Help with fusion PCR to piece together fragments - (Feb/09/2007 )
I am an undergraduate Honors biology student. My research project is two place untranslated regions of a similar gene before and after the coding sequence of a sample that I have. In my first round of PCR, I have generated the UTRs with overlapping sequence (about 16bp) with my desired gene. However, I tried to link my UTRs and gene together by combining the UTRs with my desired gene and the forward and reverse primers and it hasn't worked. Does anyone know of a way to help me with this problem?
hmm what conditions have you used?
you could tell us exactly what was done. Quantities, volumes. The PCR cycling conditions, (temp. durations, number of cycles), what is the sequence of the 16bps.
Have you tried making lots of single stranded DNA first (using only the forward primer to amplify the forward section for maybe 10 cycles, like wise for the reverse primer with the rearwards segment). Then late combine the products of the PCR reactions and continue the PCR reaction.
Off the top of my head the conditions I followed were:
1.5ul Forward Primer
1.5ul Reverse Primer
1.7ul 10x PCR buffer
0.2ul Taq Polymerase
1.0ul DNA of selected gene
45 degrees (annealing tempis two mins)
for 35 cycles
I have not tried making single stranded DNA and I really have no idea how to. I don't have much experience with molecular biology, but I am very appreciative for any suggestions!
erm the concentration of the various components are missing (primers, MgCl). I would like to know the sequence of forward and verse primers, and the 2 homology regions to calculate the tm.
how big is all this
5'UTR - gene -3'UTR
What is the size of each individual component?
Taq polymerase is not the best enzyme to use. It is quite error prone. With this many cycles and a product of any appreciable size, near all the products will have at least one mutation. Furthermore taq doesn't amplify large fragments well without a lot of optimisation (large being anything over 2kb)
I would strongly suggest you use a high fidelity enzyme. Something like KOD hifi, Vent or similar. It is possible you may have to reamplify all your fragments.
The annealing temperature is urr... too high (!?) It should be (tm - 5 Celsius) rather then (tm - 2 Celsius). Drop the annealing temperature to about 40 Celsius (?!). Still a tm of 47 Celsius is very low. I am quite uncomfortable with this. If you could redesign your primers aim for a tm of at least 58 Celsius.
ssDNA production is made via a non symertical PCR reaction. Normally in a PCR reaction both reverse and forward primers are in equal concentrations. To produce ssDNA, one primer is in excess, while the other primer is present only in small quantities. The primer in excess produces the desire ssDNA.
Rather then put all three components together. you could try putting two components first. eg 5'UTR and gene. Once you have a product (5'UTR-gene), use said product to start a second PCR reaction with (5'UTR-gene) and the 3'UTR
Thank you so much!!!
Just adding to Pernese's reply, Phusion polymerase from Finnzymes is excellent. It's a protein fusion between the DNA binding domain of Taq and the polymerase domain of Pfu (Pfu is another polymerase). This protein contains the fidelity of Pfu (which has 3` exonuclease activity that gives it high fidelity) and speed of Taq all in the one enzyme, it's a real breakthrough in PCR technology in the last few years. I've been using it for a year now and I don't think it's stuffed up once. There's are a few of these "fusion" enzymes around now, but this is the one I use and it's fine.