Download Lecture 16-LC710 Posted

710.LC GRADUATE
MOLECULAR BIOLOGY
10/31/2011
Lecture 4 Competency Test.
Enzyme
Site
Recognition
Restriction site
Palindrome
• Each enzyme digests
(cuts) DNA at a
specific sequence =
restriction site
• Enzymes recognize
4- or 6- base pair,
palindromic
sequences
(eg GAATTC)
Fragment 1
Fragment 2
5 vs 3 Prime
Overhang
• Generates 5 prime
overhang
Enzyme cuts
Common
Restriction
Enzymes
EcoRI
– Eschericha coli
– 5 prime overhang
Pstl
– Providencia stuartii
– 3 prime overhang
1) Name the five components of
a PCR reaction.
1) Template
2) Buffer
3) Primers (two of them)
4) Taq Polymerase
5) dNTPs
The PCR
Reaction
How does
it work?
Heat (94oC) to denature DNA strands
Cool (52oC) to anneal primers to template
Warm (72oC) to activate Taq polymerase, which
extends primers and replicates DNA
Repeat 35 cycles
Denaturing Template DNA
Heat causes DNA
5’
strands to separate
3’
3’
5’
Denaturation of
DNA at 94oC
5’
3’
3’
5’
Annealing Primers
Primers anneal at 52oC
Primers bind
to the
template
5’
3’
5’
3’
3’
5’
5’
3’
Taq
polymerase
recognizes 3’
end of primer
+ template
strand
Taq extends at 72oC
5’
3’
5’
3’
3’
3’
5’
5’
Taq polymerase extends…..
Cycle 1
DNA is replicated
Repeat denaturing,
annealing, and
extending 35 cycles
Cycle 2
Cycle 3
The exact-length
target product is
made in the third
cycle
2) Name two ways to synthesize
a gene.
1) Recombinant PCR
Also: Polymerase cycle assembly
2) Assembly PCR
Polymerase cycle assembly
Assembly PCR
What is Nested PCR?
3) What is the purpose of codon
optimizing genes?
To maximize the translation
to the host tRNA population
You must know single letter codes
What does
Degree of
Degeneracy
Reflect?
http://www.encorbio.com/protocols/Codon.htm
eGFP
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF
FKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*
eGFP (eucaryotic vs for bacterial expression)
4) What are the 3 common
components of plasmids used in
DNA cloning?
1) Origin [OriC] of replication
2) Selectable marker [I.e. Kan
Resistance Gene/Amp Resistance
Gene
3) Multiple Cloning Site [MCS]
5) What is the difference between
an oligonucleotide and a primer?
Nothing. It is the usage which
differs. A primer is always used
with a polymerase. An oligo
is simply a chain of nucleotides
6) Are oligonucleotides and
primers single stranded?
Yes. We use them to anneal to
other single stranded templates.
7) Do oligonucleotides and
primers have to be DNA?
No. They can be RNA.
Why do we use RNA sometimes:
Because annealing RNA to DNA
Make very stronger hybrids.
8) Name 4 parameters that affect
annealing of two single stranded
DNA chains?
1)Temperature
2) Salt concentration
3) DNA concentration
4) Length of complementarity
5) Time of re-annealing
9) What does DNA ligase do?
DNA ligase catalyzes the
Phosphodiester bond formation
between two nucleotides.
ATP is used in the reaction to donate
a phosphate.
DNA Ligase Covalently
Closes Nicks in DNA
DNA ligase forms a high energy intermediate that
Aside:
Calf Intestinal Phosphotase?
Cut with EcoR1
GAATTC
CTTAAG
G-OH p-AATTC
CTTAA-p HO-G
Calf Intestinal Phosphotase?
Cut with EcoR1
G-OH
CTTAA-p
G-OH
CTTAA-OH
p-AATTC
HO-G
HO-AATTC
HO-G
Calf Intestinal Phosphotase?
Cut with EcoR1
p-AATTCgatacagagagactcatgacgG-OH
HO-GctatgtctctctgagtactgcCTTAA-p
G-OH
CTTAA-OH
HO-AATTC
Vector won’t religate,
But will take in insert
HO-G