Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 10-1 PowerPoint to accompany Genetics: From Genes to Genomes Third

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 10-1

PowerPoint to accompany

Genetics: From Genes to GenomesThird Edition

Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres

Chapter10

Prepared by Malcolm SchugUniversity of North Carolina Greensboro

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 10-2

Reconstructing the Reconstructing the GenomeGenome

Through Through

Genetic and Molecular AnalysisGenetic and Molecular Analysis

10-3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Outline of Chapter 10Outline of Chapter 10 Challenges and strategies of genome analysisChallenges and strategies of genome analysis

Genome sizeGenome size Features to be analyzedFeatures to be analyzed Problems with DNA polymorphismsProblems with DNA polymorphisms Development of whole-genome mapsDevelopment of whole-genome maps

Insights emerging from complete genome sequencingInsights emerging from complete genome sequencing Number and type of genesNumber and type of genes Extent of repeated sequencesExtent of repeated sequences Genome organization and structureGenome organization and structure Evolution by lateral gene transferEvolution by lateral gene transfer

High throughput tools for analyzing genomes and their protein productsHigh throughput tools for analyzing genomes and their protein products DNA sequencersDNA sequencers DNA arraysDNA arrays Mass spectrophotometersMass spectrophotometers

Two paradigm changes propelled by whole-genome sequences and new tools of Two paradigm changes propelled by whole-genome sequences and new tools of genome analysisgenome analysis Systems biologySystems biology Predictive and preventative medicinePredictive and preventative medicine


The genomes of living organisms vary enormously in size.The genomes of living organisms vary enormously in size.


Genomicists look at two basic features of Genomicists look at two basic features of genomes: sequence and polymorphism.genomes: sequence and polymorphism.

Major challenges to determine sequence of each Major challenges to determine sequence of each chromosome in genome and identify many chromosome in genome and identify many polymorphisms:polymorphisms: How does one sequence a 500 Mb chromosome 600 bp at a How does one sequence a 500 Mb chromosome 600 bp at a

time?time? How accurate should a genome sequence be?How accurate should a genome sequence be?

DNA sequencing error rate is about 1% per 600 bp.DNA sequencing error rate is about 1% per 600 bp. How does one distinguish sequence errors from How does one distinguish sequence errors from

polymorphisms?polymorphisms? Rate of polymorphism in diploid human genome is about 1 in 500 bp.Rate of polymorphism in diploid human genome is about 1 in 500 bp.

Repeat sequences may be hard to place.Repeat sequences may be hard to place. Unclonable DNA cannot be sequenced.Unclonable DNA cannot be sequenced.

Up to 30% of genome is heterochromatic DNA that can not be clonedUp to 30% of genome is heterochromatic DNA that can not be cloned


Divide and conquer strategy meets Divide and conquer strategy meets most challenges.most challenges.

Chromosomes are broken into small Chromosomes are broken into small overlapping pieces and cloned.overlapping pieces and cloned.

Ends of clones sequenced and reassembled Ends of clones sequenced and reassembled into original chromosome stringsinto original chromosome strings

Each piece is sequenced multiple times to Each piece is sequenced multiple times to reduce error rate.reduce error rate. 10-fold sequence coverage achieves a rate of 10-fold sequence coverage achieves a rate of

error less than 1/10,000.error less than 1/10,000.

10-7 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayFig. 10.2


Techniques for mapping and cloningTechniques for mapping and cloning

CloningCloning Library of DNA fragments 500 – 1,000,000 bpLibrary of DNA fragments 500 – 1,000,000 bp Insert into one of a variety of vectorsInsert into one of a variety of vectors

HybridizationHybridization Location of a particular DNA sequence within the library of fragmentsLocation of a particular DNA sequence within the library of fragments

PCR amplificationPCR amplification Direct amplification of a particular region of DNA ranging from 1 bp to > 20kbDirect amplification of a particular region of DNA ranging from 1 bp to > 20kb

DNA sequencingDNA sequencing Automated DNA sequencer using Sanger method determines sequences 600 bp at a Automated DNA sequencer using Sanger method determines sequences 600 bp at a

time.time. Computational toolsComputational tools

Programs for identifying matches between a particular sequence and a large Programs for identifying matches between a particular sequence and a large population of previously sequenced fragmentspopulation of previously sequenced fragments

