am j clin nutr 1993 carroll 249s 58s

7/23/2019 Am J Clin Nutr 1993 Carroll 249S 58S

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. ini I Ii,,

.Vu ir

1993 :58 supp l :249S -58S . P r in ted in U S A . c 19 93 A m erican So cie ty fo r C lin ica l N utrition

249S

In tro duc tion to recom b inan t-D N A techno lo gy1 2

14 i/ham L C arro ll

ABS TRAC l

R ecom binan t-D N A tech no logy is n ow corn -

rn on lv used in v irtua lly every asp ec t o f the b io log ica l sc iences.

T he purpo se of th is b rie f expos ition is to p rov id e an ou tline of

the approach es used to id en tify genes , to iso la te th e gene of in -

te rest. to am plify the gene if necessa ry . and to c lone genes . A

sh ort in trod uc tio n to the princ ip les o fsepara tin g very la rg e genes

is p rov ided . a long w ith a desc rip tion o f an ap proach to p ropa-

ga ting an d c lon in g these large g en es. m m J liii Niu r

I 993 :58(supp l) :249S-58S .

K EY W O R DS P la sm id , bacter iophag e.

r ev er se tr an sc ri pt as e.

R N A polv rn erase . clon in g . res tric tion endonu c lease. po ly m erase

cha in reac tion . \eas t a rtific ia l ch ro m oso m e

Introduction

T he ov erall go al o f recom binan t-D N A tech no logy is to iden-

tify , iso la te . m an ipu la te . and re -express genes from a g iven ho st

( I -9 ). S om e of the prac tical goa ls o f su ch cu t-and-p aste tech-

n o logy is to 1) deve lop a basic un derstand ing o f the fun c tion

and regu la tion o f know n gene pro duc ts. 2 ) iden tify new g enes

w hose pro tein produc ts have no t been iso lated ( rev erse gene tics ).

3) co rrec t endo genous gene tic defec ts (eg . s ick le ce ll an em ia). 4 )

express fo re ign genes in d isease -su scep tib le h osts (eg . d isease -

resistance g en es in agricu ltu ra l c rop s). and 5) m anu fac tu re la rg e

quan tities o fa p ro tein prod uc t fo r w idespread use (eg . an tibod ies

in to bacco p lan ts ). A ny d iscussion ofth is m ethod o logy shou ld

beg in w ith a desc rip tio n ofthe un iqu e fea tu res o f th e D N A m ol-

ecu le itself. D N A is com p osed oftw o an tipa ra lle l s tran ds bou nd

by hy drogen b onds b e tw een the ir n itro genous-base side cha ins .

G en etic in fo rm atio n is p rov ided by the purine and pvrim id ine

bases. w h ich are lin ke d t o a sugar ph osp ha te backbo ne (F ig 1 .

T his s truc tu ra l supp ort is a se ries o f deoxyribo se residues linked

b p hosp hod ies te r b onds , w hich are c reated by co va len tly jo in ing

a hydroxv l g ro up a t the 3-ca rbo n pos ition o n one deoxyribo se

residue w ith a pho sph ate g roup located on the 5 ca rbon of an

ad jacen t su gar g roup . T hus D N A strands have p o la rity w ith an

unpa ired 5 pho sph ate a t one en d and a 3 hvdro xy l g roup a t th e

o ther. B y conven tion , g ene sequences a re w ritten w ith the 5 end

of the gene orien ted to the le ft and the 3 end to the righ t

F ig 2 .

G ene stru cture

The gene forms th e bas ic un it o f g en e tic in fo rm ation and o ver

a 1 genes may he located on the uninterrupted strands of

D N A tha t m ake up a m am m alian chrom o som e. G enes m ay h e

loca ted on e ithe r D N A strand and thus ind iv id ua l gen es m ay

be o rien ted in opp osite d irec tio ns o n a chro m oso m e. O ne gene

codes fo r a sing le p o lypep tide : the flow of gene tic in fo rm ation

is illu stra ted in F igure 2 . T he syn thesis o f m esseng er R N A

(m R N A ) (gene transc rip tio n ) is con tro lled by gene tic e lem en ts

tha t usua lly lie 5 t o the pro te in -cod in g p ortio n ofthe gene . T hese

,)ro,flokr e lem en ts serv e to focus the enzy m e R N A p olym erase

to the tran sc rip tion sta rt po in t. M any prom ote rs share core se -

quences tha t are essen tia l fo r e ffec tive tran sc rip tion co n tro l.

T hese co re sequen ces (bo .ves ) inc lude the thym ine-aden ine-thy-

m ine-aden ine (T A T A ) box . the cy to sine -aden ine-aden in e-th y-

m ine (C A A T ) box , an d the guan in e-cy to sine (G C ) box . T he

C A A T an d G C b oxes are lo cated fu rthe r u pstream 5 ) w hereas

the T A T A box seem s to con tro l the ac tua l s ta rt po in t fo r m R N A

sy n thesis . T he genes ofeu karyo tes a re a rran ged in ex ons. reg io ns

of the g ene represen ted in th e m atu re m R N A . E xo ns are sepa-

ra ted by in te rven ing seq uences or in trons . w hich are sp liced from

the prim ary R N A tem p la te d uring R N A p rocess in g . O ther eu-

karyo tic -m R N A p rocess in g ev en ts inc lude the ad d ition o f a

m eth y la ted gu an ine res idue con nected b y a 5-5 triphospha te

lin kage to the f irs t nu cleo tide o fthe m R N A (cap s ite ) as w ell as

the add ition of a s tre tch of po lyadeny lic acid res idues a t the 3

te rm inus . T he m R N A is trans lated in to p ro te in w ith a m eth i-

on ine se rv ing as a tran sla tio n s ta rt s ite . P ro teins a lso have po la rity

w ith an unpa ired am in o group (N H 2) a t one end and a free

carhoxy l g roup at the o ther. T he am ino-te rm ina l p ar t o f the

pro te in is encoded by the 5 end ofthe m R N A .

R es tr ic tion en do nu cleas es

T h e first b reak th roug h in recom binan t D N A techno lo gy w as

the iden tif ica tion of enzym es capab le o f c leav in g d up lex D N A

in to d iscrete rep roduc ib le f ragm ents. T h ese res ir ic iomz endomzu-

c /cases have now b een iden tified in m any pro karyo tes. and they

recognize and cleave both strands of DNA at well defined base

seq uences [usu ally com po sed of 4 , 6 . o r 8 base pa irs (bp)]. M ost

o f these c leavag e s ite s a re characte rized by an ax is o f sy m m etry

F ig 3 . C leavage m ay take p lace sym m etrica lly o n e ithe r side

F rom th e E dw ard M allinckrod t D epartm en t o f P ed ia tric s,

W ash in g ton U n iv ersity S ch oo l o f M edic in e, and th e D iv ision of

H em ato logy /O nco log y . S t L ou is C hild ren s H osp ita l. S t L ou is .

