CONTENT:

1. Examination of one or a couple of molecules

2. Examination of many molecules (genomics)

3. Complex techniques

Discussed in another lectures

CONTENT (1):

1. Molecular cloning

2. Polymerase chain reaction (PCR)

3. Gel electrophoresis

4. Detection of macromolecules

5. Gel retardation

6. Footprint analysis

7. Immunohistochemistry

8. In situ hybridization

9. FRET

10. Flow cytometry

11. FACS

1. Molecular cloning

a. Restriction endonucleases

b. DNA ligases

c. Plasmid vectors

d. Restriction/ligation cloning

e. Other enzymes

Restriction enzymes

Werner Arber Daniel Nathans Hamilton O. Smith

1978

1

EcoRI

2

5’-…GAATTC…-3’ 5’-…G-3’ 5’-AATTC…-3’

3’-…CTTAAG…-5’ 3’-…CTTAA-5’ 3’-G…-5’ 5’ protruding end: (e.g. EcoRI)

5’-…CCCGGG…-3’ 5’-…CCC-3’ 5’-GGG…-3’

3’-…GGGCCC…-5’ 3’-…GGG-5’ 3’-CCC…-5’ Blunt end: (e.g. SmaI)

5’-…GGTACC…-3’ 5’-…GGTAC-3’ 5’-C…-3’

3’-…CCATGG…-5’ 3’-…C-5’ 3’-CATGG…-5’ 3’ protruding end: (e.g. KpnI)

BEFORE CUTTING AFTER CUTTING

Restriction enzymes

5’-…GAATTC…-3’ 5’-…G-3’ 5’-AATTC…-3’

3’-…CTTAAG…-5’ 3’-…CTTAA-5’ 3’-G…-5’

CH3

CH3

X EcoRI methylase

RESTRICTION – MODIFICATION SYSTEM

EcoRI RE does not cut

One DNA molecule Two DNA molecules

DNA ligase DNA ligase

3

4

Plasmid vectors

Plasmid vectors

Recombinant plasmid

Plasmid vector

5

Molecular cloning

Original DNA molecules:

EcoRI site EcoRI site

EcoRI enzyme

Ligase

Recombinant DNA molecule:

6

Generation of recombinant

plasmid by DNA ligation

Transformation (delivery of DNA to cells)

Multiplication of plasmids

within the cells

Multiplication of

bacterial cells

A colony originates

from a single cell

Plasmid vector DNA fragment Recombinant plasmid

E. coli

Molecular cloning 7

DNA DNA lacZ lacI O lacY lacA C E P

rep

T

repressor CAP site pol-binding site operator structural genes terminator

lac operon

lactose

8

DNA DNA lacZ lacI O lacY lacA C E P rep

galactose

indoxyl

T

repressor CAP site pol-binding site operator structural genes terminator

IPTG

X-Gal

- X-Gal chromogenic substrate lacZ gene 9

DNS DNA lacZ lacI O lacY lacA C E P

trans- acetylase

permease

rep

-gal

T

pol

repressor CAP site pol-binding site operator structural genes terminator

lacZ gene - X-Gal chromogenic substrate 9

DNA DNA

lacZ lacI O lacY lacA C E P T

lacZ gene in cloning

lacZ -fragment

-fragment

lacZ- lacZ-

Foreign DNA

repressor CAP site pol-binding site operator structural genes terminator

10

5’- P P - 5’

- 5’

OH-3’

3’-HO

P

Other enzymes in cloning

- Klenow fragment

- S1 nuclease

- Alkaline phosphatase

5’-AGTGGGAATTCAAGGC-3’

3’-TCACCCTTAAGTTCCG-5’

5’-AGTGGG-3’ 5’-AATTCAAGGC-3’

3’-TCACCCTTAA-5’ 3’-GTTCCG-5’

5’-AGTGGGAATT-3’ 5’-AATTCAAGGC-3’

