TASK 1: DNA Extraction
a. Using sodium dodecyl sulfate, a detergent: Sodium dodecyl
sulfate (SDS) is used to break open the bacterial cells by
disrupting their cell membranes. This allows for the release of
the genomic DNA.
b. Adding RNase A and Proteinase K during extraction: RNase A is
added to degrade any RNA present in the sample, ensuring that
only DNA is isolated. Proteinase K helps in digesting proteins
present in the sample, which helps in removal of proteins that
could interfere with downstream applications.
c. Adding ethanol before recovering the DNA extract: Adding
ethanol helps in precipitating the DNA, causing it to come out of
solution. This step allows for the separation and recovery of the
DNA from the rest of the cellular components.
TASK 2: Polymerase Chain Reaction
a. The DNA polymerase isolated from the thermophilic bacterium
Thermus aquaticus is called Taq polymerase, which is an
abbreviation of the bacterium's name. The bacterium Thermus
aquaticus is found in hot springs and hydrothermal vents. Alice
Chien and other Chinese scientists isolated the Taq polymerase
in 1976. Taq polymerase is a thermostable enzyme that can
withstand high temperatures without losing activity. It can be
heated to 95°C repeatedly without losing much activity. Taq
polymerase is often used in the polymerase chain reaction
(PCR) to amplify short DNA segments. It automates the
repetitive step of amplifying specific DNA sequences. Taq
polymerase has many applications, including: quantifying fungal
growth; conventional reverse transcriptase (RT)-PCR; simple
sequence repeats (SSR) genotyping; amplifying genomic and
mitochondrial DNA; direct tetra-primer amplification refractory
mutation system (T-ARMS) PCR.
b. Deoxynucleotide triphosphates (dNTPs) are the fundamental
building blocks of DNA and are essential for many biological
processes: dNTPs are the precursors for DNA replication and
repair, and are incorporated into the growing DNA strand by
DNA polymerase enzymes. dNTPs are involved in ATP synthesis
and serve as energy carriers in biochemical reactions. dNTPs
are essential for DNA repair mechanisms, enabling the
correction of errors and damage in the DNA sequence. dNTPs
are necessary components of PCR mixes, and are used during
the extension phase of PCR to provide nucleotide building
blocks to the unzipped template strand of the DNA. The four
individual deoxynucleotides that make up a DNA sequence
are: Deoxyadenosine triphosphate (dATP), Deoxythymidine
triphosphate (dTTP), Deoxycytosine triphosphate (dCTP), and
Deoxyguanosine triphosphate (dGTP).
c. Forward and reverse primers are two types of primers used in
the polymerase chain reaction (PCR) to amplify DNA sequences:
Forward primers - attach to the start codon of the template
DNA, or antisense strand, which runs in the 3′ to 5′
direction. Forward primers synthesize the upper strand of DNA
using the bottom strand as a template. Reverse primers -
attach to the stop codon of the complementary strand of DNA,
or sense strand. Reverse primers are also known as 3′ primers
because they occur at the 3′ end of the PCR product. Reverse
primers synthesize the lower strand of DNA using the upper
strand as a template. Both primers are necessary for
exponential amplification to occur. If only one primer is used,
only one strand of DNA would be amplified, resulting in only one
copy being produced per cycle. The names of the primers
depend on the direction of the strand being used for
amplification. However, protocols often refer to the primers as
"senso" and "antisenso" or "forward" and "reverse".
