7/24/2019 Gene Discovery

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Application ofrecombinant DNAtechnology and

forward/reversegenetics

7/24/2019 Gene Discovery

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Applied molecular biology

Treatment of diabetes with synthetic insulin

7/24/2019 Gene Discovery

3/23

Clone the insulin gene for expression inbacteria

insulin cDNA

oriV

ampR

7/24/2019 Gene Discovery

4/23

cDNA vs genomicDNA

cDNA contains the sequences in processedmRNAExons ntrons

!romoter

"# $TR

DNA

!re%mRNA

mRNA AAAAAAAAAAA

$TR 'untranslated region(

cDNA TTTTTTTTTTT

Exons

Transcription

Reversetranscripti

on

Processing

7/24/2019 Gene Discovery

5/23

Making cDNA

solation of mRNA from other cellular RNAs

TTTTTTT

TTTTTTT

TTTTTTT

AAAAAAAAA

AAAAAAAAA

AAAAAAAAA

AAAAAAAAA

AAAAAAA

AAAAAA

AAAAAAA

AAAAAAA

AAAAAA

AAAAAAA

)ligo%dT

7/24/2019 Gene Discovery

6/23

Making cDNA

*ynthesising +rst strand of cDNA from RNA Re,erse transcriptase- an RNA%dependent DNA

polymerase

AAAAAAAAA

TTTTTTTTT

"#

)ligo%dT

primerAAAAAAAAA

TTTTTTTTT

"#

RT

TTTTTTTTTAAAAAAAAA

"#

"

#

Retro,irus

RT

.)%

Treat

withRNase

7/24/2019 Gene Discovery

7/23

Amplify insulin cDNA byPCR

"#

"#

TTTTTTTTT

dAT!/ d0T!/ dCT!/ dTT!Taq polymerase

TTTTTTTTT

TTTTTTTTT

TTTTTTTTT

7/24/2019 Gene Discovery

8/23

igate into vector and transform bacteria

7/24/2019 Gene Discovery

9/23

!ene discovery

0ene sequence0ene function

DNA sequence

!henotype

.omologous gene

Dont haveany sequenceinformation

Have somesequenceinformation

Have thegenessequence

Understand thegenes function

0enomesequenc

e!rotei

n

7/24/2019 Gene Discovery

10/23

!ene discovery " forwardgenetics

0ene sequence0ene function

DNAsequence

!henotype

.omologous

gene

Dont haveany sequenceinformation

Have somesequenceinformation

Have thegenessequence

Understand thegenes function

0enomesequenc

e!rotei

n

7/24/2019 Gene Discovery

11/23

Review# terminology ocus'pl1 loci( 2

location of a geneor other DNAsequence in a

genome !ene2 a

functional DNAsequence in a

genome Allele2 a ,ariant

of the DNA

sequence at ai,en locus

A locusThere is a gene here

Another locusThere is another gene here

And another locus

No gene exists here

An allele Another allele

An allele *ame allele

7/24/2019 Gene Discovery

12/23

Molecular markers

Markersare loci that act as indicators orsignposts Molecular markers are basically a speci+c DNA

sequence at a 3nown location on a chromosome Restriction fragment length polymorphisms 'R45!s( are

simple molecular mar3ers

CT

GC

AG

GA

CG

TC

CT

GTA

G

GA

CAT

C

CT

GC

AG

GA

CG

TC

CT

GC

AG

GA

CG

TC

CT

GC

AG

GA

CG

TC

CT

GC

AG

GA

CG

TC

!olymorphism

7/24/2019 Gene Discovery

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$orward genetics 4rom phenotype to gene 2 6classical7

genetics *tudy phenotypes of 6mutants7 to wor3 out

genetic basis8ild%type )bese

9ice- http-::www1hhmi1org:genesweshare:d;&

7/24/2019 Gene Discovery

14/23

$orward genetics >reeding experiments to +nd and con+rm

a molecular mar3er that is ?lin3ed# to thephenotype-The DNA sequence around this molecular

mar3er can then be determined to +nd the Obgene sequence

5in3age mapping

GC

TA

GC

TA

GT

CG

TA

GC

TA

GG

AT

GC

TA

CCG

ATAT

CGCG

ATA

GC

TC

TA

GG

AOb locus

D=Rc3;& locus

Determine DNA

sequence

9ice- http-::www1hhmi1org:genesweshare:d;&

7/24/2019 Gene Discovery

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!ene discovery " forwardgenetics

0ene sequence0ene function

DNAsequence

!henotype

.omologousgene

Dont haveany sequenceinformation

Have somesequence

information

Have thegenes

sequence

Understand thegenes function

0enomesequenc

e!rotei

n

di

7/24/2019 Gene Discovery

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!ene discovery " reversegenetics

0ene sequence0ene function

!rotein

DNAsequence

!henotype

.omologousgene

Dont haveany sequenceinformation

Have somesequence

information

Have thegenes

sequence

Understand thegenes function

0enomesequenc

e

7/24/2019 Gene Discovery

17/23

Reverse genetics

CGATATGGCGATACGAT

GCTACAATGCGACCACG

TAGCTATGCAGTCGTAG

TAGTAGCGGTAGCCCCC

AGTACCGGAGGGGCGGT

AGCTAGCTAGCCCCGGC

TAGTCGAGGCTAGCTGA

TCATCTGGCGGATTAGC

GGATCGATACGGCCGCGCACTATCATGCTAGCTC

GCGCGGAGCTAGCTAGT

ACGCATGACTGCATGCA

TGCATGCATGACTGCAT

9utate

*equencegenome

bidopsis images- D) ;

7/24/2019 Gene Discovery

18/23

Reverse genetics "%nding a gene

*equence the genome/ typically ofmodel organisms 9any sequencing techniques

>ene+ts of using model organisms

Easy to manipulate genetically Easy to grow/ short life cycles

8hy can#t we Bust stop at thegenome sequence A long string of A/ T/ 0/ C doesn#t

meananything

Need to +nd genes within the letters/and disco,er their function

us musculus

Drosophila

east

!" coli

#rabidopsis

R i

7/24/2019 Gene Discovery

19/23

Reverse genetics "%nding a gene

*earch the genomedata 4ind homologous

genes *earch genome using

the sequence ofanother gene:proteinof similar function

!redict new genes

by loo3ing for- *tart and stop codons

Transcription translation initiation

ATG (NNN)nTAA

TAG

TGA

0ene sequence

!rotein

.omologousgene

Have somesequence

information

Have thegenes

sequence

0enomesequenc

e

CCAATTATA

Kozak consensus sequence

Shine-Dalgarno sequence

Polyadenylaion signals

7/24/2019 Gene Discovery

20/23

Reverse genetics " from geneto function

Disco,er the gene#s function by editing it E1g1 ma3e a transcriptional fusion

!romoter for gene of interest is cloned next to a?reporter# gene

E1g1 ma3e a translational fusion 0ene of interest is

cloned next to the04! gene

C!F;