POLYMERASE CHAIN REACTION

Polymerase chain reaction (PCR) is a method widely used to rapidly make

millions to billions of copies of a specific DNA sample, allowing scientists
to take a very small sample of DNA and amplify it to a large enough amount
to study in detail. PCR was invented in 1984 by the American biochemist
Kary Mullis at Cetus Corporation. It is fundamental to much of genetic
testing including analysis of ancient samples of DNA and identification of
infectious agents. Using PCR, copies of very small amounts of DNA
sequences are exponentially amplified in a series of cycles of temperature
changes. PCR is now a common and often indispensable technique used in
medical laboratory and clinical laboratory research for a broad variety of
applications including biomedical research and criminal forensics
Almost all PCR applications employ a heat-stable DNA polymerase, such as
Taq polymerase, an enzyme originally isolated from the thermophilic
bacterium Thermus aquaticus. If the polymerase used was heat-susceptible,
it would denature under the high temperatures of the denaturation step.
Before the use of Taq polymerase, DNA polymerase had to be manually
added every cycle, which was a tedious and costly process
Applications of the technique include DNA cloning for sequencing, gene
cloning and manipulation, gene mutagenesis; construction of DNA-based
phylogenies, or functional analysis of genes; diagnosis and monitoring of
hereditary diseases; amplification of ancient DNA;[4] analysis of genetic
fingerprints for DNA profiling (for example, in forensic science and
parentage testing); and detection of pathogens in nucleic acid tests for the
diagnosis of infectious diseases.
A basic PCR set-up requires several components and reagents,including:
• a DNA template that contains the DNA target region to amplify
• a DNA polymerase; an enzyme that polymerizes new DNA strands; heat-
resistant Taq polymerase is especially common, as it is more likely to remain
intact during the hightemperature DNA denaturation process • two DNA
primers that are complementary to the 3′ (three prime) ends of each of the
sense and anti-sense strands of the DNA target (DNA polymerase can only
bind to and elongate from a double-stranded region of DNA; without
primers, there is no double-stranded initiation site at which the polymerase
can bind);[9] specific primers that are complementary to the DNA target
region are selected beforehand, and are often custom-made in a laboratory or
purchased from commercial biochemical suppliers
• deoxynucleoside triphosphates, or dNTPs (sometimes called
"deoxynucleotide triphosphates"; nucleotides containing triphosphate
hybridization of the primer to the strand, but high enough for the
hybridization to be specific, i.e., the primer should bind only to a perfectly
complementary part of the strand, and nowhere else. If the temperature is too
low, the primer may bind imperfectly. If it is too high, the primer may not
bind at all. A typical annealing temperature is about 3–5 °C below the Tm of
the primers used. Stable hydrogen bonds between complementary bases are
formed only when the primer sequence very closely matches the template
sequence. During this step, the polymerase binds to the primer-template
hybrid and begins DNA formation.
• Extension/elongation: The temperature at this step depends on the DNA
polymerase used; the optimum activity temperature for the thermostable
DNA polymerase of Taq polymerase is approximately 75–80 °C (167–176
°F), [13][14] though a temperature of 72 °C (162 °F) is commonly used with
this enzyme. In this step, the DNA polymerase synthesizes a new DNA
strand complementary to the DNA template strand by adding free dNTPs
from the reaction mixture that is complementary to the template in the 5′-to-
3′ direction, condensing the 5′- phosphate group of the dNTPs with the 3′-
hydroxy group at the end of the nascent (elongating) DNA strand. The
precise time required for elongation depends both on the DNA polymerase
used and on the length of the DNA target region to amplify. As a rule of
thumb, at their optimal temperature, most DNA polymerases polymerize a
thousand bases per minute. Under optimal conditions (i.e., if there are no
limitations due to limiting substrates or reagents), at each
extension/elongation step, the number of DNA target sequences is doubled.
With each successive cycle, the original template strands plus all newly
generated strands become template strands for the next round of elongation,
leading to exponential (geometric) amplification of the specific DNA target
region. The processes of denaturation, annealing and elongation constitute a
single cycle. Multiple cycles are required to amplify the DNA target to
millions of copies. The formula used to calculate the number of DNA copies
formed after a given number of cycles is 2 n , where n is the number of
cycles. Thus, a reaction set for 30 cycles results in 230, or 1,073,741,824,
copies of the original double-stranded DNA target region.
• Final elongation: This single step is optional, but is performed at a
temperature of 70–74 °C (158–165 °F) (the temperature range required for
optimal activity of most polymerases used in PCR) for 5–15 minutes after
the last PCR cycle to ensure that any remaining single-stranded DNA is fully
elongated
As with other chemical reactions, the reaction rate and efficiency of PCR are
affected by limiting factors. Thus, the entire PCR process can further be
divided into three stages based on reaction progress:
• Exponential amplification: At every cycle, the amount of product is
doubled (assuming 100% reaction efficiency). After 30 cycles, a single copy
of DNA can be increased up to 1,000,000,000 (one billion) copies. In a
sense, then, the replication of a discrete strand of DNA is being manipulated
in a tube under controlled conditions.[15] The reaction is very sensitive:
only minute quantities of DNA must be present.
• Leveling off stage: The reaction slows as the DNA polymerase loses
activity and as consumption of reagents, such as dNTPs and primers, causes
them to become more limited
. • Plateau: No more product accumulates due to exhaustion of reagents and
enzyme.

