Application of DNA
technology - polymerase chain reaction (PCR)
PCR Ingredients
Template - Stretch of DNA containing the target that you want
to amplify.
Deoxynucleotides - Called dNTPs (dATP, dCTP, dGTP, dTTP)
Primers - Short segments of DNA that pair up (complementary)
to stretches of DNA that bracket the region to be amplified.
Polymerase - Usually Taq polymerase but there are
others from other thermophiles with proofreading activity.
PCR buffer - Reagents
that the reaction is carried out in.
PCR Summary
Denature - separation of dsDNA at high temp. (> 94oC).
Anneal - matching up primers at moderate temp. (anywhere
from 45 - 65oC).
High enough to get specific matching but not too high.
Extension - increasing temp. to 72oC
so that Taq polymerase will find primers and extend the
DNA. Recall that Thermus aquaticus is a thermophile.
The above cycles are
repeated to exponentially amplify the target DNA. Billions of
copies may be produced to generate a workable amount of DNA.
UNIT 3 - Microbial
Genetics and Viruses
. Microbial Genetics
A. Background
B. Structure of DNA
C. DNA replication
D. DNA to protein
E. Regulation of
gene expression
1. Constituitive vs.
Inducible
2. Lac operon
F. Exchange of
genetic information
Transformation,
conjugation, transduction
Regulation of Bacterial Gene Expression
Constitutive
enzymes are expressed at a fixed rate.
Always running.
Other enzymes
are expressed only as needed
Repressible enzymes
Inducible enzymes
Lactose catabolism
Repression
Promoter - RNA polymerase binding site
Operator - Where the repressor binds
Repressor -
Regulatory protein that
controls
transcription
No repressor - transcription as normal
Repressor
present - transcription can not begin
Regulatory gene - encodes the repressor, is
always transcribed and translated.
Inducer -
binds to the repressor, indicates the presence of
lactose.
Regulates the
breakdown of lactose to galactose and glucose
1)
b
- galactosidase
2) permease
3)
transacetylase
E. coli cells grow better on
glucose than lactose.
When
given both, the lac operon is repressed until
glucose is depleted
Genotype vs. phenotype
All
E. coli cells have the genes to
metabolize lactose but do not always
express them
.
Change in the genetic
material
Mutations may be neutral,
beneficial, or harmful
Mutagen: Agent that causes
mutations
Spontaneous mutations: Occur
in the absence of a mutagen
Base substitution (point
mutation)
Missense mutation
Change in one base
Result in change in amino acid
Results in a nonsense
codon
Insertion or deletion of one or
more nucleotide pairs
Sickle cell
anemia
So, based on
what you have just
heard, sickle cell
anemia is caused
by a (pick 2)
a. point
mutation
b. frameshift
mutation
c. missense
mutation
d. nonsense
mutation
e. silent
mutation