biology lecture , chapter 5

7/23/2019 Biology Lecture , Chapter 5

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Molecular BiologyFourth Edition

Chapter 5

Molecular Tools for

Studying Genes and

Gene Activity

Lecture PoerPoint to acco!pany

Robert F. Weaver

Copyright " The McGra#$ill Co!panies% &nc' Per!ission re(uired for reproduction or display'



5#)

5'* Molecular Separations

+ften !i,tures of proteins or nucleic acids

are generated during the course of

!olecular -iological procedures

. A protein !ay need to -e purified fro! a

crude cellular e,tract

. A particular nucleic acid !olecule !ade in a

reaction needs to -e purified



5#/

Gel Electrophoresis

0 Gel electrophoresis is used to separate

different species of1

. 2ucleic acid

. Protein



5#3

42A Gel Electrophoresis

0 Melted agarose is pouredinto a for! e(uipped ithre!ova-le co!-

0 Co!- teeth6 for! slots inthe solidified agarose

0 42A sa!ples are placed inthe slots

0 An electric current is runthrough the gel at a neutralp$



5#5

42A Separation -y Agarose Gel

Electrophoresis

0 42A is negatively charged due tophosphates in its -ac7-one and!oves to anode% the positive pole

. S!all 42A pieces have little

frictional drag so !ove rapidly . Large 42As have !ore frictional

drag so their !o-ility is sloer

. 8esult distri-utes 42A according

to si9e0 Largest near the top

0 S!allest near the -otto!

0 42A is stained ith fluorescent

dye



5#:

42A Si9e Esti!ation

0 Co!parison ith standardsper!its si9e esti!ation

0 Mo-ility of frag!ents are

plotted v' log of !oleculareight ;or nu!-er of -ase pairs<

0 Electrophoresis of un7non42A in parallel ith standard

frag!ents per!its si9eesti!ation

0 Sa!e principles apply to 82Aseparation



5#=

Electrophoresis of Large 42A

0 Special techni(ues are re(uired for 42A

frag!ents larger than a-out * 7ilo-ases

0&nstead of constant current% alternate longpulses of current in forard direction ith

shorter pulses in either opposite or

sideays direction

0 Techni(ue is called pulsed#field gel

electrophoresis ;PFGE<



5#>

Protein Gel Electrophoresis

0 Separation of proteins is done using a gel!ade of polyacryla!ide ;polyacryla!ide

gel electrophoresis ? PAGE<

. Treat proteins to denature su-units ithdetergent such as S4S

0 S4S coats polypeptides ith negative charges so

all !ove to anode

0 Mas7s natural charges of protein su-units so all!ove relative to !ass not charge

. As ith 42A s!aller proteins !ove faster

toard the anode



5#@

Su!!ary

0 42As% 82As% and proteins of various!asses can -e separated -y gelelectrophoresis

0 Most co!!on gel used in nucleic acidelectrophoresis is agarose

0 Polyacryla!ide is usually used in protein

electrophoresis0 S4S#PAGE is used to separate

polypeptides according to their !asses



5#*

To#4i!ensional GelElectrophoresis

0 hile S4S#PAGE gives good resolution ofpolypeptides% so!e !i,tures are soco!ple, that additional resolution is

needed0 To#di!ensional gel electrophoresis can

-e done1

. ;no S4S< uses ) consecutive gels . Se(uential gels ith first a p$ separation%

then separate in a polyacryla!ide gel



5#**

A Si!ple )#4 Method

0 8un sa!ples in ) gels

. First di!ension separates using one

concentration of polyacryla!ide at one p$

. Second di!ension uses different

concentration of polyacryla!ide and p$

. Proteins !ove differently at different p$

values ithout S4S and at differentacryla!ide concentrations



5#*)