Programs for identifying overlaps of DNA fragmentsPrograms for identifying overlaps of DNA fragments Programs for estimating error ratesPrograms for estimating error rates Programs for identifying genes in chromosomal sequencesPrograms for identifying genes in chromosomal sequences


Making a large scale linkage mapMaking a large scale linkage map Types of DNA polymorphisms used for large-scale mapping:Types of DNA polymorphisms used for large-scale mapping:

Single nucleotide polymorphisms (SNPs) – 1/500 – 1/1000 bp across genomeSingle nucleotide polymorphisms (SNPs) – 1/500 – 1/1000 bp across genome Simple sequence repeats (SSRs) – 1/20-1/40 kb across genomeSimple sequence repeats (SSRs) – 1/20-1/40 kb across genome

2-5 nucleotides is repeated 4-50 or more times.2-5 nucleotides is repeated 4-50 or more times. Most SNPs and SSRs have little or no effect on the organism.Most SNPs and SSRs have little or no effect on the organism. Serve as DNA markers across the chromosomesServe as DNA markers across the chromosomes Must be able to rapidly identify and assay in populations from Must be able to rapidly identify and assay in populations from

100s to 1000s of individuals100s to 1000s of individuals

Fig. 10.3


Genome wide identification of Genome wide identification of genetic markersgenetic markers

Initial genetic maps used SSRs which are Initial genetic maps used SSRs which are highly polymorphic.highly polymorphic.

Identified by screening DNA libraries with Identified by screening DNA libraries with SSR probesSSR probes

Amplified by PCR and length differences Amplified by PCR and length differences assayedassayed

SNPs – millions more recently identified by SNPs – millions more recently identified by comparison of orthologous regions of cDNA comparison of orthologous regions of cDNA clones from different individualsclones from different individuals


Homologous – genes with enough sequence Homologous – genes with enough sequence similarity to be related somewhere in evolutionary similarity to be related somewhere in evolutionary historyhistory

Orthologous – genes in two different species that Orthologous – genes in two different species that arose from the same gene in the two species’ arose from the same gene in the two species’ common ancestorcommon ancestor

Paralogous – arise by duplication within same Paralogous – arise by duplication within same speciesspecies

Orthologous genes are always homologous, but Orthologous genes are always homologous, but homologous genes are not always orthologous.homologous genes are not always orthologous.


SNPs and SSRs for genome coverageSNPs and SSRs for genome coverage

Until recently, maps were constructed from Until recently, maps were constructed from about 500 SSRs evenly spaced across about 500 SSRs evenly spaced across genome (1 SSR every 6 Mb).genome (1 SSR every 6 Mb).

SNPs provide more than 500,000 DNA SNPs provide more than 500,000 DNA markers across the genome.markers across the genome.


Genome wide typing of genetic Genome wide typing of genetic markersmarkers

Two-stage assay Two-stage assay for simple for simple sequence sequence repeatsrepeats PCR PCR

amplificationamplification Size separationSize separation

Fig. 10.4


Long range physical maps: karyotypes and genomic Long range physical maps: karyotypes and genomic libraries position markers on chromosomes.libraries position markers on chromosomes.

Physical mapPhysical map Overlapping DNA fragments ordered and oriented that Overlapping DNA fragments ordered and oriented that

span each of the chromosomesspan each of the chromosomes Based on direct analysis of DNA rather than Based on direct analysis of DNA rather than

recombination on which linkage maps are basedrecombination on which linkage maps are based Chart actual number of bp, kb, or Mb that separate a Chart actual number of bp, kb, or Mb that separate a

locus from its neighborslocus from its neighbors Linkage vs. physical mapsLinkage vs. physical maps

1 cM = 1 Mb in humans1 cM = 1 Mb in humans 1 cM = 2 Mb in mice1 cM = 2 Mb in mice


Vectors used for clone large inserts Vectors used for clone large inserts for physical mappingfor physical mapping

YACs (yeast artificial chromosomes) YACs (yeast artificial chromosomes) Insert size 100-1,000,000 MbInsert size 100-1,000,000 Mb

