Biology Reference
In-Depth Information
STEP 3: DNA AMPLIFICATION
“Amplification” refers to the process of making many, many copies of a sequence of DNA.
Currently multiple amplification methods exist, including
cell cloning
where host cells are
manipulated to carry and replicate a foreign DNA sequence.
One particular method of DNA amplification has proved very important: polymerase
chain reaction, or
PCR
(
Figure 16.5
). This method is used to target limited sections of
DNA. The method relies on temperature cycling, consisting of cycles of repeated heating
and cooling of the reaction in order to “melt” the DNA (releasing of hydrogen bonds), and
replication of the DNA through the help of special biomolecules. Primers (short DNA frag-
ments) containing sequences complementary to the target region along with a DNA
poly-
merase
are key components to enable selective and repeated amplification. A DNA
polymerase is a special kind of protein molecule, called an enzyme, which works to add
complementary bases to a single-stranded DNA chain. As PCR progresses, the DNA gener-
ated is itself used as a template for replication, setting in motion a chain reaction in which the
DNA is exponentially amplified. So, in the first PCR cycle, a single target double-stranded
DNA molecule will be copied into two such molecules. In the second PCR cycle, those two
molecules will both be copied, making four copies, and the next cycle will produce eight,
then 16, then 32, and so on. The more cycles, the more copies. The reaction ultimately stops
when the chemicals in the reaction are used up.
PCR was developed in the early 1980s and was the first accessible form of DNA amplifi-
cation. Its development was responsible for the launching of the “molecular revolution” of
the late 1980s
e
1990s mentioned earlier. The method was liberating in that it allowed
researchers to easily target and analyze DNA from both living and long-dead organisms.
It does have some drawbacks, however. For example, the method potentially amplifies any
and all DNA in the extract. The method also requires you to target one or a few genomic
regions through the use of specific primers. Lastly, PCR tends to favor undamaged DNA,
which can be a problem if the DNA that you are hoping to target is damaged, as would be
the case with DNA from ancient or forensic contexts.
To give you an idea of what PCR can do, imagine that that you are interested in amplifying
DNA from a Neandertal specimen. At the end of any of your PCR runs, you will likely end up
with a mix of DNA from the Neandertal specimen (the endogenous DNA), plus that of any
excavators and museum curators (exogenous, or “contaminating” DNA) who may have
touched the samples or even shared the same space as the samples. Additionally, though
you will have thousands of copies of that DNA mixture, it will consist of short representative
sequences of the Neandertal
e
modern human mixture, and not the entirety of DNA that is
available, because classical PCR forces you to target select regions of the genome.
DNA amplification is a necessary step for providing enough DNA for further downstream
applications. After amplification, researchers can choose among various data-generating
options; a common next step is to sequence the amplification products.
STEP 4: DNA SEQUENCING
DNA sequencing involves several technologies used to determine the order of the
nucleotide bases in a molecule of DNA. Often we view sequencing results in the form of
a sequence file (
Figure 16.6
). DNA sequencers tend to be located in core laboratories with
dedicated staff.
