues to make the strand until it essentially either drops off or until the cycle is over and it is time for the cycle to repeat itself. The way you make sure to terminate a specific DNA replication at an appropriate length is to use the extension time as a control since as pointed in the above answer by @WYSIWYG the extension is largely assumed to be 1000 nt/min so if your product of interest is 500 bp, then you do 30-40 sec of extension in your PCR extension cycle program and if the product of interest is 1000 bp you do 60 sec of extension and so on.

In the training I received, it is usually not recommended to PCR DNA template which is very large such as an entire plasmid such as pUAST. The main issue even with high fidelity/proff-reading DNA polymerases is that you can get errors in replication if the strand is very long. what often happens is that you amplify a section of DNA if you want to check for the presence of something or if you need to use the amplified fragment then you design restriction sites at either end of your fragment by incorporating it in the primers itself then you go ahead with you digestion and fragment purification and then you sequence your fragment to make sure you do not have any errors although PCR has much much more applications.

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I see... incorporating restriction sites within primers sounds pretty smart. Thanks for the wider perspective.' –  Zuhaib Ali Aug 6 at 14:12

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