ASSIGNMENT NO.

01
POLYMERASE CHAIN REACTION

SUBMITTED TO : MAM SHENNEL

SUBMITTED BY : MUZZAMIL HUSSAIN
ROLL NO. : 70098818
SECTION : 5th B
DEPARTMENT OF PHARMACY
 ABSTRACT

Pathology practice is increasingly augmented with molecular tests for improved diagnostics and
patient management. The polymerase chain reaction (PCR) is foremost amongst these techniques.
This review explains the principles of PCR and the methodological factors that contribute to a
successful assay. Key PCR technique variations, such as reverse transcription (RT)-PCR and
quantitative real-time (q) PCR, are described and an overview is provided of how PCR products
are analysed. The review includes examples of PCR usage in clinical practice for the detection of
infectious and genetic diseases, for tumour diagnostics and in molecular forensic applications such
as specimen identity confirmation.
 POLYMERASE CHAIN REACTION
Introduction

Studying isolated pieces of DNA is nearly impossible. Large amounts of a sample of DNA are
necessary for molecular and genetic analyses. Sometimes called molecular photocopying,
conventional polymerase chain reaction (PCR) is a technique used to amplify (replicate) trace
amounts of DNA and RNA from a sample. A PCR thermal cycler is used to produce the large
amounts required for research.

The PCR process can be used for a wide variety of laboratory and clinical applications and
purposes. Forensic labs use it to analyze DNA samples from a crime scene. Clinical health care
labs use it to diagnose patients infected from a virus. Pharmaceutical research labs use it to analyze
and duplicate DNA and RNA samples for use in the manufacturing of drugs and vaccines.

PCR requirements needed

Four components (reagents or chemicals) are needed for the PCR process:

• A DNA or RNA sample (from saliva, blood, hair, skin scraping, etc.)
• DNA primers: short single-stranded DNA that promotes synthesis of a complementary
strand of nucleotides
• DNA polymerase: an enzyme that aids in the synthesis of a complementary strand of DNA
• Nucleotide solution mix containing adenine (A), thymidine (T), cytosine (C), and guanine
(G) used to build duplicate DNA strands

PCR process steps

The PCR process has 4 steps: collection, preparation, amplification, and post PCR clean-up. The
PCR machine steps happen in the amplification step. It begins with a segment of a DNA sample
placed in a suitable tube along with the reagents and chemicals listed above. The tube is placed
into the PCR machine or thermal cycler. The thermal cycler takes the solution through a 3-step
process: denaturation, annealing, and extension.
PCR machine steps

Step 1 - Denaturation

The solution contained in the tube is heated to at least 94°C (201.2°F) using a thermal cycler. The
heat breaks the hydrogen bonds of the original DNA sample and separates the DNA into single
strands (this is termed denaturation of double-stranded DNA).

Step 2 - Annealing

The sample mixture is then cooled to between 50 to 60°C (122 to 140°F) allowing the DNA
primers and the DNA polymerase enzyme to bind to the individual strands of DNA that were
separated by the heat (this is termed annealing of the primers). At this point, the nucleotides (A,
T, C, G) from the added mixture solution will pair with the individual separated strands of DNA
that resulted from the heating process.

Step 3 - Extension

Once joined together, they form a new complementary strand of DNA (termed extension of the
DNA). Thus, a new duplicate double-stranded DNA molecule has been formed from each of the
single strands of the original sample molecule. The temperature cycles from 95°C to 50 to 60°C.
The cycle is then repeated about 35 to 40 times using the thermal cycler which automatically
repeats the heating and cooling cycles of the process. Resulting DNA sequence is doubled each
time the heating/cooling cycle is conducted by the cycler. Thus, what started out as a single short
segment of DNA from one sample can be amplified to form millions of copies after 35 doubling
cycles.

PCR Temperature Cycle

Flow chart of the three main steps of PCR with the PCR temperature cycle, number of cycles,

and total program length.

This process provides a new duplicate double-stranded DNA molecule formed from each of the
single strands of the original sample molecule. This three-step process is done 35 to 40 times to
amplify the DNA/RNA into millions of duplicate segments.
Step 4 - Analysis with Electrophoresis

Once the PCR process is completed, the resulting amplified (replicated) segments generated can
then be compared to other nucleotide segments from a known source. The PCR-generated
nucleotide sequences are then placed next to known nucleotide sequences from humans,
pathogens, or other sources in a separating gel. Electrical current is then run through the gel, and
the various nucleotide sequences within the gel form bands that resemble a ladder, according to
their electrical charge and molecular size. This is termed gel electrophoresis. Bands or ladder-like
steps that migrate to the same levels in the gel show identity of nucleotide sequences. This method
is one of the most popular ways PCR tests are completed.

qPCR

qPCR is a variation of conventional PCR and allows analysis of the amplified/replicated DNA in
real-time during the usual 40 cycles of the procedure using fluorescent dyes. The fluorescent dyes
attach to some of the nucleotide strands allowing users to measure specific products and their
amounts during the amplification/replication cycles. Special thermal cyclers, known as real-time
thermal cyclers, are used for qPCR. As well as heating and cooling the tubes containing the PCR
reagents, they can measure the fluorescence inside the tube at every cycle. This allows users to
skip the gel electrophoresis or other secondary procedures needed for final analysis of the PCR
products, thus producing more rapid results.