2 A ddress rep rin t requ es ts to W L C arro ll, P rogram in M olecu la r

B io logy and G ene tics . U n ivers ity o f U tah . B u ild ing 53 3 . R oom 32 60 .

S a lt L ak e C ity . U T 841 12 .


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250S

C A R R O L L

--- --. .-.-. -- -.-. . -- 5 e n d W

F IG 1. T he D N A m olecu le . D N A is com po sed of t wo a nt ip ar al le l

s tran ds ho und toge ther th ro ugh hyd rogen bon d ing be tw een com ple -

m en ta ry nu cleo tides [aden ine-thym ine . gu an ine-cy tosine (A -T . G -C )J

on th e opp osite strands . T he sugar ph osp ha te backb one o fan ind iv idu a l

s t rand is lin ked tog eth er th roug h ph osp ho die ster b on ds be tw een a p ho s-

pha te g roup on the 5 c a rbon ofone res idue and a hyd roxy l g roup a ttached

to the 3 ca rb on of th e ad jacen t sugar. R ep roduced w ith perm iss ion from

J D arne ll. H L odish . and D B altim o re (10) .

o f the ax is y ie ld ing overly ing 5 o r 3 sing le -s tranded te rm in i

(s ticky ends). S om e en zy m es c leave th e tw o s trands a long the

ax is itse lf leav ing term in i w ith no s ing le s tranded overhang

(b lun t ends) (T ab le 1 R estr ic tion end onuc leases a re nam ed

accord ing to a s tand ard nom enc la tu re . T he firs t th ree le tte rs

co rrespon d to the gen us an d spec ies o f the ho st o rg an ism , fo l-

low ed by a stra in iden tifica tio n or a le tte r ind icating w h eth er

the en zym e is en co ded by an ex trachrom osom al b acte rioph ag e

or p lasm id . and th en by a R om an n um era l if m ore than o ne

res tric tion end onuc lease has been iden tified from the sam e ho st.

G ene clon ing

T o effec tive ly accom plish m any of the goa ls o f recom b inan t

D N A tech no logy , ind iv id ua l genes m u st be iso la ted from back -

g round ho st gen om ic D N A . T here a re four basic steps in gene

c lon ing and to days inves tig ato r has the cho ice of an ever-in -

c reasing num b er of d iffe ren t m etho ds fo r accom plish in g each

step (F ig 4 I n i t s sim p lest for m gene clon ing is accom plished

b y d iges ting h ost D N A w ith restric tio n en donu cleases and in -

se rting the fragm ents in to an y one of four basic vector sys tems .

w hich a llow success fu l p ro paga tio n of the gene in alte rna tive

ho sts, usua lly b ac te ria . F igu re 5 illu stra tes a genera l ex am p le o f

o ne su ch m ethod . O n e of the m ost co m m on vecto rs used to

pro paga te D N A fragm ents is p lasm id s. T h ese c lo n ing v eh icles

a re c ircu la r do ub le -strand ed D N A m o lecu les , w h ich rep licate

ex trachro m oso m ally in m any bac ter ia . C om m on fea tu res o f

cu rren tly used lab ora to ry p lasm id vec to rs in clud e an orig in of

rep lica tion . the ab ility to b e m ain tained in h igh copy num ber

per b acte ria l ce ll. a g ene th at co nfe rs a p heno type (eg . an tib io tic

res is tance) a llow in g se lection of ho st bac te ria th at have in co r-

po ra ted the p lasm id . and a c lo n ing s ite po/iIinker) con ta in ing

a la rg e n um ber of res triction endon uc lease c leavage s ite s . B o th

the v ecto r and the D N A to be c loned are d ig es ted w ith an iden-

tica l restric tio n end onuc lease , y ie ld ing m o lecu les w ith co m pat-

ib le ends . B ecause com m erc ial p lasm id vec to rs con ta in po ly -

linker sequ en ces w ith a large n um ber of restric tio n endo nuc lease

c leavage site s, these vec to rs w ill co nven ien tly accep t D N A gen-

e ra ted by d igestion w ith m any enzym es . T he o verhang ing te rm in i

o f vec to r and inse rt are ab le to annea l to o ne ano ther th rou gh

com plem en tary b ase pa iring . T he en zym e D N A ligase fo rm s

cova len t b onds b etw een D N A strand s a t these site s, resu lting in

a c losed . circu la r recom b in an t p lasm id .

T he p lasm id is th en incuba ted w ith bac te ria l cells tha t have

b een rendered competent to tak e up ex ogenou s D N A by a varie ty

o f m ethod s. B ac teria l ce lls tha t have incorpo ra ted the p lasm id

can b e se lec ted fo r by p la ting th e reac tion on an ag ar p la te con-

tam ing an an tib io tic (eg , am pic illin ). T he bac te ria l host is usua lly

suscep tib le to the an tib io tic . and on ly those ce lls tha t have taken

up the p lasm id acqu ire the gene th a t codes fo r an tib io tic res is -

tance . T h is fo rm of selec tio n d etec ts on ly b acte ria tha t can tak e

up c ircu la r p lasm id s. It does no t d istingu ish p lasm ids w ith in -

se rted D N A (reco m binan ts) from rec ircu la rized vec to r D N A

lack ing inse rts .

R ecen tly . p lasm ids b een cons truc ted su ch tha t reco m binan ts

can eas ily b e id en tified by h is tochem ical techn iqu es . T yp ica lly .

these vec to rs car ry a portion of the Escherichia co /i (1 -ga lac to -

s idase gene cod in g fo r the N H 2-te rm ina l sequ en ce of the func-

tiona l p ro tein . W ith in th is tru nca ted p lasm id g en e lies a con-

ven ien t po ly linker sequence tha t do es no t in te rrup t th e read in g

fram e bu t o n ly ad ds a few harm less am ino ac ids to the sho rten ed

1 3-ga lac to sidase pro te in . W h en these vec to rs a re inse rted in to

ho st bac te ria l stra ins tha t co de fo r th e carboxy -term in al portion

o f(3 -g alac tosidase . bo th fragm ents can assoc ia te to fo rm a func-

tiona l en zym e (alph a com plem en ta tion ). T h is type of co m ple -

m en ta tio n can b e reco gn ized because these bac te ria w ill fo rm

b lu e co lon ies w h en p la ted in the p resence of a chrom ogen ic

subs tra te . H ow ever, if fo reign D N A has been inse rted a t the

p lasm id p o ly lin ker site , the N H 2 -term in al 13 -ga lac tos idase frag -

m en t is in ac tiva ted , th ere is n o com plem en ta tion . and bac te ria

ca rry in g reco m binan t p lasm ids appear w hite .

