genomic medicine

7/24/2019 genomic medicine

1/23

Genomic Medicine:

Basic Molecular Biology

Childrens Hospital Informatics Program

Childrens Hospital BostonHarvard Medical School

Massachusetts Institute of Technology

www.chip.org

Basic Biology

functions over a lifetime

specific protein

Organisms need to produce proteins for a variety of

Enzymes to catalyze reactions

Structural support

Hormone to signal other parts of the organism

Problem one: how to encode the instructions for making a

Step one: nucleotides

Atul Butte, MD

arvard-MIT Division of Health Sciences and TechnologyST.512: Genomic Medicine

rof. Atul Butte


2/23

Basic Biology

Naturally form double helixes

Redundant information in each strand

Complementary nucleotides form base pairs

Base pairs are put together in chains (strands)

Chromosomes

chromosome

We do not know exactly how strands of DNA wind up to make a

Each chromosome has a single double-strand of DNA

22 human chromosomes are paired

In human females, there are two X chromosomes

In males, one X and one Y


3/23

What does a gene look like?

regulatory elements

There is a code for the start and end of the proteincoding portion

sequence syntax alone

Each gene encodes instructions to make a single protein

DNA before a gene is called upstream, and can contain

Introns may be within the code for the protein

Theoretically, the biological system can determinepromoter regions and intron-exon boundaries using the

Area between genes

is repeated sequences

between individuals

The human genome contains 3 billion base pairs (3000 Mb)but only 35 thousand genes

The coding region is 90 Mb (only 3% of the genome)

Over 50% of the genome

Long interspersednuclear elements

Short interspersednuclear elements

Long terminal repeats

Microsatellites

Many repeatedsequences are different


4/23

Genome size

largest genome, right?

3000 Mb

Wheat has 17000 Mb

Were the smartest, so we must have the

Not quite

Our genome contains

(~750 megabytes)

E. coli has 4 Mb

Yeast has 12 Mb

Pea has 4800 MbMaize has 5000 Mb

Genomes of other organisms

Plasmodium falciparum chromosome 2

Please see Figure 1 of Science. 1998 Nov 6; 282(5391):1126-32.

Chromosome 2 sequence of the human malaria parasite

Plasmodium falciparum. Gardner MJ, et al.


5/23

mRNA is made from DNA

proteins

The design of a proteinneeds to be duplicable

from DNA within thenucleus

cytoplasm, where theprotein is formed

Genes encodeinstructions to make

mRNA is transcribed

mRNA moves to the

Digitizing amino acid codes Proteins are made of 20

(21) amino acids

only be one of 4nucleotides

3 nucleotides to encodea single amino acid

made from mRNA

Yet each position can

Nature evolved into using

A chain of amino acids is


6/23

Genetic Code

Lander ES, et al.

Please see figure 34 of Nature. 2001 Feb 15; 409(6822): 860-921.

Initial sequencing and analysis of the human genome.

Molecular Biology

Nucleotides

Double helix tRNA

mRNA

Signal Sequence

Prefixed by

Amino Acid

Protein

Chromosome

Gene/DNA

Genome

Are in

Are in

Holds

Held in

Ribosome

Joined by

Operates on Are in


7/23

Central Dogma

Nucleotides

Double helix tRNA

mRNA

Signal Sequence

Prefixed by

Amino Acid

Protein

Chromosome

Gene/DNA

Genome

Are in

Are in

Holds

Held in

Ribosome

Joined by

Operates on Are in

Protein targeting

Works in conjunction with other cellular machinery to

The first few amino acids may serve as a signal peptide

direct protein to the right place


8/23

Transcriptional Regulation

transcription factors

Amount of protein is roughly governed by RNA level

Transcription into RNA can be activated or repressed by

What starts the process?

Transcriptional programscan start from

Hormone action on receptors

Shock or stress to the cell

New source of, or lack ofnutrients

Internal derangement of cellor genome

Many, many other internaland external stimuli


9/23

Temporal Programs Segmentation versus Homeosis: same two houses at

Please see Figure 1 of Cell. 2000 Jan 7; 100(1):27-40.

Development: the natural history of genes. Scott MP.

different times

mRNA

nucleotides per minute

transcribe

mRNA can be transcribed at up to several hundred

Some eukaryotic genes can take many hours to

Dystrophin takes 20 hours to transcribe

Most mRNA ends with poly-A, so it is easy to pick out

Can look for the presence of specific mRNA using thecomplementary sequence


10/23

Periodic Table for Biology Knowing all the genes

knowing the periodictable of the elements

our periodic table

is the equivalent of

Instead of a table,

may read like a tree

More Information

Molecular Genetics

imer/primer.pdf

Sons, 1999.

Department of Energy Primer on

http://www.ornl.gov/hgmis/publicat/pr

T. A. Brown, Genomes, John Wiley and


11/23

Gene Measurement TechniquesDNA

Sequencing

Polymorphisms

RNA

Serialanalysisofgeneexpression DNAMicroarrays

Wafers

Protein

2D-PAGE

Massspectrometry

Proteinarrays

Please refer to Annu Rev Biomed Eng.

1999; 1: 649-78.

Instrumentation for the genome project.

Jaklevic JM, Garner HR, Miller GA.

Please see Figure 1 of J Biotechnol.

2000 Jan 7;76(1): 1-31.

Sequence analysis of genes and

genomes.

Sterky F, Lundeberg J.

Sequencing Reactions

Sanger Chain Termination


12/23

Please see Buetow KH, et al.

