RAPD - close proximity of primers

[TytoAlba95]

I was reading RAPD kit manual by himedia and couldn't understand why the two primers should be at close proximity and in opposite orientation to actually amplify DNA.

'In RAPD analysis, for PCR to occur: a) the primers must anneal in a particular orientation (such that they point towards each other) and, b) they must anneal within a reasonable distance of one another. Successful primer pairs produce different banding profiles of PCR products between individuals, strains, or species when analyzed by gel electrophoresis.'

 

[BillTre]

The polymerase enzyme often used on PCR (Taq polymerase), although good at making short strands of DNA, does not generate long strands that well.
This limits most PCR amplification to shorter strands.
Thus the primers work better when close together.

 

[TytoAlba95]

Thank you so much.

 

[TytoAlba95]

Thus the primers work better when close together.

But BillTre, won't the Taq polymerase extend any primer irrespective of its orientation. All it needs are a suitable buffer, ions, temperature and dNTPs. Why should proximity be an issue when the primers are binding to two completely different strands?

 

[BillTre]

won't the Taq polymerase extend any primer irrespective of its orientation

It will extend from any primer, however the amplification requires that a primer bind to the product of a previous extention (after the process has been initiated on the first round from the starting DNA) and run back, extending the opposite strand to produce a double stranded product that will have primer binding sties on opposing strands.
Both of the strands from these will new short double stranded products will be able to bind primers and produce new product in the next round.
In this way, the number of products grow exponentially as new rounds of PCR reaction occur.
Other wise the growth of products will only be linear: a copy is made, things are denatured, new primers are bound and a new copy is made only on the starting material, not on the newer amplified strands since the binding site (the opposite strand) for the original primer has not been reproduced in the new material.

Wikipedia has a decent picture showing how this works.

 

[TytoAlba95]

In this way, the number of products grow exponentially as new rounds of PCR reaction occur.
Other wise the growth of products will only be linear: a copy is made, things are denatured, new primers are bound and a new copy is made only on the starting material, not on the newer amplified strands since the binding site (the opposite strand) for the original primer has not been reproduced in the new material.

Dear @BillTre,
This post is several months old now, but I must admit I didn't completely get your last reply.
I have tried to depict my understanding through this illustration below:

So, with one effective primer (in absence of reverse primer binding site) PCR products are not formed. Am I correct?

 

[BillTre]

With only one primer, you will get products extended from the single primer, but they will only amplify linearly not exponentially. They will also NOT be of all the same length.
On a gel, if a band is visible (due to fewer products from linear amplification), the band will be more diffuse because of the different lengths of the products.

If there are two primers that direct the polymerase TOWARDS each other, they will be predominately amplifying not the original very large pieces of starting DNA and it first products, but will be duplicating the DNA pieces from a previous round of amplification where the end of the piece being amplified is defined by the location of the first primer that was used to amplify the DNA in the opposite direction.

Again, this will only work if the primers are close enough to copy DNA extending to the location of the second primer. Many versions of the enzyme will only go so far before stopping and falling off the DNA. If the pieces produced do not enclude the second primer site, the secnd primer will not be able to bind the DNA and trigger another round of amplification.

 

[TytoAlba95]

With only one primer, you will get products extended from the single primer, but they will only amplify linearly not exponentially. They will also NOT be of all the same length.
On a gel, if a band is visible (due to fewer products from linear amplification), the band will be more diffuse because of the different lengths of the products.

I understood that the PCR products from a single primer will not be of the same length and the band observed due to those will be diffused if there's linear amplification, but I don't get how there will be a linear amplification of dsDNA.
With reference to my illustration, shouldn't the single-stranded DNA which lacks the reverse primer binding site increase linearly?

Sorry, I forgot to mention that the DNA sequence of interest lacks the reverse primer binding site.

 

[BillTre]

NOt sure I am understanding your question correctly, but does not your picture of the ssDNA amplify linearly (1 + 1 + 1 + ...) as it is copied in each round?

dsDNA in a PCR protocol would be heated to become ssDNA after which it would interact with reaction mixture.