Lecture 3 Gene Libraries Printable Version

8/16/2019 Lecture 3 Gene Libraries Printable Version

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Learning outcomes

CMB2000 – Lecture 3

Identifying the DNA1

Describe the technique for isolating DNA

Dene what Restriction endonucleasesare and how they are used to cut and

recombine DNAExplain how PR wor!s

"se electrophoresis to separate DNAfragments

Describe genetic transformationtechniques including the use of plasmidsand #ectors

Explain the selection techniques


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Uses of plasmids

1) Expression of proteins


Identifying the DNA$


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Uses of plasmids1) Expression of proteins2) Manipulation of genes e.g. site-direted mutagenesis

CMB2000 – Lecture 3 %


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Uses of plasmids

!) "ta#ilises DNA se$uenes%) Ma&ing genomi li#raries of small genomes

'although plasmids are rarely used for li#raries)

CMB2000 – Lecture 3 &

Species Genome size Number of plasmidsneeded

E Coli & x 1'( )''

() Maximum insert sie * (+,,, #ases 'aerage 2+,,,)


E Coli & x 1'( )''

*uman % x 1'+ ('','''

N =

ln (1 - P)

ln (1 – a/b)

Number

of clones

Probabilit

a !i"en !ene

is #resent

$otal si%e

of !enome &"era!e insert

si%e


E Coli & x 1'( )''

*uman % x 1'+ ('',''' -1./ million0


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Identifying the DNA(

enomi li#raries Bacterio#'a!e-λ

E coli infecting bacteriophagesbest studied

an ta!e larger inserts than

plasmids – $' !b – readily pac!aged – can infect host cells

2wo types of replication3 – Lytic pathway - #iruses lyseinfected host cell

– Lysogenic pathway- #irus nucleicacid incorporates into host cell genome

4 prophage


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Identifying the DNA)

enomi li#raries Bacterio#'a!e-λuman N&


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Identifying the DNA+

Bacterio#'a!e-λ

/onatomer


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Identifying the DNA1'


E coli infecting bacteriophagesbest studied

an ta!e larger inserts than

plasmids – $' !b – readily pac!aged – can infect host cells

2wo types of replication3 – Lytic pathway - #iruses lyseinfected host cell

– Lysogenic pathway- #irus nucleicacid incorporates into host cell genome

4 prophage

In vitro #ac,a!in! !i"es a miture of λ-#'a!e

ac' contains a 4ifferent N& insert (phage li#rary)

Blac, = #'a!e N&5

colour = forei!n N&

E.Coli in li6ui4 me4ium

&44 #'a!e

librar

&44 molten

a!ar

Pour onto

a!ar #late

Me4ium soli4ifies

coli !ro to form

a lan


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Bacterio#'a!e-λ

1


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CMB2000 – Lecture 3 1$

enomi li#raries Cosmi4sLooks like a plasmid, but can act like a

phage……… Large plasmids 5 can accept #ery large

inserts -&&!60

7.twice as large as a phage

6"2 has COS sites 5 can be packaged asa phage -and infect host E.coli0

Also has a gene for ampicillin resistance5 can be screened 8as a plasmid9

C 2000 3 1


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Identifying the DNA1%

enomi li#raries

3070002087000*8070003 10+uman

97+80*:70001087000; 10:$omato

9+8*7:00107800; 10;


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Identifying the DNA1&

enomi li#raries &rtificial C'romosomes

Modified 0-plasmid

ori ori

lac%?

C'loram#'enicol@multiple loningsite

(in4AAA7 BamA7>#'A)

0-plasmid unusual "er lar!e #lasmi4 allos !enetic ec'an!e beteen bacteria acce#ts lar!e inserts (u# to 300 b)

lo co# number (1-2 #er cell)

A/ 3 aterial Artifiial /hromosomes 4A/ 3 4east Artifiial /hromosomes

CMB2000 L t 3 1


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enomi li#raries &rtificial c'romosomes

Eletroporator 5oltage pulse6

7emporarily disrupts mem#ranes

(#ores forme4)

lectric #otential across membrane

rises b . 08-10

C'ar!e4 molecules 'DNA) drien

aross pores (electro#'oresis)

CMB2000 L t 3 1


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Identifying the DNA1(

enomi li#raries

: a collection of DNA fragments of oneorganism, each carried by a plasmid;#irus and cloned in an appropriate host

Need vectors that can hold large inserts Need a DNA probe to locate a specic

DNA se!uences in the library – "sed to be the only way to in#estigate a

genome 5 now other techniques (L05/L06) butDNA libraries still #ery important forin#estigating gene function

CMB2000 Lecture 3 1


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Identifying the DNA1/


CMB2000 Lecture 3 1


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Identifying the DNA1)

enomi li#raries

&$C$C$&$$$CC&&&&C$$C&&&&&$

&&$8

1 2 3

ector

(it' insert)

3

,b la44er

1

ector

(no insert)

2

CMB2000 Lecture 3 1


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Identifying the DNA1+

enomi li#raries

1 2 3

Buffer

el Membrane

Pa#er $oels

Membrane

CMB2000 Lecture 3 $


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Identifying the DNA$'

enomi li#raries

1 2 3

MembraneEd "outhern

4inbur!' Dni"ersit1+;0s

>out'ern blottin!

CMB2000 – Lecture 3 $


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Identifying the DNA$1

enomi li#raries

1 2 3

Membrane

>out'ern blottin!

Addition of bloc!ingreagentPre#ents non5specicbinding

"#$%&D&SA'&(N Probe

additionProbe only binds tocomplementary DNA

S'%&NG)N*#+AS")SProbe only remainshybridised tocomplementary DNA

Amplify the signalDigoxigenin and anti5digoxigenin



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CMB2000 Lecture 3

Identifying the DNA$$

enomi li#raries

$ranscri#tionene interru#te4

eons5 co4in!

introns5 non-co4in!

N& #re-m@N&

Processin!

m@N&

(mature)

$ranslation

Protein

u,arotes

(&)n

Processin! introns remo"e47 eons Eoine4 to!et'er7 #ol(&) tail a44e4 to 3F-en4

N& librariescN& libraries

Note6 1) N& is #resent in all ells at all times 2) & partiular m9NA ma onl be e#resse4 in

certain con4itions - the 79AN"/9I:7;ME



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eons5 co4in!

introns5 non-co4in!

N& #re-m@N&

Processin!

m@N&

(mature)

$ranslation

Protein

u,arotes

(&)n


CMB2000 Lecture 3

Identifying the DNA$%

enomi li#raries N& librariescN& libraries

add ri#onulease inhi#itors



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eons5 co4in!

introns5 non-co4in!

N& #re-m@N&

Processin!

m@N&

(mature)

$ranslation

Protein

u,arotes

(&)n


CMB2000 Lecture 3 $&


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CMB2000 Lecture 3

Identifying the DNA




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Method to =lone> human insulin in E coli

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Lecture 3 Gene Libraries Printable Version