Lecture 1 3
Recombina tion
# 1 Mol e c ula r C loni ng O ut line
•R e c o mb in a n t D N A te c h n o lo g y d e p e n d s o n t h e a b i lity t o p r o d u c e la r g e n u mb e r s o f id e n tic a l
D N A mo le c u le s ( c lo n e s ) •C lo n e s a r e ty p i c a lly g e n e r a te d b y p la c i n g a D N A fr a g me n t o f
in t e r e s t in to a v e c to r D N A mo le c u le , w h ic h c a n r e p l ic a te in a h o s t c e ll •W h e n a s i n g le
v e c to r c o n ta in i n g a s in g le D N A fr a g me n t is in tr o d u c e d in t o a h o s t c e l l, la r g e n u m b e r s o f th e
fr a g me n t a r e r e p r o d u c e d a lo n g w it h th e v e c to r •Tw o c o mmo n v e c t o r s a r e E. c o li p l a s mid
v e c to r s a n d b a c te r io p h a g e λ v e c to r s .
# 2 . Lig a t ion of t he s t ic k y e nds
L ig a s e r e a c tio n , o p p o s ite t o r e s t r ic tio n e n z y me s . L ig a s e r e a c tio n : o p p o s ite t o r e s t r ic tio n
e n z y me , r e q u ir e s 5 ’ - P ( fr o m D N A2 ) a n d 3 ’ - O H ( fr o m D N A1 ) .
# 3 . Lig a t ion of t he s t ic k y e nds
L ig a s e me c h a n is m: D N A lig a s e fo r ms a c tiv a te d lig a s e - AM P ( fr o m ATP o r N AD ) , AMP lin k s
to 5 ’ - P, 3 ’ - O H a tta c k s fo r min g n e w p h o s p h o d i e s te r b o n d , s e a l! Tw o D N A mo le c u le s ,
c le a v e d w ith Ec o R I a n d lig a te to fo r m r e c o mb in a n t mo le c u le s .
# 4 . Lig a t ion of t he s t ic k y e nds
Fo r ma tio n o f c o h e s iv e e n d s ( Pr o d u c in g s tic k y e n d s w ith a d a p to r s ) : a d d itio n a n d c l e a v a g e o f
a c h e mic a lly s y n th e s i z e d li n k e r ( lin k e r w a s p u t o n w ith T4 lig a s e ) .
# 5 C lo ning
Pla s mid : c ir c u la r , d s , e x t r a c h r o mo s o ma l s e lf - r e p li c a tin g D N A m o le c u l e o c c u r in g n a tu r a lly
in b a c te r ia , y e a s t a n d h ig h e r e u k a r y o ti c c e ll s , e it h e r p a r a s iti c o r s y mb io t ic , ~k b to 1 0 0 k b , a t
le a s t 1 t o th e d a u g h t e r c e l l ( d r u g - r e s i s ta n c e , tr a n s fe r g e n e s e n c o d in g p r o t e in s f o r ms
ma c r o mo le c u la r tu b e o r p il u s ) . C lo n in g v e c to r : e n g in e e r e d p l a s mid s w ith r e d u c e d s iz e ( ~3
k b ) , c o n t a in in g o n ly o r i , d r u g - r e s is ta n c e g e n e , c lo n in g s ite . Amp r e n c o d e s β - la c ta ma s e
w h i c h in a c tiv a t e s a mp ic illi n .
# 6 C lo ning
Ba s ic c lo n in g p r o to c o l: c u t a n d p a s te .
# 7 C lo ning
R e c o mb in a n t D N A : Th e p la s mi d a n d th e fo r e ig n D N A a r e c u t b y th i s r e s t r ic tio n
e n d o n u c le a s e ( Ec o R I in th is e x a mp le ) p r o d u c in g in te r m e d ia te s w ith s tic k y a n d
c o m p le me n ta r y e n d s . T h o s e t w o in t e r me d i a te s r e c o mb i n e b y b a s e - p a ir in g a n d a r e lin k e d b y
th e a c tio n o f D N A lig a s e . A n e w p la s mid c o n ta in in g th e fo r e ig n D N A a s a n in s e r t i s
o b t a in e d . A fe w mis ma tc h e s o c c u r , p r o d u c in g a n u n d e s ir a b l e r e c o mb in a n t.
