L20Biol261Genomics2014F

8/9/2019 L20Biol261Genomics2014F

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Lecture 22, Genomics

Genetic Analyses(1)FORWARD analysis began with classical transmission methods tracing the

inheritance of mutant alleles or chromosomes.

(2) REVERSEanalysis begins with mutating a reported gene sequence(knockout), tracing its inheritance and identifying its phenotypicexpression.

I find it sometimes very difficult to tell what someone means when they talk aboutgenes because we dont share the same definition, says developmental geneticistWilliam Gelbert of Harvard University in Cambridge, Massachusetts.

You have already struggled with multiple definitions of a gene, based on transmissionand cytological genetics. Now, using your understanding of molecular genetics, propose

a gene definition and explain why it would be consistent with the requirements of agene (see lecture 11), practical and comprehensive.


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Increasingly,forward genetics, change of functioninsertions, and gene knockouts or eventually

replacement, all depend on knowing something aboutsequence, and locationof a genein a genome

- TODAY Genomics and its derivatives:


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Genome: a (one) completeset of an organisms genetic

information, or usually, one complete set of chromosomes

(monoploid), sometimes, nuclear DNA content.

The original definition ofGenomics included:

(1)Mappingchromosomes

(2)Sequencingchromosomes and identifying genes

(3) Analyzing thefunctionsof entire genomes

Currently genomics is divided into several fields of study:

Structural genomics-the study of genome structure

Comparative genomics-the study of genome diversity/evolution

Bioinformatics- information from sequence structure

Functional genomics-transcriptome(complete set of RNAs

transcribed from a genome) and theproteome(the complete set

of proteins transcribed from a genome).

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But first, Sequencing the Human Genome

Clone by Clone method:Map first,

sequence later (publicly fundedHuman Genome Project)

Shotgun Method:Sequence

first, map later (Celeracorporation)

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Genetic mapsof chromosomes are based on recombination

frequencybetween markers:

Low density- limit ~ 1% recombination is a practical limit-

limited by the number of bodies you have to measure- for mostvisibly-expressed genes in eukaryotic breeding studies.

Higher densitygenetic maps use restriction sites,andgene

localization probesas landmarks.

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Genetic mapsof chromosomes start with

ordinal distance based on recombination

frequency between visible markers.

Low density Cytogenic (cytological,

ideogram) mapsare based on the location

of markers within or near cytological

features, microscopically visible.

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Low Density Genetic mapsof chromosomes are based on

recombination frequency between visible markers.

Cytogenic (cytological, ideogram) mapsare based on the location of

markers within or near cytological features.

High density mapsintegrate cytological and physical maps.

Anchor markerscorrelate the cytogenic mapsto the physical.Chromosome fragments

can beidentified by migration pattern (RFLP) , taggedusing PCRto amplify short,

unique(200-500bp)Sequence TaggedSites(STS), or short cDNAsequence probes

(ExpressedSequence Tags orEST) and Short (orSimple) Sequence Length

Polymorphisms (SSLPor short repetitive elements). These tagged fragments of known

sequence can be related to the cytogenic map (probewith complementary).

Physical maps are measured in base pairs, kilobase pairs or megabase pairs, they often

show the location of overlapping genomic fragment clones (contigs)and unique

sequences (STS).

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1996-7:Anchoring the

physical map:

2335 microsatellite (SSLP)

sites,16,000 STSmarkedloci &RFLPs used to map

1600 human genes

15Human chromosome 1 fig 4-20

Restriction

fragments

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Ordered Clone by Clone method:

map first sequence later:Screen large

clones (BAC) from a chromosome

library for known sites (restrictionsites, known genes, or other sequence

to anchor the map.

HindIII digest, agarose gel

electrophoresis on fragmented clone:

Stain, characterize fragments by

migration distance.

