12
D N A To o ls
# 1 D N A R e c om bina nt Te c hn ology
O u t lin e
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D N A e n z y mo lo g y : r e s tr ic ti o n e n z y me s , lig a s e s , p o ly n u c le o ti d e k in a s e , r e v e r s e
tr a n s c r ip ta s e s
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H y b r id iz a tio n : c o m p le me n ta r ity o f D N A a n d R N A
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R e s tr ic ti o n e n z y me a n a ly s is
Th e D N A C a r r ie r s : Pla s mid o r Vir a l Ve c t o r
Ke y c o mp o n e n ts o f D N A - b a s e d Bio te c h n o lo g y
Blo ttin g te c h n i q u e s
Se q u e n c in g
So l id - p h a s e s y n th e s is
PC R
C o m p u te r
# 2 The N obe l Priz e in Ph y s iolo gy or Me dic i ne 1 9 7 8
Wer ner A rb er 1/ 3 o f t he p ri z e Sw i tz erla nd Bio z entru m d er Uni versit ät B a sel,
Sw i tz erla nd b . 1929. Dan iel N at hans 1/ 3 o f t he p ri z e USA J o hns H o p ki ns
Uni versit y Scho o l o f M ed ici ne Ba l tim o re , M D , USA b . 1928 d . 199 9. Ham ilt on O .
Smi t h 1/ 3 o f t he p ri z e USA J o hns H o p ki ns Uni versit y Scho o l o f M ed ici ne
Ba l tim o re , M D , USA b . 1931.
# 3 The N obe l Priz e in C h e mis t r y 1 9 8 0
Pau l Berg 1/ 2 o f t he p ri z e USA Sta nf o rd Un iversi ty Sta nfo rd , C A, U SA b . 1926" fo r
h is fu n d a me n ta l s tu d i e s o f th e b i o c h e mi s tr y o f n u c l e ic a c id s , w ith p a r tic u l a r r e g a r d to
r e c o mb in a n t- D N A "
" fo r th e i r c o n t r ib u ti o n s c o n c e r n i n g th e d e te r min a ti o n o f b a s e s e q u e n c e s in n u c le i c a c id s "
Wal t er Gi lb ert 1/ 4 o f t he p ri z e USA H a rva rd Uni versit y, Bio lo g ica l L a b o ra to ri es
C a m b rid g e , M A, USA b . 1932
F re d erick Sang e r 1/ 4 o f t he p ri z e Uni ted Ki ng d o m M R C L a b o ra to ry o f M o lecu la r
Bio lo g y C a m b rid g e, Un ited K ing d o m b . 19 18.
# 4 R e s t ric t i on e nz y me s
R e s tr ic ti o n e n z y me s c u t D N A in to a r e p r o d u c ib le s e t o f r e s tr ic t io n fr a g me n t s .
R e s tr ic ti o n e n z y me s c u t D N A mo le c u le s a t s p e c ific s e q u e n c e s ( o n ly 5 ’ - - > 3 ’ is s h o w n ;
a r r o w s a r e c u tt in g s i te s ; R e s tr ic ti o n e n d o n u c le a s e s ( s e q u e n c e - s p e c ific , mo s tl y p a l in d r o m ic ,
w it h 2 - fo ld r o t a tio n a l s y mm e tr y , a r r o w s c le a v a g e s i te , s y mme tr y a x is ) .
# 5 R e s t ric t i on e nz y me s
Ec o R I , 1 s t r e s tr i c tio n e n z y me fr o m Es c h e r ic h ia c o l i , Ba m H 1 , 1 s t r e s tr ic tio n e n z y me fr o m
Ba c illu s a my lo liq u e fa c ie n s H ) .
# 6 R e s t ric t i on e nz y me s
Ex a mp le s o f r e s tr ic ti o n e n z y me s a n d th e ir c u t tin g s ite s .
# 7 R e s t ric t i on e nz y me s
R - M s y s te m ( r e s tr ic ti o n - mo d ific a t io n s y s te m w ith r e s tr ic t io n e n d o n u c le a s e a n d me t h y la s e
p a i r c o n f e r s p r o te c ti o n o f h o s t D N A fr o m c u tt in g ) .
# 8 R e s t ric t i on e nz y me s
Ec o R 1 a n d Ec o R 1 me t h y la s e ( Ec o R 1 d o e s a s ta g g e r e d c u t a t a s p e c ific 6 - b p in v e r t e d
r e p e a t a n d g iv e s s tic k y e n d s ; th e c o r r e s p o n d i n g me t h y la s e a ls o r e c o g n iz e s t h e s a m e
s e q u e n c e a n d mo d ifie s it) .