Vec to rs

A varie ty o f v ecto r system s are n ow av a ilab le to fu lfill the

needs of a particu la r exp er im en t. E ach h as particu la r advan tages

an d d isad van tages, som e o f w hich are lis ted in T ab le 2 . Bade-

riophage

vec to rs have p layed a p articu la rly im p ortan t ro le in


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P o ly A S ite

E n h a n c e r P ro m o te r e xo n I e x o n 2 e xo n 3

5.

AU G

( S t a r t )

UAG

( S t o p )

T r a n s c r i p t i o n

A A n R N A P ro c e s s in g

T r a n s l a t i o n

H2 COOH

1

start si te for thi s process.

A x is o f s y m m e try

5 -J A AT T C -3

3 - CT T : AAG - 5

A

_ _ _ _ C T T A A

{ 1 7 4 } A A T T C

RECO M B I N A N T -D N A I N T RO D U CT I ON

251 S

(r HO

P o s t T ra n s la tio na l

U

M odi f i c a t ion

FI G 2. G ene structure. A ty pi cal eukary otic gene i s com posed of ex ons. w hi ch are sequences included in the m ature

mRN A transcript, and i ntrons. or intervening sequences. w hich are spl i ced out f rom

the prim ary transcri pt duri ng

R NA pro cessi ng.

Prom oter elem ents at the 5 end of the cluster f ocus R N A pol ym erase to the start si te of transcri pti on.

Enhancer sequences increase transcri pti on and characteristi cal l y f uncti on i n ei ther ori entati on. A fter travel ing to the

cy toplasm . the m RN A is translated into protei n, w i th an adenine-uraci l -guanine A UG) codon usual l y serv i ng as the

m olecular bi ol ogy . B acteri ophages are v i ruses w i th a tropi sm f or

bacteri a. T he genom e of the prototype A -bacteri ophage vector

i s a l inear . double-stranded D N A m ol ecule approaching

50 k i l -

obases kb) i n l ength. Each term inus i s com posed of 12 bp of

si ngle-stranded com plem entary D N A . O nce inside the bacteri a

the v i rus assum es a ci rcul ar f orm w hen the term ini are jo ined

and sealed by host l i gase. T w o pathw ays of repl i cation are pos-

si bl e. D uri ng the

/C

the ci rcul ar D N A is repl i cated to a

high copy num ber and the host cel l eventual l y undergoes l ysis

w i th the rel ease of i nf ecti ous v i ral parti cles. A l ternati vel y , the

bacteri ophage D N A m ay becom e integrated into the host bac-

teri al D N A l ysogeni c pathw ay ). W hi l e i n this state, the v i ral

D N A l i es dorm ant, expressing onl y a f ew genes. B acteri ophages

have been m odi f i ed to accept and propagate exogenous D N A .

FIG 3. Restri ct ion endonucleases. Each restri ct ion endonuclease

cleaves double-stranded D N A at w el l -def ined sequences. T hese si tes

characteri sti cal l y display an ax is of sym metry . and m ost enzym es cleave

the D N A strands on ei ther side of this ax is.

T hese cloning v ectors can be classi f ied on the basis of w hether

the f orei gn D N A replaces a segm ent of nonessenti al v i ral D N A

replacement v ectors) or i s di rectl y i nserted into the v i ral genom e

insertion vectors). T he choi ce of a par ti cul ar v ector i s based on

the si ze of the f ragm ent that needs to be cloned, the restri ction

enzy m e used to generate the f ragm ent. and w hether the cloned

f ragm ent is to be expressed into protei n in the host bacteri a. I n

thi s last regard m any ex pression v ectors al l ow the cloning of

f oreign inserts nex t to a strong bacteri al prom oter. w hi ch i s nec-

essary to al l ow ef f ici ent transcri pti on i n

E co /i.

G ene iso la tion

T he i solati on of genes can be approached using tw o basic

strategies. T he gene can be isolated as i t ex i sts in the genom e

w i th i ts prom oter el em ents and m ore com plex i ntron-exon

structure. H ow ev er, genes of hi gher eukary otes contai n m any

introns and are usual l y too large to be propagated as a single

T A B L E

I

Cutt ing D N A m ol ecules: restri ct ion endonuclease nom enclature

Target

Enzym e5 H ost organi sm

sequencet

EcoR

I

Escherichia

co/i RY

13

G A A T T C

tfho I

tlo ra .v e l/a ho sL s

G T C

N ot I N o ca rd ia o ii iid is -c a ia ru n

GC1GGCCGC

Sma I

S er ra ti a i na rc es ce ns

C C C G G G

* T he enzy m e letters correspond to the ini ti al s of the host f rom w hich

i t w as isolated. Roman num erals distinguish am ong m ul tip le enzym es

i sol ated f rom the sam e host.

t G . guanine: A , adenine: T . thym ine: and C , cy tosine.


4/10

E n d o n u c l e a s e

d ig e s tio n o f

g e no m ic D N A

c D N A

s y n t h e s i s

D NA

f r a g m e n t s

I

J o i n i n g

to ve c to r

I

Introduction

in to h o s t c e ll

I

S e le c tio n o f

r e c o m b i n a n t s

I Lin ke r o r

ig a t io n o f L B I U n t e n d a d a p te r

c o h e s iv e lig a tio n a d d itio ne r m i n i _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

T r a n s f o r m a t i o n

E co i l with

re co m b in an t p la s mid

N uc le ic a c id

h y b r i d i z a t i o n

I n v itr o p a c k a n g

in to p h a ge c o a t.

T ra ns du ctio n w ith

[g e o r c o s m id

I m m u n o c h e m i c a l

T ra ns fe ctio n o f

e uk a ry o tic c e lls

w ith e xp re s s io n

v e c t o r

P C R

In v itro m R N A

s yn th e s is fro m

c DN A. In je c tio n

in to o o cy te s .

F u n c t i o n a l

a n a ly sis , b io a ss a ys

FIG 4 . S teps in D N A cloning . A n ev e r-in creasing num ber o f altern ativ es ex ist to accom m odate particu lar app roache s

in the f ou r b as ic step s in D N A iso lation and prop ag ation .

PCR .

p o ly m erase ch ain reac tion . A dap ted f rom re f e rence 11 .

