sowing pcr? - (Jun/11/2004 )
I have heard a number of people talking about sowing PCR. Yet I can't seem to find any information on it. Does anyone know what this technique is i.e. the principle behind it and where i can get more information on it, perhaps its more commonly known by another name?
Do you mean "sewing PCR", or a more common name "fusion PCR" or "PCR stitching"?
It could be called either of those two names. I am not sure. My feeling is that this technique allows two separate fragments to be "sewed together" so fusion pcr sounds like a good bet.
More official name is overlapping PCR, which is a great tool for Point Mutagenesis, Delation Mutation, and Insertion Mutation. "sewing" the PCR fragment is only one less used purpose.
The PCR is called SOEing PCR. There are different ways to perform SOEing PCR depending on how you want to use the DNA construct or product in the end. For our lab, we perform this PCR all the time to create in frame deletions of our genes of interest. We utilize four primers (A, B, C, and D), where primers A and D are about 400 base pairs before the gene and 400 base pairs behind the gene. We engineer those primers to have restriction sites on it to put it into a suicide vector. We design our primers B and C to be about 20 base pairs inside of the 5' and 3' ORF, respectively. From here, we then add on base pairs to B and C to overlap each other. SOEing PCR will be carried out in two or three steps, where the first PCR is just a reaction between A + B and C + D. You'll purify the PCR products and then perform a second reaction using the purified PCR products and adding only primers A and D. The PCR product AB and CD will overlap and because you'll have 3' hydroxyl groups on the AB and CD, the PCR will fill in to make the gene construct. Primers A and D in the reaction allow for the gene construct to be applified even more.
This procedure works well for us...especially for those who can clone will and efficiently. It can be tricky and that is why a good book on PCR techniques will be helpful on how to successfully carry out SOEing PCR. SOEing PCR can be used to modify the gene, inserting or deleting fragments of the gene. We use SOEing PCR to get make an in-frame deletion in the chromosome of the organism we study.
I hope this helps a bit!!!
en, this PCR is very useful but a little difficult sometimes.
in our lab, we ever used it to PCR a 3.5kb and a 4.5kb fragment from gemone, and then "sewing" them to get a 8.0kb fragment. it's a horrible hard work!! and it cost our one month!
but for the smaller fragment it works very well.