# 9 R e s t ric t i on e nz y me s
R e s tr ic ti o n s it e o f Ad 2 ( a d e n o v ir u s 2 , 3 6 k b g e n o me ) p r o d u c e d b y e ith e r Ec o R 1 ( G AA TTC )
o r H in d II I ( AAG C TT) f r o m H a e mo p h il lu s in f lu e n z a e ( l a b e lin g o f A - Z a c c o r d in g to s i z e ) .
# 1 0 R e s t ric t i on e nz y me s
R e s tr ic ti o n ma p : ma p p in g th e r e s t r ic tio n s ite s fo r tw o e n z y me s r e la ti v e to e a c h o th e r i n a
D N A c o n ta in in g o n e c o p y o f e a c h s ite . W ith d iffe r e n t p a i r s a d e ta ile d r e s itr ic t io n ma p c a n
b e c o n s tr u c te d .
# 1 1 R e s t ric t i on e nz y me s
SV4 0 D N A c u t w i th th r e e r e s tr ic ti o n e n z y me s . Th e h o s t D N A is p r o te c te d f r o m
e n d o n u c le a s e d i g e s tio n o f Ec o R I w it h s tr a te g ic me t h y la ti o n a t th e a d e n in e b a s e b y Ec o R I
me t h y la s e .
# 1 2 . Th e a c t i on of re s t ri c t ion e nz y me s R e s tr ic ti o n e n z y me s , a ls o c a lle d r e s tr i c tio n
n u c le a s e s ( Ec o R I in th is e x a mp le ) , s u r r o u n d s th e D N A mo le c u le a t th e p o in t it
s e e k s ( s e q u e n c e G AATT C ) . I t c u ts o n e s tr a n d o f th e D N A d o u b le h e lix a t o n e p o in t a n d t h e
s e c o n d s t r a n d a t a d i ffe r e n t, c o m p le me n ta r y p o in t ( b e tw e e n th e G a n d th e A b a s e ) . Th e
s e p a r a te d p ie c e s h a v e s in g l e s tr a n d e d " s tic k y - e n d s , " w h ic h a llo w th e c o mp le me n ta r y p ie c e s
to c o mb in e . Th e n e w ly jo in e d p ie c e s a r e s ta b i liz e d b y D N A lig a s e . Ec o R I, o n e o f ma n y
r e s tr ic ti o n e n z y me s , is o b t a in e d fr o m t h e b a c te r ia Es c h e r ic h ia c o li.
# 1 3 .R e s t ric t i on e nz y me s L ig a tio n o f r e s tr ic ti o n fr a g me n ts w ith c o mp le me n ta r y ﾒ s ti c k y
e n d s ﾓ : Stic k y e n d s tr a n s i e n tly b a s e - p a ir fo r th e a c tio n o f T4 D N A lig a s e .
# 1 4 .Lig a s e me c ha nis m
Th e a c tio n o f lig a s e , c u t a n d p a s te fo r m th e b a s is o f mo le c u la r c lo n in g .
# 1 5 . G e l Ele c t rophor e s is o f nuc l e ic a c id
G e l ma k in g , r u n n in g g e l, d e te c tio n a n d a n a ly s is .
# 1 6 . G e l Ele c t rophor e s is of nuc le i c a c id
G e l ma k in g ( v e r tic a l, h o r iz o n ta l) , s a mp le p r e p a r a ti o n ( d e n a tu r a t in g o r n a tiv e ) , Ag a r o s e g e l
w it h c o mb in s e r te d d u r in g s o lid if ic a tio n . D N A p u t i n to th e w e ll .
# 1 7 . G e l Ele c t rophor e s is of nuc le i c a c id
D N A w a s d e te c te d b y f lu o r e s c e n t d y e s ta in in g a n d g e l v is u a li z e d u n d e r u v o r b y r a d io a c ti v e
la b e lin g .
