I am facing big problems. I have got the full length gene sequence for my gene. Its 2500bp long approx. I have done 3 RACE s and got the full length by matching overlapping regions. Now I want to get the full length as one fragment. For that I have designed 3 primers from 3' UTR and 3 from 5' UTR. When I am doing PCR with deep vent, I am getting big smears even after 2-3 nested PCR. I have heated the total RNA at 65 C for 5 min and then kept it at ice and added the rest of the components for RT. The cDNA was made by oligodT primer at 42 C. Please help me regarding this as I am not getting any clue. I have also done another set of RT with high temperature RT at 48 C with one of my gene specific primers. But in that too the same story is approaching.
Thank you very much for your reply sir. I have used oligodT for the 5' and 3' RACE. I have already done a temperature gradient range of 48-61C for 8 reactions for all the reactions. I am using deep vent DNA polymerase and the extension time I am setting is 4 mins according to the protocol. I run a control PCR reaction for all the PCR sets just to confirm. I use oligodT cDNA synthesis by Fermentas kit, which is always positive in my hand. I have used a dilution of 50-500 for the template, but the same thing happened. Last day just changed the water stock, which has omitted the strong band near the wells, but still smears are there. Previously I have used Taq for smaller fragments, now I am using deep vent for 2.5 Kb.
Please suggest more sir, I am stuck in between these crucial phases of my PhD.
Prabuddha Sarkar
Visva-Bharati
India
I can suggest two directions for thinking:
1. Pessimistic: If your gene is duplicated in the genome and both copies are expessed, your full-length mRNA sequence is chimeric and doesn't exist in reality. In other words, 3' - and 5' - RACE fragments were from different transcripts from different genes and your current primers aren't compatible.
Diagnosis: Amplification of the gene from genomic DNA. If you got smears or poly-banding, you've got just the point.
Solution: New round of RACE analysis.
2. Optimistic: Sometimes 3' UTR is very long (up to 8 kb from the Stop-codon to the poly-A tail) and revertase can't reach 5' UTR during cDNA synthesis.
Solution: Try the cDNA synthesis with primer from 3' UTR... NOT with oligoDt.
Good luck.
Thanks Alexander for your answer. First thing is that I have not found any literature regarding the gene of my interest stating that it can remain in duplicated state in the genome though the gene was studied in bacteria and in one fungus only and in my case its the fungus and I have no good references regarding that. Can it happen? I mean in bacteria its a copy of the full gene, and in fungus it got divided? Another thing I would like to tell you that, I have sequenced the gene in 3 parts. 3' RACE, 5' RACE and the middle part with known primers by walking. There are good amounts of overlaps in the 3 parts. So, I think, the duplicated thing is not there. What do you think?
And 2ndly the 3' UTR is only apprx. 200 bp long.
//And 2ndly the 3' UTR is only apprx. 200 bp long.//
It's OK - the second option is ruled out.
Now, you need try to amplificate the gene from the genomic DNA in order to test your primers.
Ok, but is that possible to remain in duplicated form? As I have sequenced them in 3 parts with overlaps and by walking as I have already said. So, what may be the problems? I have 6 primers, Its very strange that all of them are not working? As I have designed them from known parts of the sequence.
I'm not an expert in fungi genomes, Prabuddha.
You may search your gene in here: http://genome.jgi-psf.org/pages/blast.jsf?db=fungi
Perhaps it may give some ideas on gene multiplication in this group.
Any thing is possible, from outdated cDNA synthesis kit... to the problem of sequence missinterpretation.
In your case, I see two most common potential problems: wrong primers and wrong cDNA synthesis kit. The first looks as easiest to be tested. Try your primers in DNA PCR - just add a very small piece of your fungi colony (touch with a tip) directly to the master mix and do PCR as usual. You'll get an answer already in two hours.
Sorry I am not an expert in cDNA synthesis. However if you want the full length with overlapping fragments, mix them both add your 5' and 3' primers and start a PCR (dNTPs, enzyme ... as usual). You can also try few cycles (5 to 10) with your 2 overlapping fragments then add your 5' and 3' primers and continue PCR. For site-directed mutagenesis that was working for me.
Thanks for your replies. May be these suggestions will help me a lot. Very good ideas. Will try and get back.
Dear Prabuddha,
With all due respect did you try out using some other reverse transcriptase enzyme like may be Superscript III etc. If yes!
What was the RTase enzyme you had used?
Also if you have the sequence the ORF region I request you to check the GC content somehow. High GC content is another big reason for irregular RT results.
With regards,
Gaurab Banerjee
Biogene India
I was using RT from fermentas and from finnzymes,robust I.Yes,I have a GC content issue towards the 5' end.I thought of that and used Robust.but there still was problems.so will try to do what
Dear Prabuddha,
I do not know how much the GC rich part is creating problems for you but you can always try out with a better RTase enzyme for such experiment.
A modified MMLV has the benefit of Temperature stability and avoiding problems like this.
I would like to suggest you the following:
Superscript III is a highly recommended RT enzyme from Invitrogen. Also ThermoScript RT as it can stay stable at 65°C.
For Fermentas REVERTAID First Strand cDNA synthesis kit (According to protocol)
If the RNA template is GC-rich or contains secondary structures, mix gently, centrifuge briefly and incubate at 65°C for 5 min. Chill on ice, spin down and place the vial back on ice.
Please use betaine in the PCR reaction buffer. It will be helpful getting better amplification.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC146979/pdf/253957.pdf
With regards,
Gaurab Banerjee
Also Try to use Thermus thermophilus DNA Polymerase. It is also a good solution at times.
http://www.promega.com/products/pcr/rt-pcr/tth-dna-polymerase/
MasterAmp™ Tth DNA Polymerase
Epicentre Biotechnologies
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