We have a PCR assay whose product is later sent for simple DNA sequencing. Because we were restricted with our primer design (high A-T content) we were only able to design 1 set to use for both PCR amplification and sequencing.
When reviewing all our sequence data we notice that the chromatograms rarely capture the beginning of our region of interest. Is there a way of ensuring that our primers capture this? Someone briefly mentioned "adding extra tail on PCR primers", but I'm not sure what that means.
I should also mention that increasing concentration of our sample is not really an option. We are working with really small amount.
Adding an extra tail usually means adding an m13 sequence or sp6 or t3 sequence to the 5'endof your specific primers then you can always sequence any amplifier with sp6 or t7 antisense sequencing primers but that will only give you about 20 more bases at each end. Sequencing with the reverse primer should give you sequence close to the forward primer and vise versa. If no more sequence exists to design better primers sequencing after cloning would give more sequence
Thank you, Paul!
Follow-up question:
1- what are m13, sp6, and t3 sequences? Are these commercially available primers that I add to my master mix or do I need to physically add the extra sequences to the primer sequence of both F and R?
2- If I add these tails to my primers, will it be used in both amplification and sequence steps, or will it only be needed for one or the other?
T3might be added to the 5'end of one primer and sp6 to the 5'end of the reverse primer. Only the specific sequence will be used to calculate tm so the primers will be 34 bases not just 20 bases then after pcr the whole primer sequence is incorporated into the amplifier which can now be sequenced with short t3or sp6 primers. The advantage is that all of your amplified sequences can be sequenced with just 2 sequencing primers. The disadvantage is that all of your pcr primers will be about 35bases long so more expensive
I totally agree with Paul. However, one simple way is that you may extend your primers 20-50bp before and after the area of your interest.
1- Is there a preference over using M13, sp6, or T3? I did a quick search and cloning vectors seem to be used with T3 and sp6, (but I'm not cloning). Is that correct?
2- To be clear, I cannot use, for example, M13 forward sequence tailed to my f primer and a M13 reverse tailed to my reverse primer? It has to be M13 tailed in 1, and T3 or sp6 on the other primer?
It does have to be a different sequence at each end. The exact sequence does not matter so long as it does not form hairpins etc with your sequence. How long is your pcr product and where is the region of interest in relation to either end of the sequence please.if the amplifier is very long and the region of interest is near one end you can design a sequencing primer in the middle of the sequence pointing in the direction of your ROI and sequencing should be fine
I have a few assays I would like to apply this method to and they vary in size. A couple of them are about 400bp. I couldn't design separate PCR and sequencing primers because the size of the gene for 2 of them isn't that long, it's rich in A-T, and I had to avoid SNP sites.
I do have 2 separate primer sets (1 for PCR and the other for sequencing) for another assay, and that one is about 900bp long.
I agree with Paul. However, I wish to mention that adding standard sequences to the primers will still not be able to give the complete terminal sequences. I strongly recommend that the amplicons be cloned in a T/U-A PCR cloning vector, followed by sequencing using the standard primers that are integrated in the vector sequence. This will give complete sequence of the inserted amplicon, and the quality of the sequence will also be better than amplicon sequencing.
best wishes,
SD
@Paul- forgot to answer your other question: My PCR primers land right before the region of interest. Not much room.
@Sibnarayan- I don't have experience cloning, but someone told me that because I have hundreds of samples, mostl from different sources, cloning might not be a good solution. I'm not sure how true that is, again, since I don't know what it entails to clone.
I should add that I am learning a lot thanks to all of you who have responded!
The 400base fragments should sequence well from either end depending on where your ROI is located and if your ROI is at 350bases just sequence from the longer end and if your ROI is ,say, at 850 bases from one end of the long amplifier then a sequencing primer at about 600 bases pointing towards your ROI should give you a strong sequencing signal right up to the end of your amplimer
Since you say you have a small amount of starting material, clone your PCR product into a plasmid and sequence using primers that anneal to the plasmid DNA and flank your region of interest. Cloning means you won't have to worry about running out of material to sequence.
Good luck!
why not to perform sequencing with both forward and reverse primer, initial region of both sequencing reads is messy but later region is usually clear, at least upto 600-700bp. so from the end of one primer sequencing u can easily get clear read for initial region of other primer
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