# 8 C lo ning
Th e fir s t r e c o m b in a n t D N A e v e r p r o d u c e d in th e la b : Sta n l e y C o h e n a n d H e r b e r t Bo y e r c u t
2 p la s mid s w ith Ec o R I, lig a te a n d s c r e e n fo r E.c o li c l o n e s t h a t a r e r e s i s ta n t to b o t h
a n t ib io ti c s s in c e th e y h a r b o r th e r e c o mb in a n t p la s mid s .
# 9 C lo ning
p BR 3 2 2 p l a s mid : h o s t c o n ta i n in g p BR 3 2 2 w ith i n s e r t a t d if fe r e n t r e s tr ic tio n s ite s c a n b e
s e l e c te d . Th e r e a r e m a n y s i te s c a n b e u s e d .
# 1 0 C lo ning
C lo n in g a t th e Ps t I s ite : ma k e c o n s tr u c t, tr a n s fo r m b a c te r i a , s c r e e n fo r te tr a c y c li n e r e s is ta n t b u t a mp ic illi n - s e n s itiv e c lo n e s u s in g r e p li c a p la tin g ( ma k e a r e p li c a p la te tr e a tin g
w it h a mp ic illi n , s e n s itiv e c lo n e s a r e r e c o v e r e d fr o m th e o r ig i n a l p l a te ) .
# 1 1 C lo ning
Po l y lin k e r s : c h e mic a l ly s y n th e s iz e d p o l y lin k e r c o n t a in in g o n e c o p y o f s e v e r a l d if fe r e n t
r e s tr ic ti o n s it e s in in tr o d u c e d i n to th e v e c t o r to fa c ili ta te c lo n in g a n d d ir e c ti o n a l c lo n in g .
# 1 2 C lo ning
( i) MC S: m u ltip l e c lo n in g s i te . M C S is in s e r t e d in t o a g e n e e n c o d in g th e N - te r min a l p a r t o f
β - g a l a c to s i d a s e . C lo n e s h a r b o r in g th e v e c to r p lu s a n in s e r t r e ma in w h i te . ( ii) p U C s e r ie s :
d e r iv e d f r o m p B R 3 2 2 ( 4 0 % d e le te d ) , th e MC S is in th e la c Z’ ( N - te r mi n a l o f β - g a l a c to s i d a s e ,
th e h o s t c o n ta i n s th e C - te r min u s p o r tio n , MC S s ta r t s a fte r th e 7 th c o d o n , r e ta in s th e O R F) ,
s o n o in s e r t b l u e ( w i th X- g a l) , w ith in s e r t w h ite .
# 1 3 Mol e c ula r C lo ning
Th e g e n e r a l p r o c e d u r e fo r c lo n in g w ith p la s mi d v e c t o r s : p la s mid w ith Amp r a n d in s e r t is
tr a n s fo r m e d in t o h o s t w ith C a C l 2 o r o th e r me th o d s , g r o w in a mp ic illi n p la t e , s u r v iv in g c e lls
fo r m c o lo n y . Pl a s mid c lo n in g p e r m its is o la tio n o f D N A fr a g me n ts fr o m c o mp le x mix t u r e s :
Ea c h c o lo n y is d e r iv e d fr o m a s in g le c e ll c o n ta in in g th e s a me p la s mid .
# 1 4 C lo ning S t ra t e g y
Pr o c e s s b y w h ic h a p l a s mid is u s e d to i mp o r t r e c o mb in a n t D N A in to a h o s t c e ll fo r
c lo n in g .I n D N A c lo n in g , a D N A fr a g me n t th a t c o n ta in s a g e n e o f in te r e s t is in s e r t e d in t o a
c lo n in g v e c to r o r p la s mid . Th e p l a s mid c a r r y i n g g e n e s fo r a n tib io tic r e s is t a n c e , a n d a D N A
s tr a n d , w h ic h c o n ta in s th e g e n e o f in te r e s t, a r e b o th c u t w ith th e s a me r e s tr ic ti o n
e n d o n u c le a s e . T h e p la s mid i s o p e n e d u p a n d th e g e n e is fr e e d fr o m its p a r e n t D N A s tr a n d .
Th e y h a v e c o mp l e me n ta r y " s t ic k y e n d s ." Th e o p e n e d p la s mid a n d t h e fr e e d g e n e a r e mix e d
w it h D N A lig a s e , w h ic h r e fo r ms th e t w o p ie c e s a s r e c o m b in a n t D N A.