To#4i!ensional Gel

Electrophoresis 4etails A to process !ethod1

0 &soelectric focusing gel1 !i,ture of proteins

electrophoresed through gel in a narro

tu-e containing a p$ gradient

. 2egatively charged protein !oves to its

isoelectric point at hich it is no longer

charged . Tu-e gel is re!oved and used as the sa!ple

in the second process



5#*/

More To#4i!ensional Gel

Electrophoresis 4etails0 Standard S4S#PAGE1

. Tu-e gel is re!oved and used as the sa!ple at

the top of a standard polyacryla!ide gel

. Proteins partially resolved -y isoelectric

focusing are further resolved according to si9e

0 hen used to a co!pare co!ple, !i,tures

of proteins prepared under to differentconditions% even su-tle differences are

visi-le



5#*3

&on#E,change Chro!atography

0 Chromatography originally referred to the

pattern seen after separating colored

su-stances on paper

0 &on#e,change chro!atography uses a

resin to separate su-stances -y charge

0 This is especially useful for proteins

0 8esin is placed in a colu!n and sa!ple

loaded onto the colu!n !aterial



5#*5

Separation -y &on#E,change

Chro!atography0 +nce the sa!ple is

loaded -uffer is passedover the resin sa!ple

0 As ionic strength ofelution -uffer increases%sa!ples of solutionfloing through the

colu!n are collected0 Sa!ples are tested forthe presence of theprotein of interest



5#*:

Gel Filtration Chro!atography

0 Protein si9e is a valua-le property that can -e used as a

-asis of physical separation

0 Gel filtration uses colu!ns filled ith porous resins that

let in s!aller su-stances% e,clude larger ones

0 Larger su-stances travel faster through the colu!n



5#*=

Affinity Chro!atography

0 &n affinity chro!atography% the resin containsa su-stance to hich the !olecule ofinterest has a strong and specific affinity

0 The !olecule -inds to a colu!n resincoupled to the affinity reagent . Molecule of interest is retained

. Most other !olecules flo through ithout-inding

. Last% the !olecule of interest is eluted fro! thecolu!n using a specific solution that disrupts the

specific -inding



5#*>

5') La-eled Tracers

0 For !any years la-eled6 has -een

synony!ous ith radioactive6

0 8adioactive tracers allo vanishingly

s!all (uantities of su-stances to -e

detected

0 Molecular -iology e,peri!ents typically

re(uire detection of e,tre!ely s!all

a!ounts of a particular su-stance



5#*@

Autoradiography

Autoradiography is a !eans ofdetecting radioactiveco!pounds ith aphotographic e!ulsion

. Preferred e!ulsion is ,#ray fil! . 42A is separated on a gel and

radiola-eled

. Gel is placed in contact ith ,#ray fil! for hours or days

. 8adioactive e!issions fro! thela-eled 42A e,pose the fil!

. 4eveloped fil! shos dar7-ands



5#)

Autoradiography Analysis

0 8elative (uantity of

radioactivity can -e assessed

loo7ing at the developed fil!

0 More precise !easure!entsare !ade using densito!eter

. Area under pea7s on a tracing

-y a scanner

. Proportional to dar7ness of the

-ands on autoradiogra!



5#)*

Phosphori!aging

This techni(ue is !ore accurate in (uantifying

a!ount of radioactivity in a su-stance

. 8esponse to radioactivity is !uch !ore linear

. Place gel ith radioactive -ands in contact ith

a phosphori!ager plate

. Plate a-sor-s β electrons that e,cite !olecules

on the plate hich re!ain e,cited until plate isscanned

. Molecular e,citation is !onitored -y a detector



5#))

Li(uid Scintillation Counting

8adioactive e!issions fro! a sa!ple createphotons of visi-le light are detected -y aphoto!ultiplier tu-e in the process of li(uid

scintillation counting . 8e!ove the radioactive !aterial ;-and fro!

gel< to a vial containing scintillation fluid

. Fluid contains a fluor that fluoresces hen hitith radioactive e!issions

. Acts to convert invisi-le radioactivity intovisi-le light



5#)/

2onradioactive Tracers

0 2eer nonradioactive tracers no rivalolder radioactive tracers in sensitivity

0 These tracers do not have ha9ards1 . $ealth e,posure

. $andling

. 4isposal

0 &ncreased sensitivity is fro! use of a!ultiplier effect of an en9y!e that iscoupled to pro-e for !olecule of interest



