Lecture 2 DNA Amplification Printable Version

8/16/2019 Lecture 2 DNA Amplification Printable Version

1/40

1

Learning outcomes

CMB2000 – Lecture 1

Isolating DNA

Explain how PCRworks


2/40

ATGGCTGCTATTTCCAAAACTGTCAGAGAGGGAAGTC

TACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

1. Denature 94°C

CMB2000 – Lecture 2 2


3/40



1. Denature 94°C

2. Anneal by cooling

TTGACAGT

Hybridisation

Kary Mullis (1987)

awarded N!le "ri#e$ 199%

Polymerase Chain eaction !PC"

Taq DNA #olymerase

CTCTCCCTTCAG

$. %&tension



4/40



1. Denature 94°C

Polymerase Chain eaction !PC"CMB2000 – Lecture 2 4


5/40


CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCA

ACCG AGGG

1. Denature 94°C

2. Anneal #rimers



6/40



ACCG

AGGG

1. Denature 94°C

2. Anneal #rimers



7/40


CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCA AGGG

1. Denature 94°C

2. Anneal #rimers

ACCG



8/40


CTACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCA AGGGGATGGCTGCTATTTCCAAAACTGTCAGAG

ACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

Taq DNA #olymerase

ACCG

1. Denature 94°C

2. Anneal #rimers$. %&tension

Cycles

C&ies

1

2



9/40



AGGG ATGGCTGCTATTTCCAAAACTGTCAGAG

ACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

1. Denature 94°C


Cycles

C&ies

1

2



10/40



AGGG ATGGCTGCTATTTCCAAAACTGTCAGAG

ACCGACGATAAAGGTTTTGACAGTCTCTCCCTTCAG

ACCG

AGGG

ACCG

AGGG

ACGATAAAGGTTTTGACAGTCTCTCCC

ACGATAAAGGTTTTGACAGTCTCTCCC

TGGCTGCTATTTCCAAAACTGTCAGAG

TGGCTGCTATTTCCAAAACTGTCAGAG

1. Denature 94°C


Cycles

C&ies

1

2

2

'


0


11/40

1. Denature 94°C


Cycles

C&ies

2

'


1

1


12/40

1. Denature 94°C


Cycles

C&ies

2

'

%

8


2

C 2000 2 1


13/40

%

8

1. Denature 94°C


Cycles

C&ies

'

1


3

CMB2000 L t 2 1


14/40

'

1

1. Denature 94°C


Cycles

C&ies

10

102'

%0

107%7'182'

$ $ $


4

CMB2000 L t 2 1


15/40


'imits• 'og am#li(ication #hase starts to #lateau

• DNA #olymerase ma)es mista)es

• Insu((icient reagents to com#lete e&tension

• *ust be ultra+sterile in your lab s)ills

• Problem se,uences


CMB2000 L t 2 1


16/40


• 'og am#li(ication #hase starts to #lateau

• DNA #olymerase ma)es mista)es

• Insu((icient reagents to com#lete e&tension

• *ust be ultra+sterile in your lab s)ills

• Problem se,uences

Melt a& *rat +e,erated !y -A./MAK/ s*tware *r te

3C e4, swi,+ !ase &siti, a+ai,st te te&eraturere5uired t re,der 76 i, elical *r at tat &siti,


CMB2000 Lecture 2 1


17/40


PC

-asic

research

Phylogeny

Diagnostics

Agricultural

management orensics

Drug

de/elo#ment


CMB2000 Lecture 2 1


18/40


•-u((er (c,tai,i,+ M+)

• 0em#late :NA

• 2 #rimers tat *la,; te *ra+e,t * :NA t !e

a&li*ied• our nucleotides (dAT"$ dGT"$ dTT"$ dCT")

• 0hermostable DNA Polymerase


CMB2000 Lecture 2 1


19/40


• 0hermostable DNA Polymerase

5’-3’ polymerase activity

Taq DNA #olymerase I *r Thermus aquaticus• wr;s &tially at 2oC

Pfu DNA Polymerase *r Pyrococcus furiosus•

as %ity (re accurate)• wr;s &tially at oC


CMB2000 Lecture 2 2


20/40


9 ' ?C 6 2 ?C 7 2 ?C




21/40


0hermocyclers

9 ' ?C 6 2 ?C 7 2 ?C




22/40


Primers• 3ynthetic oligonucleotides 1+$5 bases long• Le,+t ,ly as l,+ as re5uired *r s&eci*icity

"rier 1 = ACCGTTACGCGGGC

• Pre/ent #rimer dimer (ormation

"rier 2 ATGCGCCCGGTTAAG

CMB2000 Lecture 2 22



23/40



• Pre/ent #rimer dimer (ormation

• Pre/ent sel( annealing

• A!se,ce * @air&i,s – , long sel(+com#limentary

se,uences i, a &rier ( % !&)