Th i s r e c o mb in a n t D N A s te w i s a llo w e d to tr a n s fo r m a b a c te r ia l c u ltu r e , w h ic h is t h e n
e x p o s e d t o a n ti b io tic s . All th e c e lls e x c e p t th o s e w h ic h h a v e b e e n e n c o d e d b y th e p la s m id
D N A r e c o m b in a n t a r e k ille d , le a v i n g a c e ll c u ltu r e c o n ta i n in g t h e d e s ir e d r e c o mb i n a n t D N A.
D N A c lo n i n g a ll o w s a c o p y o f a n y s p e c if ic p a r t o f a D N A ( o r R N A ) s e q u e n c e t o b e s e le c te d
a mo n g ma n y o th e r s a n d p r o d u c e d in a n u n limite d a mo u n t. Th is te c h n iq u e is th e fir s t s ta g e
o f mo s t o f th e g e n e ti c e n g i n e e r in g e x p e r ime n t s : p r o d u c tio n o f D N A lib r a r ie s , PC R , D N A
s e q u e n c in g , e t a l.
# 1 5 Mol e c ula r C loni ng
Th e g e n e r a l p r o c e d u r e fo r c lo n in g w ith p la s mi d v e c t o r s : p la s mid w ith Amp r a n d in s e r t is
tr a n s fo r m e d in t o h o s t w ith C a C l2 o r o th e r me th o d s , g r o w in a mp ic illi n p la t e , s u r v iv in g c e lls
fo r m c o lo n y . Pla s mid c lo n in g p e r mi ts is o la tio n o f D N A fr a g me n ts fr o m c o mp le x mix tu r e s :
Ea c h c o lo n y is d e r iv e d fr o m a s in g le c e ll c o n ta in in g th e s a me p la s mid .
# 1 6 Mol e c ula r C loni ng
Sc h e ma tic d ia g r a m ill u s tr a t in g mo le c u la r c lo n in g in to p la s mid .
# 1 7 D N A libra rie s
C o n s tr u c t in g a D N A li b r a r y w ith l a mb d a p h a g e a n d o t h e r c l o n in g v e c to r s : •C lo n in g a ll o f th e
g e n o mic D N A o f h ig h e r o r g a n is ms i n to p l a s mid v e c to r s is n o t p r a c tic a l d u e t o th e r e la ti v e ly
lo w tr a n s fo r ma t io n e f fic ie n c y o f E . c o l i a n d th e s ma ll n u mb e r o f tr a n s fo r me d c o lo n ie s th a t
c a n b e g r o w n o n a ty p ic a l c u ltu r e p la te •C lo n in g v e c to r s d e r iv e d fr o m b a c te r io p h a g e d o n o t
s u f fe r fr o m s u c h limi ta tio n s •A c o lle c t io n o f c lo n e s th a t in c lu d e s a l l th e D N A s e q u e n c e s o f a
g iv e n s p e c ie s i s c a ll e d a g e n o mic lib r a r y •A g e n o mic lib r a r y c a n b e s c r e e n e d fo r c lo n e s
c o n ta in in g a s e q u e n c e o f in te r e s t .
# 1 8 B a c t e riop ha ge
EM o f b a c te r io p h a g e λ v ir io n . (ii) Sim p lifie d v ie w o f b a c te r io p h a g e g e n o me :a b o u t 6 0 g e n e s ,
r e p la c e a b le r e g io n n o t e s s e n tia l a n d c a n b e r e p la c e d b y f o r e ig n D N A u p to 2 5 k b . L a r g e
s e g me n t o f th e 4 8 k b D N A o f th e l p h a g e a r e n o t e s s e n t ia l fo r p r o d u c tiv e in fe c tio n a n d c a n
b e r e p la c e d w it h in s e r ts .
# 1 9 B a c t e riop ha ge
As s e mb ly o f b a c te r io p h a g e l v ir io n : d u r in g la te s ta g e o f λ in f e c tio n c o n c a to me r s a r e
fo r me d , N u 1 a n d A p r o t e in p u s h 1 c o p y o f λ fr o m c o n c a to me r in to th e h e a d , t h e n ta il a d d e d
a n d th e y a r e r e a d y to g o to th e w a r .
