Joining two PCR products together
kl...@med.usyd.edu.au
I came across your answer regarding how to join 2 pieces of PCR
products together in a single cycle PCR. Please advice me if this is
the right way to produce a single piece of DNA fragment from two PCR
products. Here is my experiment:
1. From cDNA, I have generated two PCR products using two different
primer sets. One product has a size of 500bp (A), while another one is
600bp (B). A and B has around 10-15 homologous bases.
2. Consequently, I need to generate single PCR product using the
forward primer of A and reverse primer of B, to produce a final
product of approximately 1100bp. I have repeated this stage for many
times but still failed. What I get is just smear. I only performed
normal PCR without the 2 quasi-exponential PCR as what you have
mentioned before.
3. Do I need to perform 2 quasi-exponential PCR for the initial PCR;
to produce A and B? Can you please explain the PCR parameters in
detail? Can I replace it with a normal PCR, i.e. initial denaturation
(98C, 2min), followed by 35 cycles of (98, 30sec-> 55C, 30sec-> 72C,
30sec) and finally final extension at 72C, 8min.
4. With the initial PCR product, do you recommend me to use it at a
small quantity (how much would be sufficient?) to perform a single
cycle PCR without adding the primers at high Tm? Does that only
include these steps: 98C, 30sec-> 70C, 30sec and 72C, 30sec, for one
cycle? Do I need to add DNA polymerase at this step?
5. Do I then use this single cycle PCR product from above as a
template for exponential PCR or just add in appropriate primers and
run 15-cycles exponential PCR for this product at primers Tm? Can you
explain the exponential PCR here in details?
Thank you so much for your help!!
Cheers,
Katherine
Kolling Institute of Medical Research
Royal North Shore Hospital
St Leonards, NSW, Australia, 2065
Peter Ellis
> 1. From cDNA, I have generated two PCR products using two different primer
> sets. One product has a size of 500bp (A), while another one is 600bp (B). A
> and B has around 10-15 homologous bases.
To confirm: the first 10-15 bases of your A reverse primer are
complementary to the first 10-15 bases of the B forward primer?
May I ask what you're trying to do in the first place? Are you trying to
construct a fusion gene?
>
> 2. Consequently, I need to generate single PCR product using the forward
> primer of A and reverse primer of B, to produce a final product of
> approximately 1100bp. I have repeated this stage for many times but still
> failed. What I get is just smear. I only performed normal PCR without the 2
> quasi-exponential PCR as what you have mentioned before.
To join these fragments, the general principles are as follows:
1) Amplify and purify fragments A and B. This gives you your two
starting segments of DNA with no remaining primers. It's important to
make sure all the starting primers are removed, otherwise you'll just
re-amplify the two small fragments rather than the one large fragment
2) Mix A and B, denature and allow to anneal. This allows the 10-15 base
overlap to bind.
3) Extend the annealed products to form the "joined" A-B molecule.
4) Amplify the "joined" construct using the A forward and B reverse
primers.
Steps 2-3 will be the difficult bit - with only 10-15 base paire of
overlap, the annealing will be very inefficient if not negligible at
anything approaching a normal PCR temperature.
>
> 4. With the initial PCR product, do you recommend me to use it at a small
> quantity (how much would be sufficient?) to perform a single cycle PCR without
> adding the primers at high Tm? Does that only include these steps: 98C,
> 30sec-> 70C, 30sec and 72C, 30sec, for one cycle? Do I need to add DNA
> polymerase at this step?
You need DNA polymerase at this step, and you need to lower the Tm to
ensure that the two fragments bind even though they have only a small
region of complementarity. Tm for a 15-bp duplex is in the region of 50
degrees C, while Tm for a 10-bp duplex is in the region of 30 degrees C.
If you try to use an ordinary PCR protocol, your fragments will just melt
apart from each other and you won't get any extension - this is why you've
been getting nothing but smears.
To be honest, I'm not sure a thermostable polymerase will work for you,
you may want to try Klenow instead and run the extension reaction at 37
degrees or even lower.
It might also be useful to add one of the two fragments in large excess
relative to the other. If you add A in excess, then when you allow
them to re-anneal after denaturing, the (lower concentration) strands of B
will be forced to anneal to the (higher concentration) strands of A,
rather than the complementary B-strand.
>
> 5. Do I then use this single cycle PCR product from above as a template for
> exponential PCR or just add in appropriate primers and run 15-cycles
> exponential PCR for this product at primers Tm? Can you explain the
> exponential PCR here in details?
After you've carried out the extension reaction, you would then use this
as a template for an exponential PCR using A-forward and B-reverse. The
PCR part isn't the step that will cause problems though!
Peter
nazmi...@gmail.com
I am also facing a problem. Actually, I want to join two templates a) 1 kb and b) 2 kb hpt fragment which should give a 3 kb product but these two templates lacking homologous overhangs. Whenever I am doing a normal PCR I am getting smear. Please help me!!!