24/40






25/40



•

Annealing tem#erature must be similar (oreach #rimer in the #air

• Ideally ha/e a 6 or a C at the $7+end

• A/oid a 0 because most common to mismatch

6


26/40


PC cloning

• Adding additional se,uences to the endsthat 8ill be recognised by %

• Designing #rimers 8hich ha/e cut sites

• Produces #ieces o( DNA 8ith stic)y ends

GAATTCCTTAAG

GAATTCCTTAAG

GAATTCCTTAAG

GAATTCCTTAAG

6

2CMB2000 – Lecture 2


27/40

Describe ene!ic !rans"or#a!ion!echni$%es incl%&in !he %se o" plas#i&san& 'ec!ors

Learning outcomes

Isolating DNA7



28/40

GAATTCCTTAAG

GAATTCCTTAAG

GCTTAA

AATTCG

GCTTAA

AATTCG

Cut withEco

RI

DNA ligase(+ ATP)

GCTTAA

AATTCG

Recombinant DNA molecule

8



29/40

A A T T C

G

G C T T A

A

Eco RI

DNA ligaseATP

Plasmid vecto

DNA to inset

AATTC

G

G

CTTAA

Recombinant !lasmid

9

3


30/40

0

0rans(ormation



31/40

D heat shoc)

Plasmids and ectors

0rans(ormation

1 Cld CaCl21 Cld CaCl2



32/40

D heat shoc)

Plasmids and ectors

0rans(ormation

1 '2?C 5uic;ly

33



33/40

Plasmids and ectors3

Vector ( an) !)pe o" plas#i&* 'ir%s or+ar!i,cial chro#oso#e- !ha! has beenenineere& !o accep! !he inser!ion o"+"orein- D./ an& can be !aken %p b) li'in

cells an& be replica!e&* !ranscribe& an&!ransla!e&

34



34/40


Originof Replication

Ampr

gen

e 2.7 kb

pIC19

H

"electable ma#e

35



35/40


Originof Replication

2.7 kb

pIC19

H

"electable ma#e

Allows onl$ bacteial (E% coli ) cells containingthe !lasmid to suvive unde s!eci&icconditions (!esence o& antibiotic)%e%g% gene coding &o am!icillin esistance

Am!

Plate onam!icillin

36



36/40

"electable ma#e


Ampr

ene

2.7 kb

pIC19

H

N

Cl

Br

H

O

CH2OH

O

OH

OH

HO

O

CH2OH

O

OH

OH

HOH N

HO

Cl

Br

H

E.coli lacZ-

strain

Truncated β-galactosidase (lacks α-peptide). Inactive

pUC

β-galactosidaseα-peptide. Inactive

Non-covalent association

of two inactive components

giving active β-galactosidase

X-gal

urt!er reaction to

"lue insolu"le d#e

E coli tat +rw will!e !lue

37



37/40


!ultiple Cloning"ite

#pol$linker%Lac &

geneOriginof Replication

Ampr

ene

2.7 kb

pIC19

H

38



38/40


Lac &


Ampr

ene

2.7 kbp'C1( estictionen'$me sites in

C"

A#r

LacZ si#e a&&r4

2700!&EcoRI

A#r

LacZ

A#r

LacZ

A#r

si#e a&&r4

%200!&

&EC19F &lasid

&!lt101 rec!i,a,t

-lue colonies

-ite cl,ies

39



39/40


Lac &


Ampr

ene

2.7 kbp'C1( estictionen'$me sites in

C"

A#r

LacZ

si#e a&&r4

2700!&

EcoRI

A#r

LacZ

A#r

LacZ

A#r

si#e a&&r4

%200!&

&EC19F &lasid

&!lt101 rec!i,a,t

-lue colonies

-ite cl,ies

-lue : 8hite selection

Am!

DNA inset ecombinant

Plate onam!icillin!lus *gal

All colonies ae Am!

,ut onl$ some ae ecombinant (white ones)

Bl / hit s l ti n fCMB2000 – Lecture 2


40/40

Blue/white selection of

recombinant E. coli clones

D -!e!tide o& betagalactosidase e.!essed &om!lasmid (o&ten temed the /ac0 gene)

D 1!e!tide e.!essed &om host (E% coli ) genome

D E% coli containing !lasmid without DNA inset(nonecombinant) metabolise *gal to a blue!oduct

D Inset in vecto disu!ts !oduction o& betagalactosidase -!e!tide thus no active en'$me!oduced thus colon$ a!!eas white not blue

Documents

Lecture 2 DNA Amplification Printable Version