1. Polymerase
Chain
Reaction
(PCR)

2. I. Overview Two types of directed DNA Synthesis:
1. recombinant DNA vector cloning - using mRNA, and a bacteria or virus as a host
2. PCR - best for replicating/synthesizing large quantities of a particular gene - or when the source is a fragment or impure
a. DNA synthesis in vitro
b. designed as a way to produce enough copies of a specified segment of DNA to do meaningful analysis - given current technologies
c. highly specific target sequences
1. custom primers isolate specific regions
2. Compared to cellular DNA replication - some of the process is similar and some is different

3. II. Similarities to in vivo DNA replication
A. RNA Primers - molecular point of origin for DNA polymerase
B. DNA Polymerase - builds new segment
C. dNTP’s (deoxynucleoside triphosphates)
D. the system is buffered

4. III. Differences from in vivo DNA replication
A. No natural points of origin, replication forks, bubbles, or any of the related enzymes
B. Heat instead of Helicase is used to denature DNA
C. Cooling - allows primers to anneal to the DNA
D. Mg2+ is added in the form of MgCl2 to satisfy the cofactor needs of DNA Polymerase
E. A thermostable polymerase from the bacterium Thermus aquaticus a.k.a. Taq is used
F. Both strands are copied continuously
Thermal cycler - a machine where a single cycle involves ramping between 3 specific temperatures
1. multiple cycles are pre-programmed and managed by the thermal cycler

5. that will ultimately be analyzed via gel electrophoresis
IV. The Mechanics of PCR
A. Each Cycle involves 3 steps:
1. Heating to denature DNA
2. Cooling to allow primers to anneal
3. Heating to optimize polymerase activity
The 5´ ends of the primers are “tagged” with nucleotide sequences that define the target region
as a result, three cycles must be completed before two “tagged” complimentary 5´ to 3´ sequences of copied target DNA can anneal and form the short product
that will ultimately be analyzed via gel electrophoresis

6. 1st 3 cycles
Fig pg. 382

7. The End