Cloning PCR products by addition of restriction sites to their termini

목차

1. Introduction of restriction sites
2. MATERIALS
3. METHOD
4. REFERENCES

본문내용

● Introduction of restriction sites

It is possible to introduce restriction site sequences into PCR products by having these sequences incorporated into the 5' end of the PCR primer(s).

◦ The short restriction site sequence on the 5' end of the PCR primer will not hybridize, but as long as the 3' hybridizing region is long enough (i.e. its Tm is high enough; ~20mer; Tm 값은 binding하는 부분에 한해서 결정) the primer will specifically bind to the appropriate site.
◦ The PCR product will thus have an additional DNA sequence at the 5' end which will contain the endonuclease restriction site.
◦ A similar or different restriction site sequence can be added via the other PCR primer.
◦ If the other primer has a different restriction sequence then the PCR fragment can be inserted in a directional dependent manner in a host plasmid.

참고자료

· Sambrook and Russell. 2001. Molecular cloning (the 3rd edition). CSHL press. vol.2, pp8.37 - 8.41
· http://wine1.sb.fsu.edu/bch5425/lect24/lect24.htm
· http://www.medigenomix.de/molbio6techinfo.html