I have recently started cloning a gene of 2.6Kb in TOPO-Zero blunt vector. I amplified my gene with KOD FX Neo and got the correct size band, then proceeded to clone into TOPO blunt vector. After transformation with DH5 E.coli, I got a few colonies and did colony PCR with gene specific primers (since I am in need of CDS) and I could get 3 positive colonies with expected 2.6Kb size. However, when I send them for sequencing, my sequencing results show something totally different. There is an insert but Not my gene of interest. I have checked the specificity of my primers using BLAST, and they can only anneal to my gene of interest. What could be happening? How is it possible to get positive results in all the steps and sequencing gives a strange sequence? Considering my primers are very specific to only this gene of interest? Your suggestions will be welcome.
As your amplimer is high GC it may be capable of forming secondary structure loops where 2 gc regions anneal and sometimes the sequencing reaction reads across the neck of loop to give 2 sequences on top of each other....both a long proper sequence and the shorter looped out sequence. You may need to sequence in presence of 4-6%DMSO or 1Molar betaine. If you are getting your sequencing done commercially then make sure that you tell the company that they are high GC sequences
what sequencing primer that you used? from the result there is a probability that there is something wrong with the primer. if you are confident that there is nothing wrong with the cloning process. You could also do a restriction using a restriction enzyme and compare between empty vector and the one with insert for another confirmation methods.
Thank you Fiona,
the sequencing is not very good. The real peaks are at intensity about 250 and there is another sequence showing under the strong sequence at intensity of 100plus in many areas. You may need to clean up your pcr with increasing annealing temperature, dmso or better primers but try to get a cleaner amplimer. The sequence will BLAST badly because of the additional peaks from the high background. I deleted the first 100 bases and much of the other end of the sequence and read some of the middle of the sequence by eye to get rid of some of the additional peaks and the sequence BLASTs as either zea mays or sorghum on a similarity BLAST not a complete homology Blast. If this is what you wanted then just clean up your pcr method but if it is not what you wanted you need to look closely at your primers that are amplifying your GOI and make sure you are amplifying the correct sequence possibly by sequencing the pcr product before inserting into the vector
Wahyu Handoko I also checked with restriction and it shows that I have a fragment of similar size to my GOI but its not actually my GOI. I have also tried making new primers but since I want to clone the whole coding sequence, I am limited in how I can design my primers, I guess?
Paul Rutland Oh thank you, I will try to clean up my PCR more, and sequence the product before cloning. Yes, I am working with sorghum and it has high GC content. I am not sure if this can make my primers anneal to the wrong place? Even though the primers are only specific to my GOI..
I agree with you since its for cloning your primer design is limited.. then i guess it could be because of your sequencing primer or your sample purity. For your sequencing primer you design it by yourself?
Wahyu Handoko yes I use the same primers used for gene amplification, then I design other primers within the gene by myself. Then I check them with Genetyx for properties like self annealing and formation of secondary structures and they seem alright...
i see... what is the primer sequence of your sequencing primer? usually for sequencing primer design i use this parameter from this site https://dnacore.mgh.harvard.edu/new-cgi-bin/site/pages/sequencing_pages/primer_design.jsp
its never fail me during sequencing.
- Primer length should be in the range of 18 and 24 bases.
- The primer should have a GC content of about 45-55%.
- The primers should have a GC-lock (or GC "clamp") on the 3' end (i.e. the last 1 or 2 nucleotides should be a G or C residue).
- The primer should have a melting temperature (Tm) greater than 50°C but less than 65°C.
- The primer should not include homopolymeric runs of more than 4-5 nucleotides.
- Avoid primers with secondary structures or the potential to self-hybridize.
- Avoid designing primer upstream of homopolymeric or heteropolymeric regions (A, C, G or T repeats).
- Check primer for specificity in annealing to template (= lack of secondary priming site).
- Primer should be located at least 50-60 bases upstream of your sequence of interest.
As your amplimer is high GC it may be capable of forming secondary structure loops where 2 gc regions anneal and sometimes the sequencing reaction reads across the neck of loop to give 2 sequences on top of each other....both a long proper sequence and the shorter looped out sequence. You may need to sequence in presence of 4-6%DMSO or 1Molar betaine. If you are getting your sequencing done commercially then make sure that you tell the company that they are high GC sequences
Wahyu Handoko thank you very much. I think I will try to redesign my primers following these guidelines. Paul Rutland thank you very much too. I understand it and I will give it a more informed shot now. Wish me luck!
good luck to you Fiona and please let us know how it works out for you
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