Say you have three fragments that have overlaps with each other. During PCR, I'm assuming that since these fragments could physically overlap with each other they will anneal. My question is will the fragments amplify, even without the help of primers? Can't the dntp just bind to the fragment ends next to where the overlap takes place? I would like an explanation of the mechanisms behind this - I know that primers are necessary for pcr - I just don't see why we can't see PCR product from overlapped fragments alone.
If you have overlapping fragments and denature then anneal the likelihood is that much of the reannealing will be to the complementary individual strand again but some will anneal to the overlap and the polymerase will extend ,as you say, from the 3' end to fill in the single strand and make it double stranded. The difference between this and pcr is that when you have overlapping fragments most of the fragments will be used up in the first cycle and no further amplification will take place. In pcr the primer is always in huge excess so when the product denatures there is always more primer to anneal to each strand and complete the next cycle of the pcr. Sometimes you do see concatenation of product in pcr when too many cycles or re-amplification of a product is over amplified because then the primer is not in large excess compared with the product and the product can self anneal and produce long concatemers.
If you have overlapping fragments and denature then anneal the likelihood is that much of the reannealing will be to the complementary individual strand again but some will anneal to the overlap and the polymerase will extend ,as you say, from the 3' end to fill in the single strand and make it double stranded. The difference between this and pcr is that when you have overlapping fragments most of the fragments will be used up in the first cycle and no further amplification will take place. In pcr the primer is always in huge excess so when the product denatures there is always more primer to anneal to each strand and complete the next cycle of the pcr. Sometimes you do see concatenation of product in pcr when too many cycles or re-amplification of a product is over amplified because then the primer is not in large excess compared with the product and the product can self anneal and produce long concatemers.
If you didn't see the PCR product, its because the low concentration. If you dont have primers, you could have, as maximum product quantity, the substrate quantity that you put in the PCR.
In that case i recomend you make a PCR without primers 5 cycles (to the assemby) and them add it (to amplify)
Other option is use gibson assambly reaction.
You need primers binding to the outside most fragments to drive the PCR reaction. Without primers, the mechanics of PCR become very complicated.
Primers are typically in far excess of the number of eventual PCR products that are produced efficiently by the polymerase. These primers bind very well and quickly to denatured ssDNA from amplicons. Without the excessive amount of primers, homology search for fragments of DNA takes quite a long time.
It is also likely that most of your DNA fragments bind to their reverse complement counter parts, in preference to the homology of other DNA pieces. The stability of an exact reverse compliment to the entire DNA fragment is much more stable than the binding to smaller region of homology needed to extend the DNA fusion product. It follows that it is hard or impossible to find an appropriate PCR cycle condition that selects for the fusion product formation instead of many cycles of annealing and denaturing without extension.
Using outside driving primers is the best way to produce such a fusion PCR product. It can be done without primers, but the yields of desired product will be very low typically.
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