it is a brief ppt file about Genomics and the technical methods .

1. Genomics
Presented by: Samaneh.Rasoulinejad
Fall 2013 - 2014

2. What is Genomics?
•
•
Genomcis is the study of all genes present in an
organism
In 1986 mouse geneticist Thomas Roderick used
Genomics for “mapping, sequencing and
characterizing genomes”

3. Introduction
• Genomics built on recombinant-DNA technology
(developed since early 1970s)
• Thorough understanding of recombinant-DNA
techniques
• Prerequisite for understanding genomics
technologies
• Differences between genomics and
recombinant-DNA technology
• Genomics is high throughput approaches to allow
more analyses in parallel
• Genomics is dependent on computational
analysis due to larger data sets

4. Sequence the entire genome by cutting it into small,
manageable pieces (fragments)
Assemble the entire genome from the pieces
(fragments)
Make sense of the genome
Understand how gene expression takes place?
How life processes are networked?
Understand life??

5. Technical Foundations of
Genomic and cDNA libraries
Genomics
DNA
Hybridization and Northern blots
Subcloning in vectors
Restriction-enzyme mapping
DNA sequencing
PCR amplification
Genomics and Medicine

6. What we hope to gain from
genomics
-Drug, diagnostics, and prognostics development
- Genotyping to predict patient susceptibility to
disease
- Personalized healthcare based on an individual’s
genomic features
genome
decision support systems
genotype
molecular profile
patient history
knowledge base
drugs diagnostics prognostics
health

7. Over 1,000 disease
genes were
characterized by 2000

8. How to make a genomic library
ori
total genomic DNA
ampR
genomic
DNA
restriction
enzyme
anneal
and ligate
ori
ampR
ori
plasmid (black)
ampR
ori
ampR
ori
ampR
same
restriction
enzyme
transform E. coli;
select for
Amp resistance

9. selected
colonies
tissue or cell
membrane
mRNA
polyA
stationary support
polyT
Radioactive
probe
plasmid
E. Coli
bacteria
hybridization
X-ray film
cDNA
library
Clone 1
2
3
4
5

10. Colony picking
microtiter

11. Microarrays
• Basis of microarrays for determining gene expression
• Process by which complementary strands find each
other
• A–T and C–G base pairing
• speed and fidelity: dependent on temperature, salt,
sequence, and concentration (High temp and low
salt)

12. •
•
•
•
Microarrays permit the simultaneous analysis of the
RNA expression of thousands of genes.
For fully sequenced genomes, microarrays can be
used to analyze the expression of every gene.
Prior to the introduction of microarrays, RNA
abundance was usually analyzed through
hybridization to RNA bound to filters. These Northern
blots normally had no more than 20–40 lanes, and no
more than three probes could be used
simultaneously.
In contrast, microarrays can interrogate 30,000
genes at the same time, vastly increasing our ability
to analyze RNA expression.

13. Northern blot and microarray
0 2 5 6 7 hrs
0 2 5 6 7 9 11 hrs
DMC1 –
DMC1 –
SPS1 –
DIT1 –
SPS1 –
SPS100 –
0 2 5 6 7 9 11 hrs
DIT1 –
SPS100 –
fold
repressed
fold
induced
>20 10x 3x | 3x 10x >20
1:1
Identify genes whose expression was induced during
sporulation in yeast

14. Cross-hybridization
•
•
•
•
Hybridization to a related, but not identical,
sequence = cross-hybridization
Example: A probe from one member of a gene family
is likely to hybridize to all other members
Problem in microarrays, particularly cDNA arrays
Oligonucleotide arrays prescreened to eliminate
sequences likely to cross-hybridize

15. Improved disease diagnostics from
genomics
•
Microarray analysis of
gene expression from four
different types of tumors

16. Microarrays and cancer
•
•
Histology not always effective tool for prognosis and
diagnosis
Microarrays distinguish cancerous tissues on the basis
of a gene expression profile
•
Use in diagnosis (presence)
•
•
Example: characterizing acute lymphoblastic leukemia. Also
breast cancer.
Use in prognosis
•
Example: assessing the likelihood of metastasis in
medulloblastoma (brain tumor in children)

