The document discusses several key steps in polymerase chain reaction (PCR) and DNA analysis: 1. PCR involves copying specific DNA sequences through cycles of heating and cooling DNA samples. This allows for amplification of target DNA regions marked by primers. 2. DNA extraction is used to isolate DNA from samples prior to PCR. Library preparation then ligates adaptors to DNA fragments to make them suitable for sequencing. 3. The techniques of PCR, DNA extraction, and library preparation are applied in a research laboratory studying international viral samples to amplify and analyze DNA from viruses like adenovirus, cytomegalovirus, HIV, and HPV. This helps compare patterns and susceptibilities between different viruses.

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The Extraction and Amplification of International Viral DNA
Polymerase Chain Reaction (PCR)
The Steps of the Polymerase Chain Reaction (PCR)
NucliSENS® easyMAG® Nucleic Acid Extraction
ACKNOWLEDGEMENTS / REFERENCES
Emily Shuangyue Cao
1. Separating the Target DNA – Denaturation
For the first step of PCR, which is acknowledged as denaturation, the tube
that contains the sample DNA is heated to a temperature that is greater than
90 degrees Celsius, or 194 degrees Fahrenheit. This allows for the separation
of double-stranded DNA into two separate strands. The condition has to be a
high temperature due to the fact that it breaks the relatively weak bonds
between the nucleotides from the DNA code.
2. Binding Primers to the DNA Sequence – Annealing
PCR is not supposed to copy all of the DNA in a sample; It is only supposed
to amplify a very specific sequence of the genetic code, targeted by the PCR
primers. For instance, a specific virus such as Adenovirus possesses a unique
pattern of nucleotides specific to that bacteria. The PCR will only copy that
specific DNA sequence present in Adenovirus and absent from other bacterial
species. In order to accomplish this task, PCR utilizes primers, or man-made
oligonucleotides (Short pieces of synthetic DNA) that bind, or anneal, only to
sequences on either side of the target DNA region.
Only two primers are utilized for this step, one for each of the newly
separated DNA strands. The primers bind to the beginning of the sequence
that will be copied, which thus marks the sequence for the subsequent step.
For step two, the tube is cooled and the primer binding occurs between 40
and 60 degrees Celsius, or 104 and 140 degrees Fahrenheit.
3. Making a Copy – Extension
The third and final phase increases the temperature to approximately 72
degrees, or 161.5 degrees Fahrenheit. Beginning at the regions marked by the
primers, nucleotides in the solution are added to the annealed primers by the
DNA polymerase, which then creates a new strand of DNA that is
complementary to each of the single template strands.
Once this step is complete, two identical copies of the original DNA should
be created.
Henry M. Gunn Senior High School
The polymerase chain reaction, PCR is an efficient and cost-effective way of
copying,
or amplifying small segments of DNA or RNA. PCR occurs outside of the body in a
laboratory, and it is based on the natural process of DNA replication. The reaction
occurs when a DNA sample and a DNA polymerase, nucleotides, primers, and other
reagents such as man-made chemical compounds are added to a sample tube. The
reagents facilitate the reaction needed to copy the DNA code.
PCR enables the monitoring of the amount of a virus present in a person’s body. In
diseases such as hepatitis C or human immunodeficiency virus (HIV) infections, the
viral load in a human body is an excellent indicator of how far the disease has
advanced and can also determine the effectiveness of an individual’s medicine.
Furthermore, PCR can help physicians determine when they can start an
individual’s personalized treatment.
There are approximately three steps in a PCR cycle, which approximately doubles
the
amount of target DNA. Prior to initiating PCR, DNA must be isolated from the
sample.
Viral DNA Library Preparation
Library preparation is essentially the ligation of specific adaptor oligos to
fragments of the DNA to be sequenced. The DNA is firstly fragmented to be the
optimal length determined by the downstream platform. Due to the fact that DNA
fragment does not result in a homogeneous pattern, end repair is necessitated in
order to ensure that each molecule is free from overhang and contains both 5’
phosphate in addition to 3’ hydroxyl groups.
Incorporation of a non-templated deoxyadenosine 5’-monoposphate (dAMP) onto
the 3’ end of blunted DNA fragments, a process otherwise acknowledged as dA-
tailing is necessary because it prevents concatamer formation during downstream
ligation. This also enables DNA fragments to be ligated to adaptors with
complementary dT-overhangs.
How this Contributes to the Research Project
My work at Dr. Pinsky’s laboratory at the Stanford University School of Medicine’s
Pathology Department is further accentuated by the primary techniques I have
learned: the polymerase chain reaction (PCR), DNA extraction, and the library
preparation:
1. The laboratory receives samples in plasma mediums from an international
context: countries such as Zimbabwe, Brazil, Kenya, and many others are
included in the experiments. First, we run these samples through the easyMAG
extraction machines by putting magnetic bases into them and then receiving a
nucleic acid eluate.
1. After the DNA extraction, we run them through a polymerase chain reaction with
RotorGene that exposes the samples to a variety of temperatures in order to
amplify and duplicate the original amount of DNA.
1. Following the polymerase chain reaction, we apply the DNA library preparation
in order to make the nucleic acid eluate ready for sequencing, which allows the
laboratory to analyze the patterns, distinctions, and susceptibilities of a specific
virus, such as ADV, CMV, HIV, or HPV in comparison to one another.