Introduction, principle and applications of genomic and proteomics .

1. NAME : AISHWARYA SADANAND TELI
TOPIC NAME : GENOMICS AND PROTOMIC
CLASS : M. PHARMACY - I
DEPARTMENT : PHARMACOLOGY

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

3. Genomics
1. It is the study of genome includes various events takes place in genome and alterations
made to it.
2. It includes various tools and techniques perform.to understands these events.
3. Genome could be of studies in two ways:
 Structural genomics
 Functional genomics

4. PROTEOMICS
1. It is the study of "PROTEOME".
2. The word "PROTEOME" is a blend of "PROTEIN" and "GENOME".
3. Large scale studies of proteins particularly their STRUCTURE and FUNCTIONS.

5. DNA GEL ELECTROPHORESIS
WHAT IS GEL ELECTROPHORESIS?
 Electro=flow of electricity,
 Phoresis from the Greek to carry across
 A gel 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.

6. REASONS FOR SELECTING 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.

7. WORKING:
 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
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.

8. POLYMERASE CHAINREACTION(PCR)
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.

9. 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).
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 analyses 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.

10. REVERSE TRANSCRIPTION PCR (RT-PCR)
• 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.
• 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.

11. 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 GI from eukaryotic into
prokaryotic.
• Disease diagnosis and management-e.g. viral quantification.
• Food testing-percent genetically modified food.
• Animal and plant breeding-Gene copy number.
• Forensics- sample identification and quantification

12. DNA Sequencing
What is DNA sequencing?
 Determining the precise order of nucleotides in a piece of DNA.
 DNA sequencing methods have been around for 40years, 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 standard DNA.

13. Sequencing methods
• Two basic methods for DNA sequencing :
1. 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.
 B-Maxam and Gilbert method

14. 2.Chemical modification in the DNA :
 Here purified DNA is used
 The fragments are radioactively labeled at 5' end. Chemical treatment generates the
nucleotide bases
 DNA is cleaved by hot piperidine at modified base
 Conc. modifying chemicals is applied to the DNA
 Generates radiolabeled DNA fragments
 Electrophoresis and visualized in X-rays films

15. Principle involved in Enzymatic method(Sanger,1981) ssDNA
Enzymatic synthesis of complementary polynucleotide chains
Termination at specific nucleotide positions
Separate by Gel Electrophoresis
Read DNA Sequence

16. Recent methods of chain termination sequencing
 Thermal cycler sequencing
 Automated DNA sequencing
 Pyro sequencing
 Sequencing by hybridization.

17. MICROARRAY
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.

18. MICROARRAY TECHNIQUE

19. APPLICATIONS:
a) Primary analysis: In this step, the quality of the data obtained from each array is
verified by checking if hybridization, labeling, scanning, etc., are done properly. Here, all
the unnecessary and low-quality data are eliminated.
b) Scaling and Normalization: These are the two methods that are involved in regulating
the data collected from each arrays. This is done to make the comparison efficient and
easier.
Per-chip normalization scaling/per-chip normalization is a method in which the
overall fluorescence of each array is adjusted to an average intensity so that the brightness
of every sample becomes the same.
Per-gene normalization/normalization is a process in which the sources of variations
that can affect the measured expression levels of gene are removed. There are many
methods for normalization, but it is difficult to decide which is the best.

20. • In-depth analysis is the third step in analyzing microarray data. Based on the nature of
the experiment, tests dependent on statistics and filters are applied here to categorize
genes whose expressions are modified in various samples. Simple analysis is done for
fewer samples while for large numbers of samples, more sophisticated “clustering and
classification” is used.
• The simplest analysis: Filtering is the method used to analyze the data of fewer samples.
“Filter on flags” and “filter on fold change” are the two main approaches used in filtering.
• The flag is a qualitative measure that is accompanied by raw expression score. It verifies
the statistical differences of the genes from the background and allows filtering of only
accurately measurable genes.
• ‘Filter on fold change’ is a basic filtering method done by comparing fold change. It is
used to identify genes that are at least two-fold different in the experimental conditions.

