Take the 2-minute tour ×
Biology Stack Exchange is a question and answer site for biology researchers, academics, and students. It's 100% free, no registration required.

I'm having difficulty finding an explanation of how single primer amplification of DNA works in the literature available to me, can anyone explain the methodology and what it accomplishes?

share|improve this question
add comment

2 Answers

up vote 1 down vote accepted

This process is called sequence-independent single primer amplification (or in short SISPA). Its used to amplify unknown sequences for amplification, for example in unknown viruses. The primers used for this process contain random sequences which should bind to the DNA to be amplified and also known sequences, which can be used a adapters. In the first rounds of PCR only the random primers give rise to specific products, which are amplified exponentially in the later rounds of PCR since the primers fit perfectly then. This is described in detail in this publication:"Random priming PCR strategy to amplify and clone trace amounts of DNA" Another interesting paper describing the method is:"Sequence-independent, single-primer amplification (SISPA) of complex DNA populations."

share|improve this answer
add comment

single primer amplification is used to find unknown regions of DNA if you have no idea what to use as a primer further upstream. It works on the idea that any primer given a certain temperature will misprime. That it, it will anneal to a non specific location. So it is PCR, but at this temperature, the five prime region primer will bind specifically to it's known location. On the three prime end up to 3500 bases away, the low annealing temperature will allow this specific primer to bind to something resembling its complementary sequence but not exactly. This way you will still get a successful PCR reaction. Then as the cycles continue, the 3' end binds specifically to your one primer and you can raise the temperature to make it more specific. The primer design and temperatures have to be considered very carefully.

Sources

Roth Lab

share|improve this answer
add comment

Your Answer

 
discard

By posting your answer, you agree to the privacy policy and terms of service.

Not the answer you're looking for? Browse other questions tagged or ask your own question.