Antib io t ic

R I d ig e s tio n

A A T T

T T A A

A n n e a y

T A B L E 2

R e c o m b i n a n t

P l a s m i d

S

R R O L L

I D N A Lig a se

0

I T ra ns fo rm B a cte ria

R e c o m b in a n t An tib io tic s e le c tio n

P l a s m i d I

r

c D ) . ( . .L B a c t e r i a l

c

( , ,> ) c h r o m o s o m e

FIG 5 . G eneration o f reco mb inan t DN A m olecu les . G enom ic D N A and

v ec to r are

cleav ed w ith

re s triction endo nuc lease . T he tw o

sp ec ie s are in cu bate d

to ge th er. allow ing com patib le term in i to ann eal to on e ano ther and becom e

co valently lin ked by the enz y m e DN A ligase. Bacter ia are re nd ere d c om p ete nt

to in te rnaliz e the f o re ig n DN A . an d cells that gain th e p lasm id are se lec ted

by an tib io t ic resistan ce. R I. co R I re stri ctio n e nd on uc le ase .

u nit. O f ten the g ene is clo ned labo rio usly as o v e rlappin g f rag-

m en ts . H ow ev er, it is po ssib le to m ak e a do ub le -strand ed cop y

[com p lem en tary DN A (eD N A )] o f the m R N A transcrip t by use

o f the enz y m e rev erse tran scrip tase . B ecause th e m R N A is dev o id

o f in trons. it con tain s on ly the pro tein -co d ing sequence w ith

un tran slated portion s lim ited to the 5 and 3 en ds. T h e sy n thesis

o f cDN A is illu strated in Fig ure 6 .

D igested to tal g enom ic D N A or eD N A rep resen tativ e o f the

ce lls to tal m R N A can be in se rted in to a v ariety o f v ec to r sy stem s

creating a lib rary w hose co n ten ts co n tain a hos t o f d if f e ren t genes .

W hen one w ishes to iso late a particu lar gene f rom these lib rarie s,

it is necessary to sc reen the lib rary to iden tif y th e spec ies o f

in te re st. Figure 7 illu s trate s a com m on strategy . In th is ex am p le,

eD N A is c loned in a bac te riophage v ec to r that is th en pack aged

in to in f ectiou s v iral partic le s. E ach v iru s co n tain s a d if f e ren t

V ec to rs u sed in D N A clon in g

Library

S iz e o f

V ecto r insert

e DNA

G enom ic C om m en ts

kh

Plasm id


5/10

0 0

0 0 0

cDNA

Ec o RI d i g e s t i o n

s i z e s e l e c t i o n

rI I r I

D N A lig a s e

A T P Lin k e rs

T D NA p oly m e ra s e

5 3

5 AAAA 3 R. T .

. -T T T T d N T P

I

_ _ _ _ _ _ _ _ _ _ _ _ _ _ 3 5

TTTT

I

R ECOM B IN A N T-D N A IN TR OD U CTIO N 5 S

R N A is o la tio n

AAAA AAAA

/\

p o l y A

s e l e c t i o n

# { 1 4 9 }ve r o lig o d T c e llu lo s e

- - -

R N A H /D N A p O l 1

.

T T T T

FIG 6 . eD N A sy nthesis. A D N A co py o f the m R N A transcript can be sy nthesized by use o f the enzy m e rev erse

transcriptase . m RN A is iso lated by binding o f the po ly -A tail to an o lig o dT affinity co lum n. A n o lig o dT prim er is

used to initiate first-s trand eD N A sy nthes is in a 5 to 3 direc tio n anlisense ) fro m the m R N A tem plate . The RN A

strand o f the resulting he tero duplex is partially deg raded by RN ase H RN A H ). and D N A po ly m erase I initiate s

seco nd-s trand D NA sy nthes is

sense

strand). w ith the final pro duc t be ing a do uble -stranded D N A co py o fthe m RN A .

To ensure that the pro duc t has flush term ini, the enzy m e T7 D N A po ly m erase w ill po lish unev en ends. This enzy m e

p o sse sse s both 5- to-3 polm erase ac tiv ity and a 3 -to -S ex o nuc lease ac tio n that acts e spec ially w e ll o n o v erhang ing

s ing le -stranded D N A term ini. The eD N A can then be m o dified to inc lude any restric tio n s ite s at its term ini by the

additio n o f linker frag m ents. Linkers are sho rt, blunt-ended. do uble -s tranded frag m ents w ith co nv enient internal re -

s tric tio n site s. These are added to the D N A by D N A lig ase i e . blunt-ended lig atio n) and after dig e stio n w ith the

appro priate re stric tio n endo nuc lease . y ie ld co hesiv e term ini capable o f be ing lig ated into the v ecto r o f cho ice . To

ensure that this last dig e stio n step do es no t cleav e the eD N A itse lf if it happens to co ntain the sam e res tric tio n site

inc luded in the linker, the eD N A m ay be m ethy lated be fo re the additio n o f linkers so that it is re sistant to enzy m e

dig estio n. N ew er v ers io ns o f linkers called adapters o bv iate the need fo r these later s teps. R.T., rev erse transcriptase:

dN TP. deo x ynuc leo tide tripho sphate.

phag e w ith a unique g ene insert. These v iruse s are m ix ed w ith

ho st bac teria and plated o n nutrient ag ar m edia. B acteria that

hav e been infec ted w ith an indiv idual phag e lead to ev entual

ly s is o r decreased g ro w th o f the ho st. w hich appears as a ho le ,

orp/aque. o n the law n o fbac teria. A sam ple o feach plaque can

be replicated o nto a piece o f nitro ce llulo se filte r paper. The paper

abso rbs v iral nuc le ic ac ids as w e ll as pro teins sy nthesized by the

v irus. The f ilte rs are screened w ith a radio ac tiv e pro be that is a

seg m ent o f D N A ho m o lo g o us to the desired g ene . The pro be

m ay be a piece o f the g ene itse lf, iso lated prev io usly fro m a

dif ferent ce ll ty pe. a c lo se ly re lated g ene fro m a fam ily o f s im ilar

pro te ins. the sam e g ene fro m a different ho st spec ie s. o r a sy n-

the tic D N A frag m ent based o n the pro tein sequence fro m a

sm all po rtio n o f the pro te in itself. The filte r is incubated w ith

the radio activ e ly labe led pro be. B ecause o f sequence co m ple -

m entarity , the pro be w ill bind, o r

/ i rb r id i :e

to plaques co ntain-

ing s im ilar sequences . A fter ex po s ing the filte r to x -ray film ,

candidate plaques co ntaining the g ene o f intere st can be identified

and iso lated fro m the o rig inal m aster plate .

