CSB349H1 Lecture Notes - Lecture 2: Green Fluorescent Protein, Photobleaching, Mitosis
14 May 2018
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Lecture 2(d): Genes & Genome Structure
Photobleaching:
• Fluorescent proteins expressed in a cell have a limit to how much fluorescence it can emit
o Shooting the fluorescent protein with a laser will allow it to emit it’s color, however
shooting it long enough will actually cause the fluorescent protein to degrade and it
will no longer emit any fluorescence
§ Degrading the fluorescent protein with a laser is known as photobleaching
• Labeling histones with two fluorescent proteins (red & green) = yellow
o Photobleaching the YFP marker (green) focused only on half
the nucleus causes the green fluorescence to disappear, however
the red fluorescence remains
ð Observing the cell undergo replication after photobleaching displays that:
o Chromosome territories are persevered through mitosis
Chromosome Conformation Capture (3C):
• Major technique used to study genome organization and structure
o Ligate together pieces of the genome that are near each other in a 3-D conformation
• Once a genome forms a 3D structure (i.e. DNA loop) there are regions in the genome that
will be found near each other in the 3D conformation however, are not near each other in the
primary DNA sequence
3C Technique:
• Try to capture parts of the DNA that are close together
in a 3D conformation and make them close together in
a primary sequence
How is This Done?
• If the nucleus contains a DNA loop we will cross-link
o “chemical glue” that is mixed into the cell (kills the cell) & glues molecule together
(DNA region that are close together forming the DNA loop is glued together)
• Restriction enzymes will then be used to cut the DNA sequence allowing us to isolate the
small region that are in close contact to form the loop
• Following is a ligation step that will connect the free ends of the two piece of DNA together
• Another chemical will be added to reverse the function of the cross-link, therefore, no longer
gluing the two DNA regions together making it linear
o Will still remain intact together due to the ligation step – so now we have a two parts
of the DNA that were initially far apart in the normal genome sequence
• We can later sequence that linear DNA and allow us to identify two parts of the genome that
are not close together in the normal genome sequence, but must be together when forming a
DNA loop