# 1 8 . G e l Ele c t rophor e s is of nuc le i c a c id
Po l y a c r y l a mid e g e l e l e c tr o p h o r e s i s ( 1 n t to 2 k b )
# 1 9 . G e l Ele c t rophor e s is of nuc le i c a c id
Ag a r o s e g e l e le c tr o p h o r e s is ( Ag a r o s e : r e p e a t in g 1 , 3 lin k e d b - D - g a l a c to p y r a n o s e a n d 1 ,4
lin k e d 3 , 6 a n h y d r o - a- L - g a la c t o p y r a n o s e , p o r e la r g e r f o r 5 0 0 b p to 2 0 k b ) b a n d d e t e c te d b y
e it h e r a u to r a d i o g r a p h y o r d y e s ta in in g .
# 2 0 . G e l Ele c t rophor e s is of nuc le i c a c id
Ele c tr o p h o r e tic mo b il ity o f a D N A fr a g m e n t is p r o p o r tio n a l to t h e lo g o f it s s iz e .
# 2 1 . G e l Ele c t rophor e s is of nuc le i c a c id
( a ) D iffe r e n t p la s mid c lo n e s d ig e s te d w ith Ec o R I, H in d III o f Ad 2 w a s u s e d a s mo le c u la r s i z e
ma r k e r . ( b ) Au t o r a d io g r a m o f 3 2 P- la b e le d fr a g me n ts s e p a r a te d o n p o l y a c r y l a mid e g e l.
# 2 2 . G e l Ele c t rophor e s is of nuc le i c a c id
Pu l s e d - fi e ld g e l e le c tr o p h o r e s is c a n s e p a r a te D N A f r o m 2 0 k b to 1 0 Mb . Pu ls e d e le c tr ic
fie ld fo r c e s D N A d o e s th r o u g h s tr e tc h e d - c o ile d mo ti o n , w h ic h s i g n ific a n tly s e p a r a te s la r g e r
D N A o f d i ffe r e n t s iz e s . ( A ) L a n e 1 , y e a s t c h r o mo s o ma l D N A; la n e 2 , c o n c a to me r s o f λ D N A
( 4 8 .5 k b ) ; la n e 3 , N o t I o f E.c o li c h r o mo s o me . ( B ) Ye a s t c h r o mo s o me s r a n g in g fr o m 0 .2 Mb
to 2 .2 Mb . Th e g e l i s 1 3 x 1 2 .5 c m ( r e p r o d u c ib ility ) .
# 2 3 . A r e s t ric t ion m a pping e x pe r ime nt
( i) d e te r m in e th e p o s i tio n o f a Ba m H 1 s ite . ( ii) d e te r mi n e th e o r ie n ta tio n o f a 1 .6 k b H in d II I
fr a g me n t.
# 2 4 . A r e s t ric t ion m a pping e x pe r ime nt
R e s tr ic ti o n ma p p in g o f a n u n k n o w n D N A.
# 2 5 . B lo t t ing t e c hni que s f or na l y z ing s pe c if ic nuc le ic a c ids i n c omp le x mi x t ure
A s p e c ifi c D N A s e q u e n c e is o la te d b y c lo n in g c a n s e r v e a s a p r o b e to d e te c t th e p r e s e n c e
a n d th e a mo u n ts o f c o mp le me n ta r y n u c le i c a c id s in c o mp le x mix tu r e s in c lu d i n g to t a l
c e l lu la r D N A o r R N A. ( i) Th e me mb r a n e - h y b r i d iz a ti o n a s s a y ( Bl o ttin g ) ( d s D N A d e n a tu r e o r
me l t b in d o n to filte r p a p e r , in c u b a te w ith la b e ld s s D N A p r o b e , c o mp le m e n ta r y D N A
h y b r id iz e s , w a s h a w a y n o n - b o u n d l a b e le d D N A, p e r fo r m a u to r a d io g r a p h y ) .( ii) D e te c t in g
n u c le ic a c id w i th a n o n - r a d io a c ti v e p r o b e ( r e p lic a t e th e p r o b e D N A in th e p r e s e n c e o f
d U T P- b io t in , d e n a tu r e , h y b r id iz e w ith t a r g e t D N A, m ix w it h b if u n c tio n a l r e a g e n t a v i d in - AP ,
a lk a lin e p h o s p h a ta s e c a n c le a v e th e P- r e a g e n t a n d g e n e r a te c h e milu mi n e s c e n c e fo r
a u t o r a d io g r a p h i c d e te c tio n .
# 2 6 . Sou t he rn blot t i ng
So u th e r n b lo tti n g ( E.M .So u th e r n , J MB 9 8 , 5 0 8 , 1 9 7 5 ) : D N A s e p a r a te o n g e l , d e n a tu r e b y
b a s e , tr a n s fe r to me m b r a n e ( b lo t) v ia d iffu s i o n o f e le c tr o p h o r e s is , b lo c k w ith n o n - s p e c ific
D N A o r p r o te in , h y b r i d iz e w ith th e p r o b e a n d d e te c t th e p o s itio n a n d a mo u n t o f ta r g e t D N A.