BACs (bacterial artificial chromosomes)BACs (bacterial artificial chromosomes) Insert size 50 – 300 kbInsert size 50 – 300 kb More stable and easier to purify from host DNA More stable and easier to purify from host DNA

than YACsthan YACs


How to determine order of clones How to determine order of clones across genomeacross genome

Overlapping inserts help align cloned Overlapping inserts help align cloned fragments.fragments. Bottom-up approach – overlapping sequences Bottom-up approach – overlapping sequences

of tens of thousands of clones determined by of tens of thousands of clones determined by restriction site analysis or sequence tag sites restriction site analysis or sequence tag sites (STSs)(STSs)

Top-down approach – insert is hybridized Top-down approach – insert is hybridized against karyotype of entire genome.against karyotype of entire genome.


Identifying and isolating a set of overlapping Identifying and isolating a set of overlapping fragments from a libraryfragments from a library

Two approaches:Two approaches: Linkage maps used to derive a physical mapLinkage maps used to derive a physical map

Set of markers less than 1 cM apartSet of markers less than 1 cM apart Use markers to retrieve fragments from library by Use markers to retrieve fragments from library by

hybridization.hybridization. Construct contigs – two or more partially overlapping cloned Construct contigs – two or more partially overlapping cloned

fragments.fragments. Chromosome walk by using ends of unconnected contigs to Chromosome walk by using ends of unconnected contigs to

probe library for fragments in unmapped regionsprobe library for fragments in unmapped regions Physical mapping techniques:Physical mapping techniques:

Direct analysis of DNADirect analysis of DNA Overlapping clones aligned by restriction mappingOverlapping clones aligned by restriction mapping Sequence tag segments (STSs)Sequence tag segments (STSs)


Physical mapping by analysis of STSsPhysical mapping by analysis of STSsBottom-up approachBottom-up approach

Each STS represents a unique segment of the genome amplified by

PCR.

Fig. 10.5


Human KaryotypeHuman Karyotype

(a) Complete set (a) Complete set of human of human chromosomes chromosomes stained with stained with Giemsa dye Giemsa dye shows bands.shows bands.

(b) Ideograms (b) Ideograms show idealized show idealized banding pattern.banding pattern.

Fig. 10.6 a, b


Chromosome 7 at three levels of resolutionChromosome 7 at three levels of resolution

Fig. 10. 6 c


FISH protocol for top-down approachFISH protocol for top-down approach

Fig. 10.8


Sequence maps show the order of Sequence maps show the order of nucleotides in a cloned piece of DNA.nucleotides in a cloned piece of DNA. Two strategies for sequence human genome:Two strategies for sequence human genome:

Hierarchical shotgun approachHierarchical shotgun approach Whole-genome shotgun approachWhole-genome shotgun approach

Shotgun – randomly generated overlapping Shotgun – randomly generated overlapping insert fragments:insert fragments: Fragments from BACsFragments from BACs Fragments from shearing whole genomeFragments from shearing whole genome

Shearing DNA with sonicationShearing DNA with sonication Partial digestion with restriction enzymesPartial digestion with restriction enzymes


Hierarchical shotgun strategyHierarchical shotgun strategyUsed in publicly funded effort to sequence human genomeUsed in publicly funded effort to sequence human genome

Shear 200 kb BAC clone Shear 200 kb BAC clone into ~2 kb fragmentsinto ~2 kb fragments

Sequence ends 10 timesSequence ends 10 times Need about 1700 plasmid Need about 1700 plasmid

inserts per BAC and about inserts per BAC and about 20,000 BACs to cover 20,000 BACs to cover genomegenome

Data form linkage and Data form linkage and physical maps used to physical maps used to assemble sequence maps assemble sequence maps of chromosomesof chromosomes

Significant work to create Significant work to create libraries of each BAC and libraries of each BAC and physically map BAC physically map BAC clonesclones

