A common method to clone PCR products involves digestion with restriction enzymes that recognize sites introduced into PCR primers. Its drawback is that the cleavage efficiency close to the ends of a DNA fragment is not well defined what makes appropriate digestion of a PCR product difficult.
This problem is overcome in an original and efficient method "Directional PCR Product Cloning using BpiI, Eco3I and Esp3I" by using one of the three well-characterized type IIS restriction enzymes to digest a PCR fragment leaving sticky ends of 4 bases that can be ligated with complementary DNA ends generated by more than 45 restriction enzymes.
All three restriction enzymes cut outside their recognition site leaving 4-base 5'-overhangs that are predetermined by a downstream sequence, i.e.:
BpiI (BbvII)
5'...G A A G A C N N^...3' 3'...C T T C T G N N N N N N^...5'5'...G G T C T C N^...3' 3'...C C A G A G N N N N N^...5'5'...C G T C T C N^...3' 3'...G C A G A G N N N N N^...5'As a result, only a desired PCR product with ends complementary to chosen restriction enzyme(s) can be directionally inserted into any suitably digested vector. The information about the activity of enzymes near the ends of PCR products is given here.
Features
Can be used with PCR products generated using any DNA polymerase.
- Efficient - >90% of the resulting clones contain the target DNA in correct orientation.
- PCR fragments can be cloned directionally.
- Selection from the three restriction enzymes increases the probability that at least one enzyme will have no targets within the PCR fragment of interest.
- Each restriction enzyme generates sticky ends which can be ligated with DNA fragments produced by any one of more than 45 restriction enzymes.
- Primers can be designed such that the vector site is either retained or destroyed when a PCR product is ligated into the vector.
PCR Product Cloning using Eco31I.
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Updated lapkričio 08, 2006 11:34
