Genetic mapping Doc. MUDr. Ing. RNDr. Peter Celec, DrSc., MPH - PowerPoint Presentation

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Genetic mapping

Doc. MUDr. Ing. RNDr. Peter Celec, DrSc., MPHpetercelec@gmail.comwww.imbm.sk

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Linus

Carl

Pauling

(1901 – 1994)

Nobel

prize1954 –

Chemistry1962 – Peace

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Francis

Crick

(1916 – 2004)

Nobel

p

rize

1962 –

Physiology

or

m

edicine

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James

Dewey

Watson

(1928 – )

Nobel

prize

1962 –

Physiology

or

m

edicine

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Frederick

Sanger

(1918 –

2013)

Nobel

Prize

1958 –

Chemistry

1980 –

Chemistry

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Kary

Banks

Mullis

(1944 – )

Nobel

prize

1993 –

Chemistry

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John Craig Venter

(1946

-

)

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Francis

 

Sellers

 

Collins

(1950

- )

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DNA

is

information

!!!

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Genetic mapping – Why

?Diseases and traits

Identification

and

localisation

of

genesCave! HeritabilityTwin studies

Cystic fibrosis, fracture, intelligence...Mendelian inheritance

Genetic

vs

physical

mapscM vs. MbpMeiosis... Many

meioses

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Genetic mapping – How

?Linkage analysis

Co-segregation

Rare

–

large

effect sizeAssociation studies

GWASCommon – low effect sizeMarkers linked

with

genes of interest

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Genetic mapping – How?

Recombinants vs non-recombinants

Frequency

of

recombinations

vs. genetic distanceGenetic maps

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Gene mapping

LinkedGenes with recombination frequencies less than 50 percent are on the same chromosome

Linkage group

all

known genes on a chromosome

Unlinked

Genes with a recombination frequency of 50 percent

are located on nonhomologous chromosomes or far apart on the same chromosomeInterferenceCrossover in one

region

decreases the propability

of a crossover in a nearby region

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Recombination

1 Mbp = 1 cM... 0,5-50 cM

Thomas

Hunt

Morgan

Hot spots – 1-2 kbp – 10 bpDesserts

Sex differencesHaldane-Huxley ruleHeterogametic sexAchiasmate meiosis

Suppression

from

gonosomesY chromosome

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Genetic markers

PolymorphismsMendelian inheritance

More

markers

–

denser

mapDenser map – less meioses are

needed for linkage evidenceHeterozygosity – informative meiosis

RFLP

Minisatellite

– VNTR

Microsatellite – STR

SNP

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RFLP

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LOD

Logarithmus of oddsLogarithmus of the

ratio

...

Propability

of a linkagePropability of no linkageLod > 3 Linkage

Lod < -2No linkage

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Multipoint mapping

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Autozygosity

Homozygosity for markers identical by

descent

/state

Recent

common acestorInbreedingRecessive diseasesAlready

few affected families might be informative

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Physical mapping

Restriction mapping – low

resolution

Endonucleases

FISH –

low

resolutionFluorescent in situ hybridizationShort

tagged site (STS) mappingShort sequences mapped by PCR

Sequencing

based

on known gene

markers

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Positional cloning

Identification of a gene responsible

for

a

trait

Linkage

analysisGenomic associationsBioinformaticsNo

biochemistryFine mappingcDNA

library

Zoo-

Blot

– evolution of conserved

regionsUnmethylated CpG Islands – near genes

Exon

trapping

–

expression

vecors

–

mRNA

length

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Top-down approach

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Bottom-up approach

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Contig(uous)

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Arrays

Expression cDNA arraysSNP arrays

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Outcomes?

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Individualized medicine

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Personalized medicine

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Personalized medicine

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23andme

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23andme

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Reverse genetics

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Nobel Prize in Physiology or Medicine 2007

Mario R. Capecchi

Sir Martin J. Evans

Oliver

Smithies

1/3 of the prize

1/3 of the prize

1/3 of the prizeUSA

United Kingdom

USA

University of Utah

Salt Lake City, UT, USA; Howard Hughes Medical Institute

Cardiff University

Cardiff, United Kingdom

University of North Carolina at Chapel Hill

Chapel Hill, NC, USA

b. 1937

(in Italy)

b. 1941

b. 1925

(in United Kingdom)

"for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells"

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GM mice

Knock-outHomologous recombination

Knock

-in

Transposons

Homologous

recombinationTissue-specific expressionInsulin receptor

AlbuminInducible expressionVEGFMECP2

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Knock-in mice

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Knock-in mice

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Cre-loxPcyclization

recombination – Cre locus of X-over

P1 –

phage

Similar

to FLP-FRT

recombinationFlippaseFlippase recognition target

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Tetracycline system

Tetracycline-controlled transactivatorTamoxifen

Conditional

knock-out

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Animal models of diseases

Surgical Pharmacological

Genetic

Interpretation

Interspecies

differencesReproducibilitystrainsValidityAutism

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Genome

editing

Zinc

finger

nucleases (ZFN)

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Genome editing

Transcription activator-like effector nuclease

(TALEN)

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CRISPR-Cas9

Jennifer Doudna (1963-)

Feng

Zhang

(1982-)

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CRISPR-Cas9

Bacterial

immunity

against

phagesClustered Regularly Interspaced Short Palindromic Repeats

CRISPR-associated nucleaseTrans-acivating CRISPR RNA

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CRISPR-Cas9 applications

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CRISPR-Cas9

Addgenehttps://www.addgene.org/CRISPR/guide/Animations

https://

www.youtube.com/watch?v=2pp17E4E-O8

Doudna

J

https://www.youtube.com/watch?v=SuAxDVBt7kQZhang Fhttps://

www.youtube.com/watch?v=EjolOzkYNlk

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Genetic modifications in humans

?

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RNA interference

Silencing of genesPost-transcriptional regulation of gene expressionsiRNA, shRNA, miRNA...Gene function

Pathogenesis

Therapy

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Nobel Prize in Physiology or Medicine 2006

Andrew Z. Fire

Craig C. Mello

1/2 of the prize

1/2 of the prize

USA

USA

Stanford University School of Medicine

Stanford, CA, USA

University of Massachusetts Medical School

Worcester, MA, USA

b. 1959

b. 1960

"for their discovery of RNA interference - gene silencing by double-stranded RNA"

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RNA interference

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www.imbm.sk

petercelec@gmail.com