Introduction, Working Principle and applications of genomic and proteomic tools

1. APPLICATION OF GENOMIC AND
PROTEOMIC
TOOLS
PRESENTED BY : RAJU PAUDEL
M.PHARM : PHARMACOLOGY (I SEM)
DEPARTMENT OF PHARMACOLOGY
ISF COLLEGE OF PHARMACY, MOGA (PUNJAB)

2. CONTENTS
INTRODUCTION TO GENOMIC AND PROTEOMIC
WORKING PRINCIPLE AND APPLICATIONS OF
GENOMIC AND PROTEOMIC TOOLS

3.  IT IS THE STUDY OF GENOME INCLUDES VARIOUS EVENTS
TAKES PLACE IN GENOME AND ALTERATIONS MADE TO IT.
 IT INCLUDES VARIOUS TOOLS AND TECHNIQUES PERFORM
TO UNDERSTAND THESE EVENTS.
 GENOME COULD BE OF STUDIES IN TWO WAYS:
 1.STRUCTURAL GENOMICS
 2. FUNCTIONAL GENOMICS
Genomics

4. PROTEOMICS
• IT IS THE STUDY OF “PROTEOME”.
• THE WORD "PROTEOME" IS A BLEND OF "PROTEIN" AND "GENOME“.
• LARGE SCALE STUDY OF PROTEINS..
• PARTICULARLY THEIR STRUCTURES AND FUNCTIONS.

5. DNA Gel Electrophoresis

6. WHAT IS GEL ELECTROPHORESIS?
• ELECTRO = FLOW OF ELECTRICITY, PHORESIS, FROM THE GREEK = TO CARRY
ACROSS
• A GEL IS A COLLOID, A SUSPENSION OF TINY PARTICLES IN A MEDIUM,
OCCURRING IN A SOLID FORM, LIKE GELATIN
• GEL ELECTROPHORESIS REFERS TO THE SEPARATION OF CHARGED PARTICLES
LOCATED IN A GEL WHEN AN ELECTRIC CURRENT IS APPLIED
• CHARGED PARTICLES CAN INCLUDE DNA, AMINO ACIDS, PEPTIDES, ETC

7. WHY ELECTROPHORESIS?
• TO SEPARATE DNA
FRAGMENTS FROM
EACH OTHER
• TO DETERMINE THE
SIZES OF DNA
FRAGMENTS
• TO DETERMINE THE
PRESENCE OR AMOUNT
OF DNA
• TO ANALYZE
RESTRICTION
DIGESTION PRODUCTS

8. HOW DOES IT WORK?
• DNA IS AN ORGANIC ACID, AND IS NEGATIVELY CHARGED (REMEMBER, DNA FOR
NEGATIVE)
• WHEN THE DNA IS EXPOSED TO AN ELECTRICAL FIELD, THE PARTICLES
MIGRATE TOWARD THE POSITIVE ELECTRODE
• SMALLER PIECES OF DNA CAN TRAVEL FURTHER IN A GIVEN TIME THAN LARGER
PIECES

9. Application of Gel Electrophoresis
1. Estimate DNA molecule size after restriction enzyme
digestion (RFLP).
2. Check PCR amplified product.
3. Preparation of DNA to be used in other techniques such
as Southern blotting.
4.To separate DNA fragments from each other.

10. (PCR)
Polymerase Chain Reactio

11. Definition and developer
• The polymerase chain reaction (PCR) is a molecular
biology technique to amplify a single or a few copies
of a piece of DNA up to several orders of magnitude
of a particular DNA sequence.
• This automated process bypasses the need to use
bacteria for amplifying DNA.
• Nobel Prize in Chemistry (1993) to the developers,
Kary Mullis along with Michael Smith

12. Principle
• Most PCR methods typically amplify DNA
fragments of up to ~10 kilo base pairs (kb) (some
techniques up to 40 kb)
• A basic PCR set up requires several components
and reagents in a reaction volume of 10–200 μl in
small reaction tubes (0.2–0.5 ml volumes)

13. The
scheme of
a cycle
and a
3step-

14. Extreme applications
• PCR can be used for forensic analysis, when only a
trace amount of DNA is available as evidence.
• PCR-based techniques have been successfully used
to analyse ancient DNA (tens of thousands of years
old), such as a forty-thousand-year-old mammoth
• Also on human DNA, in applications ranging from
the analysis of Egyptian mummies to the
identification of family tree of a Russian tsar.

15. REVERSE
TRANSCRIPTION
PCR (RT-PCR)

16. Invention of RT-PCR
 The idea of RT-PCR is based upon
retroviruses.
 Retroviruses have an RNA rather than
DNA genome.
 So they must use reverse
transcriptase (RT) to covert their RNA
genomes into DNA.
 Human Immunodeficiency Virus (HIV)
is an example of a retrovirus.

17. WHAT IS REAL-TIME PCR?
REAL-TIME PCR A SPECIALIZED TECHNIQUE THAT ALLOWS A PCR REACTION TO BE
VISUALIZED “IN REAL TIME” AS THE REACTION PROGRESSES.
BY USING RT- PCR WE CAN MEASURE MINUTE AMOUNTS OF DNA SEQUENCES IN A SAMPLE!