3’-TCACCCTTAA-5’ 3’-TTAAGTTCCG-5’

5’-AGTGGG-3’ 5’-CAAGGC-3’

3’-TCACCC-5’ 3’-GTTCCG-5’

5’-AGTGGG-OH-3’ 5’-P-AATTCAAGGC-3’

3’-TCACCCTTAA-P-5’ 3’-HO-GTTCCG-5’

EcoRI cleavage

5’-AGTGGG-OH-3’ 5’-AATTCAAGGC-3’

3’-TCACCCTTAA-5’ 3’-HO-GTTCCG-5’

dephosphorylation

11

1993

2. PCR: polymerase chain reaction

1. cycle

2. cycle

3. cycle

denaturation annealing synthesis

Forward primer

Reverse primer

12

PCR

Denaturation (Separation of

2 DNA strans)

Heating to

94 - 96 C°

Annealing (primer binding to

single-stranded DNA

Cooling down to

60 - 65 C°

Synthesis (Synthesis of new DNA

strand fro m

the primer by the DNA pol)

Heeting to

68 - 72 C°

Each time the cycle is repeated, the amount of

DNA is duplicated (in optimal circumstances)

Primer New DNA

OldDNA

13 Components needed for PCR:

1. DNA template

2. DNA polymerase

3. Two primers

4. dNTPs

5. Buffer

3. Gel electrophoresis

a. Agarose gel electrophoresis

b. Polyacrilamide gel electrophoresis (PAGE)