5#)3

4etecting 2ucleic Acids ith a

2onradioactive Pro-e



5#)5

5'/ Dsing 2ucleic Acid

$y-ridi9ation0 $y-ridi9ation is the a-ility of one single#

stranded nucleic acid to for! a dou-le

heli, ith another single strand of

co!ple!entary -ase se(uence

0 Previous discussion focused on colony

and pla(ue hy-ridi9ation

0 This section loo7s at techni(ues for

isolated nucleic acids



5#):

Southern Blots1 &dentifying

Specific 42A Frag!ents0 4igests of geno!ic 42A are separated on

agarose gel

0 The separated pieces are transferred to

filter -y diffusion% or !ore recently -y

electrophoresing the -ands onto the filter

0 Filter is treated ith al7ali to denature the

42A% resulting ss42A -inds to the filter

0 Pro-e the filter using la-eled c42A



5#)=

Southern Blots0 Pro-e c42A hy-ridi9es and

a -and is generated

corresponding to the 42A

frag!ent of interest

0 isuali9e -ands ith ,#rayfil! or autoradiography

0 Multiple -ands can lead to

several interpretations

. Multiple genes

. Several restriction sites in the

gene



5#)>

42A Fingerprinting and 42A

Typing0 Southern -lots are used in forensic la-s to

identify individuals fro! 42A#containing!aterials

0 Minisatellite 42A is a se(uence of -asesrepeated several ti!es% also called 42Afingerprint .

&ndividuals differ in the pattern of repeats ofthe -asic se(uence

. 4ifference is large enough that ) people haveonly a re!ote chance of having e,actly the

sa!e pattern



5#)@

42A FingerprintingProcess really ust a Southern

-lot

0Cut the 42A under study

ith restriction en9y!e

. &deally cut on either side of

!inisatellite -ut not inside

08un digest on a gel and -lot

0Pro-e ith la-eled

!inisatellite 42A and i!aged . 8eal sa!ples result in very

co!ple, patterns



5#/

Forensic Dses of 42A

Fingerprinting and 42A Typing0 hile people have different 42A fingerprints%

parts of the pattern are inherited in a Mendelian

fashion

. Can -e used to esta-lish parentage . Potential to identify cri!inals

. 8e!ove innocent people fro! suspicion

0 Actual pattern has so !any -ands they can

s!ear together indistinguisha-ly . Forensics uses pro-es for ust a single locus

. Set of pro-es gives a set of si!ple patterns



5#/*

&n Situ $y-ridi9ation1 Locating

Genes in Chro!oso!es0 La-eled pro-es can -e used to hy-ridi9e to

chro!oso!es and reveal hich chro!oso!econtains the gene of interest . Spread chro!oso!es fro! a cell

. Partially denature 42A creating single#strandedregions to hy-ridi9e to la-eled pro-e

. Stain chro!oso!es and detect presence of la-el onparticular chro!oso!e

0 Pro-e can -e detected ith a fluorescentanti-ody in a techni(ue called fluorescence insitu hy-ridi9ation ;F&S$<



5#/)

&!!uno-lots

&!!uno-lots ;also called estern -lots< use

a si!ilar process to Southern -lots

. Electrophoresis of proteins

. Blot the proteins fro! the gel to a !e!-rane

. 4etect the protein using anti-ody or antiseru!

to the target protein

. La-eled secondary anti-ody is used to -indthe first anti-ody and increase the signal



5#//

estern Blots



5#/3

42A Se(uencing

0 Sanger% Ma,a!% Gil-ert developed )

!ethods for deter!ining the e,act -ase

se(uence of a cloned piece of 42A

0 Modern 42A se(uencing is -ased on the

Sanger !ethod



5#/5

Sanger Manual Se(uencing

Sanger 42A se(uencing !ethod usesdideo,y nucleotides to ter!inate 42Asynthesis

. The process yields a series of 42A frag!entshose si9e is !easured -y electrophoresis

