Real-Time PCR: Current Technology and Applications

Dominique McCoy

4/25/14

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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