A lternativ e ly . eD N A m o lecule s m ay be inserted into v ec to rs

that allo w efficient pro te in pro duc tio n in

E

co/ i. T he se e xpre ss io n

v ec to rs co ntain a s tro ng bac terial pro m o ter that direc ts fo re ig n-

g ene transcriptio n. In m o st sy stem s a po rtio n o fthe

/ac

operon

is used, w hich inc ludes the pro m o ter and a po rtio n o f the g ene

fo r . g alac to sidase /acZ). The eD N A insert is c lo ned in the

sam e translatio nal reading fram e as is the 3 -g alac to s idase g ene,

creating a fusiOt i gene. Thus , the m R N A pro duced fro m this

g ene co ntains a sho rt 1 3 -g alac to sidase sequence at its 5 end, w hich

allo w s ribo so m e binding and e ffic ient translatio n in the pro -

kary o te ho st. The fus io n pro te in pro duced by these reco m binant

v ec to rs can be de tected by radio labe led antibo dy screening o f

nitro ce llulo se filte rs in a fashio n sim ilar to that described fo r

nuc leic ac id pro bes.

N ew er m etho ds o fscreening hav e been dev e lo ped to circum -

v ent so m e co m m o n pro blem s enco untered w ith the traditio nal

techniques described abo v e . Ex press io n-v ec to r c lo ning is lim ited

because antibo die s direc ted ag ainst the nativ e pro te in m ay no t

reco g nize a fus io n pro tein, o r the bacterial ho s t m ay no t m o dify

the translated pro duc t sim ilarly to the w ay eukary o tic ce lls do .

S uch im po rtant po s ttranslatio nal ev ents inc lude g ly co sy latio n.

One m etho d dev elo ped fo r the iso latio n o f cD N A s co ding fo r

cell-surface m o lecule s is diag ram m ed in Fig ure 8 . Plasm id v ec-


6/10

c o s

In fe c t h o s t E . c o li

S V 4 O o n

sp l ice + A n

I

D N A fro m a n tig e n

p os itiv e tis s ue

p B R 3 2 2 o n

E u k a r y o t i c

.

tra ns crip tio n u n it

I

2 5 4 S

C A R R O L L

c D N A

lig a te to p h a g e a rm s

p a c k a g e p h a g e

Lift p h a g e s o n to

I

n itro ce llu lo s e . S c re e n

w ith ra d ia c tiv e p ro be

E ID

FIG 7. eD N A clo ning . The eD N A w ith the additio n o f co m patible

ends is no w lig ated into the appro priate v ec to r. In this ex am ple the v ec to r

is a bac terio phag e . The phag e is packag ed into an infec tio us v iral partic le

and is used to infec t bac teria. B ecause the v irus w ill ev entually lead to

ly sis o f the hos t. ho les w ill appear on the law n o f bacteria w he re a

single bacterial ce ll has picke d up a re com binant virus. A portion o f

the se v iral partic le s can be replicated o nto a piece o f filte r paper. The

paper can then be screened w ith a pro be to identify the c lo ne co ntaining

the D N A seg m ent o f intere st.

to rs capable o f replicating w ithin eukary o tic ce lls o f hig her o r-

g anism s hav e no w been dev elo ped. These v ec to rs inc lude v iral

o rig ins o f replicatio n. usually fro m the S V 4 O v irus o r the Epste in-

B arr v irus, w hich allo w the ir e ffec tiv e pro pag atio n in eukary o tic

ce lls . These plasm ids can be intro duced into eukary o tic ho s ts

such as C OS cells . S tro ng pro m o ter sequences induce the

ex pressio n o fm RN A fro m eD N A inserts . Trans latio n pro ceeds ,

e ffec tiv e po s ttranslatio nal m o dificatio n takes place . and m o le-

cule s are transpo rted to the ce ll surface . Ce lls co ntaining the

eD N A o f intere st can be enriched by s tepw ise binding to anti-

bo die s reco g niz ing the cell-surface pro te in.

In so m e c ircum stances a pro te in m ay be asso ciated w ith a

w ell-de fined bio lo g ical ac tiv ity but m ay no t hav e been purified

to allo w the g eneratio n o fantibo die s o r lim ited peptide -sequence

analy s is . O ne such ex am ple w as the sero to nin recepto r. It w as

kno w n that these recepto rs are ex pressed in the cho ro id plex us ,

w hich is a brain tissue asso c iated w ith the pro ductio n o f cere -

bro spinal fluid. m RN A iso lated fro m this tis sue w hen injec ted

into fro g o o cy tes w as able to pro duce an inw ard e lec trical current

in the presence o f sero to nin, an ac tiv ity that o ccurs in v iv o w ith

sero to nin binding . Researchers g enerated eD N A fro m the cho -

ro id plex us and inserted it into a bac terio phag e v ec to r that has

pro m o ters fo r RN A po ly m erases flanking the c lo ning site

F ig

9 ) . This perm its the pro duc tio n o f m R N A fro m eD N A inserts

in v itro . Plasm id clo nes w ere div ided into po o ls, m RN A w as

o s pro duced, and the resulting transcripts w ere injected into o o cy te s .

O nce inside the o o cy te the m R N A w as trans lated into pro te in

and o o cy tes co ntaining the m RN A fo r the sero to nin recepto r

ex hibited an e lectrical current in the presence o fsero to nin. The

eD N A po o l fro m w hich the transcript w as pro duced w as further

div ided until a s ing le re spo ns ible c lo ne w as ev entually iso lated.

A fter iso lating g enes thro ug h these techniques , their nuc le ic

ac id sequence can be de term ined and the co m ple te identity o f

the translated pro te in can be deduced. Clo ned g enes can be used

as pro bes them se lv e s to analy ze g ene struc ture in different ho s ts

o r in certain patho lo g ical states . The bas ic technique used in

such surv ey s is called S o uthern blo tting and is illus trated in Fig

u r e 10. G enom ic D NA is p r epar ed and d igested w i t h a r est r i ct i on

endo nuc lease . The dig es ted D N A is then e lec tro pho resed o n an

ag aro se g el. w hich separate s the frag m ents acco rding to size

s m aller frag m ents m ig rate fas ter to w ard the po s itiv e elec tro de ).

B ecause the enzy m e c leav es to tal g eno m ic D N A at m any s ite s ,

a br oad sm ear of DN A is seen . T he DN A is t hen t r an sfer r ed

fro m the g e l to a piece o f f ilte r paper. A s described earlier, a

seg m ent o f the g ene is radio ac tiv e ly labe led and is incubated

w ith the filter. A fter w ashing aw ay backg ro und radio activ ity , the

lo catio n o f the D N A frag m ent is seen thro ug h auto radio g raphy .

