the strands and the cycle repeated.

Each new strand then acts as a template for the next cycle

of synthesis. Thus amplification proceeds at an exponential

(logarithmic)rate,i.e. amountofDNAproduceddoubles at

each cycle. 30–35 cycles of amplification can yield around

1 µg DNA of 2000 bp length from 10–6 µg original template

DNA. This is a million-fold amplification.

Initially, the 3 different stages at 3 different temperatures were carried out in separate water baths but

nowadays, a thermal cycler is used (a machine that

automatically changes the temperature at the correct time

for each of the stages and can be programed to carry out a

set number of cycles).

A typical thermal cycle might be as follows:

Heat denaturation at 94oC for 20 seconds

Primer annealing at 55oC for 20 seconds

Primer extension at 72oC for 30 seconds

Total time for one cycle = approx. 4 minutes.

DNA is denatured

Primers attach to each strand. A new DNA strand is

synthesized behind primers on each template strand.

Another round

DNA is denatured, primers are attached, and the number

of DNA strands are doubled.

Continued rounds

Continued rounds of amplification swiftly produce large

numbers of identical fragments. Each fragment contains

the DNA region of interest.

Limitations/Difficulties

While a very powerful technique, PCR can also be very

tricky. The polymerase reaction is very sensitive to the

levels of divalent cations (especially Mg2+) and nucleotides,

and the conditions for each particular application must

be worked out. Primer design is extremely important

for effective amplification. The primers for the reaction

must be very specific for the template to be amplified.

Crossreactivity with non-target DNA sequences results

in nonspecific amplification of DNA. Also, the primers

must not be capable of annealing to themselves or each

other, as this will result in the very efficient amplification

of short nonsense DNAs. The reaction is limited in the size

of the DNAs to be amplified (i.e. the distance apart that the

primers can be placed). The most efficient amplification

is in the 300–1000 bp range, however, amplification

of products up to 4 Kb has been reported. Also, Taq

polymerase has been reported to make frequent mismatch

mistakes when incorporating new bases into a strand.

The most important consideration in PCR is

contamination. If the sample that is being tested has even

the smallest contamination with DNA from the target,

the reaction could amplify this DNA and report a falsely

positive identification. For example, if a technician in a

crime lab sets up a test reaction (with blood from the crime

scene) after setting up a positive control reaction (with

blood from the suspect) cross contamination between the

samples could result in an erroneous incrimination, even

if the technician changed pipette tips between samples. A

few blood cells could volatilize in the pipette, stick to the

plastic of the pipette, and then get ejected into the test

sample. The powerful amplification of PCR may be able to

detect this cross contamination of samples. Modern labs

590 Concise Book of Medical Laboratory Technology: Methods and Interpretations take account of this fact and devote tremendous effort to

avoiding this problem.

Types of PCR

RT-PCR

This is reverse transcriptase-PCR and is a two-stage

procedure used for the amplification of RNA. The first

stage employs an enzyme called reverse transcriptase,

which synthesises a DNA strand complementary to the

RNA of interest by using one of the PCR primer as its

primer. The complementary DNA is then used in the

second stage as the starting material for PCR amplification

by a conventional thermostable DNA polymerase.

Nested PCR

It is a PCR done in two steps, a primary PCR reaction and a

nested reaction.  The primary (or first) reaction uses a set of

primers to generate a product that serves as the template for

the nested (or second) reaction. The nested reaction uses a

set of PCR primers specific for a region within the amplified

product from the first reaction. Therefore, the nested

reaction often serves as a confirmation for the specificity of

the PCR products amplified in the primary reaction.

Real-Time PCR

Combines PCR amplification and detection into a single

step.  The basic principle of real-time quantitative PCR is

the detection of target sequences using a fluorogenic 5’

nuclease assay (often called ‘TaqMan’). The advantages

of this system include high reproducibility, the capability

of handling large numbers of samples, the potential for

quantitative results, and decreased turnaround time. The

disadvantages include high instrument cost and the

requirement for technical proficiency.

Multiplex PCR

It is a PCR designed to detect more than one target

sequence in a single PCR reaction. The assay uses two or

more sets of primers.  Each set of primers is specific for a

different target sequence.  The assay is most commonly

used for simultaneous detection of multiple viral genes

and differentiation of genotypes or subtypes of related

microorganisms.

Differential PCR

Differential PCR can sometimes be used to distinguish

closely related targets.  Differential PCR is done either in

a multiplex format using two or more sets of primers or by

running two separate PCR assays.

RIA

Radioimmunoassay (RIA) combines the high specificity of

an antigen-antibody reaction with the great sensitivity of

detection and quantification of compounds tagged with a

radioactive “label” atom.

If there is, in a solution, a mixture of three components,

i.e. a “natural”, or unlabeled, antigen, the same antigen

with one of its atoms carrying a radioactivity label,

and a quantity of antibody specific for the antigen that

is insufficient to bind all the unlabeled and labeled

antigen molecules present, the two forms of the antigen

will compete for the available binding sites. Thus, if the

number of labeled and unlabeled molecules is the same,

each type has an equal chance of finding a free binding

site, half the available antibody-binding sites will carry

labeled antigen and half will carry unlabeled antigen. If

the number of unlabeled antigen molecules is greater than

the number of labeled ones, a large number of antibodybinding sites will become occupied by unlabeled antigen

molecules. Thus, the larger the number of unlabeled

antigen molecules in the mixture, the smaller the

fraction of the original quantity of labeled antigen that

will become bound by antibody. Since the firmly bound

combination of antigen and antibody can be separated

from the remaining components of the original mixture

and its radioactivity determined and compared with that

of the original labeled antigen addition; and since the

relative amounts of bound and free labeled antigen will

depend upon the number of unlabeled antigen molecules originally present, a calibration curve can be made

by adding known amounts of unlabeled antigen to the

system of labeled antigen and antibody, separating

and determining the ratio of radioactivity of bound to

original labeled antigen, and plotting this ratio against the

known amounts of added unlabeled antigen. If a sample

containing an unknown amount of natural (unlabeled)

antigen is then mixed with the same amounts of labeled

antigen and antibody as in the calibration curve mixture,

the antigen-antibody complex separated and the ratio of

its radioactivity determined when compared with that of

the original amount of labeled antigen, this ratio, usually

expressed as a percentage, when referred to the calibration

curve, will give the amount of unlabeled (natural) antigen

in the sample.

The unique combination of specificity and sensitivity of

the RIA principle makes it particularly suitable for the assay

of substances such as insulin, growth hormone, thyroxine,

testosterone, progesterone, angiotensin, aldosterone, and

drugs such as digoxin in serum or plasma at the level of

nanogram per mL.

The procedures involved in RIA differ in the radioactive

element used as the label,