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