I am trying to amplify an 8KB linear cDNA template with primers annealing just in both ends of the sequence with no success, can anyone suggest tips for PCR optimization or a method to elongate both ends to improve annealing and PCR efficiency? Thanks in advance.
If you use the search function on RG you will find several similar questions and many suggestions how to solve your problem.
e.g.
https://www.researchgate.net/post/Does_anybody_know_a_good_enzyme_for_high-fidelity_long-range_PCR
https://www.researchgate.net/post/Is_there_a_Taq_or_other_DNA_polymerase_enzyme_that_can_be_used_to_amplify_large_fragments_20-30_kb_and_does_not_have_5-3_exonuclease_activity
https://www.researchgate.net/post/How_to_amplify_large_fragment_by_PCR
https://www.researchgate.net/post/Long_Range_PCR_30_kb_Is_it_possible_from_human_DNA1
https://www.researchgate.net/post/problem_with_cloning_PCR_cant_amply_the_full-length_cDNA_with_PCR
You can try to decrease the annealing temperature, about -5 degree C.
Moreover, for amplification of long-template PCR, I often use enzyme Herculase, it is a good choice for this purpose.
With the long template from Roche, I do this program :
10 cycles with 8 to 10 min of annealing.
25 cycles with 8 to 10 min of annealing and incremental adding of 30sec.
PCR were very long but works well.
If you use the long template from Roche, try the 3 buffers.
And last, for some difficult templates, with this enzyme, I use a speed of annealing around 500 bases per min (for 8Kb, 16 min of annealing !!)
Is the gene known or you are the first one trying to clone it?
Are you sure your cDNA contains the mRNA?
Did you check if you can amplify a smaller part (~1 KB) of the gene?
Did you check the quality of your cDNA by trying to amplify a housekeeping gene?
If the cDNA is good, the sequence is known and the mRNA is expressed, try to clone it in two steps and ligate the two pieces, more wet work but less frustrating.
If you use the search function on RG you will find several similar questions and many suggestions how to solve your problem.
e.g.
https://www.researchgate.net/post/Does_anybody_know_a_good_enzyme_for_high-fidelity_long-range_PCR
https://www.researchgate.net/post/Is_there_a_Taq_or_other_DNA_polymerase_enzyme_that_can_be_used_to_amplify_large_fragments_20-30_kb_and_does_not_have_5-3_exonuclease_activity
https://www.researchgate.net/post/How_to_amplify_large_fragment_by_PCR
https://www.researchgate.net/post/Long_Range_PCR_30_kb_Is_it_possible_from_human_DNA1
https://www.researchgate.net/post/problem_with_cloning_PCR_cant_amply_the_full-length_cDNA_with_PCR
Be sure that (1) your RNA is intact and contains your gene of interest (detecting it by PCR amplification of an smaller region) and (2) that you are using a reverse transcriptase suitable for cDNA transcription of long mRNAs such as RevertAid H Minus Reverse Transcriptase from fermentas. For the cDNA amplification, I agree with the previous suggestion: Q5 from NEB is really great for long templates.
Good lucky!
I would extend the primers or re-design/try several combos. The template (RNA) quality is important as well as the selection of the optimal RT. I would not waste a whole lot of time/money on trying to amplify the entire CDS in a single PCR reaction. What if the transcript is partially degraded? I would divide the predicted sequence into 2-3 kb regions, order overlapping recursive PCR primers and run several reactions alongside with the aim of hooking them together into full-length CDS in the final PCR reaction. This way it will at least be obvious that you may be missing parts of the transcript you are pursuing.
Thank you all very much for your answers. The sequence of my interest is a whole RNA viral genome, within a total RNA preparation from infected cells. I'd strongly prefer to avoid the option of amplifying and cloning smaller (2-3 Kb) fragments and further overlapping, because probably by doing so I will amplify fragments from very distinct RNA templates, and the final construct may not fit virus requirements for replication. The fact that viral RNA polymerases lack proofreading activity leads to many single nucleotidic changes throughout the genome, that can ultimately be fixed or cis-complemented by other mutations elsewhere. I need to amplify 8 Kb. In addition, primers for PCR hibridize only by 50% in both sequences ends, because I need to incorporate a promoter and a restriction site.
Try extending polymerization time by 10 sec after each cycle, reduce denaturation time (94C for 10secs, that helps increase enzyme half life) and add Betaine to the reaction mix. All these help a lot and I have been able to amplify ~12.0 kb.
Hi Angel, maybe you can try with some middle-sequence primers.
And, as Raul says, try all the mentioned!
Good luck!
For long range PCR use specific enzymes with high processivity, low annealing temp and longer extension time. But you are likely to get spurious products in this case which can be avoided by fine tuning the PcR conditions.
In addition to the mentioned Q5 polymerase you may try Phusion (from Thermo). I believe Q5 is more or less a copy of Phusion, both having a single strand DNA binding domain fused to the polymerase, so they should be quite similar. Another option is DyNAzyme EXT (Thermo), which is specifically designed for long range polymerase.
In case you need additional overlapping bases, do the following:
1) 20 microl PCR reaction with primers matching exactly the 5' and 3' end of your gene
2) Check it on a agarose, keep >1 microl aside
3) If you get good amplification, use 1 microl from the previous reaction as a template for another PCR reaction with a primer set which includes the desired overhangs
First try to see if you can amplify your gene with perfectly matching primers and then add whatever you need.
Whenever I tried to include the restriction sites directly in the first pair of primers I never worked, for all the genes I cloned I used this two step strategy.
Please keep us posted about your results, I'm sure more people encountered the same problem.
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