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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