# 2 7 . Sou t he rn blot
An o th e r v ie w o f So u th e r n b l o t a n a ly s is . Au to r a d io g r a p h y
# 2 8 . D N A f inge rprint ing
C u t D N A w ith H a e III in to 8 fr a g me n ts , o n ly 3 c o n t a in s min is a te l lite s ; e le c t r o p h o r e s is ,
d e n a tu r e , So u th e r n b l o t, h y b r id iz e w ith r a d io a c tiv e p r o b e .
# 2 9 . D N A f inge rprint ing
D N A fin g e r p r in t : la n e 1 - 9 , 9 u n r e la te d w h ite s u b je c ts ; la n e s 1 0 a n d 1 1 mo n o z y g o ti c tw in s .
# 3 0 . D N A f inge rprint ing
D N A ty p in g : Fo r e n s ic s c ie n t is t u s e s a u n iq u e p r o b e th a t h y b r id i z e s to s in g l e v a r i a b le D N A
lo c i. Th is is c a lle d D N A t y p in g . Su s p e c t B w a s g u i lty .
# 3 1 . D N A f inge rprint ing
D N A fr o m th e b l o o d s ta in s o n d e fe n d a n t’ s s h ir t, je a n s , ma tc h e d D N A fr o m th e v i c tim, g u ilty .
# 3 2 . N or t he rn blot
A b lo ttin g me th o d fo r d e te c tin g s p e c ifi c R N A in th e mix tu r e o f R N A s .
# 3 3 . N or t he rn blot
Sc h e ma ti c d ia g r a m il lu s tr a tin g N o r th e r n b lo t tin g m e th o d . ( i) R N A d e n a tu r e d ( e .g . H C H O ) ,
s e p a r a te d b y g e l e le c tr o p h o r e s is , tr a n s fe r to n itr o c e llu l o s e fi lte r , e x p o s e d to a la b e l e d D N A
p r o b e , a u to r a d i o g r a p h y . W id e ly u s e d to c o mp a r e th e a mo u n t s o f a p a r ti c u la r mR N A i n c e ll s
u n d e r d if fe r e n t c o n d i tio n s . ( ii) N o r t h e r n b lo t o f β - g lo b in mR N A in e r y th r o le u k e mia c e lls
b e f o r e a n d a fte r d iff e r e n ti a tio n .
# 3 4 . N or t he rn blot
N o r th e r n b lo t: Po ly ( A ) + R N A fr o m r a t ti s s u e s , e q u a l a mo u n ts o f R N A w e r e e le c tr o p h o r e s e d
a n d N o r th e r n b l o tte d . H y b r id iz e d to r a t g ly c e r a ld e h y d e - 3 - p h o s p h a te d e h y d r o g e n a s e .
# 3 5 . N ul e a s e p rot e c t ion a s s a y
S1 n u c le a s e p r o te c tio n a s s a y . N PA s a r e th e me th o d o f c h o i c e fo r th e s imu lta n e o u s
d e t e c tio n o f s e v e r a l R N A s p e c ie s . D u r in g s o lu tio n h y b r id i z a tio n a n d s u b s e q u e n t a n a ly s is ,
in d iv id u a l p r o b e /ta r g e t in t e r a c ti o n s a r e c o mp le te ly in d e p e n d e n t o f o n e a n o t h e r . T h u s ,
s e v e r a l R N A ta r g e ts a n d in t e r n a l c o n tr o ls c a n b e a s s a y e d s imu lt a n e o u s ly ( u p to tw e lv e
h a v e b e e n u s e d in th e s a me r e a c ti o n ) , p r o v id e d th a t th e p r o te c t e d fr a g me n t o f in d iv id u a l
p r o b e s a r e o f d iffe r e n t le n g th s .
# 3 6 . N ul e a s e p rot e c t ion a s s a y
N PA s a r e a ls o c o mmo n l y u s e d to p r e c is e l y ma p mR N A t e r min i a n d in t r o n /e x o n ju n c tio n s .
H e r e s h o w s th e ma p p in g o f a 1 .7 k b fr a g me n t o n th e 3 6 0 k b a d n o v ir u s g e n o m e .
