The document explains the Polymerase Chain Reaction (PCR), a technique used to amplify specific DNA fragments from various sources, highlighting its steps: denaturation, annealing, and extension. It covers multiplex PCR for amplifying multiple targets simultaneously and reverse transcriptase PCR (RT-PCR) for RNA amplification, as well as quantitative PCR (qPCR) for real-time monitoring of the amplification process. The text also emphasizes the role of primer design and bioinformatics in ensuring specificity and efficiency in PCR applications.

1. Diagnostic Molecular biology
Lecture 3

2. 5. The Polymerase Chain Reaction (PCR)
• The PCR is used to amplify a precise fragment of DNA from a complex mixture of
starting material, usually termed the template, which may be DNA from
microbes, mouth swabs, blood, urine, tissue biopsy, etc.
• It requires the knowledge of some DNA sequence information flanking the
fragment of DNA to be amplified (target DNA).
• From this sequence information, two oligonucleotide primers are chemically
synthesised, each complementary to a stretch of DNA to the 3′ side of the target
DNA.
• The result is an amplification of a specific DNA fragment that obviates the need
for more time-consuming cloning procedures.
• In some respects, the PCR can be regarded as analogous to molecular cloning
since it results in the generation of new DNA molecules based exactly upon the
sequence of existing ones.

4. Mechanism and Steps in the PCR
• The PCR consists of three well-defined times and temperatures
termed steps:
• (i) denaturation at high temperature,
• (ii) annealing of primer and target DNA and
• (iii) extension in the presence of a thermostable DNA polymerase.

6. Multiplex PCR
It is possible to amplify more than one target sequence in a single reaction by including multiple
sets of primer pairs in a process termed multiplex PCR.
The main advantage of the technique is that it is not only cost-effective but also allows information
to be obtained from limited amounts of template. In general, there are two types of multiplex PCR,
one using a single template with a number of primer pairs and the other employing multiple
templates with primer pairs.
Multiplex PCR has been used effectively in many areas of nucleic acid analysis such as microbial
identification, mutation analysis and single nucleotide polymorphism (SNP) genotyping.
One main consideration of the technique is the design of the primers and, although the same basic
rules are followed as for singleplex PCR, the primers need to be specific and not interfere or form
dimers.
The annealing temperature is also a critical feature with multiple primer sets. Bioinformatic
assistance in the primer design process using software such as PrimerPlex is essential and is able
to design and optimise primer sets while minimizing potential mismatches to ensure high specificity
and specific
amplification take place.

7. Reverse Transcriptase PCR (RT-PCR)
RT-PCR is an extremely useful variation of the standard PCR that essentially allows the amplification
of RNA molecules, such as mRNA transcripts via a cDNA reaction, from very limited sample
amounts.14
This may be undertaken using a two-step process following RNA extraction or, in some cases, a
single-step process in which both reactions are performed in a single tube.
One advantage of the two-step process is the ability to archive the original sample for later analysis
and the removal of aliquots when required. In some cases, the need for the rigorous extraction
procedures associated with mRNA purification for conventional cloning purposes is obviated.13
For single-step PCR the dNTPs, buffer, Taq polymerase, oligonucleotide primers, reverse
transcriptase (RT) and RNA template are added together to the reaction tube.
The reaction mixture is heated to 37 °C, thus allowing the RT to work, and permits the production of
a cDNA copy of the RNA strands that anneal to one of the primers in the mixture.
Following ‘first strand synthesis’, a normal PCR is carried out to amplify the cDNA product, resulting
in ‘second strand synthesis’, and subsequently a dsDNA product is amplified as usual.

9. Quantitative or Real-time PCR (qPCR)
An important evolution of the PCR method is the development of quantitative PCR (qPCR).
This method has been gaining popularity for many applications because of the rapidity of the method
compared with conventional PCR amplification while simultaneously
providing a lower limit of detection and greater dynamic range.16
It is possible to track the reaction in real time and the method allows assessments of any problems with the
amplification process without the need for end-point analysis. Early qPCR methods involved the comparison
of a standard or control DNA template amplified with separate primers at the same time as the specific target
DNA.
These types of quantification rely on the reaction being exponential and so any factors that affect the
reaction may also affect the result. Other methods have involved the incorporation of a radiolabel through
the primers or nucleotides and their subsequent detection following purification of the amplicon.
In its simplest form, a DNA binding dye such as SYBR Green is included in the reaction. As amplicons
accumulate, SYBR Green binds the dsDNA proportionally. Fluorescence emission of the dye is detected
following excitation.
The binding of SYBR Green is non-specific but most qPCR thermal cycler machines will produce a melt
curve, which allows a degree of identity of the amplicon through its melting temperature profile