Download - Dominique McCoy PCR paper
The field of molecular biology has been revolutionized by the development of
polymerase chain reaction. Polymerase chain reaction has been and still is used for a
plethora of applications. These applications involve both novel procedures and
modifications from existing methods [Griffin 1994].Polymerase Chain Reaction was
developed by Kary Mullis in the 1980s. It is based on using the ability of DNA
polymerase to synthesize new strands of DNA complementary to the offered template
strand [Griffin 1994].
The process of the polymerase chain reaction process is quite complex. The PCR
process requires five crucial components. 1) DNA template-is the sample DNA that
contains the target sequence. Initially, high temperature is applied to the original strand to
separate the strands. 2) DNA polymerase- is an enzyme that catalyzes template-
dependent synthesis of DNA from its deoxyribonucleoside 5’-triphosphate precursors.
There are two important types of precursors. A) Taq DNA polymerase is the most
commonly used, and B) Pfu DNA polymerase is widely used because of its higher
fidelity when copying DNA. Pfu DNA polymerase has two actions. Pfu DNA polymerase
generates new strands of DNA using DNA templates and primers; and it helps assist in
heat resistance. Therefore, both types of precursors are suitable for polymerase chain
reaction. 3) Nucleotides (dNTPs) are base units of A, T, C, and G are the building blocks
for new DNA strands. 4) RT-PCR or,reverse transcription polymerase chain reaction, is a
sample of RNA into codon cDNA with enzyme reverse transcriptase[Espy 2006].
How does the polymerase chain reaction work with its necessary components?
First, denaturation takes place. Denaturation is the initial process where the original
strands of DNA separate with a heat shock of 94-96°C which results in two separate
strands of DNA. Second, Annealing takes place where primer binding of both the
forward and reverse primers bind to the end of the DNA strands. Then, DNA extension
occurs. Extension leads to the synthesis of new DNA. This is known as the first cycle.
The last step is exponential amplification where the cycles keep continuing. The second
cyle equals 8 total, and so forth until the 35th cycle.
Figure 1. Polymerase Chain Reaction Process
Real time polymerase chain reaction consists of real time probe technologies for
the different applications. There are three types of nucleic acid detection methods such as
1) the 5’ nuclease (Taqman probe). The 5’nuclease activity of Taq polymerase can be
used to detect amplification of the target specific product because of the cleavage of the
probe in polymerase chain reaction [Heid 1996]. This is prevalent in the Taqman Real
Time Quantitive Reverse Transcription Polymerase Chain Reaction which allows for
reliable detection and measurement of products generated during every cycle of PCR [].
Figure 2. Taq Polymerase
The second type of nucleic acid detection used in polymerase chain reaction
applications is a molecular beacon. Molecular beacons do not cleave at 5’nucleases like
Taq polymerases do. This probe has a fluorescent dye on the 5’ end, and a quencher on
the 3’end. A molecular beacon forms a hairpin structure. It is used mainly to detect
polymerase chain reaction amplifications product. The selection of appropriate PCR
temperature and or extension of probe length will bind to the target PCR product when an
unknown nucleotide polymorphism is present [Heid 2006].
Figure 3. Molecular Beacon
Finally, the third type of nucleic acid detection used in polymerase chain reaction
applications are FRET hybridization probes. These probes are also known as Light
Cycler Probes. They are designed to anneal next to each other in a head-to-tail
configuration on the PCR product upstream has a fluorescent dye on the 3’end, and the
downstream has an acceptor dye on the 5’end [Espy 2006].
Figure 4. FRET hybridization probe
The applications used by these nucleic acid detection methods are numerous. The
first application is PCR in prenatal diagnosis. Over the years, many experts turned to
conventional methods in prenatal diagnosis of fetal chromosomal abnormalities. It is
commonly determined by cultured aminocytes, chronic villi, or fetal blood. However,
using quantitative fluorescent polymerase chain reaction (QT-PCR) is a newer technique
that detects common aneuploidies that has been reported by a number of investigators. It
also has an advantage of providing rapid results for the diagnosis.
This guideline promotes the use of a rapid aneuploidy DNA test for women at increased
risk of having a pregnancy affected by a common aneuploidy. This will have the benefit
of providing rapid and accurate results to women at increased risk of fetal Down
syndrome, trisomy 13, trisomy 18, sex chromosome aneuploidy or triploidy. It will also
promote better use of laboratory resources and reduce the cost of prenatal diagnosis.
However, a small percentage of pregnancies with a potentially clinically significant
chromosomal abnormality will remain undetected by QF-PCR but detectable by
conventional cytogenetics. Recommendations 1. QF-PCR is a reliable method to detect
trisomies and should replace conventional cytogenetic analysis whenever prenatal testing
is performed solely because of an increased risk of aneuploidy in chromosomes 13, 18,
21, X or Y[Lakshmi 2011].
Another application used by polymerase chain reaction is PCR detecting viruses.
One main virus that is being used by PCR is HIV. Nucleic acid tests that detect HIV
infection at an early phase are available and have been applied on individual dried blood
spot (DBS). The present study was undertaken with an aim to evaluate the feasibility of
performing PCR for HIV-1 DNA on pools of DBS as an alternative to individual testing
[Lakshmi 2011].
