Polymerase Chain Reaction (PCR)

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

Procedure:

1.  Heat denaturation at about 95oC. 
2.  Primers bind to the denatured DNA by base pairing as the temperature is gradually cooled to about 60o 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.

 Site of Interest:

Animation of PCR - From Cold Spring Harbor Lab.

 Book Sections: 

Basic Features and Applications of PCR - From Human Molecular Genetics, 1999.

Review article: 

Real-Time PCR Technology for Cancer Diagnostics - Clinical Chemistry, 2002.

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