Fig. 10.9


Whole-genome shotgun sequencingWhole-genome shotgun sequencing

Private company Celera used to sequence whole human genome.Private company Celera used to sequence whole human genome. Whole genome randomly Whole genome randomly

sheared three timessheared three times Plasmid library constructed Plasmid library constructed

with ~ 2kb insertswith ~ 2kb inserts Plasmid library with ~10 kb Plasmid library with ~10 kb

insertsinserts BAC library with ~ 200 kb BAC library with ~ 200 kb

insertsinserts Computer program assembles Computer program assembles

sequences into chromosomes.sequences into chromosomes. No physical map constructionNo physical map construction Only one BAC libraryOnly one BAC library Overcomes problems of repeat Overcomes problems of repeat

sequencessequences

Fig. 10.10


Limitations of whole genome Limitations of whole genome sequencingsequencing

Some DNA can not be cloned.Some DNA can not be cloned. e.g., heterochromatine.g., heterochromatin

Some sequences rearrange or sustain Some sequences rearrange or sustain deletions when cloned.deletions when cloned.

Future large genome sequencing will use Future large genome sequencing will use both shotgun approaches.both shotgun approaches.


Sequencing of the human genomeSequencing of the human genome

Most of draft took place during last year of Most of draft took place during last year of project.project. Instrument improvements – 500,000,000 bp/dayInstrument improvements – 500,000,000 bp/day Automated factory-like production line Automated factory-like production line

generated sufficient DNA to supply sequencers generated sufficient DNA to supply sequencers on a daily basis.on a daily basis.

Large sequencing centers with 100-300 Large sequencing centers with 100-300 instruments – 150,000,000 bp/dayinstruments – 150,000,000 bp/day


Integration of linkage, physical, and Integration of linkage, physical, and sequence mapssequence maps

Provides check on the correct order of each Provides check on the correct order of each map against other twomap against other two

SSR and SNP DNA linkage markers readily SSR and SNP DNA linkage markers readily integrated into physical map by PCR integrated into physical map by PCR analysis across insert clones in physical mapanalysis across insert clones in physical map

SSR, SNP (linkage maps), and STS markers SSR, SNP (linkage maps), and STS markers (physical maps) have unique sequences 20 (physical maps) have unique sequences 20 bp or more, allowing placement on sequence bp or more, allowing placement on sequence map.map.


Changes in biology, genetics and genomics from Changes in biology, genetics and genomics from human genome sequencehuman genome sequence

Genetics parts listGenetics parts list Speeds gene-finding and gene-function analysisSpeeds gene-finding and gene-function analysis

Sequence identification in second organism through Sequence identification in second organism through homologyhomology

Gene function in one organism helps understand Gene function in one organism helps understand function in another for orthologous and paralogous function in another for orthologous and paralogous genesgenes

Genes often encode one or more protein domainsGenes often encode one or more protein domains Allows guess at function of new protein by comparison of Allows guess at function of new protein by comparison of

protein sequence in databases of all known domainsprotein sequence in databases of all known domains Ready access to identification of known human Ready access to identification of known human

polymorphismpolymorphism Speeds mapping of new organisms by comparisonSpeeds mapping of new organisms by comparison

e.g., mouse and human have high similarity in gene content and e.g., mouse and human have high similarity in gene content and orderorder


Major insights from human and Major insights from human and model organism sequencesmodel organism sequences

Approximately 25,000 human genesApproximately 25,000 human genes Genes encode noncoding RNA or proteins.Genes encode noncoding RNA or proteins. Repeat sequences are > 50% of genome.Repeat sequences are > 50% of genome. Distinct types of gene organization:Distinct types of gene organization:

Gene familiesGene families Gene rich regionsGene rich regions

Combinatorial strategies amplify genetic information and Combinatorial strategies amplify genetic information and increase diversity.increase diversity.

Evolution by lateral transfer of genes from one organism to Evolution by lateral transfer of genes from one organism to anotheranother

Males have twofold higher mutation rate than females.Males have twofold higher mutation rate than females. Human races have very few unique distinguishing genes.Human races have very few unique distinguishing genes. All living organisms evolve from a common ancestor.All living organisms evolve from a common ancestor.