I f t h e r est r i ct i on endonuclease cleaves the gene in t er n al l y at m an y

site s, m o re than o ne band m ay be de tec ted. A lternativ e ly , if the

r a n s f e c t n t o

C O S c e l l s

C ells th a t e xp re ss

a n tig e n b in d to

M a b o n p la te

C e lls h a rv e s te d a n d p la s m id s

re c o v e re d fo r a d d itio n a l ro u n d s o f s e le c tio n

F I G

8 . M o lecular c lo ning by ce ll-surface ex pressio n o f pro te in. The

eD N A is c lo ned into a v ec to r that eg , S V 4 0 o ri v irus ) that is able to

replicate in eukary o tic cells . The eD N A is transcribed into m RN A and

translated into pro te in w ithin the ce ll. The pro te in is m o dified and ex -

po rted to the ce ll surface , w here it can be detec ted by a m o no c lo nal

antibody

Mab). A n, po ly A -additio n site : pB R 3 2 2 o ri, plasm id o rig in

o f re p li c at io n .


7/10

R E C O M B I N A N T -D N A I N T R O D U C T I O N 2 55S

Co n s t r u c t i o n o f

c D NA e xp re s s io n lib ra ry

f r o m p o s i t i v e f r a c t i o n

Di v i d e

c D N A

c l o n es

i n t o po ol s

I I I

C AP - R N A

In je ctio n o f o o c y te s

w ith c D N A tra n s crip ts

a n d m e a s u re m e n t o f

ac t iv i ty

5

3

Cy c l e 1

D e n a t u r a t i o n ,

P rim e r B in din g

D N A

S y n t h e s i s

A m p l if ie d

S e g m e n t

C y c le 2

G e l

A c i d ( D ep u r i r ia t i o n )

1 T r e a t -

B a s e (D e n a tu ra tro n ) o f G e lm e r i t

T n , ( N e ut ra liz a tio n )

I

P a p e r

T o w e l s

Filter

G el

S p o n g e

p o s i t i v e

Gr a d i e n t

F rac t iona t ion .

o f RNA _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

T r a n s c r i p t i o n

o f c DNA

p oo l s

n v tro w ith T 7

R N A p o ly m e ra s e

Pu r i t y c DNA b y

s e q ue n tia l d ilu tio n

o f c D N A p o o ls

F ra c tio n N u m b e r

Sm a l l L a r g e

R N A R N A

N o t

A T 7 c D N A T 3 A

Im

U

u 1 O O n A

FIG 9. Functional analysis of eD NA clones. The eD NA is inserted

into a vector system w here the cloning site is flanked by prom oters for

T3 and T7 R NA polym erase. m R N A is m ade in vitro from eD N A pools

and is injected into oocytes. W ithin the oocyte the transcripts are trans-

lated into protein. T he oocytes containing transcripts for the receptor

are recognized because a current is generated in the presence of bound

ligand. Figure adapted from ref4. C A P. term inus ofeukaryotic m R N As

containing 7-m ethvlguanylate: V. voltage: I. current.

gene is sm all or ifonly a segm ent ofthe gene is used as a probe,

a single hand m ay em erge. A disruption in gene structure caused

by a deletion or rearrangem ent w ith other chrom osom e segm ents

m ay produce a different pattern by this technique. The local-

:: ) /

A g e r o s e - -

G el

_ -

7 4

1234

x-

H y b r i d i z e

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Fi lm

/

Ex p o s e t o F i l m

- _ _

FIG 10. Southern blot analysis. G enom ic D N A is digested w ith a

restriction endonuclease yielding a broad sm ear of D N A on agarose gel

electrophoresis. The gel is treated with an acid solution depurinated to

fragm ent the D N A and assist transfer of it to a section of filter paper.

T he D N A is also denatured before it is transferred to allow binding of

com plem entary radioactively labeled probe D N A . R eprinted w ith per-

m ission from W L C arroll and A L Schw artz 2 .

ization ofsueh abnorm alities and the fine m apping ofthe norm al

gene are accom plished by use of a series of restriction endonu-

cleases and different portions ofthe gene as probes. A variation

ofthis blotting procedure called N orthern blotting analyzes R N A

instead of D NA . Processed R N A is m uch sm aller than native

D N A and cleavage is not necessary before eleetrophoresis. H ow -

ever, the single-stranded character of R N A allow s this species

to form m any hairpin-loop structures. w hich alter its m igration

during electrophoresis. T herefore. these gels are run under strong

denaturing conditions.

A lthough the construction of new er. m ore-efficient vector-

cloning system s have m ade these techniques less labor intensive,

a considerable am ount of laboratory effort is spent isolating a

single gene. It is even m ore form idable to isolate the sam e gene

from different sources different species or m any m em bers of

gene fam ilies eg. im m unoglobulin genes . In addition. biological

m aterial m ay be in short supply. R ecently. a technique has

em erged that has greatly sim plified m any com m on recom binant

D N A techniques 6 . The polym erase chain reaction PC R al-

low s the efficient am plification of gene segm ents from exceed-

ingly sm all am ounts of D N A Fig I I . S in g le -s t r an d ed o ligom er s

bind to opposite strands of D NA at a point flanking the gene

segm ent of interest. Substrate D N A is heated to a high tem per-

ature to allow the strands to denature. The tem perature is then

low ered to allow the single-stranded oligonucleotide prim ers to

anneal to the target D NA . The annealing tem perature is high

3.

5 3

.

5

* -

. - - . ,

0

F IG 11 . Polymerase chain reaction. Single-stranded oligonucleotide

prim ers bind to opposite strands of denatured D N A under stringent

conditions at high tem perature. T aq polym erase functions at high tem -

perature to faithfully reproduce the gene segm e nt of interest. M ultiple

cycles am plify the target segm ent. R eproduced w ith perm ission from

W L C arroll and A L Schw artz 12 .


8/10

C o n v e n t i o n a l

( < 2 0 K b )

. .

Pu l s e d F i e l d

( 4 O K b - l 4 0 0 K b )

. .

I

#{ 14 9 } I I

+ + +

I

+

FI G I 2. G el e lec troph oresis o f D NA . In conv en tion al elec tropho resis

the elec tric f ie ld is cons tan t and is orien ted in a parallel d irec tion . Pu lsed -

f ie ld ge l e lec trop horesis co nsis ts o f perpend icu larly o rien ted . non un if o rm .

alternate ly pu lsed elec tric f ie ld s.

en ough to allow prim er b in d ing to sequences o f h igh com p le -

m en tarity on ly , th u s m in im iz ing non spec if ic b ind ing . A new ly

iso lated heat-s tab le po ly m erase f rom the o rgan ism Thermos

aqualicus in itiates n ew DN A copies f rom th e bound p rim ers.

