bs - nucleic acid

8/20/2019 BS - Nucleic Acid

1/46


2/46

http://bcs.whfreeman.com/lehninger5e/


3/46

DNA - Deoxyribonucleic Acid

RNA – Ribonucleic Acid


4/46


5/46


6/46


7/46


8/46


9/46


10/46


11/46


12/46

Discovering the structure of DNA

• Structure was discovered in 1953 by James

Watson and rancis !ric"


13/46


14/46


15/46


16/46


17/46


18/46


19/46


20/46


21/46


22/46


23/46


24/46

Outline for ReplicationReplication

A. Initiation

B. Priming

C. Elongation

D. Proofreading and Termination


25/46

5’ 3’


26/46

Identicalbase

sequences

5

5’

3

3’ 5’

5’3’

3’

Waton!Cric" propoed mec#anim of D$A replication


27/46

1955: Arthur Kornberg

Worked with E. coli .Discovered the mechanisms of DNA synthesis.

our com!onents are re"uired:

1. dN#$s: dA#$% d##$% d$% d'#$(deo)yribonuc*eoside 5+,tri!hos!hates-(sugar,base / !hos!hates-

0. DNA tem!*ate

/. DNA !o*ymerase (Kornberg enzyme-

. 2g 0 (o!timi3es DNA !o*ymerase activity-

1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NU)


28/46


29/46

/

$o*ymerase 444

5 + / +

eading strand

base !airs

5+

5+

/+

/+

6u!ercoi*ed DNA re*a)ed by gyrase 7 unwound by he*icase !roteins:

8e*icase

4nitiator $roteins

A#$

66 $roteins

NA $rimer

!rimase

0$o*ymerase 444

agging strand

;ka3aki ragments1

NA !rimer re!*aced by !o*ymerase 4

7 ga! is sea*ed by *igase


30/46

Se#uencing

• Se#uencing is the $rocess by which youdetermine the e%act order of the nuc&eotides ina given region of DNA'

• Dideo%ynuc&eotide se#uencing is done throughcom$&ementary chain synthesis and ear&ytermination'

• (he synthesi)ed chains are visua&i)ed by

methods using* + ,adioactive &abe&s' + Nonradioactive &abe&s'


31/46

,e#uirements for Sanger-

!ou&son Se#uencing• DNA to be se#uenced must be in sing&e strand

form'

• (he region to be se#uenced must be 3. f&an"ed

by "nown se#uence'• ,eagents needed are*

+ A $rimer com$&ementary to the "nown region to directchain synthesis'

+ DNA $o&ymerase' + / deo%ynuc&eotide tri$hos$hates 0dN(s2'

+ / dideo%ynuc&eotide tri$hos$hates 0ddN(s2'


32/46

Dideoxynucleotides

dATP ddATP

The 3’ hydroxyl has been changed to a hydrogen in ddNTP’s,

which terminates a DNA chain because a phosphodiester bond

cannot form at this 3’ location

Here is an example comparing dATP and ddATP:

N

NN

N

N4

-

-

-

-

N

NN

N

N4

-

-

-

-


33/46

DNA polymerase

catalyzed

nucleophilic attack

of the 3’-OH on a

phospho-anhydride

** Since the 3’ –OH is changed to a –H in ddNTPs, it is unable

to form a phosphodiester bond by nucleophilic attack on the

phosphate, and it will cause a termination in the DNA chain

Mechanism of DNA polymerization

: :

-

-

-

6ase

6ase

6ase

- -

-

5’

3’

-

-

-

-

6ase

-

-

-

6ase

6ase

6ase

-

6ase

-

-

5’

3’

-

-

-


34/46

Se#uencing 7isua&i)ation 8ethods

• (wo forms of &abe&ing*

+ ,adioactive

• rimer &abe&ed 034 or 332

• dN( &abe&ed 035S2

+ Nonradioactive

• rimer &abe&ed

• ddN( &abe&ed 0big dye terminator2


35/46

Radioactive Primer Labeled Sequencing

4. dNTP’sdATP, dGTP, dCTP, and dTTP

ddGTPddATP ddCTP ddTTP

6. One type of ddNTP per reaction

Remember each reaction has manymolecules each one incorporating

its respective ddNTP and stopping

at a different length.

7. DNA polymerase

3. Complementary primer,

5’end-labeled with 32P or 33P

5’

2. with region of known sequence

3’

1. Unknown fragment

5’

Reaction 2Reaction 1 Reaction 3 Reaction 4

5. Four separate reactions

5’ 5’

5’3’3’3’ 5’ 5’

5’

8. ddNTP incorporation

-stops chain synthesis

3’3’ 3’3’

& S i


36/46

e& Se$aration• (he reaction mi%tures are se$arated on a denaturing $o&yacry&amide

ge&'

+ Denaturing to $revent the DNA from fo&ding u$ on itse&f whi&e ittrave&s through'

+ o&yacry&amide to se$arate the strands which differ in &ength byon&y one nuc&eotide'

• :ach band corres$onds to a se#uence of DNA which was terminatedby a $articu&ar ddN('

• (his ddN( is identified by &ane in the radioactive method and byco&or in the f&uorescent method'

• (he &owest band on the ge& is the shortest' (he shorter the strand; theear&ier in the synthetic reaction the ddN( was incor$orated'

• (he &owest band on the ge& is at the 5. end of our synthesi)ed strandand is com$&ementary to the 3. end of our un"nown fragment'


37/46

e& 7isua&i)ation

• ,adioactive method which re#uires four ge&&anes; one for each reaction vesse&'

+ ,eadout is done by hand or with a

densitometric scanner'• Nonradioactive f&uorescence se#uencingre#uires on&y one ge& &ane because eachnuc&eotide has a distinct co&or'

+ (he readout $rocess is done by &aser scannerand recorded by com$uter'


38/46

ddGTP ddATP ddTTP ddCTP

Radioactive vs.Gel Electrophoresis and Readout of Reaction Products:

S e q u

e n c e o f u n k n

o w n f r a g m e n

t

Shortest synthesized band = 5’ end of synthesized strand

Longest synthesized band =

3’ end of synthesized strand


39/46

Polymerase ChainPolymerase Chain

Reaction (PCR) and ItsReaction (PCR) and Its

Applications Applications


40/46

What is !,


41/46

What is !,< *What is !,< *

Why =o&ymerase><

It is called )%oly"erase* because the

only eny"e used in this reaction isDN' %oly"erase.

Wh i !,


42/46


Why =!hain><

It is called )chain* because the

%roducts of the first reaction beco"esubstrates of the followin& one, and

so on.

Wh t i !,


43/46


(he =,eaction> !om$onents(he =,eaction> !om$onents

%& Target D$A ' contain t#e e(uence to )e amplified.

*& Pair of Primer ' oligonucleotide t#at define t#e e(uence

to )e amplified.

3& d$TP ' deo+,nucleotidetrip#op#ate- D$A )uilding )loc".

& T#ermota)le D$A Pol,merae ' en/,me t#at

catal,/e t#e reaction

5& 0g11 ion ' cofactor of t#e en/,me

2& Buffer olution maintain p4 and ionic

trengt# of t#e reaction olution uita)le for t#e

actiit, of t#e en/,me


44/46

(he ,eaction(he ,eaction

T4ER0OC6C7ER PCR tu)e


45/46

Denature 8#eat to

95oC&

7o:er temperature to 52oC

Anneal :it# primer

Increae temperature to

;*oC D$A pol,merae 1

d$TP


46/46

bs - nucleic acid

Documents