Dr Ambika Jawalkar

POLYMERASE CHAIN REACTION

Kary Mullis in 1983, Noble Prize in Chemistry

in 1993

A scientific technique in Molecular Biology

Amplification of a single or a few copies of DNA

across several orders of magnitude

Can generate thousands to millions of copies of a

desired DNA sequence within few minutes

PRINCIPLES OF PCR:

• Thermal cycling

• Selective & repeated amplification with help of

Primers

• Taq Polymerase isolated from Thermus aquaticus

• As the reaction progresses the DNA generated is

itself used as a template

PROCEDURE

• Mostly amplify DNA fragments up to 10kbs but

some allow amplification of fragments up to 40kbs size

• Carried out in a reaction volume of 10-200µl in small

reaction tubes of 0.2-0.5ml volumes

• Reaction tubes are placed in a thermal cycler

COMPONENTS

DNA template / target DNA

Primers

Taq Polymerase

dNTPs

Buffer solution

Magnesium Chloride salt solution

STEPS:

Each cycle consists of 3 discrete temperature

steps

1. Denaturation step - @ 95ºc for 20 to 30 sec

2. Annealing Step – 50 to 65ºc for 20 to 40 sec

3. Extension / Elongation step

STAGES OF PCR

a. Exponential amplification

b. Leveling off stage

c. Plateau

CLINICAL APPLICATIONS

Role in diagnosis of Infectious diseases

Role in Cancer diagnostics

Genetic diseases & Paternity testing

VARIATIONS / MODIFICATIONS OF

BASIC PCR TECHNIQUE:

Reverse Transcription PCR (RT-PCR)

Quantitative PCR (Q-PCR)

Nested PCR

Asymmetric PCR

Multiplex PCR

DNA MICROARRAY TECHNOLOGY-The Diagnostics of Future

Introduction:

Central Dogma of Life

DNA

mRNA

Protein

Transcription

Translation

This technology measures the activity of genes at a

transcriptional level.

The information that can be obtained by sequencing a gene is,

Sequence of protein it encodes

Can guess the function of the gene

Can look for presence of mutations

Can compare the gene sequence & the protein it encodes

in different animal species

Can study evolution of genes

STEPS :

1. Sample Preparation

- isolation of total RNA

- reverse transcription

- labeling

2. Hybridization

- binding between the targets & probes

- washing

3. Detection

- chip reading

4. Data acquisition & analysis

- collection & summary of raw data

- statistical analysis of the data

DNA Microarrays / DNA chips : basic concept

• Small solid supports onto which the sequences from

thousands of different genes are immobilized or

attached at fixed locations.

• Are usually glass microscope slides or silicon chips

or nylon membranes

• DNA is printed, spotted or synthesized directly on

to the glass slide

• Each spot represents a particular gene sequence

• Spots can be DNA, cDNA or oligonucleotides

Dimensions of a gene chip

Principle:

Hybridization Probing –a technique that uses

fluorescently labeled nucleic acid molecules to identify

complementary molecules

PROCEDURE

Interpretation of gene chip array

Types of Microarrays:

3 basic types of samples can be used to construct

DNA microarrays

Two are genomic

Transcriptomic

Advantages: Follow activity of many genes at the same time

Fast results

Comparing the activity of many genes in diseased &

healthy cells

Categorize diseases into subgroups

Limitations / Drawbacks:

× too much data at once

× results may be too complex to interpret

× results are not always reproducible

× still too expensive

Microarray applications (in brief)

o Expression analysis

drug development, drug response &

therapy development

o Mutation / Polymorphism analysis

drug development, therapy development

& tracking disease progression

CONCLUSION

THANK YOU