My idea is comparing the S gene (>4kb) of the new strain of PED virus with the novel strain (sequence already available on GeneBank). Most of the papers I have reviewed were cloning the S gene to a plasmid. I wonder can we just send directly the purified PCR product to the company? Because right now my lab does not have a suitable vector and also considering the time and cost for cloning.
Thank you for your help.
The Sanger sequencing provider we use is able to sequence amplicons for us. The typical read length is about 500-700nt, so if you provided both primers used to create the amplicon, you would be able to sequence about ~500nt at either side of the amplicon with missing information in the middle. If you need the sequence of the whole amplicon, this can be done by sequencing it repeatedly, using primers spaced every ~500nt along the length of the amplicon. If you're interested in doing that, you should ask your sequencing provider for recommendations about primer design and spacing.
Hi there,
Have a look at the dia 9 of the following presentation:
https://www.slideshare.net/faridmusa1/introduction-to-next-generation-sequencing
Yes you can send the purified pcr product for sequencing to the company as you mentioned.
If you are looking to clone a full gene fragment, cloning it into the plasmid is a better option. While you can get good sequence reads from purified PCR product, you can only get about 500-700 bp reads as a previous person mentioned - this isn't the limitation of sequencing, it is the limitation of pcr. Unless you are using a high fidelity taq polymerase designed for long range amplifcation (i.e. Takara PrimeStar GXL) you won't be able to amplify fragments bigger than ~1,000 bp. The only way to get full sequence coverage of a gene would be to make sets of primers that amplify overlapping regions, so you could get sequencing data of 500-700 bp fragments and then use snapgene or similar software to assemble these fragments. This method would require you already having a rough sequence though, which I am assuming you do not have? Molecular cloning honestly isn't a super time intensive procedure, and if you can find a lab at your institute that has a plasmid they are willing to share, you may be able to find a suitable vector for your application. Cloning also reduces the "unreadable" ends of the sequence that you find in PCR amplicon sequencing, which is a bonus.
Thu Thi Nguyen Yes, you can do that. I have done that before. I sequenced from both ends of the DNA. You might need more sequencing primers to cover the whole >4k DNA stretch.
As the others have pointed out, yes you can sequence directly from PCR product without problem. We have done it frequently. However it is very important that your PCR product is purified away from the PCR primers otherwise you are likely to get background signal from any primers still present.
Yes, you can directly sequence from a PCR product. As others have said, you'll need to do some sort of cleanup step to remove excess dTNPs and primers. There are several options (PCR cleanup kit, gel purification, etc.). If you have one bright band, I'd use Exo-SAPit reagent. It's the cheapest & easiest cleanup method.
Yes, and I agree with the previous recommendation, but if you want to have a good sequencing result you need to do clean up or use the purified product .
Thu Thi Nguyen One advantage of cloning the PCR product is that you get the 5' and 3' end of the amplicon sequenced properly as cloning allows you to use a plasmid specific primer that flanks the GOI (in your case S gene). Sanger sequencing has a limitation that the nucleotides present just after the primer binding site (around 30 to 70 bp) is not of good quality hence, we cannot trust that sequence. To circumvent this problem without the cloning would be designing overlapping primers in both forward and reverse direction to get the 5' and 3' end of the amplicon properly sequenced. Thus, to answer your question, yes you can use purified PCR product and have to design more overlapping primers to get the sequence done properly. Later, CAP3 is an online program that you may want to use to get the full 4kb sequence by combining different overlapping amplicons.
Thank you for all your answers. The detail explaination in those did helped me a lot.
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