. Last -ase in each frag!ent is 7non as thatdideo,y nucleotide as used to ter!inate the

reaction . +rdering the frag!ents -y si9e tells the -ase

se(uence of the 42A



5#/:

Sanger 42A Se(uencing



5#/=

Auto!ated 42A Se(uencing

0 Manual se(uencing is poerful -ut slo

0 Auto!ated se(uencing uses

dideo,ynucleotides tagged ith different

fluorescent !olecules

. Products of each dideo,ynucleotide ill

fluoresce a different color

. Four reactions are co!pleted% then !i,edtogether and run out on one lane of a gel



5#/>

Auto!ated 42A Se(uencing



5#/@

8estriction Mapping

0 Prior to start of large#scale se(uencing

preli!inary or7 is done to locate

land!ar7s

. A !ap -ased on physical characteristics is

called a physical !ap

. &f restriction sites are the only !ap features

then a restriction !ap has -een prepared0 Consider a *': 7- piece of 42A as an

e,a!ple



5#3

8estriction Map E,a!ple

0 Cut separate sa!ples of the original *':

7- frag!ent ith different restriction

en9y!es

0 Separate the digests on an agarose gel to

deter!ine the si9e of pieces fro! each

digest

0 Can also use sa!e digest to find the

orientation of an insert cloned into a vector



5#3*

Mapping E,peri!ent

D i 8 i i M i i h



5#3)

Dsing 8estriction Mapping ith

an Dn7non 42A Sa!ple



5#3/

Mapping the Dn7non

S th Bl t d 8 t i ti



5#33

Southern Blots and 8estriction

Mapping



5#35

Su!!ary

0 Physical !ap tells a-out the spatial arrange!entof physical land!ar7s6 such as restriction sites . &n restriction !apping cut the 42A in (uestion ith )

or !ore restriction en9y!es in separate reactions

.Measure the si9es of the resulting frag!ents . Cut each ith another restriction en9y!e and!easure si9e of su-frag!ents -y gel electrophoresis

0 Si9es per!it location of so!e restriction sitesrelative to others

0 &!prove process -y Southern -lotting frag!entsand hy-ridi9ing the! to la-eled frag!ents fro!another restriction en9y!e to reveal overlaps

P t i E i i ith Cl d



5#3:

Protein Engineering ith Cloned

Genes1 Site#4irected Mutagenesis

0 Cloned genes per!it -ioche!ical !icrosurgery

on proteins

. Specific -ases in a gene !ay -e changed

. A!ino acids at specific sites in the protein product!ay also -e altered

. Effects of those changes on protein function can -e

o-served

0 Might investigate the role of phenolic group ontyrosine co!pared to phenylalanine

Sit 4i t d M t i ith



5#3=

Site#4irected Mutagenesis ith

PC8



5#3>

Su!!ary

0 Dsing cloned genes% can introduce changes atill to alter a!ino acid se(uence of protein

products

0 Mutageni9ed 42A can -e !ade ith1

. 4ou-le#stranded 42A

. To co!ple!entary !utagenic pri!ers

. PC8

0 4igest the PC8 product to re!ove ild#type42A

0 Cells can -e transfor!ed ith !utageni9ed 42A

5 3 M i d tif i



5#3@

5'3 Mapping and uantifying

Transcripts0 Mapping ;locating start and end< and

(uantifying ;ho !uch transcript e,ists at

a set ti!e< are co!!on procedures

0 +ften transcripts do not have a unifor!

ter!inator% resulting in a continuu! of

species s!eared on a gel

0 Techni(ues that specific for the se(uence

of interest are i!portant



5#5

2orthern Blots

0 Hou have cloned a c42A

. $o actively is the corresponding gene

e,pressed in different tissuesI

. Find out using a 2orthern Blot0 +-tain 82A fro! different tissues

0 8un 82A on agarose gel and -lot to !e!-rane

0 $y-ridi9e to a la-eled c42A pro-e . 2orthern plot tells a-undance of the transcript

. uantify using densito!eter



5#5*

S* Mapping

Dse S* !apping to locate the ends of 82As andto deter!ine the a!ount of a given 82A in cells ata given ti!e