ELECTROPHORESIS
Electrophoresis is an old established method of analytical chemistry. It is
based on the principle that the individual components of the colloidal
solution migrate in a liquid at different speeds when subjected to an electric
field. Separations are possible, because particles of similar geometry but
different charge, and particles of like charge but different geomeiry migrate
at different rates towards an oppositely charged electrode. Therefore, when
the current is passed for a certain time through such a solution, various
components present in the solution would move through different distances
in their effort to migrate towards the electrodes. Therefore, a substance
which may be a mixture, is thus separated into its components along the
migration distance, according to a definite law/Measurement of the
concentration along this migration distance, therefore, would provide the
quantitative result of the analysis. Historically, some of the earliest reports
described characteristic electrophoretic mobilities of bio-colloids, such as
proteins or enzymes. However, the technique received little note until 1937,
when Tiselius published a paper introducing the moving boundary concept.
The moving boundary method utilises the migration of particles in free
solution and observation of the various molecular boundaries through
sensitive refractometric techniques, With this, the value of electrophoresis in
obtaining distinct and measurable fractions of a variety of substances got
well established, particularly in clinical laboratories.
Normally, with moving boundary method/only two components of a
mixture, one with the highest mobility and the other with the lowest mobility
can be separated in pure form, If it is desired to recover components other
BASIC COMPONENTS OF A GAS CHROMATOGRAPHY
TYPES OF LIQUID CHROMATOGRAPHY
• A flow cytometer, despite its name, does not necessarily deal with
cells; it deals with cells quite often, but it can also deal with
chromosomes or molecules or many other particles that can be
suspended in a fluid.
Flow Cytometer Principle
The basic principle of flow cytometer is based on the measurement of
light
scattered by particles, and the fluorescence observed when these
particles are passed in a stream through a laser beam.
Types of Flow Cytometry
There are different types of flow cytometers based on the purpose and
precision of the process
1. Traditional flow cytometers
• The traditional cytometers are the
common cytometer using sheath fluid for focusing the sample stream.
• The most common lasers used in
traditional flow cytometers are 488 nm (blue), 405m (violet), 532nm
(green), 552nm (green), 561 nm
(green-yellow), 640 nm (red) and 355 nm (ultraviolet).
2. Acoustic Focusing Cytometers
• in these cviometers. ultrasonic waves
are used to focus the cells for
analysis.
• This prevents sample clogging and also allows higher sample inputs.
3. Cell sorters
• Cell sorters are a category of traditional flow cvtometers which
allows the user to collect samples after processing.
• The cells that are positive for the
desired parameter can be separated
from those that are negative for the
valameers
• The cells that are positive for the
desired parameter can be separated from those that are negative for the
parameters.
4. Imaging flow cytometer
• Imaging cytometers are traditional cytometers combined with
fluorescence microscopy.
• Imaging cytometer allows for rapid analysis of a sample for
morphology and multi-parameter fluorescence at
containing the cell dilution, creating an electrical circuit between the two
electrodes. Current will flow from one electrode to the other through the
orifice. When the cell suspension is drawn through the orifice, cells will
displace their own volume of electrolyte and cause a resistance change,
which is converted to a voltage change, and is amplified and displayed.

In practice, the cell suspension is drawn through the orifice by means of a

mercury manometer. This manometer includes two platinum wire contacts
(A and B) set through the glass walls. Contact A will start the count and
contact B will stop it when precisely 0.5 ml of the dilution has passed
through the orifice tube. Thus, it provides a count of the number of particles
in a fixed volume of suspension. Figure 16.5 shows the sequence of building
up the pulse in terms of increase in resistance at different positions of the
cell with respect to the orifice. To enable the instrument to count only those
pulses, which fall within certain preset size limits, the threshold facility is
required. The threshold is also necessary to enable the instrument to ignore
any electronic noise, which may be present in the system. The lower
threshold sets an overall voltage level, which must be exceeded by a pulse
before it can be counted. The upper threshold will not allow pulses to be
counted which exceed its preset level. The Coulter counters are usually
provided with an oscilloscope monitor to display the pulse information,
which has passed through the amplifier, and acts as a visible check on the
counting process indicating instantaneously any malfunctions such as a
blocked orifice. In particular, it provides information regarding (i) relative
cell size, (ii) relative cell size distribution, (iii) settings of the threshold level
control, and (iv) means to check the performance of the instrument for