# 2 0 B a c t e riop ha ge
λ p h a g e v e c to r : C h a r o n p h a g e 4 ta k e s 2 0 k b , r e mo v e th e s tu ffe r , lig a te th e in s e r t, mi x w ith
th e in v i tr o p a c k a g in g e x tr a c t, i n fe c t c e lls , th e i n s e r t h a s to b e b e tw e e n 1 2 k b a n d 2 0 k b .
# 2 1 B a c t e riop ha ge
Alt e r n a ti v e in f e c tio n mo d e s fo r l p h a g e : ly t ic o r ly s o g e n ic p a th w a y .
# 2 2 G e n omic L ibra ry
A s e t o f la mb d a ( o r p la s mid ) c lo n e s th a t c o ll e c tiv e ly c o n ta in e v e r y D N A s e q u e n c e in th e
g e n o me o f a p a r tic u la r o r g a n is m. N e a r ly c o mp l e te g e n o mic lib r a r ie s o f h ig h e r o r g a n is ms
c a n b e p r e p a r e d b y la mb d a c lo n in g . C o n s tr u c t io n o f a g e n o mic l ib r a r y : Sa u 3 A a n d Ba mH 1
g e n e r a te c o mp le me n ta r y s tic k y e n d s .
# 2 3 G e n omic L ibra ry
Sc h e ma tic d ia g r a m ill u s tr a t in g th e g e n e r a l a p p r o a c h in g fo r c o n s tr u c ti n g g e n o mic lib r a r y .
# 2 4 G e n omic L ibra ry
( i) Pla q u e h y b r i d iz a ti o n : s e le c tio n o f p o s itiv e c lo n e s , fi lte r r e p lic a te s th e d is h , p la q u e
c o n ta in in g p h a g e D N A d e n a tu r e d b y b a s e , b lo c k th e f ilte r w ith n o n - s p e c ific D N A o r p r o te in ,
h y b r id iz e w ith s p e c if ic g e n e p r o b e , a u t o r a d io g r a p h y to s e le c t t h e ta r g e t .g e n e . ( ii ) Th e mo s t
c o m mo n a p p r o a c h to id e n tify in g a s p e c if ic c lo n e in v o lv e s s c r e e n in g a lib r a r y b y
h y b r id iz a tio n w ith r a d io a c t iv e ly la b e le d D N A o r R N A p r o b e s . Id e n tific a tio n o f a s p e c ifi c
c lo n e fr o m a l p h a g e li b r a r y b y me m b r a n e h y b r id iz a tio n . Id e n tify in g , a n a ly z i n g , a n d
s e q u e n c in g c lo n e d D N A
# 2 5 C os mid v e c t or
C o s mid v e c to r : L a r g e r D N A f r a g me n ts u p to 4 5 k b c a n b e c l o n e d i n c o s mid s a n d o th e r
v e c to r s , c o s mid h a s c o h e s iv e e n d s fo r p a c k a g in g a n d p l a s mid o r i fo r r e p lic a tio n a s
p la s mid s in b a c te r ia . C o s mid s c a n n o t r e p lic a te a s p h a g e s b u t th e y a r e s ti ll in f r c tio u s .
# 2 6 M1 3 Pha ge
a f ila me n to u s v ir u s 9 0 0 n m lo n g a n d 9 n m w id e , s in g le s tr a n d 6 . 4 k b c i r c le D N A p r o te c te d b y
2 7 1 0 id e n tic a l p r o te i n s , g e ts in t o E. c o li v i a s e x p il u s , r e p lic a t e to R F, o n l y ( +) is p a c k e d
in t o v ir u s p a r t ic le . U s e fu l fo r s e q u e n c in g .
# 2 7 C om ple me n t a ry D N A ( c D N A ) lib ra rie s
c D N A a r e p r e p a r e d fr o m is o l a te d m R N As Po l y ( A) + R N A. o li g o - d T c o lu mn to p u r ify m R N A.
# 2 8 C om ple me n t a ry D N A ( c D N A ) lib ra rie s
Fo r ma tio n o f c D N A d u p l e x : R T, a lk a l i d ig e s tio n , o li g o ( d G ) ta ili n g , o li g o ( d C ) p r ime r , D N A
p o l , a n d d N T Ps .
