Single primer PCR - (Dec/01/2006 )
If I utilize a single primer to perform a PCR, could I get a single strand DNA?
And if my template is more than 5Kb, the extension time: 3min, to surppose my Pfu efficency is 1Kb per min. , then can I get a 3Kb fragment?
Thank U much!
Yes. You will get a single stranded DNA. However the ssDNA will amplify at a linear rate rather then a geometric rate.
So it will go : 1, 2, 3, 4, 5, 6, 7 molecules and so forth.
(Normal PCR goes : 1, 2, 4, 8, 16, 32, 64, molecules and so forth)
Single primer PCR is thus highly dependent on the number of template molecules. ssDNA PCR is usually used to make big primers from shorter primers.
Yes.....
However I have a feeling that you intend to use a single primer to proform PCR on your 5kb template... in which case you will get a smear. Like all things in biology, 1kb per min is the average speed. On average this is how fast the enzyme moves... but not all molecules.. some will move faster some slower. The geometric expension of normal PCR is what keeps main product the product that you designed the primers to amplify.
Furthermore as you don't have the use of the second primer... and thus geometric expension... miss priming become a bigger problem.
If I utilize a single primer to perform a PCR, could I get a single strand DNA?
Yes. You will get a single stranded DNA. However the ssDNA will amplify at a linear rate rather then a geometric rate.
So it will go : 1, 2, 3, 4, 5, 6, 7 molecules and so forth.
(Normal PCR goes : 1, 2, 4, 8, 16, 32, 64, molecules and so forth)
Single primer PCR is thus highly dependent on the number of template molecules. ssDNA PCR is usually used to make big primers from shorter primers.
in. , then can I get a 3Kb fragment?Yes.....
However I have a feeling that you intend to use a single primer to proform PCR on your 5kb template... in which case you will get a smear. Like all things in biology, 1kb per min is the average speed. On average this is how fast the enzyme moves... but not all molecules.. some will move faster some slower. The geometric expension of normal PCR is what keeps main product the product that you designed the primers to amplify.
Furthermore as you don't have the use of the second primer... and thus geometric expension... miss priming become a bigger problem.
when I use a single primer to perform my PCR, I really get a smear.
Thank you so much fo ur reply!
Sure u will have ssDNA but in order to get high numbers of product u have to increase the cycle number (but it will be problem for 5kb -->most probably more bands or smear).
To solve it you can also use assymetric pcr meaning primer 1 with low amount of primer 2 (100:1 , 50:1 u have to optimize this) u will see 2 bands in gel; one representing ssDNA and another for dsDNA.
Also u can make it some easy by designing primers that differ in length by adding polyA for example or a modification(biotin for example) that can cause to run them in different rates in gel.
But the most easy solution (according to me:) you carry out normal pcr but with a biotin on one primer. Than in a solution containing urea use streptavidin magnetic beads to hold biotin modified strands and take remanining solution and da da u have single stranded DNA in your hand.
Also there are some other techniques that use terminator primers but i think it is ok for u. 