3.

4 DNA sequencing: the Sanger method

Four separate polymerization reactions are performed

3.4 DNA sequencing: the Sanger (dideoxy) method

3.4 The polymerase chain reaction

3.5 Centrifugation can separate molecules that differ in mass or density

3.5 Electrophoresis separates molecules according to their charge:mass ratio

SDS-polyacrylamide gel electrophoresis

3.5 Highly specific enzymes and antibody assays can detect individual proteins

3.5 Two-dimensional electrophoresis separates molecules according to their charge and their mass

3.5 Separation of proteins by size: gel filtration chromatography

3.5 Separation of proteins by charge: ion exchange chromatography

3.5 Separation of proteins by specific binding to another molecule: affinity chromatography

10

3.5 Gas Chromatography

E A

D B

Gas Chromatograph
C

Sample: mixture of volatile liquids (~1L)

Gas Chromatogram B A C E

Abundance

D
0 5 10 Time (minutes) 15 20

11

3.5 Gas Chromatography

Injection Port

Detector Capillary Column

Data System or Recorder

Carrier Gas Supply

Oven

12

3.5 Mass Spectrometry

O H 3 C C N C O N H C C C H 3 N C H N Mass Spectrometer

Typical sample: isolated compound (~1 nanogram)

Mass Spectrum

194

67

109

Abundance

55 82 42

94 40 60 80 100 120

136 140 160

165 180 200

Mass (amu)

13

3.5 Mass Spectrometry

Sample Introduction

Data Output

Inlet

Data System

Ion Source

Mass Analyzer

Ion Detector

Vacuum Pumps

14

3.5 Mass Spectrometry

~70 Volts

Electron Impact Ionization Source

Electron Collector (Trap)

Positive Ions Repeller Neutral Molecules

Inlet + + +

_ _
+ + + to Analyzer

e-

e_

eElectrons Extraction Plate

Filament

15

3.5 Mass Spectrometry

Magnetic Sector Mass Analyzer
ion trajectory in register ion trajectory not in register (too light)

S Ion Source N Electromagnet Detector ion trajectory not in register (too heavy)

16

3.5 Mass Spectrometry

Quadrupole Ion Filter
resonant ion non-resonant ion

+ _ _ +
Ion Source

Detector

DC and AC Voltages

17

3.5 Time-of-flight mass spectrometry measures the mass of proteins and peptides

18

3.5 X-ray crystallography is used to determine protein structure

Other techniques such as cryoelectron microscopy and NMR spectroscopy may be used to solve the structures of certain types of proteins
19

4.1 Gene knockout in mice

20

4.1 Cell-type-specific gene knockouts in mice

21

5.1 Stem cells

What are stem cells? Unique capacity to renew itself Give rise to specialized cell types Are uncommited and remain uncommitted until they recieve a signal for developing into a specialized cell Are pluripotent Deff.: Have the ability to give rise to cell types that develop from the 3 germ layers Differentiation potential of stem cells - basic concept Fertilized egg/zygote = totipotent Pluripotent cells: give rise to cells derived from all three embryonic germ layers Unipotent cells can only develop along one lineage
22

5.1 Stem cells

23

5.1 Stem cells: embryonic stem cells

Derived from the inner cell mass Cultivated in vitro their development differ from cells developing in vivo (in the embryo) long-term self-renewal Maintain a stabel and normal karyotype Capable of integrating into all fetal tissues during development Capable of colonizing the germ line and giving rise to egg or sperm cells Express TF Oct-4 Can be induced for proliferation or differentiation

24

5.1 Stem cells: adult stem cells

undifferentiated cells in differentiated tissue
Hematopoietic stem cells mesenchymal stem cells adult stem cells in other tissues

Renew itself Divide to generate progenitor or precursor cells which then differentiate and develop into mature cells Difficult to identify; Are rare Do not replicate indefinitely in culture Sources: bone marrow, blood stream, cornea, retina, liver skin, gastrointestinal tract, pancreas Primary function to maintain the steady state functioning of a cell Why there are undifferentiated stem cells in differentiated tissue is unclear (maybe they are restrained by development) 25

5.1 Stem cells

Celltypes derived from hematopoietic stem c e l l s

erythrocytes monocytes, macrophages lymphoid progenitor cell eosinophil

natural killer cell

basophil

B-cells

myeloid progenitor cell T-cell Hematopoietic stem cell platelets

neutrophil

26

5.1 Stem cells

Celltypes derived from m esenchymal stem cell
neural cells skeletal muscle, tendon

bone, cartilage mesenchymal stem cell

adipocytes

marrow connective tissue

hepatocytes

cardiomyocytes

27

5.1 Stem cells

Stem cell applications in the future

28