Conserved Conserved segments of segments of

syntenic blocks syntenic blocks in human and in human and

mouse genomesmouse genomes

Fig. 10.12


Noncoding RNA genesNoncoding RNA genes

Transfer RNAs (tRNAs) – adaptors that translate Transfer RNAs (tRNAs) – adaptors that translate triplet code of RNA into amino acid sequence of triplet code of RNA into amino acid sequence of proteinsproteins

Ribosomal RNAs (rRNAs) – components of Ribosomal RNAs (rRNAs) – components of ribosomeribosome

Small nucleolar RNAs (snoRNAs) – RNA Small nucleolar RNAs (snoRNAs) – RNA processing and base modification in nucleolusprocessing and base modification in nucleolus

Small nuclear RNAs (sncRNAs) - spliceosomesSmall nuclear RNAs (sncRNAs) - spliceosomes


Protein coding genes generate the Protein coding genes generate the proteome.proteome.

Proteome – collective translation of 30,000 protein Proteome – collective translation of 30,000 protein coding genes into proteinscoding genes into proteins

Complexity of proteome increase from yeast to Complexity of proteome increase from yeast to humans.humans. More genesMore genes Shuffling, increase, or decrease of functional modulesShuffling, increase, or decrease of functional modules More paralogsMore paralogs Alternative RNA splicing – humans exhibit significantly Alternative RNA splicing – humans exhibit significantly

moremore Chemical modification of proteins is higher in humans.Chemical modification of proteins is higher in humans.


Protein coding genes generate the proteomeProtein coding genes generate the proteomeHow transcription factor protein domains have expanded How transcription factor protein domains have expanded

in specific lineagesin specific lineages

Fig. 10.11


Examples of domain accretions in Examples of domain accretions in chromatin proteinschromatin proteins

Fig. 10.13


Number of distinct domain architectures in Number of distinct domain architectures in four eukaryotic genomesfour eukaryotic genomes

Fig. 10.14


Repeat sequences fall into five classes.Repeat sequences fall into five classes.

Transposon-derived repeatsTransposon-derived repeats Processed pseudogenesProcessed pseudogenes SSRsSSRs Segmental duplications of 10-300 kbSegmental duplications of 10-300 kb Blocks of repeated sequences at centromere, Blocks of repeated sequences at centromere,

telomeres and other chromosomal featurestelomeres and other chromosomal features


Repeat sequences constitute more Repeat sequences constitute more than 50% of the genome.than 50% of the genome.

Fig. 10.15


Gene organization of genomeGene organization of genome

Gene familiesGene families Closely related genes clustered or dispersedClosely related genes clustered or dispersed

Gene-rich regionsGene-rich regions Functional or chance events?Functional or chance events?

Gene desertsGene deserts Span 144 Mb or 3% of genomeSpan 144 Mb or 3% of genome Contain regions difficult to identify?Contain regions difficult to identify?

e.g., big genes – nuclear transcript spans 500 kb or e.g., big genes – nuclear transcript spans 500 kb or more with very large introns (exons < 1% of DNA)more with very large introns (exons < 1% of DNA)


Genome has a distinct organization. Genome has a distinct organization. Gene family – olfactory receptor gene familyGene family – olfactory receptor gene family


Class II region of human major Class II region of human major histocompatibility complex contains histocompatibility complex contains

60 genes in 700 kb60 genes in 700 kb

Fig. 10.17


Combinatorial strategiesCombinatorial strategies

At DNA level – T-cell receptor genes are encoded by a multiplicity of At DNA level – T-cell receptor genes are encoded by a multiplicity of gene segments.gene segments.

At RNA level – At RNA level – splicing of exons in splicing of exons in different ordersdifferent orders

Fig. 10.19a

Fig. 10.18


Lateral transfer of genesLateral transfer of genes

> 200 human genes may arise by transfer > 200 human genes may arise by transfer from organisms such as bacteria.from organisms such as bacteria.

Lateral transfer is direct transfer of genes Lateral transfer is direct transfer of genes from one species into the germ line of from one species into the germ line of another.another.


Twofold higher mutation rate in Twofold higher mutation rate in malesmales

Comparison of X and Y chromosomesComparison of X and Y chromosomes Same may be true for autosomes, but Same may be true for autosomes, but

difficult to measure.difficult to measure. Majority of human mutations arise in Majority of human mutations arise in

males.males. Males give rise to more defects, but also Males give rise to more defects, but also

more diversity.more diversity.


Human races have similar genes.Human races have similar genes.

Genome sequence centers have sequenced Genome sequence centers have sequenced significant portions of at least three races.significant portions of at least three races.