Nature Genetics 21:323 (1999).


perday

PHRAP:assemblessequencedatausingbase-qualityscoresinto

sequencecontigs

Assembly-qualityscores

Mostofthegenomewas

sequencedover12months

Highestthroughputcenterat

Whitehead:100,000sequencing

reactionsper12hours

Robotspick100,000colonies,sequence60millionnucleotides



13/23

Assembly Contaminationfromnon-humansequencesremoved

Clonesoverlaidonphysicalmap

High-qualitysemiautomaticsequencingfrombothendsofverylargenumbersofnumbersofhumangenomefragments

Overlapstakememory:Drosophila600GBRAM

Human104-processor4GBand16-processor64GB,10KCPUhrs

Genome Browsers Genomebrowsers:UniversityofCaliforniaatSantaCruzand

EnsEMBL

Overlapsequence,cytogenetic,SNP,geneticmaps

Overlapannotations,diseasegenes


14/23

Single Nucleotide

Polymorphisms

Threestepapproach

First,findthegenesyouareinterestedin

Second,catalogallthepolymorphismsina

gene(bysequencing)

Third,measurethose

polymorphismsina

largerpopulation

Clinical use of SNPs

NewpublicationwithassociationofSNPwithdiseaseisalmostadailyoccurrence

Please see Gao, X. et al.

Effect of a

single amino acid change in MHC

class I molecules on the rate ofprogression to

AIDS. N EnglJMed344,

1668-75 (2001).


15/23

SNPs and pharmacogenomics

side-effects

Please see Sesti F. PNAS 97:10613, 2000.

Geneswillhelpusdeterminewhichdrugstouseinparticulardiseasesubtypes

Geneswillhelpuspredictthosewhoget

Please refer to Madden, S. L., Wang,C.J. &

Landes, G.Serial analysisof gene expression:

from gene discoveryto target identification.

Drug Discov Today 5, 415-425 (2000).

Serial Analysis of Gene

Expression


16/23

RNA expression detection chips

Tissueor

Tissueunderinfluence

RNA cDNATagged

withfluorcopy

cDNAspottedonglassslideor

oligonucleotidesbuiltonslide

Quantitative,absoluteorrelative

GeneschosenarbitrarilySchenaM,etal.PNAS93:10614(1996).

NeedsfunctionaltissueNatureGenetics,21:supplement(Jan1999).

Please see Lockhart, DJ. Winzeler,

EA. Nature 405, 827-36 (2000).

roarray of genes aka

gene chips


17/23

Experiment Design

QuantitatespecificRNA

expressionbeforeandafter

anintervention

Compareexpression

betweentwotissuetypes

Compareexpressionbetweendifferentstrainsorconstructedorganisms

Compareexpression

betweenneighboringcells

Please see Luo L, et al. Nature Medicine;

5: 117(1999).

Validation

Insituhybridization

Real-timePolymeraseChainReaction

Microarrays in DiagnosisPlease see Figure 3b of Science. 1999 Oct 15; 286(5439):

531-7. Molecular classification of cancer: class discovery andclass prediction by geneexpression monitoring.

Difficulty

Golub TR, Slonim DK, Tamayo P, Huard C,

distinguishing

Gaasenbeek M, Mesirov JP, Coller H, Loh ML,

betweenleukemias

Downing JR, Caligiuri MA, Bloomfield CD,

Microarrayscanfind

Lander ES

genesthathelpmake

thediagnosiseasier


18/23

Microarrays in Prognosis

Pleasesee Nature. 2000 Feb 3; 403 (6769):503-11. Distinct

types of diffuselarge B-cell lymphoma identified by gene

expression profilling.Alizadeh AA, et al.

PatientswithseeminglythesameB-cell

lymphoma

Lookingatpatternofactivatedgenes

helpeddiscovertwosubsetsof

lymphoma

Bigdifferencesinsurvival

After microarrays comes wafers Chromosome21has21millionbase-pairs

Each5inchsquarewafers(Perlegen)hold60millionprobes

Cansequenceanentirechromosomeinoneexperiment

Eachscantakesuparound10terabytes

CansequenceallSNPswithinahumanin10days

Please see Patil N. Science 2001, 294:1719.


19/23

2D-PAGE

Twoaxis=two

propertiesofproteins:

pHversusmass

Globalviewof

proteins Patternscanbe

scanned,savedand

searched

Spotsneedtobe

pickedfor

identification

Unfortunately,not

veryquantitative

Please see Gygi, S. P., Rochon, Y., Franza,

B. R. & Aebersold, R. Correlation between

protein and mRNA abundance in yeast.

Mol Cell Biol

19, 1720-30 (1999).

Please see Gygi, S. P. & Aebersold,

R.Proteomics: A Trends Guide. (2000)


20/23

Please see Gygi, S. P. & Aebersold, R. Mass

spectrometry and proteomics.

Curr

Opin

Chem

Biol

4, 489-94 (2000).


21/23

Clinical uses for

proteomics

Petricoin,etal.,usedthistechnique

onserum

Findingmarkersdistinguishingovariancancerversusnon-neoplasia

Questforbiomarkers

Please see Petricoin, E. F. et al. Use of

proteomic patterns in serum to

identify ovarian

cancer. Lancet

359, 572-7. (2002).

Quantitative proteomics

Theexamplessofardemonstrateidentification,not

quantification

Onecantakeadvantageoftheextremesensitivityof

detectionofmassspectrometry

Addtotheproteinsaknownamountoflabel


22/23

Protein chips

Function

Please see Williams, D. M. & Cole, P. A.Kinasechips hit the proteomics era.

Trends BiochemSci26, 271-3 (2001).

Detectionvs.

Kinasechips


23/23

Protein

Detection

Specificantibodies

Antibodiesneedtobeavailable

Gene Measurement TechniquesDNA

Sequencing

Polymorphisms

RNA

Serialanalysisofgeneexpression

DNAMicroarrays

Wafers

Protein

2D-PAGE Massspectrometry

Proteinarrays

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