. La-el a ss42A pro-e that can only hy-ridi9e totranscript of interest

. Pro-e !ust span the se(uence start to finish

. After hy-ridi9ation% treat ith S* nuclease hichdegrades ss42A and 82A

. Transcript protects part of the pro-e fro! degradation . Si9e of protected area can -e !easured -y gel

electrophoresis



5#5)

S* Mapping the 5J End



5#5/

S* Mapping the /J End

S !!ar



5#53

Su!!ary0 &n S* !apping% a la-eled 42A pro-e is used to

detect 5J# or /J#end of a transcript0 $y-ridi9ation of the pro-e to the transcript protects

a portion of the pro-e fro! digestion -y S*nuclease% specific for single#stranded

polynucleotides0 Length of the section of pro-e protected -y the

transcript locates the end of the transcript relativeto the 7non location of an end of the pro-e

0 A!ount of pro-e protected is proportional toconcentration of transcript% so S* !apping can -e(uantitative

0 82ase !apping uses an 82A pro-e and 82ase



5#55

Pri!er E,tension

0 Pri!er e,tension or7s to deter!ine e,actlythe 5J#end of a transcript to one#nucleotideaccuracy

0 Specificity of this !ethod is due toco!ple!entarity -eteen pri!er and transcript

0 S* !apping ill give si!ilar results -ut li!its1 . S* ill ni--le6 ends of 82A#42A hy-rid

. Also can ni--le6 A#T rich regions that have!elted

. Might not co!pletely digest single#strandedregions



5#5:

Pri!er E,tension Sche!atic0 Start ith in vivo

transcription% harvestcellular 82A containingdesired transcript

0 $y-ridi9e la-eled

oligonucleotide K*>nt;pri!er<

0 8everse transcriptasee,tends the pri!er to the5J#end of transcript

0 4enature the 82A#42Ahy-rid and run the !i, ona high#resolution 42A gel

0 Can esti!ate transcriptconcentration also

8un +ff Transcription and G



5#5=

8un#+ff Transcription and G#

Less Cassette Transcription0 &f ant to assess1

. Transcription accuracy

. $o !uch of this accurate transcription

0 Si!pler !ethod is run#off transcription

0 Can -e used after the physiological start

site is found -y S* !apping or pri!er

e,tension

0 Dseful to see effects of pro!oter !utation

on accuracy and efficiency of transcription



5#5>

8un#+ff Transcription

0 42A frag!ent containinggene to transcri-e is cut ithrestriction en9y!e in !iddleof transcription region

0 Transcri-e the truncatedfrag!ent in vitro using la-elednucleotides% as poly!erasereaches truncation it runs off6the end

0 Measure length of run#offtranscript co!pared tolocation of restriction site at/J#end of truncated gene



5#5@

G#Less Cassette Assay

0 ariation of the run#off techni(ue% instead

of cutting the gene ith restriction

en9y!e% insert a stretch of nucleotides

lac7ing guanines in nonte!plate strand ust donstrea! of pro!oter

0 As pro!oter is stronger a greater nu!-er

of a-orted transcripts is produced

Sche!atic of the G Less



5#:

Sche!atic of the G#Less

Cassette Assay0 Transcri-e altered

te!plate in vitro ithCTP% ATP and DTP one ofhich is la-eled% -ut no

GTP0 Transcription ill stophen the first G isre(uired resulting in ana-orted transcript of

predicta-le si9e0 Separate transcripts on a

gel and !easuretranscription activity ithautoradiography

S



5#:*

Su!!ary

0 8un#off transcription is a !eans of chec7ing

efficiency and accuracy of in vitro transcription . Gene is truncated in the !iddle and transcri-ed in vitro in

presence of la-eled nucleotides

. 82A poly!erase runs off the end !a7ing an inco!plete

transcript . Si9e of run#off transcript locates transcription start site

. A!ount of transcript reflects efficiency of transcription

0 &n G#less cassette transcription% a pro!oter is fused

to ds42A cassette lac7ing Gs in nonte!plate strand . Construct is transcri-ed in vitro in a-sence of of GTP

