University of Lagos
Question
Asked 6th Nov, 2013
Can anyone please recommend a software to design long range PCR primers?
I want to sequence whole nuclear genes.
Most recent answer
It will be better if you use a master mix specifically designed for long range PCR, Dream taq green can amplify up to 20kb. Good luck
Popular answers (1)
Umm Al-Qura University
Hi Razan,
The best effective way to design the long-range PCR primers, is to use NCBI website.
choose Gene database and type your gene (NDUFA1). Then, choose (Homo Sapiens, human) depending on your case. The next page will give you more information with regards your gene of interest. Go to Genomic regions, transcripts, and products and choose FASTA. The nucleotide sequence of your gene will appear in the next screen.
You notice that the first line written (Showing 6.36kb region from base 119004999 to 119011362.)
So it is better to add nucleotides upstream and downstream for your gene from (Change region Shown) and click (update view), so the target region now is getting bigger depending on how many nucleotides you added to it.
Then you are ready to start, so from the right list choose (Pick Primers). This will open a new window (Primer-Blast). In the (Primer Parameters) choose a suitable PCR product size (5200-5700 bp) to get the whole gene in a single product. From (Primer Pair Specificity Checking Parameters Specificity check) You need to be sure to select the right choice depending on your application and if you are working on humans. If so please choose (Genome, reference assembly for selected organism) and the number 9606 (human) will appear for the organism.
And finally, click on GET PRIMERS.
This is the most effective way and easiest to design primers especially the long-range ones.
You need to play around with the numbers to get your suitable products.
One more interesting thing that you can check your target by go to UCSC website and do in silico PCR, so you can see where your primers are locating within the genomes.
If you get your primers so we can then discuss the options of polymerases available in the market.
All the best,
Amr
10 Recommendations
All Answers (10)
Sapienza University of Rome
In my experience to run a long-range PCR you need highly processive DNA polymerases and special conditions, but you don't need special primers. You can use any primer designing tool, such as primer3 plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/ ), a free web-based tool, which I recommend.
4 Recommendations
King Faisal Specialist Hospital and Research Centre
Thank you for your reply. I tried using primer3 to design LR primers amplifying the NDUFA1 gene( 5174 bp) but it did not work! Probably because I chose a big region!
Are there certain guidelines of how big the PCR product should be?
Los Alamos National Laboratory
Long PCR products, greater than 1,000 bases long, are very much more difficult to produce than short products. There is a lot of "art" as well as theory involved in amplifying more than 2 or 3KB in a single amplicon. I am not sure if the difficulty goes up linearly or exponentially with length, but if you fail to get a long product the primers are not the most likely problem. http://www.whitelabs.org/Lab%20Protocols/PCR%20Protocols/kyle.htm
etc...
Ruhr-Universität Bochum
I once had to amplify fragments of 6-8 kb to check mouse cell clones for knock out of a gene. I actually had good results (i.e. identical to a later Southern blot) using Phusion polymerase, and playing with the buffer (GC or HF) and addition of betain. If the genome of your organism is known, try primerblast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) for the primer design. This tests for unspecific binding to other region of the genome, and you can set the maximum length of the likely wrong product. Primerblast is based on Primer3, actually.
2 Recommendations
Bhabha Atomic Research Centre
Yes, primers have nothing to do with long range PCR. You need to have specific DNA polymerases/kit. Once I had to clone a 6.8 Kb long gene. I struggled for getting full length amplicon even after using LongRange PCR kit from Qiagen as it contained tandem repeats. I used 5% DMSO in PCR rxn and this solved the problem.
gd luck,
Sudhir,
1 Recommendation
Umm Al-Qura University
Hi Razan,
The best effective way to design the long-range PCR primers, is to use NCBI website.
choose Gene database and type your gene (NDUFA1). Then, choose (Homo Sapiens, human) depending on your case. The next page will give you more information with regards your gene of interest. Go to Genomic regions, transcripts, and products and choose FASTA. The nucleotide sequence of your gene will appear in the next screen.
You notice that the first line written (Showing 6.36kb region from base 119004999 to 119011362.)
So it is better to add nucleotides upstream and downstream for your gene from (Change region Shown) and click (update view), so the target region now is getting bigger depending on how many nucleotides you added to it.
Then you are ready to start, so from the right list choose (Pick Primers). This will open a new window (Primer-Blast). In the (Primer Parameters) choose a suitable PCR product size (5200-5700 bp) to get the whole gene in a single product. From (Primer Pair Specificity Checking Parameters Specificity check) You need to be sure to select the right choice depending on your application and if you are working on humans. If so please choose (Genome, reference assembly for selected organism) and the number 9606 (human) will appear for the organism.
And finally, click on GET PRIMERS.
This is the most effective way and easiest to design primers especially the long-range ones.
You need to play around with the numbers to get your suitable products.
One more interesting thing that you can check your target by go to UCSC website and do in silico PCR, so you can see where your primers are locating within the genomes.
If you get your primers so we can then discuss the options of polymerases available in the market.
All the best,
Amr
10 Recommendations
King Faisal Specialist Hospital and Research Centre
Thank you all that was really helpful..
Amr, thank you for the detailed explanation, that was really helpful :)
I followed the steps you mentioned and I got some primers designed..I blasted them in both UCSC and ensemble and I got the same product in both..Have you ever tried LR multiplex PCR?
Umm Al-Qura University
Hi Razan,
I tried the multiplex PCR for products range from ~ 300bp to 1200bp, but not for the long-range ones.
Similar questions and discussions
Related Publications
Ispol'zovanie «standartnyh» diagnosticheskih panelej, dayushchih vozmozhnost' opredelyat' lish' neskol'ko naibolee rasprostranennyh v populyacii mutacij v genah BRCA1 i BRCA2, mozhet privodit' k poyavleniyu lozhnootricatel'nyh rezul'tatov iz-za nalichiya drugih povrezhdenij v kodiruyushchih oblastyah dannyh genov, chto, v svoyu ochered', mozhet pri...
Ziel dieses handlichen, deutschsprachigen Laborhandbuches ist es, die Standard- und Spezialanwendungen der PCR in praxisnaher und verständlicher Form darzustellen. Es wendet sich an Diplomanden, Doktoranden, Wissenschaftler und TAs, die die Möglichkeiten der PCR für ihre molekularbiologischen oder diagnostischen Fragestellungen nutzen möchten.
In v...