# 3 7 . N ul e a s e p rot e c t ion a s s a y
Tr a n s c r ip tio n s ta r t s ite s c a n b e ma p p e d b y S1 p r o te c tio n a n d p r ime r e x te n s i o n : L a b e le d
D N A h y b r i d iz e d w ith m R N A, S 1 n u c l e a s e d ig e s ti o n , d e n a tu r e a n d e le c tr o p h o r e s e , d is ta n c e
g iv e s th e in iti a tio n s ite . L a b e l e d p r i me r e x te n d e d w ith R T, d e n a tu r e a n d e le c tr o p h o r e s is ,
a u t o r a d io g r a p h y , th e d is ta n c e g iv e th e in itia tio n s ite .
# 3 8 . N ul e a s e p rot e c t ion a s s a y
N u l e a s e p r o te c t io n a s s a y fo r q u a n tify in g s p e c ific m R N As i n a mi x tu r e a n d ma p p in g th e m:
( a ) e x c e s s D N A p r o b e , th e p r o te c t e d h y b r id a l c o h o l p r e c ip ita te d , e le c tr o p h o r e s e d a n d
a u t o r a d io g r a p h e d , th e b a n d in te n s ity g i v e s th e r e la tiv e a mo u n ts . S1 p r o te c tio n , fo llo w e d b y
fo u r d iff e r e n t r e s tr i c tio n e n z y me s a llo w s ma p p in g o f mR N A s ite .
# 3 9 . Sou t he rn blot v s . N or t he rn blot .
Ty p ic a l d a ta fr o m N o r th e r n a n d So u th e r n b lo t a n a ly s is . N o te th e u s e o f r R N A o r t o ta l D N A
s ta in in g a s lo a d in g c o n tr o l .
# 4 0 . Sa n ge r’ s D N A Se q ue nc in g
Sa n g e r ’ s m e th o d : C o n tr o lle d in te r r u p tio n o f e n z y ma ti c r e p l ic a tio n , s o th a t t h e s iz e o f t h e
fr a g me n t d e p e n d s o n t h e la s t b a s e in th e s e q u e n c e . Th e k e y is t h e 2 ’ ,3 ’ - d id e o x y
n u c le o tid e .
# 4 1 . Sa n ge r’ s D N A Se que nc ing
Sa n g e r r e a c tio n : Fo u r s e p a r a te p o ly me r i z a tio n r e a c t io n s a r e p e r fo r me d ( d N T Ps a n d d d N TP.
1 0 0 :1 ) , d d G TP r e a c tio n ,
# 4 2 . Sa n ge r’ s D N A Se que nc ing
Sc h e ma tic d ia g r a m s h o w in g h o w th e fo u r p o ly me r iz a ti o n r e a c tio n p r o c e e d .
# 4 3 . Sa n ge r’ s D N A Se que nc ing
An e x a mp l e o f S a n g e r ’ s m e th o d in a c t io n . Sa n g e r s e q u e n c i n g : th e r e a d in g 5 ’ ATG ATAC G G TC T fr o m th e p r ime r e x te n s io n u s in g Kle n o w fr a g me n t g iv e s th e s e q u e n c e o f
th e te mp l a te a s 5 ’ - AG A C C G TAT C AT.
# 4 4 . Sa n ge r’ s D N A Se que nc ing
( le ft) th e r e a d i n g 5 ’ - ATG ATAC G G TC T fr o m th e p r ime r e x te n s io n . ( r ig h t) A t y p ic a l
s e q u e n c in g film r e a d a s C AA AAAAC G G ...
# 4 5 . Ma x a m- G il be rt s e que nc ing
Ma x a m- G il b e r t M e th o d : Br e a k in g th e e n d - la b e le d D N A s tr a n d a t s p e c ific b a s e s u s in g b a s e s p e c ific r e a g e n ts . R a d io la b e l, me t h y la ti o n w it h d im e th y l s u lfa t e , tr e a t w it h p ip e r id in e ,
r e m o v e b a s e , b r e a k D N A a t t h e a p u r in ic s ite , le a v e s ‘ - p h o s p h a t e o n t h e n t p r e c e d e d th e G .
All G - r e m o v e d f r a g me n ts c a n b e s e p a r a te d b y g e l.
