BIMM110 LECTURES 1 - 3
A.
Introduction to the Course
B.
Molecular/Genetic Technology – Review
The following
subjects should be familiar to you:
Chapter
1: DNA Structure and Gene Expression
-
structure
of DNA
-
structure
of RNA
-
DNA
replication
-
Transcription
-
Splicing
(exons and introns)
-
5’
and 3’ UTRs of mRNA
-
poly(A)
tails of mRNA
-
translation
of mRNAs
- the material in chapters 5 and 6:
- in every case you should understand a basic principle, and
also know precisely what the ultimate objective of a method is
characterizing DNA sequence
making lots
of DNA of a particular sequence (gene)
finding out
if a gene is expressed and how much
finding out
whether a particular protein is expressed and how much
how does
the change of a single amino acid (mutation) affect the function of a protein
what is a
genomic library
what is a cDNA library
a)
restriction
enzymes
b)
electrophoretic analysis: regular gels
and FIGE (pulsed field)
a)
restriction
fragments from DNA of an organism
b)
cDNA fragments with added ends containing
restriction sites
a)
denaturation and renaturation
of DNA; melting curves, Tm
b)
conditions,
stringency
c)
annealing
d)
tolerance
for mismatches
e)
probes:
radioactive and nonradioactive
f)
detection:
autoradiography, x-ray film, non-radioactive methods (chemiluminescence,
fluorescence)
g)
dot
blots
h)
Southern
and Northern blots
i)
microarrays
a)
plasmid
vectors
b)
phage/cosmid vectors
c)
retroviral
vectors
d)
yeast
artificial chromosomes
a)
amplification
in host A requires origin that is operative in A and selective marker
b)
expression
in host B requires appropriate choice of promoter, poly(A) site,
c)
transient
vs permanent/stable expression from expression
plasmid; may require selective marker
d)
use
of internal ribosomal entry sites to make polycistronic
transcripts
e)
inducible
systems
f)
a)
expression
in bacteria to make large amount of protein
b)
expression
in mammalian cells to study function and other biological questions
c)
transient
vs stable transfectants
d)
a)
preparation
of RNA and electrophoresis
b)
preparation
of specific probe
c)
transfer/blotting
and hybridization
d)
wash
e)
detection
systems
f)
a)
sequence
must be known
b)
selection
of primers
c)
steps
n the cycle
d)
overhanging
ends of A
e)
incorporation
of restriction sites into the primers
f)
a)
SDS
PAGE
b)
BN
gel electrophoresis
c)
Transfer
to “paper”
d)
Detection
with specific antisera