21. • Advanced Analysis: Clustering and classification are the methods that can be used to
analyze extremely complex microarray data. However, as the data analyzed by these
methods are too large in quantity, it is better to filter the data first and limit it as per the
needs.
• Cluster Analysis: This method that involves various supervised/unsupervised techniques
of clustering divides the genes into different groups, especially when the sample consists
of different types of genes. It is a famous technique used for analyzing data matrix of
gene expression

22. • The three common clustering methods are as follows:
i. Hierarchical Clustering: An unsupervised technique in which clusters of genes are built
with approximately same patterns of expression by grouping genes together that are
greatly related in expression measurements. All genes are represented in the form of
leaves on a branching tree in the dendrogram.
ii. K-Means Clustering: This is a data mining algorithm that is used in clustering the data
into groups with no prior information on the relationships.
iii.Self-Organizing Maps (SOM): A neural network-based non-hierarchic clustering
approach that works like K-means clustering.

23. Enzyme Linked Immunosorbent Assay(ELISA)
 Enzyme Linked Immunosorbent Assay (ELISA),term Was Coined By Engvall and
Pearlmann in 1971
 Different Types
A) Sandwich
B) Indirect
C)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

24. METHODS:

25. Basic principle of ELISA
• Use an enzyme to detect the binding of antigen (Ag) antibody (Ab).
• The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the
presence of Ag: Ab binding.
• An ELISA can be used to detect either the presence of Antigens or antibodies in a sample
depending how the test is designed.
• ELISA was developed in 1970 and became rapidly accepted
APPLICATIONS OF ELISA
• Hormones
• Proteins
• FOR GMO (Genetically modified organism)
• Infectious Agent (Viral, Bacterial, Parasitic, Fungal)
• For Rapid Test
• Drug Markers
• IgG, IgM, IgA
• In New Born Screening
• Tumor Markers, ETC

26. BLOTTING
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)
 western blotting (for protein)

27. WESTERN BLOTTING
 APPLICATIONS
1. The confirmatory HIV test employs a western blot to detect anti-HIV antibody in
a human serum sample. Proteins from known HIV-infected cells are separated and
blotted on a membrane. Then, the serum to be tested is applied in the primary
antibody incubation step; free antibody is washed away, and a secondary anti-
human antibody linked to an enzyme signal is added. The stained bands then
indicate the proteins to which the patient's serum contains antibody.
2. A western blot is also used as the definitive test for Bovine spongiform
encephalopathy (BSE, commonly referred to as 'mad cow disease').
3. Western blot can also be used as a confirmatory test for Hepatitis B infection.

28. Electrophoresing the protein sample
Assembling the Western blot sandwich
Transferring proteins from gel to nitrocellulose paper
Staining of transferred proteins
Blocking nonspecific antibody sites on the nitrocellulose paper
Probing electroblotted proteins with primary antibody
Washing away nonspecifically bound primary antibody
Detecting bound antibody by horseradish peroxidase-anti-Iq conjugate and formation of a
diaminobenzidine (DAB) precipitate
Photographing the immunoblot
Flow chart of Western blotting

29. SDS PAGE
Principle
 Separates protein in an electric field.
 Migrate through a liquid or semisolid medium when subjected to an electric
field from anode to cathode terminal.
 Molecules flow at different rates depends on the molecular size of proteins.

30.  SDS-coated large proteins migrate slowly through the gel matrix and small
proteins migrate quickly through the matrix.
 The nearer the band to the well, the larger the molecular size of protein.
SDS: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-
PAGE)
 negatively charged detergent sodium dodecyl sulfate (SDS)
 used to denature and linearize the proteins
 coated the proteins with negatively charged

31.  PAGE:
 SDS-PAGE is differentiated into two system
1. Continuous sds-page
2. Discontinuous sds-page
 Polyacrylamide is used to form a gel, a matrix of pores which allow the molecules
migrate at different rates.
 Applications
1. Determine purity of protein samples
2. Determine molecular weight of protein
3. Identifying disulfide bonds between protein
4. Quantifying proteins
5. Blotting applications.