# 4 6 . Ma x a m- G il be rt s e que nc ing
( i) d s D N A la b e le d a t th e 5 ’ - e n d , th e la b e l is r e mo v e d fr o m o n e e n d , d e n a tu r e d , e x p o s e fo u r
s a m p le s t o d iff e r e n t c h e mic a l r e a c tio n s ( c o n t r o lle d s o th a t e a c h la b e le d c h a in w i ll b r e a k
o n l y o n c e ) , e le c tr o p h o r e s e d a n d a u t o r a d io g r a p h e d . ( ii ) Th e r e a d in g o f th e s e q u e n c e 5 ’ C TT TTTTG G G C TTAG C …., g iv e s a d ir e c t r e a d in g o f t h e s e q u e n c e u p t o 5 0 0 n t.
# 4 7 . A ut oma t e d s e que nc ing
( i) Sa n g e r ’s r e a c ti o n , e x c e p t t h a t fo u r d if fe r e n t ly la b e le d p r ime r s a r e u s e d fo r e a c h d d N TP
e x t e n s io n r e a c t io n , s a mp le s e le c tr o p h o r e s e d a n d s c a n n e d w ith la s e r , th e c o lo r f r o m th e
flu o r e s c e n t ta g s a r e r e c o r d e d in th e c o mp u te r , Sa mp le p r i n to u t o f a n a u to ma te d s e q u e n c i n g
e x p e r ime n t. ( ii ) A s c h e ma ti c v ie w o f th e flu o r e s c e n t d e te c tio n o f th e fr a g m e n t p r o d u c e d b y
d id e o x y m e th o d .
# 4 8 . G e n ome s e que nc i ng
Th e fir s t g e n o m e s e q u e n c e o b ta in e d in 1 9 9 5 .
Sci ence, 269, 496, 1995
# 4 9 . O li gonuc l e ot ide Sy nt h e s is
Sma ll D N A mo le c u le s c a n b e c h e mic a lly s y n th e s iz e d .
Sy n th e tic D N A i s u s e f u l fo r : g e n e r a tin g p o l y lin k e r s e q u e n c e s , s e q u e n c in g D N A is o la tin g
c lo n e s o f in te r e s t c r e a tin g s ite - s p e c if ic mu t a tio n s . So li d - p h a s e s y n t h e s is o f o li g o me r
Th e a c tiv a te d m o n o me r is u s e d to a d d to th e th e 5 ’ - O H o f a r e s in a tta c h e d n u c le o s id e . Th e
5 ’ - O H a n d t h e 3 ’ - p h o s p h o r y l g r o u p a r e p r o te c te d .
# 5 0 . O li gonuc l e ot ide Sy nt h e s is
Th e k e y c o mp o u n d , a d e o x y r ib o n u c le o s id e 3 ’ - p h o s p h r a m id ite w ith p r o te c t in g g r o u p s .
# 5 1 . The poly m e ra s e c ha in re a c t i on
( PC R ) c a n b e u s e d to a mp lify r a r e s p e c if ic D N A s e q u e n c e s f r o m a c o mp le x mix t u r e w h e n
th e e n d s o f th e s e q u e n c e a r e k n o w n . PC R a mp li fic a ti o n o f mu ta n t a lle l e s a ll o w s d e te c tio n
o f h u ma n g e n e ti c d is e a s e s . D N A s e q u e n c e s c a n b e a mp lif ie d b y PC R f o r u s e in c l o n in g , a s
p r o b e s , a n d in fo r e n s ic s
# 5 2 .The poly m e ra s e c ha in re a c t i on
Sc h e ma tic d ia g r a m ill u s tr a t in g th e fir s t s e v e r a l c y l c e o f PC R r e a c t io n .
# 5 3 . Pla s mid
Pla s mid s a r e c i r c u la r , d o u b le - s tr a n d e d D N A mo le c u le s th a t e x is t in b a c te r ia a n d i n th e
n u c le i o f s o me e u k a r y o tic c e lls . Th e y c a n r e p lic a t e in d e p e n d e n tly o f th e h o s t c e ll. T h e
s iz e o f p la s mid s r a n g e s fr o m a fe w k b t o n e a r 1 0 0 k b ( s e e Pla s mid D a ta b a s e a t N C BI) .
# 5 4 . Pla s mid f or s a l e
To c lo n e b lu n t- e n d e d PC R fr a g me n t s a mp l ifie d w ith P la tin u m Pfx ,
w h i c h c a n a mp li fy g e n o mic t e mp la t e s fo r u p to 1 2 k b a n d p la s mid te mp l a te u p to 2 0 b p .
Se e w w w .i n v itr o g e n .c o m