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.

Materials required:

• 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 (> 60^o C)
• A large number of free deoxynucleotides (dNTPs)
• The target DNA fragment.

Procedure:

1. Heat denaturation at about 95^oC. 
2. Primers bind to the denatured DNA by base pairing as the temperature is gradually cooled to about 60^o C.
3. Extend primers with Tag polymerase.
4. Repeat the above process.  The number of copies doubles in each cycle.  Typically 20 to 30 cycles are sufficient for effective DNA amplification. 

Advantages:

• Much faster than using vectors.
• Only very small amount of target DNA is needed.

Disadvantages:

• 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.