3.4 DNA sequencing: the Sanger method

Transcription

1 3.4 DNA sequencing: the Sanger method Four separate polymerization reactions are performed DNA sequencing: the Sanger (dideoxy) method 2

2 3.4 The polymerase chain reaction Centrifugation can separate molecules that differ in mass or density 4

3 3.5 Electrophoresis separates molecules according to their charge:mass ratio SDS-polyacrylamide gel electrophoresis Highly specific enzymes and antibody assays can detect individual proteins 6

4 3.5 Two-dimensional electrophoresis separates molecules according to their charge and their mass Separation of proteins by size: gel filtration chromatography 8

5 3.5 Separation of proteins by charge: ion exchange chromatography Separation of proteins by specific binding to another molecule: affinity chromatography 10

6 3.5 Gas Chromatography E A D Gas Chromatograph C Sample: mixture of volatile liquids (~1μL) B Gas Chromatogram A B C E Abundance D Time (minutes) Gas Chromatography Injection Port Detector Capillary Column Data System or Recorder Carrier Gas Supply Oven 12

7 3.5 Mass Spectrometry O C H 3 H 3 C O N C C N H C C N N C H Mass Spectrometer Typical sample: isolated compound (~1 nanogram) Mass Spectrum Abundance Mass (amu) Mass Spectrometry Sample Introduction Data Output Inlet Data System Ion Source Mass Analyzer Ion Detector Vacuum Pumps 14

8 3.5 Mass Spectrometry ~70 Volts Electron Impact Ionization Source Electron Collector (Trap) Repeller Neutral Molecules Inlet _ Positive Ions to Analyzer e - e - e - _ Electrons Filament Extraction Plate Mass Spectrometry Magnetic Sector Mass Analyzer ion trajectory not in register (too light) ion trajectory in register S Ion Source N Electromagnet ion trajectory not in register (too heavy) Detector 16

9 3.5 Mass Spectrometry Quadrupole Ion Filter non-resonant ion resonant ion Detector Ion Source DC and AC Voltages Time-of-flight mass spectrometry measures the mass of proteins and peptides 18

10 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 Gene knockout in mice 20

11 4.1 Cell-type-specific gene knockouts in mice 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

12 5.1 Stem cells 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

13 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) Stem cells Celltypes derived from hematopoietic stem cells natural killer cell erythrocytes monocytes, macrophages eosinophil B-cells lymphoid progenitor cell basophil T-cell Hematopoietic stem cell myeloid progenitor cell neutrophil platelets 26

14 5.1 Stem cells Celltypes derived from mesenchymal stem cell neural cells skeletal muscle, tendon mesenchymal stem cell bone, cartilage adipocytes marrow connective tissue hepatocytes cardiomyocytes Stem cells Stem cell applications in the future 28