Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
Does anyone know if it is posible to do a PCR amplification of a small fragment with only one primer (that anneals only at one site)?
Plus, ideas to separate two possible types of PCR products are more than welcome
Thanks a lot!
Well, you see, PCR is used to make an exponential amount of copies of a specific gene. Since genes usually have a complimentary bases in order to work, it would seem impossible to successfully do PCR with one primer on one strand of the DNA.
A way to seperate two types of products is the gel electrophoresis method (or it may be called "gel electrolysis").
On a side note, it may be very beneficial to you to know that my knowledge on the subject so far only extends to Grade 12 Academic Biology.
eheh I know what PCR and electrophoresis are!
Just asked because i could be missing some points and nowadays there are several types of PCR for different purposes.
about electrophoresis... I forgot to mention that I want to separate two products with exactly the same size...
You can amplify a gene with one primer, if you prime with a sequence located in invrted repeats urrounding your fragment of interest; It is not that frequent though. And in all other case you are going to produce little DNA , and of plenty of different sizes, so it is not going to be very useful.
As for separating fragments of identical sizes, but different sequences, it can be done using gradient gel electrophoresis (DGGE or TGGE) protocols can easily be googled.
Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
that's what I thought... PCR with one primer that anneals at only one site is generally used for sequencing, but not to amplify fragments.
I really didn't know those types of electrophoresis.
Thank you very much
Depends on what is a "small fragment". If it's a small fragment of a chromosome, it could still be quite large. If it's a PCR product or restriction fragment of reasonable size, you can do a PCR-like reaction with one primer to linearly (not exponentially) amplify one strand of the fragment. This is commonly done to, for example, incorporate a label into the amplified strand for detection (as in one method of SSCP).
i think that if you want to seperate two pcr products with exactly the same size the next step after electrophoresis is to do a digestion of the pcr products with a restriction enzyme that cut the two sequences to different sites (so different fragments) and then do electrophoresis of the uncuts and the cuts...so you will know what each product is ...(this of course you know it before you do your pcr) but this is a way to persuade someone else what each product is...and of course an other way if the size is not exactly the same but somes bp different is to use not an agarose gel but a polyacrylamide gel....
unfortunately the size is exactly the same and the sequence only differs in about 10 nucleotides (in a 1,8kb fragment) and there won't be new restriction sites.
well... I think I just found another way to do what I want, by linearly amplify one strand and purify it... then, after the PCR, I will only get one type of product (I haven't tried it yet, though )
Thanks for your help anyway!
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