DNA Replication

DNA Replication

postulate replication
Francis Crick proposed 3 models for how DNA might replicate. Credit: Jeremy Seto (CC-BY 3.0)

Meselson & Stahl Experiment

Meselson and Stahl
Meselson & Stahl revealed semi-conservative replication as the method through use of radiolabeling DNA in bacteria. Credit: Jeremy Seto (CC0)

 

Replication Machinery

DNA replication split

Proteins Involved in Replication

Polymerase Chain Reaction (PCR)

The¬†Polymerase¬†Chain Reaction¬†(PCR)¬†is a method of rapidly amplifying or copying a region of DNA in a¬†tube. As the name implies, the technique uses a thermostable DNA¬†Polymerase¬†enzyme to mimic in a tube what happens within a cell during DNA¬†replication. The chain reaction permits us to rapidly copy DNA from¬†very minute source material in an exponential way. This technique is¬†used in forensic science, genetic testing and cloning of rare genes.¬†Because of the exponential copying process, a stray cell left behind¬†can provide enough genetic material to make billions of copies of¬†this DNA. The process of PCR can be observed in an animation found at¬†Cold Spring Harbor Laboratory’s DNA Learning Center website
(http://www.dnalc.org/resources/3d/19-polymerase-chain-reaction.html).

 

primers & reverse complement
Primers are reverse complimentary to one of the 2 strands of DNA. They flank the area of interest and become incorporated into the replicated product. Credit: Jeremy Seto (CC-BY 3.0)

As with any DNA replication process, one needs to start off with a template.The template is the source material that is meant for duplication. In this process, scientists are not interested in copying the entirety of the genome, just a small segment of interest. DNA polymerases require primers to begin the polymerization process. Primers are designed as small oligonucleotide segments that flank the area of interest (interactive for designing primers) . These are short strands of DNA that reverse complement to the DNA area of interest so that the DNA polymerase has a starting point and is guided only to the DNA segment of interest. These primers tend to be about 18-24 bases long.

Polymerase chain reaction

However, a double stranded¬†DNA molecule is already base paired together into a double helix so¬†our primers can not interact. The first step of PCR is to separate¬†the double-stranded DNA molecule by denaturing¬†the H-bonds using high heat (95¬įC). The primer concentrations are¬†much higher than the original template. The next step of PCR is¬†called annealing.¬†During this step, the temperature is reduced to a temperature of¬†about 55¬įC.¬†This temperature is still hot by our standards, but is necessary to¬†enhance the stringency of the correct base pairing of the primers to¬†their targets on the template. The DNA Polymerase used in this¬†process is derived from a bacteria that lives in very high¬†temperatures and does not denature as other proteins would under such¬†conditions (thermostable). The original enzyme was isolated from an¬†organism called Thermus¬†aquaticus,¬†so we call the enzyme Taq polymerase or just Taq¬†for¬†short. This bacteria lives in hot springs where the temperatures are¬†about 50¬įC¬†but it thrives at a range between 50-80¬įC.¬†The temperature is raised again to a higher temperature of 72¬įC¬†for the polymerase to extend¬†(also called elongation)¬†or¬†continue the polymerization step from the primer. ¬†


PCR is accomplished by cycling rapidly between these three steps: denature, anneal, extension. The rate limiting step is the extension which limits the length of DNA to be copied. If the original template is only a single copy, then after the completion of a cycle, we would have 2 copies. The subsequent cycle would have 4 copies, then 8, then 16, 32, and so on. The doubling process is exponential so from 1 copy undergoing 30 cycles; we would have 230 or 1,073,741,824 copies. This is over a billion copies in a few hours of time. 

exponential amplification
Exponential amplification by PCR. Credit: Jeremy Seto (CC-BY 3.0)

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