. Transcription a-orts at end of cassette for a predicta-lesi9e -and on a gel

5 5 Measuring Transcription



5#:)

5'5 Measuring Transcription

8ates in ivo0 Pri!er e,tension% S* !apping and

2orthern -lotting ill deter!ine theconcentration of specific transcripts at a

given ti!e0 These techni(ues do not really reveal the

rate of transcript synthesis asconcentration involves -oth1 . Transcript synthesis

. Transcript degradation



5#:/

2uclear 8un#+n Transcription

0 &solate nuclei fro! cells% allo the! toe,tend in vitro the transcripts alreadystarted in vivo in a techni(ue called run#on

transcription0 82A poly!erase that has already initiated

transcription ill run#on6 or continue toelongate sa!e 82A chains

0 Effective as initiation of ne 82A chains inisolated nuclei does not generally occur



5#:3

8un#+n Analysis

0 8esults ill sho transcription rates andan idea of hich genes are transcri-ed

0 &dentification of la-eled run#on transcripts

is -est done -y dot -lotting . Spot denatured 42As on a filter

. $y-ridi9e to la-eled run#on 82A

. &dentify the 82A -y 42A to hich it hy-ridi9es0 Conditions of run#on reaction can -e

!anipulated ith effects of product can -e!easured

2uclear 8un +n Transcription



5#:5

2uclear 8un#+n Transcription

4iagra!



5#::

8eporter Gene Transcription

0 Place a surrogate reporter gene under control ofa specific pro!oter% !easure accu!ulation ofproduct of this reporter gene

0 8eporter genes are carefully chosen to haveproducts very convenient to assay . lacZ produces β#galactosidase hich has a -lue

cleavage product

. cat produces chlora!phenicol acetyl transferase

;CAT< hich inhi-its -acterial groth . Luciferase produces che!ilu!inescent co!pound

that e!its light

Measuring Protein Accu!ulation



5#:=

Measuring Protein Accu!ulation

in ivo0 Gene activity can -e !onitored -y

!easuring the accu!ulation of protein

;the ulti!ate gene product<

0 To pri!ary !ethods of !easuring

protein accu!ulation

. &!!uno-lotting estern -lotting

. &!!unoprecipitation



5#:>

&!!unoprecipitation

0 La-el proteins -y groing cells ith /5S#la-eled a!ino acid

0 Bind protein of interest to an anti-ody

0 Precipitate the protein#anti-ody co!ple,ith a secondary anti-ody co!ple,ed toProtein A on resin -eads using a lo#

speed centrifuge0 4eter!ine protein level ith li(uidscintillation counting

5 : Assaying 42A#Protein



5#:@

5': Assaying 42A#Protein

&nteractions

0 Study of 42A#protein interactions is of

significant interest to !olecular

-iologists0 Types of interactions often studied1

. Protein#42A -inding

. hich -ases of 42A interact ith aprotein



5#=

Filter Binding

Filter -inding to !easure 42A#protein

interaction is -ased on the fact that dou-le#

stranded 42A ill not -ind -y itself to a filter%

-ut a protein#42A co!ple, ill . 4ou-le#stranded 42A can -e la-eled and

!i,ed ith protein

. Assay protein#42A -inding -y !easuring thea!ount of la-el retained on the filter

2itrocellulose Filter#Binding



5#=*

2itrocellulose Filter#Binding

Assay0 ds42A is la-eled and !i,ed ith protein

0 Pour ds42A through a nitrocellulose filter

0 Measure a!ount of radioactivity that passed

through filter and retained on filter

Gel Mo-ilit Shift



5#=)