Range of polymorphisms within a race can Range of polymorphisms within a race can be much greater than the range of be much greater than the range of differences between any two individuals of differences between any two individuals of different races.different races.

Very few genes are race specific.Very few genes are race specific. Genetically, humans are a single race.Genetically, humans are a single race.


All living organisms are a single All living organisms are a single race.race.

All living organisms have remarkably All living organisms have remarkably similar genetic components.similar genetic components.

Life evolved once and we are descendents Life evolved once and we are descendents of that event.of that event.

Analysis of appropriate biological systems Analysis of appropriate biological systems in model organisms provides fundamental in model organisms provides fundamental insight into corresponding human systems.insight into corresponding human systems.


In the future, other features of chromosomes In the future, other features of chromosomes will become increasingly important.will become increasingly important.

Chemical modification of basesChemical modification of bases Understand DNA methylation nowUnderstand DNA methylation now Others may be discoveredOthers may be discovered

Interaction of various proteins with chromosomeInteraction of various proteins with chromosome Three dimensional structure of proteins in nucleusThree dimensional structure of proteins in nucleus

May determine interactions of chromosomal regions May determine interactions of chromosomal regions with regions of nuclear envelopewith regions of nuclear envelope

More effective tools need to be developed to More effective tools need to be developed to examine chromosome features.examine chromosome features.



High-throughput instrumentsHigh-throughput instrumentsDNA sequencerDNA sequencer

Fig. 10.20


High-throughput High-throughput instrumentsinstruments

e.g, microarrayse.g, microarrays

Fig. 10.21


Two color - DNA microarray

Fig. 10.22


Analysis of genomic and RNA Analysis of genomic and RNA sequencessequences

Quantitative analysis of mRNA levelsQuantitative analysis of mRNA levels Serial analysis of gene expression (SAGE)Serial analysis of gene expression (SAGE)

Small cDNA tags of 15 bp from 3’ ends of mRNA are Small cDNA tags of 15 bp from 3’ ends of mRNA are linked and sequenced.linked and sequenced.

Massively parallel signature sequence (MPSS)Massively parallel signature sequence (MPSS) Transcriptome – population of mRNAs expressed in Transcriptome – population of mRNAs expressed in

a single cell or cell typea single cell or cell type MPSS allows identification of most of cell’s rarely MPSS allows identification of most of cell’s rarely

expressed mRNAsexpressed mRNAs


Lynx therapeutics sequencing Lynx therapeutics sequencing strategy of MPSSstrategy of MPSS

Fig. 10.24


Systems Biology – the global study of multiple components Systems Biology – the global study of multiple components of biological systems and their interactionsof biological systems and their interactions

New approach to studying biological New approach to studying biological systems has made possible:systems has made possible: Sequencing genomesSequencing genomes High-throughput platform developmentHigh-throughput platform development Development of powerful computational toolsDevelopment of powerful computational tools The use of model organismsThe use of model organisms Comparative genomicsComparative genomics


Human Genome Project has changed the Human Genome Project has changed the potential for predictive/preventive medicine.potential for predictive/preventive medicine.

Provided access to DNA polymorphisms Provided access to DNA polymorphisms underlying human variabilityunderlying human variability Makes possible identification of genes predisposing to Makes possible identification of genes predisposing to

diseasedisease Understanding of defective genes in context of Understanding of defective genes in context of

biological systemsbiological systems Circumvent limitations of defective genesCircumvent limitations of defective genes

Novel drugsNovel drugs Environmental controlsEnvironmental controls Approaches such as stem-cell transplants or gene therapyApproaches such as stem-cell transplants or gene therapy


Social, ethical, and legal issuesSocial, ethical, and legal issues

Privacy of genetic informationPrivacy of genetic information Limitations on genetic testingLimitations on genetic testing Patenting of DNA sequencesPatenting of DNA sequences Society’s view of older peopleSociety’s view of older people Training of physiciansTraining of physicians Human genetic engineeringHuman genetic engineering

Somatic gene therapy – inserting replacement genesSomatic gene therapy – inserting replacement genes Germ-line therapy – modifications of human germ lineGerm-line therapy – modifications of human germ line

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