S o u rc e D N A

I R e s t r i c t i o n

e n d o n u c l e a s e

R ig h t a rm

# {1 4 9 }-H :4 1 : 1 -c 1 V

Lef t

a r m

TEL T RP1 ARS1 CEN4

L i g a se

In s e rt D N A

URA3 T EL

FI G I 3. Y eas t art if ic ial ch rom osom e (Y AC ) c lon ing sy stem . T he v ec tor cons ists o f thre e y eas t elem ents e ssential

f or chrom o som e f unc tio n: a cen trom ere (CEN 4 ). seq uence s th at f o rm te lom eres in v iv o (T E L ). and an au tonom ou s

rep licatio n sequence (A R S I). In addition . p lasm id sequ en ces that allow g row th and se lect ion in E co /i a re r ep re s en t ed

by the th in line. T w o se lectable m ark ers (T R PI and UR A 3 ) allow sele ction of y east co lonies con tain in g both v ecto r

arm s. In terruption of th e clon ing site (S U P4 ) leads to a red colony . R eprin ted w ith p erm iss ion f rom D T B urk e, G F

Carle . an d M V O lson (3 ).

2 5 6 S

R R O L L

R ep eated round s o f th is basic reaction lead to lo garithm ic am -

p lif ication o f targe t DN A . S ev eral m o d if ication s o f th is p rocedure

has led to a num ber o f new app lication s too num erou s to inc lude

in th is rev iew . T he PCR reaction has had a trem endou s im pact

on rou tine recom b in an t DN A techn iqu es .

.

+ T he m e thods d iscu ssed abov e are capab le o f an aly z ing DN A

f ragm en ts u p to a m ax im um s iz e o f 50 k b . H ow ev er, it is n ow

apparen t that the genes o f h ig her o rgan ism s m ay span hu ndreds

o f k ilob ases. For ex am p le , th e gene encod ing the b loo d -c lo ttin g

com pon en t f ac to r 8 . w h ich is de f ic ien t in p atien ts w ith h em o -

philia. approach es 200 k b . T h e gen e encod ing the I 5-k b tran-

sc rip t that is de f ec tiv e in patien ts w ith D u ch en nes m uscu lar dy s-

trophy is e s tim ated to cov er

106 bp ( 1000 k b ). E f f ectiv e an aly s is

o f th e se larg e gene c lu ste rs can no t be approached us ing conv en -

tio nal techn iqu es . In som e c ircum stances a gene m ay be lo caliz ed

to a particu lar band in standard k ary o ty p e an aly sis bu t its ev en -

tual locatio n m ay lie a s ign if ican t d istance aw ay . W alk ing

tow ard the gene o f in te re s t w ith cu rren t v ec to r sy stem s is im -

p rac tical. In recen t y ears con siderab le in te re s t has dev e lop ed in

m ap p ing the genom es o f m any w e ll-stud ied o rgan ism s . in c lu d ing

hum ans . T o ef f ectiv e ly analy z e larger D N A f ragm en ts new er

m etho ds to iso late , m ap . an d clon e large D N A sam p les are be in g

deve loped .

T he generation o f larg e D N A f ragm en ts can be accom p lish ed

by restriction endonuclease diges tio n w ith enz ym es th at recog-

n iz e seq uences that app ear relativ e ly in f requ en tly in the genom e.

CEN 4

T R P 1 S f1 1 /N o t I Sm a I,c lo n in g s i t e

I / No t I

B ar n H I

B arn H I

S r n a

I

p h o s p h a t as e ________ __________ _


9/10

G e n o m i c E nd R es cu e

PC R

4,

R ECOM B IN A NT -DN A

YA C

T r p l/A rs l C en 4 S u p 4 S u p 4 u r a 3

I N T RODUCT ION 257S

m igrate th ro ugh ge l pores in the d irec tion o f n ew cu rren t. B y

v ary in g the tim e in terv al (o r sw itch in te rv al) be tw een chang es

in curren t d irec tion . DN A m olecu le s be tw een 50 and > 1000

kb c a n b e separated e f f ec tiv e ly . N ew er g el techn iques based on

1= II

L e f t A rm

R gh t A rm

th e abov e concep t hav e been in troduced and hav e f eatu red m ore

un if o rm e lectric f ield s w ith f ew er artif ac ts d ue to d is to rtion o f

4 , th e f ie ld .

= = = tf k Ai

I

Liga t ion

T he generation o f su eh large f ragm en ts g reatly aid s m app ing

stud ie s bu t the ir p ropagatio n in v ec to rs rem ain s a prob lem be -

cause th e larges t f ragm en t accom m od ated by trad itional co sm id

v ec to rs ap proaches 50 k b . O ne sy stem now av ailab le f o r c lon ing

d f ut e c o nd it io n s)

4 ,

s u c h la rg e f ragm en ts is th e y east artif icial ch rom o som e (Y AC )

(3 , 8 ). T he bas ic f unc tional un its o f y east ch rom osom es. th e

sequences that allow segregation and rep lication . w ere p rev iou s ly

iden tif ied and iso lated . T hese e lem en ts w ere com b ined w ith

p lasm id sequen ces to y ie ld a hy brid v ec to r capab le o f rep licating

as a p lasm id in I: : c o/ i (Fig 1 3 ). A f ter the ap prop riate en z y m e

d igestion . th e circu lar v ecto r y ie ld s tw o linear D N A arm s ab le

to accep t a large segm en t o f f o re ign DN A . A recom b inan t c lone

can be used to trans f o rm y east sphero p las ts. B ecau se each arm

con tain s a se lec tab le m ark er, th e trans f o rm an ts are p lated on

de f ined m ed ia, w h ich allow s selec tiv e o u tg row th o f y east eon -

tam ing bo th v ec to r arm s. In add ition . th e in se rtio n o f a DN A

f ragm en t be tw een bo th v ec to r arm s in terrup ts a g ene seq uence ,

w h ich lead s to d is tin ctiv e red co lon ie s. T h is sy stem has now

been show n to f aith f u lly p ropagate eno ugh larg e D N A f ragm en ts

to allow con struc tion o f com prehens iv e lib rarie s. T h is strategy

4

P C R

P r o d u c t

* k g . m

a o

r .oa.

has already led to the iso lation o f large DN A f ragm en ts eon -

tam ing m ultip le gene c lu ste rs .