18. APPLICATIONS OF REAL-TIME PCR
REAL-TIME PCR IS WIDELY USED FOR:
• GENE EXPRESSION ANALYSIS (E.G. CANCER AND DRUG RESEARCH)
• GENE INSERTION (E.G. GENOME STUDY WITH RT-PCR AND G.I FROM EUKARYOTIC INTO PROKARYOTIC)
• DISEASE DIAGNOSIS AND MANAGEMENT
• VIRAL QUANTIFICATION
• FOOD TESTING
• PERCENT GENETICALLY MODIFIED FOOD
• ANIMAL AND PLANT BREEDING
• GENE COPY NUMBER
• FORENSICS
• SAMPLE IDENTIFICATION AND QUANTIFICATION

19. WORKING OF
REAL-TIME PCR
19
5’
5’
3’
3’
d.NTPs
Thermal Stable
DNA Polymerase
Primers
Denaturation
Annealing
Add to Reaction Tube

20. Extension
5’ 3’
5’3’
Extension Continued
5’ 3’
5’3’
Taq
Taq
3’
5’3’
Taq
Taq
Repeat

21. 5’3’
3’
3’
3’
5’3’
3’
5’3’
3’
Cycle 2
4 Copies
Cycle 3
8 Copies
3’
3’
5’3’
3’
5’3’
3’
5’3’
3’
5’3’
3’
5’3’
3’
5’3’
3’
5’3’
3’

23. DNA Sequencing

24. What is DNA sequencing?
-Determining the precise order of nucleotides in a
piece of DNA
-DNA sequencing methods have been around for 40
years, and since the mid-1970s
-It includes any method or technology that used to
determine the order of four bases (A,G,C,T) in

25. Two basic methods for DNA sequencing :-
A- Chemical cleavage method (Maxam and Gilbert,
1977)
- Base-specific cleavage of DNA by certain chemicals
- Four different chemicals, one for each base
- A set of DNA fragments of different sizes
- DNA fragments contain up to 500 nucleotides
- Electrophoresed using acrylamide gel for size separation
- Expose to x-ray to visualize the fragments from which sequence
may be
guess.
Sequencing methods

27. B- Enzymatic method (Sanger, 1981)

28. Recent methods of chain termination
sequencing
Thermal cycler sequencing
Automated DNA sequencing
Pyrosequencing
Sequencing by hybridization

29. An automated sequencer
The output

30. CURRENT TRENDS IN SEQUENCING
It is rare for labs to do their own sequencing:
--costly, perishable reagents
--time consuming
--success rate varies
Instead most labs send out for sequencing:
--You prepare the DNA (usually plasmid, M13, or PCR product),
supply the primer, company or university sequencing center
does the rest
--The sequence is recorded by an automated sequencer as an
“electropherogram”

32. MICROARRAY-BASED
APPROACHES
A microarray is a pattern of ssDNA probes which are immobilized on a surface called a chip
or a slide.
 Microarrays use hybridization to detect a specific DNA or RNA in a sample.
 DNA microarray uses a million different probes, fixed on a solid surface.
 Microarray technology evolved from Southern blotting.
The concept of microarrays was first proposed in the late 1980s by Augenlicht and his
colleagues.

33. MICROARRAY TECHNIQUE

34. APPLICATIONS OF MICROARRAY
TECHNOLOGY
MICROARRAY AS A
GENE EXPRESSION
PROFILING TOOL
MICROARRAY AS A
COMPARATIVE
GENOMICS TOOL
DISEASE
DIAGNOSIS
DRUG DISCOVERY
TOXICOLOGICAL
RESEARCH

35. Enzyme
Linked
Immunosorb
ent Assay
(ELISA)

36. Enzyme Linked Immunosorbent Assay (ELISA),term Was
Coined By Engvall and Pearlmann in 1971
Different Types
Sandwich
Indirect
Competitive
Similar To Radio Immuno Assay (RIA), except radiolabel
molecule
Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs
ELISA

41. Applications
 Human anti-IgG, double antibody sandwich ELISA
 Because the ELISA can be performed to evaluate either the presence of antigen
or the presence of antibody in a sample, it is a useful tool for
determining serum antibody concentrations (such as with the HIV test or West
Nile virus). It has also found applications in the food industry in detecting
potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs and
as serological blood test for coeliac disease. ELISA can also be used in
toxicology as a rapid presumptive screen for certain classes of drugs.

42. BLOTTING

43. INTRODUCTION
• BLOTTING IS A TECHNIQUE BY WHICH A MACROMOLECULE SUCH AS DNA, RNA, OR
PROTEIN IS RESOLVED IN A GEL MATRIX, TRANSFERRED TO A SOLID SUPPORT, AND
DETECTED WITH A SPECIFIC PROBE.
• THESE POWERFUL TECHNIQUES ALLOW US TO IDENTIFY AND CHARACTERIZE SPECIFIC
MOLECULES IN A COMPLEX MIXTURE OF RELATED MOLECULES.
• SOME OF THE MORE COMMON TECHNIQUES INCLUDE:
• SOUTHERN BLOTTING (DNA)
• NORTHERN BLOTTING (RNA)
• AND WESTERN BLOTTING (FOR PROTEIN)

44. WESTERN BLOTTING

47. THANK YOU