Trouble with Inverse PCR - (Aug/07/2005 )

Dear all,

I am having a lot of trouble with inverse PCR to clone a breakpoint in a gene. I use normal human genomic DNA as control to test the self-ligation conditions. I digest the control DNA with PST1, purify the DNA with Qiagen nucleotide removal kit and then ligate the DNA at a concentration of 1 ng/ul at 16'c overnight. I purify the DNA again after ligation using QIagen kit and then do the PCR. IN control DNA there should be a band about 1 kb but I cannot get anything at all. THe PCR condition I use is 94C 4min
94C 15 sec
72C 3 min 5 cycles
94C 15 sec
68C 3 min 30 cycles

Is there any special requirement for PCR condition, PCR primers or something else in inverse PCR??

Is this a control reaction specified by a kit manufacturer? Are the primers they supplied supposed to work with these cycling conditions? You are not explicitly doing an primer annealing phase in your PCR, which may be ok if the primers are GC rich enough and long enough. But, if you have designed the primers, or are winging the PCR conditions, then I would suggest that you think about lowering the PCR annealing temperature. Try something like this:

94 C 4 min
< 94 C 30 sec
60 C 30 sec
68 C 1 min > 35 cycles

You'll need to extend the 68C extension time for longer products, but for a 1KB product, that should be plenty.

You might think about a control PCR only reaction on an existing, linear DNA fragment to test that your PCR reagents, machine,and protocol are all working.

Thanks! I am not using a kit. The primers I am using are long (29 mer) but not so rich in GC. I did a control PCR , the condition and reagents worked fine. I think I will try as you suggest and see if it works. Thanks.

You may have lost your DNA. Can you do a control PCR that you know will amplify on your inverted DNA? Also do you know how large your inverse PCR product should be? It might be very large.

Daniel

Better DNA sequencing

I did that control too so I know my DNA is there. Because I am currently working on the control DNA ( HUMAN genomic DNA) to test the PCR conditions, I know the size should be about 1 KB. I also tested if the fragments are self-ligated or they ligate to other digested fragments. It seems they ligated to other fragments. So I am wondering what conditions I should use to promote self-ligation.

Are you using PEG in your ligase buffers? This can promote intermolecular ligation. Other than this you might want to try using an even low DNA concentration on you could try a different approach by ligating linkers and amplifying that way.

Daniel

DNA sequencing software

Is that PEG-800? What concentration do you use?

For your application (intramolecular ligation) you don't want PEG. It is in some ligase buffers so I thought that you should check.

Daniel

Longer automated DNA sequencing reads

Daniel, thank you so much for all this information !!!!