17. Microarrays in the prognosis of metastasis
(childhood cancer: medulloblastoma)
•
•
•
•
Identified 85 genes with
different levels of expression in
metastatic (M+) and nonmetastatic tumors (M_)
59 up and 26 down
72% accuracy in predicting
metastasis
Identified genes induced in
metastasis
• Could serve as potential
drug targets for in vitro
experiments
• platelet derived growth
factor receptor alpha
(PDGFRα). Antibodies
prevent migration.
M–
M+
green =
down
regulated
red = up
regulated

18. Cancer genome projects
One in three people will suffer from cancer in his or her lifetime.
Cancer Genome Anatomy Project (CGAP)
Established 1997 by National Cancer Institute (USA)
Specializes in EST sequencing
Human Cancer Genome Project (HCGP)
Established 1999 by Brazilian research groups
Cancer Genome Project (CGP)
Established 2000 by Wellcome Trust and Sanger Institute
(United Kingdom)
Specializes in genomic mutations leading to cancer
Funding: $15 million to $60 million

19. Classes of microarrays
•
Custom/spotted/two-color
microarrays (cDNAs, BACs)
•
High-density oligonucleotide
arrays (GeneChip, Affymetrix)
•
Long oligonucleotide microarrays
- Agilent (25-60 bases)
- Illumina (50 bases)
- Nimblegen (50-75 bases)

20. Affymetrix oligonucleotide arrays
The array elements
are a series of 25mer oligos designed
from known sequence
and synthesized
Directly on the surface
The entire array is
formed by >500,000
cells, each containing
a different oligo

21. subcloning
• Propagating fragments
of cloned DNA
• Used for sequencing
and protein production
• Plasmid vectors
• Replicate in bacteria
• Resistant to antibiotics
• Cloning sites
ORI
Region
into which
DNA can
be inserted
Plasmid
cloning
vector
ampr

22. Subcloning: vector and fragment
•
Vector and fragment to be
DNA
fragment
inserted must have compatible
ends
•
Sticky ends anneal
•
Enzyme ligase makes covalent
cloning
vector
bond between vector and
fragment
•
Use of recombination instead of
restriction sites
recombinant
plasmid
restriction
enzymes

23. Recombination cloning
•
•
•
•
Uses site-specific
recombination for
subcloning
DNA fragment flanked by
recombination sites
Add recombinase
“Clonase®”
Moves fragment from
one vector to another

24. DNA sequencing
•
Most current sequencing projects use the chain
termination method
•
•
Based on action of DNA polymerase
•
•
Also known as Sanger sequencing, after its inventor, Fredrick
Sanger
Adds nucleotides to complementary strand
Requires template DNA and primer

25. Chain terminates
H
dideoxyribonucleotide

26. Chain-termination sequencing
•
Dideoxynucleotides stop
synthesis
•
Chain terminators
Included in amounts so as
to terminate every time the
base appears in the
template
• Use four reactions
•
•
One for each base:
A,C,G, and T

27. –
A
T
C
G
–
+
CAGTCAGT
+

28. Sequence detection
To detect products of sequencing
reaction
Include labeled nucleotides
Formerly, radioactive labels were
used
Now fluorescent labels
Use different fluorescent tag for
each nucleotide
Can run all four reactions in
same lane

29. Pyrosequencing
•
•
•
based on the sequencing by synthesis principle.
it relies on the detection of pyrophosphate release on
nucleotide incorporation
The technique was developed by Mostafa Ronaghi
and Pål Nyrén at the Royal Institute of Technology in
Stockholm in 1996

32. PCRs
•
•
•
•
•
•
•
•
•
•
Colony PCR
Helicase PCR
Hot- start
In situ
Intersequence specific
Inverse
Multiplex
Quantitative
Touch down
......

33. Resources
•
Bioinformatics, Genomics, and Proteomics (Ann Finney Batiza, Ph.D.)
•
SciencePages
•
Functional Genomics (Michael Kaufmann and Claudia Klinger
Private Universitt, Witten/Herdecke gGmbH, Witten, Germany)
Introduction to Genomics by Arthur M. Lesk, 2007, Oxford University Press

34. Thank you