Principles
of
Genetic
Engineering
&
Biotechnology
In Principle:
Genetic
Engineering
involves the laboratory manipulation
of DNA (MGA2 8-2)
What does a particular region of DNA do?
This may involve isolation of a "gene
of
interest"
in
vivo or in
vitro "cloning" of the
gene
analysis of the cloned gene
gel electrophoresis
nucleic
acid
"blotting"
DNA sequencing
mRNA expression
Biotechnology:
“The use of biological systems to
create goods & services"
Restriction
enzymes
[Nobel Prize 1978]
DNA sequencing
[Nobel
Prize
1980]
Polymerase
Chain Reaction (PCR)
[Nobel
Prize
1993]
For more details, see
Bio2250 - Principles of Genetics
DNA
sequencing
in vitro DNA replication reaction copies one
strand repeatedly
Provides complete order of bases in
a DNA
fragment
DNA primer is complementary to 3'
end of
gene of interest
primers are end-labelled
with 32P
or 35S
dideoxynucleotide
terminators (ddNTPs)
(MGA2 8-23)
stop
strand
growth
during
replication
four
separate
reactions
terminate
at
ddA, ddC, ddG,
or
ddT (MGA 8-22a)
Sequencing gel
shows series of partial DNA replications
Sequencing
"ladder" is
read from bottom to top in an autoradiogram (MGA2
8-22b)
[click
here
for an
of autoradiographic DNA sequencing]
Automated
DNA
sequencer uses laser fluorometry
ddNTPs are
attached to fluorescent dyes
scanning
laser
&
fluorometer
"see"
fluorescence
colours (A
C
G
T)
computer
compares
colours,
"calls
sequence" (MGA2 8-24)
Alternative separation methods: Capillary electrophoresis
[click
here
for
an
of automated
DNA sequencing]
Bio4241 - Sanger et al. 1977
Microarray
DNA
sequencing - "DNA
Chips"
DNA
Re-Sequencing Chips for iterative sequencing of populations [PPT presentation]
The ArkChip for
multiplex species populations studies
All
text material © 2010 by
Steven M. Carr