Share a partial fragment ? = overlap

(different clones share sequence)

use overlaptoorient the clone

fragments into a map

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Physical maps, are built by reconstructing the order of fragments cut by restriction enzymes. The

first cloning vector is usually a YAC. For example, five YACS were known to hybridize to 1

chromosome band ( 17q2 ). A restriction enzyme cutting an 8 base palindrome sequence having a

low sequence probability (on average 48 or every 66,000 bases) was used to cut the chromosome

fragment. The fragments were denatured and the 5 single stranded radioactive YACSwerehybridized to the blots of the digest to visualize the target chromosome fragments. The

autoradiogram is below. Order of band fragments?

1

2

3

Chromosome

band map of

RFLP fragments

The exposed

photographic image of

lanes (columns) corr-

esponding to the same

chromosome DNA

tested with 5 different

YAC probes.

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+

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Map first sequence

later: clone by clone,or ordered clone

approach - public.

Minimum tiling path-fewest clones

necessary to get a

complete sequence

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Whole genome Shotgun method

Overlap: - the overlap

is determined directly

by sequence,not

indirectly by fragment

length.

Most of the genome is

sequenced many

(10-15) times to get the

correct overlap

Small insert clones

are prepared

directly from DNA

& sequenced

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(2)Paired end reads:each

clone isprimed fromtwo different ends of a

vector, which is known

and PCR the

intervening sequence,

producing an endtagged linear sequence.

End tagged, multiple

inserts may then be

overlapped to produce

a sequence contig

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Where do the fragments overlap ?


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The overlapof sequences in regions of identity can be

used to make contiguous sequences.

(1) GATCTCGCCGCGTTGGAGAAGGACTACGAGGAGGTTGGCTCTGAGTCCGAC

TCTGAGTCCGACCCGTATCC

(2) ATGATGATGATGAGGATGGCGATGATGGTGACGAGTACTAG

AGGATGGCGATGATGGTGACGAGTACTAGAGGAGTCGTCGTCGTCTGGGGGCT

(3) TGATGTTCTGTGTGTCAAGGCCTGATTGATAACTGCTGCTATCCCATGATCTGCCAGTGT

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Problem?Repetitive sequence gaps,

Using clones containing

fragments of different sizes

(different restriction enzymes)

there will be overlap

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Sequencing DNA fragments


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DNA SEQUENCING- Sanger Method: A cloned fragment of DNA

is sequenced by using:

(1)aspecificprimerpiece of DNA(oligonucleotide) to replicate theDNAfrom a known, pre-defined starting point.

(2)a spikeof radioactive dideoxy-nucleotides(ddATP or ddCTP,

ddGTP, ddTTP) are incorporated with excess normaldATP, dCTP,

dGTP, dTTP , + DNA polymerase,

The ddNTPs are randomly

incorporated and terminate

the strand elongation

(3)electrophoresis, visualizethe DNA

(4)read the fragment order (by size)

(5)reconstruct the complementaryoriginal sequence

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Automated Sequencing

uses flourescent tags for

each ddNTP reaction.

The sequencing reaction

can be done in a tube, and

it is read by a light

detector

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Pyrosequencingrequires single strand DNA (template), a DNA

primer DNA polymerase and dNTP. Read the sequence by the

chemiluminescence, powered by ATP produced by sulfurlase.

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The basic techniques for sequencing entire genomes:

(1) libraries (whole genome) (2) cloning vectors (3) PCR (4) DNA

sequencing machines (5) chromosome maps (6) computers

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The DNA sequence is the base for computer - assisted analyses

Structural genomicsinvolves the analysis of gene

sequence, gene number, order andphysical nature of chromosomes.

Comparative Genomics- similarity and divergence among genes with a

similar function in different species

Bioinformaticsis the use of computer analysis forstructural or functional genomics.

Proteomics the study of all the proteins of an organism.

Transcriptomics - transcript studies

Functional genomicsstudies the function of genes

gene expression, interactions between gene and proteins, and

between proteins

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Genomics is the study of genomes in their entirety.

Most Bacteria are now known by their sequence or

partial sequence, viruses can be sequenced in a day or two

Over 100 eukaryotic genomes have been sequenced including

Human MouseYeast Several fungi

Malaria parasite Mosquito

Arabidopsis Rice

Poplar tree

Many other species have a great deal of cDNA and gene sequences

especially ESTs - partial sequences of cDNA clones.

