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it is a brief ppt file about Genomics and the technical methods .
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Genomics
Presented by: Samaneh.RasoulinejadFall 2013 - 2014
• 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”
What is Genomics?
• 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
Introduction
Sequence the entire genome by cutting it into small, manageable pieces (fragments)
Assemble the entire genome from the pieces (fragments)
Make sense of the genomeUnderstand how gene expression takes place?How life processes are networked?Understand life??
Genomic and cDNA librariesDNA Hybridization and Northern blotsSubcloning in vectorsRestriction-enzyme mappingDNA sequencingPCR amplification
Technical Foundations of Genomics
Genomics and Medicine
-Drug, diagnostics, and prognostics development- Genotyping to predict patient susceptibility to disease- Personalized healthcare based on an individual’s genomic features
What we hope to gain from genomics
decision support systems
genotype
molecular profile
patient history
knowledge base
drugs diagnostics prognostics
genome health
pharma R&D patient care
· Over 1,000 disease genes were characterized by 2000
How to make a genomic library total genomic DNA
plasmid (black)
ampR oriampR ori
oriori
ori
ampR
ampR
ampR
restrictionenzyme
samerestriction
enzyme
annealand ligate
transform E. coli;select for
Amp resistance
genomic DNA
stationary supportpolyT
mRNApolyA
tissue or cell
E. Colibacteria
plasmidRadioactive
probe
selectedcolonies
membrane
hybridization
X-ray film
cDNAlibrary
Clone 1 2 3 4 5
Colony picking
microtiter
• 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)
Microarrays
• 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.
Northern blot and microarray0 2 5 6 7 hrs
0 2 5 6 7 9 11 hrs
DMC1–
SPS1–
DIT1–
SPS100–
0 2 5 6 7 9 11 hrs
DMC1– SPS1– DIT1–
SPS100–
foldrepressed
foldinduced
>20 10x 3x | 3x 10x >20 1:1
Identify genes whose expression was induced during sporulation in yeast
• 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
Cross-hybridization
• Microarray analysis of gene expression from four different types of tumors
Improved disease diagnostics from genomics
• 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)
Microarrays and cancer
• Identified 85 genes with different levels of expression in metastatic (M+) and non-metastatic 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.
Microarrays in the prognosis of metastasis(childhood cancer: medulloblastoma)
M+M–
green = downregulated
red = upregulated
· 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
Cancer genome projects One in three people will suffer from cancer in his or her
lifetime .
• 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)
Classes of microarrays
The array elementsare a series of 25-mer oligos designedfrom known sequenceand synthesizedDirectly on the surface
The entire array isformed by >500,000cells, each containinga different oligo
Affymetrix oligonucleotide arrays
• Propagating fragments of cloned DNA
• Used for sequencing and protein production
• Plasmid vectors• Replicate in bacteria• Resistant to
antibiotics• Cloning sites
subcloning
Regioninto whichDNA can
be inserted
Plasmidcloningvector
ORI
ampr
• Vector and fragment to be
inserted must have compatible
ends
• Sticky ends anneal
• Enzyme ligase makes covalent
bond between vector and
fragment
• Use of recombination instead of
restriction sites
Subcloning: vector and fragment
restrictionenzymesDNA
cloningvector
fragment
recombinantplasmid
• Uses site-specific recombination for subcloning
• DNA fragment flanked by recombination sites
• Add recombinase “Clonase®”
• Moves fragment from one vector to another
Recombination cloning
• Most current sequencing projects use the chain termination method
• Also known as Sanger sequencing, after its inventor, Fredrick Sanger
• Based on action of DNA polymerase• Adds nucleotides to complementary strand
• Requires template DNA and primer
DNA sequencing
H
dideoxyribonucleotide
Chain terminates
• 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
Chain-termination sequencing
–
+C A G T C A G T
A T C G
+
–
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
• 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
Pyrosequencing
• Colony PCR• Helicase PCR• Hot- start• In situ• Intersequence specific• Inverse• Multiplex• Quantitative • Touch down• ......
PCRs
• 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
Resources
.Thank you