With the absence of availability of any publications on HIV PCR on pooled DBS,
there has been a need of standardization and evaluation of the feasibility of performing
PCR for HIV-1 DNA from pooled DBS. The present study was undertaken with an aim
to evaluate the feasibility of performing PCR for HIV-1 DNA on pools of DBS as an
alternative to individual testing [Lakshmi 2011]. HIV-I and II and HTLV-I and II are
both looked at in presumptive cases and tuberculosis. Primers SK145 and SKCC1B
supplied in the commercial kit of Amlicor HIV-1 DNA test, version 1.5 are used to
amplify a 155-nucleotide sequence of the HIV-1 gag gene RT and downstream PCR
primer SKCC1B complementary to nucleotides 1485–1512 of HIV-1HXB2 is (5′-
TACTAGTAGTTCCTGCTATGTCACTTCC-3′) and upstream primer SK145 (5′-
AGTGGGGGGACATCAAGCAGCCATGCAAAT-3′) [Lakshmi 2011].
Quantitative real-time PCR can determine gene duplications or deletions.
Furthermore, melting curve analysis immediately after PCR can identify small mutations,
down to single base changes. These techniques are becoming easier and faster and can be
multiplexed. Real-time PCR methods are a favorable option for the analysis of cancer
markers [Bernard 2002].
Figure 5. PCR Cancer Therapy
A very important application of polymerase chain reaction is forensics.
Polymerase chain reaction can be viewed through gel electrophoresis of DNA samples.
This is usually done for blood stains because law enforcement has to figure out the source
of a crime. The expression of polymorphic proteins, and the possibility of detecting
change in the physical property of a protein.
It is usually insufficient and degraded DNA that is resolved by polymerase
chain reaction. However, this process decreases by one day when compared to the
Southern blot analysis. It is suitable as a substrate for amplification as long as the DNA
fragment present encompassing the two primer binding sites.
Both conventional cytogenetics and QF-PCR should be performed in all
cases of prenatal diagnosis referred for a fetal ultrasound abnormality (including an
increased nuchal translucency measurement > 3.5 mm) or a familial chromosomal
rearrangement. (II-2A) 3. Cytogenetic follow-up of QF-PCR findings of trisomy 13 and
21 is recommended to rule out inherited Robertsonian translocations. However, the
decision to set up a back-up culture for all cases that would allow for traditional
cytogenetic testing if indicated by additional clinical or laboratory information should be
made by each centre offering the testing according to the local clinical and laboratory
experience and resources. (III-A) 4. Other technologies for the rapid detection of
aneuploidy may replace QF-PCR if they offer a similar or improved performance for the
detection of trisomy 13, 18, 21, and sex chromosome aneuploidy. (III-A) [Langlois
2011].
Figure 6. Forensic Analysis of PCR
There is also no large amount of DNA for polymorphisms. It is possible for other
material concerns. There is also a risk of accidental contamination such as over
amplificated DNA. It can be presented as a false positive. As a consequence a false
positive results can be obtained which could lead to incorrect conclusions and in an
extreme case might exclude or include suspects from being involved in a crime. It is
Therefore; of extreme importance to take the necessary precautions in order to avoid
contamination. This is not only true for forensic laboratories but also for clinical settings
where PCR is used as a routine diagnostic tool. The carryover of PCR products can be
prevented by physical separation of the areas for DNA extraction, setting up of the
amplification reactions, and analysis of the PCR products [Descorte 1993].
The application of PCR to ancient DNA (aDNA) experiments often requires
significant modification to standard protocols. The degraded nature of most aDNA
fragments requires targeting shorter fragments, performing replicate amplifications,
incorporating multiple negative controls, combating PCR inhibition, using specific DNA
polymerases to deal with damaged bases, working in a separate aDNA facility, and
modifying the PCR recipe to deal with damaged and low copy-number target DNA
[Fulton 2012].
DNA can also be copied in a process called DNA copying. DNA cloning by PCR
can be performed in a few hours, using relatively unsophisticated equipment. Typically, a
PCR reaction consists of 30 cycles containing a denaturation, synthesis and reannealing
step, with an individual cycle typically taking 3–5 min in an automated thermal cycler.
This compares favorably with the time required for cell-based DNA cloning, which may
take weeks. Clearly, sometime is also required for designing and synthesizing
oligonucleotide primers, but this has been simplified by the availability of computer
software for primer design and rapid commercial synthesis of custom oligonucleotides.
Once the conditions for a reaction have been tested, the reaction can then be repeated
simply [Stratchan 1999].
In conclusion, the entire process of polymerase chain reaction seems to be simple
enough in terms of figures, but when completely explained it is quite complex. The basic
way to explain PCR is taking a sample of DNA, adding DNA polymerase to amplify it
and make the DNA sample larger. PCR contains many different elements such as
important probes that aid in the process especially during the many applications. There
are numerous applications of PCR such as ancient DNA, genetic cloning, prenatal
diagnosis, forensics, and even identifying viruses and important ones such as HIV. PCR
is more than just a biochemical process, it is constantly revolutionizing the biological
world one application at a time.
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