science of living system ab (part of unit 2)

7/31/2019 Science of LIVING SYSTEM AB (Part of Unit 2)

1/46

Professor A. BasakDepartment of ChemistryEmail:[email protected]


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 Francis Crick


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 Replication

A. InitiationB. PrimingC. ElongationD. Proofreading and Termination


25/46

5 3


26/46

Identicalbase sequences

5

5

3

3 5

5 3

3

Watson/Crick proposed mechanism of DNA replication


27/46

1955: Arthur Kornberg

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

Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5 -triphosphates)(sugar-base + 3 phosphates)

2. DNA template

3. DNA polymerase ( Kornberg enzyme )

4. Mg 2+ (optimizes DNA polymerase activity)

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


28/46


29/46

3

Polymerase III

Leading strand

base pairs

5

5

3

3

Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

Helicase+

Initiator Proteins

ATP

SSB Proteins

RNA Primer

primase

2Polymerase III

Lagging strand

Okazaki Fragments1

RNA primer replaced by polymerase I& gap is sealed by ligase


30/46

Sequencing

Sequencing is the process by which youdetermine the exact order of the nucleotidesin a given region of DNA.

Dideoxynucleotide sequencing is donethrough complementary chain synthesis andearly termination.

The synthesized chains are visualized by

methods using: Radioactive labels. Nonradioactive labels.


31/46

Requirements for Sanger-Coulson Sequencing

DNA to be sequenced must be in single strandform.

The region to be sequenced must be 3 flankedby known sequence.

Reagents needed are: A primer complementary to the known region to direct

chain synthesis. DNA polymerase. 4 deoxynucleotide triphosphates (dNTPs). 4 dideoxynucleotide triphosphates (ddNTPs).


32/46

Dideoxynucleotides

dATP ddATP

The 3 hydroxyl has been changed to a hydrogen in ddNTPs,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

NH 2

O

HH

HH

HH

OPO

O-

O

POP-O

O

O-

O

O-

N

NN

N

NH 2

O

HOH

HH

HH

OPH

O-

O

POP-O

O

O-

O

O-


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 unableto form a phosphodiester bond by nucleophilic attack on thephosphate, and it will cause a termination in the DNA chain

Mechanism of DNA polymerization

: :

O

HO

HH

HH

PO

O-

O-

O

HO

HH

HH

PO O-

Base

Base

O

HOH

HH

HH

OBase

O

P-O O-

O-

5

3

O

HOH

HH

HH

OPO

O-

O

POP-O

O

O-

O

O-

Base

O

HO

HH

HH

PO

O-

O

O-

O

HO

HH

HH

PO O-

Base

Base

O

HO

HH

HH

OBase

PO O-

O

HOH

HH

HH

OBase

O

P-O

O-

5

3

OP-O

O-

O

OHP

O-

O


34/46

Sequencing Visualization Methods

Two forms of labeling: Radioactive

Primer labeled ( 32 P or 33 P)

dNTP labeled ( 35 S) Nonradioactive

Primer labeled

ddNTP labeled (big dye terminator)


35/46

Radioactive Primer Labeled Sequencing

4. dNTPsdATP, dGTP, dCTP, and dTTP

ddGTPddATP ddCTP ddTTP

6. One type of ddNTP per reaction

Remember each reaction has manymolecules each one incorporatingits respective ddNTP and stoppingat a different length.

7. DNA polymerase

3. Complementary primer,5end -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


36/46

Gel Separation The reaction mixtures are separated on a denaturing

polyacrylamide gel. Denaturing to prevent the DNA from folding up on

itself while it travels through. Polyacrylamide to separate the strands which

differ in length by only one nucleotide.

Each band corresponds to a sequence of DNA whichwas terminated by a particular ddNTP. This ddNTP is identified by lane in the radioactive

method and by color in the fluorescent method. The lowest band on the gel is the shortest. The

shorter the strand, the earlier in the synthetic reactionthe ddNTP was incorporated.

The lowest band on the gel is at the 5 end of our synthesized strand and is complementary to the 3

end of our unknown fragment.


37/46

Gel Visualization

Radioactive method which requires four gellanes, one for each reaction vessel. Readout is done by hand or with a

densitometric scanner. Nonradioactive fluorescence sequencing

requires only one gel lane because eachnucleotide has a distinct color.

The readout process is done by laser scanner and recorded by computer.


38/46


39/46

Polymerase ChainReaction (PCR) and Its

Applications


40/46

What is PCR?

It was invented in 1983 by Dr. KaryMullis, for which he received the NobelPrize in Chemistry in 1993.

PCR is an exponentially progressingsynthesis of the defined target DNA sequences in vitro .


41/46


42/46

What is PCR? :Why Chain?

It is called chain because the

products of the first reaction becomesubstrates of the following one, andso on.


43/46


44/46

The Reaction

THERMOCYCLER PCR tube


45/46

Denature (heat to95 oC)

Lower temperature to 56 oCAnneal with primers

Increase temperature to72 oC DNA polymerase +

dNTPs


46/46

science of living system ab (part of unit 2)

Documents