# 2 9 Pr e p a r a tio n o f a b a c te r io p h a g e l c D N A lib r a r y
# 3 0 Ma k ing a c D N A Libr a ry
R e v e r s e T r a n s c r ip ta s e , R N a s e H ( d e g r a d e R N A in R N A - D N A h y b r id ) , D N A p o ly me r a s e I
( u s in g R N A a s p r ime r a n d p e r fo r m n ic k t r a n s la tio n ) , Te r mi n a l d e o x y n u c l e o tid y l tr a n s fe r a s e
( a d d in g d C T P to t h e c D N A d u p l e x , a n d d G T P to t h e v e c to r ) , L ig a s e a n d p o l I in th e h o s t
p e r fo r m lig a tio n R N A r e mo v a l, a n d s e a lin g ) .
# 3 1 C lo ne a s ingle c D N A
U s e R T- PC R to c lo n e a s in g le c D N A if t h e s e q u e n c e o f mR N A is k n o w n .
# 3 2 Pha ge mid
Ph a g e mid : p BS o r p Bl u e s c r i p t, MC S in s e r te d i n to la c Z’, o r i o f th e s s p h a g e f1 , T3 a n d T 7
p h a g e R N A p o ly m e r a s e p r o mo t e r s , u s e fu l fo r c D N A c lo n in g .
# 3 3 Ex p re s s io n v e c t or
Th e e x p r e s s io n v e c to r c o n ta in s a fr a g me n t o f th e E. c o li c h r o mo s o me c o n ta in in g th e la c
p r o mo te r a n d th e n e ig h b o r in g la c Z g e n e . In t h e p r e s e n c e o f th e la c to s e a n a lo g IPT G , R N A
p o l n o r ma l ly tr a n s c r ib e s th e la c Z g e n e , p r o d u c in g la c Z mR N A , w h ic h is t r a n s la te d to th e
e n c o d e d p r o te in , b - g a l a c to s i d a s e . Fo r min g f u s io n p r o te i n in p la s mid v e c to r : p U C a n d p BS
v e c to r s p la c e i n s e r te d D N A u n d e r th e c o n tr o l o f th e la c p r o mo te r . If th e D N A is i n fr a m e
w it h th e la c Z’ g e n e , a fu s io n p r o te in w il l b e e x p r e s s e d .
# 3 4 Ex p re s s io n v e c t or
E. c o li e x p r e s s i o n s y s te ms c a n p r o d u c e f u ll- le n g th p r o te in s : Pr o d u c in g h ig h le v e ls o f
p r o te in s fr o m c lo n e d c D N As . Ma n y p r o te in s a r e n o r ma lly e x p r e s s e d a t v e r y lo w
c o n c e n tr a tio n s w ith in c e lls , w h ic h ma k e s is o l a tio n o f s u f fic ie n t a mo u n ts fo r a n a l y s is
d if fic u lt . To o v e r c o m e th is p r o b l e m, D N A e x p r e s s io n v e c to r s c a n b e u s e d to p r o d u c e la r g e
a mo u n ts o f fu ll le n g t h p r o t e in s . L a c p r o mo te r .
# 3 5 Ex p re s s io n v e c t or
Tw o - s te p s y s te m : T7 R N A p o l y me r a s e a n d T7 la t e p r o m o te r , Ev e n l a r g e r a mo u n t s o f a
d e s ir e d p r o te in c a n b e e x p r e s s e d w ith a tw o - s te p s y s te m, 1 0 - 7 0 % o f th e to ta l p r o t e in
s y n th e s iz e d b y th e s e c e lls a fte r IPTG i s th e p r o te i n o f i n te r e s t.
# 3 6 Ex p re s s io n v e c t or
Fo r min g f u s io n p r o te i n in p h a g e v e c to r : lg 1 1 w ith la c c o n tr o l r e g io n fo llo w e d b y la c Z g e n e ,
c lo n in g s ite s a r e lo c a te d w ith in th e la c Z g e n e , d ir e c t d e t e c tio n o f p r o te in w ith a n tib o d y .
# 3 7 D e g e ne ra t e prob e : s c r e e n fo r th e c o r r e c t c D N A o r g e n o mic c lo n e fr o m th e li b r a r y .
Sc r e e n o f c D N A c lo n e s , Pr e p a r e th e c D N A e x p r e s s io n p la s m id a n d p r o d u c e th e d e s ir e d
r e c o mb in a n t p r o te in .