An app lication o f the PC R reac tion illu strated in Figure 14

allow s the rap id iso lation o f the ends o f th ese larg e in se rts c lon ed

FIG I 4 . Inv erted PC R to iso late en ds o f Y A C c lones . T he Y AC c lone

is d iges ted w ith enz y m e T aq I. w h ich c leav es the le f t v ecto r arm at a

k now n site ad jacen t to the c lon in g site and the in sert at an unk now n

s ite w ith in the c lone . T he T aq I f ragm en ts are ligated tog e th er to f o rm a

c irc le. T h is lin e s up tw o prim ers in to pos itio n to be ab le to am plif y in se rt

DN A by the PCR reaction . R eprin ted w ith perm iss io n f rom GA S ilv e r-

in to Y A C s. T hese ends can th en be used as probes to iso late

con tiguou s c lo nes f rom th e Y A C lib rary . T hu s, on e can e f f ic ien tly

jum p to one end o f a f ragm en t and u se the ends as p rob es to

rap id ly w alk tow ard a gen e o f in te re st (9 ). B y scann ing these

larg e f ragm en ts f o r open -read in g f ram es and abnorm alities that

occur in certain d isease s . n ew gen es can b e iso lated w hose pro te in

m an . R D Y e, K M Po llock . JE S ad le r. and S i K orsm ey er (9 ).

p rodu cts hav e no t been iden tif ied . T h is ty pe o f analy s is, o r reverse

genet ics, w ill g reatly aid o ur unders tan d ing o f a v arie ty o f g en etic

DN A f ragm en ts o f 100 to > 1 000 k b m ay be seen af te r d iges tio n

w ith enz y m es such as

1/ u

I, No t I, and X/zo I. B ecau se D N A o f

th is siz e has a tendency to shear w hen standard d iges tion s in

so lu tion are used , it is necessary to carry th ese reac tion s o u t in

agaro se . T hese agaro se b lock s are then loaded d irectly in to s lo ts

at th e o rig in o f horiz on tal agaro se ge ls . N ew er elec troph oresis

techn iques allow separatio n o f su ch larg e D N A m o lecu le s . In

con v en tional ge l e lee trop hores is the sep aration o f DN A is b ased

o n the s iev ing properties o f th e ge l m atrix . A sm aller m o lecu le

eas ily nego tiates the pores o f the ge l w h ereas larger m o lecu le s

hav e g reate r d if f icu lty . H ow ev er, under the in f lu ence o f an e lee -

tiv e f ield , th e D N A m olecu le m ay change its shape b y assum ing

d isease s an d w ill allow m o re e f f icien t m app in g o f large genom es.

T h is artic le p rov ides a s im p le ov erv iew o f conv en tion al re -

com b inan t DN A techn o logy and in trodu ces som e m ore recen t

m etho do log ical adv ances to allow m o re e f f icien t, re liab le gene

c lon ing . T he p ow er o f the se techn iq ues m ak es it m andato ry f o r

ev ery lif e sc ien tis t to be f am iliar w ith th is f ield . T h is k now ledge

w ill no t necessarily p rov ide inv es tigato rs w ith th e right ques -

tio n s bu t w ill lik e ly so lv e b io log ical p ro b lem s n o t appro ach ed

by prev iou s tech n iques . T h e thoug h tf u l app lication o f th is

k now led ge in th e f ie ld o f b io techn o logy w ill undo ub ted ly lead

to so lu tion s to the p rob lem o f an increasing f oo d dem and f rom

a p lane t w hose re sou rces are alread y strained .

# {163 } 3

an e lo ngated co n f igu ration paralle l to the elec tric f ie ld , a p rocess

re f e rred to as replalion. In c on ve ntio nal co ntin uo us -e le ctric -f ie ld

ge ls, rep tating D N A m olecu les abo v e a certain th re sho ld s iz e

R eferences

>

3 0 k b ) all m ig rate toge ther independ en t o f th e ir leng th . Pulsed-

fie ld g e / e/ectrophoresis in v o lv e s alte rnating the d irec tion o f th e

elec tric cu rren t (Fig 1 2 ). D N A m olecu le s reo rien t th em selv e s in

1 . A ru f f o A . S eed B . M o lecu lar clon ing o f a CD 28 eDN A by a h igh -

e f f ic iency co s cell ex p re ss ion sy s tem . Proc N atl A cad Sc i US A

1987 :84 :8573 -7 .

re sponse to th is change in curren t d irec tion . L arger m o lecu le s

2.

Berger

S L , K im me l A R . ed s. G u ide to m olecu lar c lon ing techn iques .

tak e a lon ger tim e to reo rien t th em se lv e s be f o re th ey ef f ectiv e ly M e thod s E nz y m o l. S an D iego . C A : A cadem ic Press , l98 7 .

I

T e l o m e r e s

-

Y ea s t D NA

=

p B R 3 2 2 DN A

J

I n s e r t DN A

--+

L e ft A rm S e n s e P r i m er

. . . .>

L ef t A rm A nt i -S en se P r i m er

RE D i g e s t i o n

.9. .

T a q I )


10/10

258S

C A R R O L L

3. B u rke D T . C arle G F . O lson M . C lon ing of la rge segm en ts o f ex-

o genous D N A in to yeast by m eans o fa rtif ic ia l ch rom osom e v ec to rs .

S ci en ce 1 98 7: 23 6: 80 6- 12 .

Ju lius D . M acD erm ott A B . A x e R . Jesse TM . M olecu lar ch arac -

te riza tio n of a fun ctiona l eD N A en cod ing the se ro ton in Ic recep to r.

S ci en ce 1 98 8: 24 1: 55 8- 64 .

5. L ew in B . G enes IV . N ew Y ork : O xfo rd U nivers ity P ress . 199 0 .

6 . S a ik i R K . G elfan d D N . S to ffe l S . e t al. P rim er d irected enzym atic

am plifica tio n o f D N A w ith a therm ostab le D N A

polymerase.

Science

1988 :239 :487-9 .

7. S am b rook I. F ritsch E F . M ania tis T . M olecu lar clon in g . A lab ora to ry

m anua l. C o ld S pring H arbor. N Y : C old Sp ring H arbor L ab ora to ry

P ress . 198 9.

8 . S chw artz D C . C an to r C R . Separa tion of y eas t ch rom osom e sized

D N A s by pu lsed f ie ld grad ien t ge l elee tropho resis. C ell 1 984 :37 :6 7-

75 .

9 . S ilve rm an G A . Y e R D . Po llock K M . S ad le r JE , K orsm eyer S i. U se

ofyeast artific ia l ch ro m oso m e c lones fo r m app ing and w alk in g w ith in

hu m an chrom osom e segm ent 18q 2 I .3 . P roc N atI A cad S e i U SA

1989 :86 :7485-9 .

10 . D arne ll J, L od ish H . B altim ore D . M o lecu la r ce ll b io logy . N ew Y ork :

F reem an W H , 19 86 .

1 . O ld R W , Prim ro se S B . Princ ip les o f gene m anipu la tion . C am bridg e ,

U K : B laekw ell Sc ien tific Pub lica tions . 19 85 .

12 . C arro ll W L . Schw artz A L . M olecu lar onco logy . In : Fern bach D i,

V ietti T i. eds . C lin ical p ed ia tric onco log y . S t L o u is: C V M o sby .

1 9 9 1 :2 9 -6 8.

am j clin nutr 1993 carroll 249s 58s

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