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93% gene similarity - many mutations are rearrangements


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Bioinformatics: (broadly)computational challenges

in biologyor (narrowly) the information content of

the genome. A first objective being theidentification of binding sites or thefunctional

elements

gene annotation.

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Bioinformatics(the information content) collates multiple sources of

information, including comparative genomic (BLAST search), cDNA

sequence and ORF to annotate a candidate gene sequence.

Sequenceinformation

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A codingtranscriptome(ics) represents that small percentage of thegenetic code that is (apparently) transcribed into RNA moleculesestimated to be less than 5% of thegenome in humans Adams J. (2008)Nature Education 1:1

It now appears that the majority of the human genome is transcribed(introns and intervening sequence), and the vast majority of sequences are non-protein coding (Frith et al.,2005). The proportion of transcribed sequencesthat are non-protein-coding appears to be greater in mammals compared tonematodes or drosophila.

Frith et al., 2005. E.J.H.G. 13:894-897


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The ENCODE PROJECT: identify and map all the transcribed regions of the human

genome including regulatory regions, replication origins, DNA methylation and histone

methylation sites etc..

The pilot project found among other interesting findings:

On average- per coding region:

- There are 5.4 different transcriptsper coding region, over half showed transcription

from both strands-exact reverse direction complements.

- 63% of the mouse genome is transcribed, 1-2% have recognizable exons- 41% span introns, 22% span intergenic regions.

-Majority of the human genome is transcribed

-Genome- islands of protein coding sequence - interwoven and overlapping

-transcription units spanning the genome

Gene definition ?Micro rNA - regulatory gene ?

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Proteomicsthe study of the proteome, the sequence andexpression of all proteins

25-35,000

genes,

100,000-30

0,000

differentproteins ?

(alternativesplicing, RNAediting, oralternative

transcriptioninitiation andterminationsites).Unknown fraction

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Genomic sequencing has made possible a new approach to genetics called

functional genomics, which focuses on genome-wide patterns of geneexpression and the mechanisms by which gene expression is coordinated

DNA microarray (or chip) - a flat surface about the size of a postage stampwith up to 100,000 distinct spots, each containing a different immobilizedoligotide DNA sequence, of all the known genes in a genomeor all the knowncDNA from a genomesuitable for hybridization with DNA or RNA isolated from cellsgrowing under different conditions

Functional Genomics- study of expression patterns

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unknown

known

Relate - which genes are

active in the tissue

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Every yeastgene was

cloned and

sampled are

spotted onto

glass slides


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Binding (color) intensity indicates RNA concentration- gene activity


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DNA chips use synthetic DNA, oligonucleotides, that

can be spotted at a density of 106/cm2 so fragments

from all human or other eukaryotic organisms can beannealed to the chip.

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29Transcriptional

regulation of ~

2500 genes

showingsignificant

changes during

the first 2.75

hours of C.

elegansdevelopment

Time minutesbefore (-2) to minutes

after (165) the 4 cell stage in

development (gastrulation)

Transcript

abundance relative

to a non-dividing

cell 37


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A glossaryof types of DNA sequence:

1. Full length cDNA- complement of the mRNA

2. Full length (eukaryotic) gene clone(exons, introns, flanking regions).

3. Restriction fragments, Restriction Fragment Length Polymorphisms4. SNPs - Single Nuclear Polymorphism(s)- nucleotide polymorphism

5. PCR clone- partial gene sequence

6. Large genomic clones:BACs or YACs may have the sequence of

many adjacent genes

7. Satellite DNA- mid and highly repetitive DNA including VNTRs,mini and microsatellite DNA

8.STS(sequence tagged sites)short unique sequences used to hybridize

to chromosomes

9. Expressed sequence tags ESTs -partial sequence of cDNAs used as

probes to ID chromosome locations, correlate RFLP and cytologicalmaps.

10. SSLP (short (simple) sequence length polymorphisms)-short

repetitive sequences use to anchor a map

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L20Biol261Genomics2014F