PCR is a cell-free method of DNA cloning. It is much faster and more sensitive than cell-based cloning.
Figure 9-E-1. Polymerase Chain Reaction (PCR). Primers are in green color.
- Two primers, each about 20 bases long with sequence complementary to the sequence immediately adjacent to the DNA segment of interest.
- DNA polymerase (e.g., Tag polymerase) which can sustain high temperature (> 60o C)
- A large number of free deoxynucleotides (dNTPs)
- The target DNA fragment.
- Heat denaturation at about 95oC.
- Primers bind to the denatured DNA by base pairing as the temperature is gradually cooled to about 60o C.
- Extend primers with Tag polymerase.
- Repeat the above process. The number of copies doubles in each cycle. Typically 20 to 30 cycles are sufficient for effective DNA amplification.
- Much faster than using vectors.
- Only very small amount of target DNA is needed.
- To synthesize primers, we need to know the sequence flanking the DNA segment of interest.
- Applies only to short DNA fragments, typically less than 5 kb.