Gel Mo-ility Shift

0 42A !oves through a gel faster hen it is not

-ound to protein

0 Gel shift assays detect interaction -eteen

protein and 42A -y reduction of the

electrophoretic !o-ility of a s!all 42A -ound toa protein



5#=/

Footprinting

0 Footprinting shos here a target lies on

42A and hich -ases are involved in

protein -inding

0 Three !ethods are very popular1

. 42ase footprinting

. 4i!ethylsulfate footprinting

. $ydro,yl radical footprinting



5#=3

42ase Footprinting

Protein -inding to 42A coversthe -inding site and protects fro!attac7 -y 42ase

0 End la-el 42A% * strand only

0 Protein -inds 42A0 Treat co!ple, ith 42ase &

!ild conditions for average

of * cut per !olecule

0 8e!ove protein fro! 42A%separate strands and run on

a high#resolution

polyacryla!ide gel



5#=5

4MS Footprinting

0 4i!ethylsulfate

;4MS< is a

!ethylating agent

hich can fit into 42Anoo7s and crannies

0 Starts as 42ase% then

!ethylate ith 4MS

at conditions for *!ethylation per 42A

!olecule

Su!!ary



5#=:

Su!!ary0 Footprinting finds target 42A se(uence or

-inding site of a 42A#-inding protein0 42ase footprinting -inds protein to end#la-eled

42A target% then attac7s 42A#protein co!ple,ith 42ase

0 42A frag!ents are electrophoresed ith protein-inding site appearing as a gap in the patternhere protein protected 42A fro! degradation

0 4MS% 42A !ethylating agent is used to attac7the 42A#protein co!ple,

0 $ydro,yl radicals . copper# or iron#containingorgano!etallic co!ple,es generate hydro,ylradicals that -rea7 the 42A strands

5 = Finding 82A Se(uences That



5#==

5'= Finding 82A Se(uences That

&nteract ith +ther Molecules0 SELEN is syste!atic evolution of ligands -y

e,ponential enrich!ent

0 SELEN is a !ethod to find 82A se(uences that

interact ith other !olecules% even proteins . 82As that interact ith a target !olecule are selected

-y affinity chro!atography

. Convert to ds42A and a!plify -y PC8

. 82As are no highly enriched for se(uences that-ind to the target !olecule



5#=>

Functional SELEN

0 Functional SELEN is a variation here the

desired function alters 82A so it can -e

a!plified

0 &f desired function is en9y!atic%!utagenesis can -e introduced into the

a!plification step to produce variants ith

higher activity



5#=@

5'> Onoc7outs

0 Pro-ing structures and activities of genes

does not anser (uestions a-out the role

of the gene in the life of the organis!

0 Targeted disruption of genes is nopossi-le in several organis!s

0 hen genes are disrupted in !ice the

products are called 7noc7out !ice

S * f h O 7 M



5#>

Stage * of the Onoc7out Mouse

0 Cloned 42A containing the !ouse gene to -e7noc7ed out is interrupted ith another gene thatconfers resistance to neo!ycin

0 A thy!idine 7inase gene is placed outside the targetgene

0 Mi, engineered !ouse 42A ith ste! cells sointerrupted gene ill find ay into nucleus andho!ologous reco!-ination ith altered gene andresident% intact gene

0 These events are rare% !any cells ill need to -escreened using the introduced genes

Ma7ing a Onoc7out Mouse1



5#>*

Ma7ing a Onoc7out Mouse1

Stage *

St ) f th O 7 t M



5#>)

Stage ) of the Onoc7out Mouse

0 &ntroduce the interrupted gene into a hole!ouse

0 &nect engineered cells into a !ouse -lastocyst

0 E!-ryo into a surrogate !other ho gives -irthto chi!eric !ouse ith patchy coat

0 True hetero9ygote results hen chi!era !ates

ith a -lac7 !ouse to produce -ron !ice% half

of hich ill have interrupted gene

Ma7ing a Onoc7out Mousse1



5#>/

Ma7ing a Onoc7out Mousse1

Stage )

O 7 t 8 lt



Onoc7out 8esults

0 Phenotype !ay not -e o-vious in theprogeny% -ut still instructive

0 +ther cases can -e lethal ith the !ice

dying -efore -irth

0 &nter!ediate effects are also co!!on and

!ay re(uire !onitoring during the life of

the !ouse

biology lecture , chapter 5

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