14

Agarose gel electrophoresis

Molecular weight marker

16

Fluorescence of DNA bands

UV UV UV

Soaking in Ethidium bromide

Agarose gel electrophoresis 16

10 nucleotides

40 nucleotides

gel

Molecular weight marker

Polyacrylamide gel electrophoresis 17

Protein with two subunits

joined by a disulfide bridge Single subunit protein C

Heated with SDS and mercaptoethanol

C

B

A

SH

HS Negatively charged

SDS molecules

-S-S-

A B C

SDS-PAGE 18

4. Detection of macromolecules 19

a. Southern blot

b. Northern blot

c. Western blot

d. Eastern blot

Cleave with

restriction enzymes

DNA G

el

ele

ctr

op

ho

resis

Alkaline solution

Capillary action transfers

DNA from gel to nitrocellulose

Nitrocellulose Autoradiogram

Hybridize with

labeled DNA

probe

Filter paper Nitrocellulose

Gel

Southern blot

Sir Edwin Southern

20

(A) Transfer of DNA from gel to membrane

(B) Hybridization analysis

Nylon membrane

Nylon membrane Autoradiograph

Hybridizing

bands

Labeled DNA probe

DNA

markers

Restricted

DNA

Agarose gel Support

Gel

Nylon membrane

Paper tower

Buffer

Southern blot 21

P

Extract

RNA Cells

tissues

Filter paper Nitrocellulose

Gel

RNA

Agarose gel

electrophoresis

Blotting

Hybridization and

autoradiography

DNA probe

Hybridized to an

RNA transcript

RNA sample

Marker

RNA bands

Northern blot 22

Cells

Extract RNA

Denaturing agarose gel electrophoresis

Blotting, northern hybridization,

autoradiography

RNA bands

DNA probe hybridizes to

a single RNA transcript

Northern blot 23

Electroblot

Incubate with

Ab1( ) and then

wash excess Ab1

Incubate with enzyme-linked

Ab2( ) and then wash

excess Ab2 then activate

color reaction

(A) Electrophoresis/transfer (B) Antibody detection

(C) Chromogenic detection

Add

substrate

Electric

current

SDS-

Polyacrylamide gel

Porous

Membrane sheet

Western blot 24

Retarded band

DNA markers

Restriction fragments Restriction fragments

+ nuclear protein

5. Gel retardation analyis Detection of DNA/protein interaction

25

Footprint

End-label End-labeled restriction fragments

+ nuclear extract - nuclear extract

Limited DNase I digestion

Digest protein,

gel electrophoresis,

autoradiography Gel electrophoresis,

autoradiography

DNA-binding protein

6. Footprint analysis Detection of DNA/protein interaction

26

Footprint

7. Immunohistochemistry

Y Y Y

nucleus nucleus

cytoplasm

Cell surface antigenes

anti-C antibody

Fluorescent dye

Rabbit anti-C

Primary antibody

Goat anti-rabbit

Secondary antibody

Direct method Indirect method

B

A B

B B

B

B

A C C

- immunofluorescence

Citoplasmic antigenes

cytoplasm

D

E

Cytoplasmic antigenes

D

E

Cell surface antigens

27

peroxidase

Y Y

nucleus

cytoplasm

Cell surfice antigens

peroxidase Rabbit anti-C

primary antibody

Goat anti-rabbit

secondary antibody

Indirect methods

B

B

B

A C

Y peroxidase

peroxidase

peroxidase

Y

avidin

- peroxidase

- biotin – avidin – peroxidase

Anti-rabbit antibody (secondary antibody)

- immunoperoxidase-based methods

DAB + H2O2 Ni-DAB + H2O2

cytoplazmic antigens

anti-rabbit antibody (secondary antibody)

D

E

Y anti-C antibody

Y

anti-C antibody

7. Immunohistochemistry 28

biotin

biotin

avidin

ABC method (ABC = avidin-biotin complex)

anti-rabbit antibody (secondary antibody)

Y

peroxidase

Y

primary antibody

antigen

7. Immunohistochemistry - immunoperoxidase-based methods

29

DAB + H2O2 Ni-DAB + H2O2

Y Y

Y Y

nucleus

cytoplasm

Alkaline phosphatase Rabbit anti-C

primary antibody

Goat anti-rabbit

secondary antibody

Indirect method

B

B

B

A C

- alkaline phosphatase

- digoxigenin – anti-digoxigenin – alkaline phosphatase

alk. phosph. Y

Sheep anti-rabbit antibody

alk. phosph

Y

DIG

anti-C antibody

Cell surfice antigens

cytoplasmic antigens

D

E

Y

Rabbit anti-C antibody

30

Y

alk. phosph

DIG

anti-C antitbody

Y

anti-rabbit antibody

7. Immunohistochemistry

8. In situ hybridization

DIG anti-DIG-peroxidase

Radioactive

Enzymatic Fluorescent

Labeling of probe:

1.Radioactive (S35)

2. Non-radioactive

- fluorescent

- enzymatic

- detection of DNA or RNA

streptavidin

fluorescent dye

probe (DNA or RNA)

biotin

Target DNA

target DNA

probe

DIG

Anti-DIG antitest

fluoreszcens festék

fluoreszcencia

FISH peroxidase

DIG

biotin

avidin

anti-DIG

antibody

S35

31

slide

Dividing cells

Denaturation of DNA

Fluorescent

labeling

Fluorescent in situ hybridization

Metaphase chromosome

32

Fluorescence resonance energy transfer

Protein X Protein Y

No protein interaction Protein interaction

UV light

excitation

Blue light

emission Blue light

excitation

Yellow light

emission

Yellow light UV light UV light Blue light

Blue fluorescent

protein

Yellow fluorescent

protein

9. FRET 33

Sample

Sorter

Laser Flow cell

Scatter detectors

Laser

Band pass filters

Dichroic

filters

Photomultipliers

PMT4

PMT3

PMT1

PMT2

10. Flow cytometry 34

Fluorescence activated cell sorting

Waste

Sample

Collection tubes

Sheath fluid

Vibrating

flow cell

Deflection plates

Charging

collar

Laser

Beam

splitters

Red

fluorescence

Green

fluorescence

900 light

scatter

(granularity) Forward

light

scatter

(size)

11. FACS 35