This document discusses polymerase chain reaction (PCR), a technique used to amplify DNA. It describes how PCR was developed by Kary Mullis in 1983 and involves thermal cycling to selectively amplify target DNA sequences using primers and Taq polymerase. The key components and steps of PCR are outlined, including denaturation, annealing and extension. Clinical and other applications of PCR like diagnosis of diseases, cancer detection, and genetic testing are mentioned. Variations of PCR like quantitative PCR and nested PCR are also summarized.

1. Dr Ambika Jawalkar

2. 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

3. 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

4. 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

5. COMPONENTS
 DNA template / target DNA
 Primers
 Taq Polymerase
 dNTPs
 Buffer solution
 Magnesium Chloride salt solution

6. 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

9. STAGES OF PCR
a. Exponential amplification
b. Leveling off stage
c. Plateau

11. CLINICAL APPLICATIONS
 Role in diagnosis of Infectious diseases
 Role in Cancer diagnostics
 Genetic diseases & Paternity testing

12. VARIATIONS / MODIFICATIONS OF
BASIC PCR TECHNIQUE:
 Reverse Transcription PCR (RT-PCR)
 Quantitative PCR (Q-PCR)
 Nested PCR
 Asymmetric PCR
 Multiplex PCR

13. DNA MICROARRAY TECHNOLOGY
-The Diagnostics of Future
Introduction:
Central Dogma of Life
DNA
mRNA
Protein
Transcription
Translation

15.  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

16. 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

17. 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

18. Dimensions of a gene chip

20. Principle:
 Hybridization Probing –a technique that uses
fluorescently labeled nucleic acid molecules to identify
complementary molecules

21. PROCEDURE

24. Interpretation of gene chip array

26. Types of Microarrays:
3 basic types of samples can be used to construct
DNA microarrays
 Two are genomic
 Transcriptomic

27. 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

28. Microarray applications (in brief)
o Expression analysis
drug development, drug response &
therapy development
o Mutation / Polymorphism analysis
drug development, therapy development
& tracking disease progression

29. CONCLUSION

30. THANK YOU