710.LC GRADUATE MOLECULAR BIOLOGY 9/15/2010
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Transcript of 710.LC GRADUATE MOLECULAR BIOLOGY 9/15/2010
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710.LC GRADUATE MOLECULAR BIOLOGY
9/15/2010
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Lecture 4 Competency Test.
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Enzyme Site Recognition • Each enzyme digests
(cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)
Palindrome
Restriction site
Fragment 1 Fragment 2
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5 vs 3 Prime Overhang
• Generates 5 prime overhang
Enzyme cuts
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Common Restriction Enzymes
EcoRI– Eschericha coli– 5 prime overhang
Pstl– Providencia stuartii– 3 prime overhang
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1) Name the five components of a PCR reaction.
1) Template2) Buffer3) Primers (two of them)4) Taq Polymerase5) dNTPs
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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
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Denaturing Template DNA
Heat causes DNA Heat causes DNA strands to separatestrands to separate
3’
5’
5’
3’
Denaturation of Denaturation of DNA at 94DNA at 94ooCC
5’
3’
3’
5’
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Annealing Primers
Primers bind to Primers bind to the templatethe template
Taq Taq polymerase polymerase recognizes 3’ recognizes 3’ end of primer end of primer + template + template strandstrand
Primers anneal at 52Primers anneal at 52ooCC
5’
3’
3’
5’
5’
3’
3’
5’
3’5’3’ 5’
3’ 5’3’5’
Taq Taq extendsextends at 72at 72ooCC
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Taq polymerase extends…..
DNA is replicatedDNA is replicated
Repeat denaturing, Repeat denaturing, annealing, and annealing, and extending 35 cyclesextending 35 cycles
Cycle 1
Cycle 2
Cycle 3The exact-length target product is made in the third
cycle
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2) Name two ways to synthesize a gene.
1) Recombinant PCRAlso: Polymerase cycle assembly2) Assembly PCR
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Polymerase cycle assembly
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Assembly PCR
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What is Nested PCR?
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3) What is the purpose of codon optimizing genes?
To maximize the translationto the host tRNA population
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You must know single letter codes
What doesDegree of DegeneracyReflect?
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http://www.encorbio.com/protocols/Codon.htm
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eGFP
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*
eGFP (eucaryotic vs for bacterial expression)
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4) What are the 3 common components of plasmids used in
DNA cloning?
1) Origin [OriC] of replication2) Selectable marker [I.e. KanResistance Gene/Amp ResistanceGene3) Multiple Cloning Site [MCS]
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5) What is the difference between an oligonucleotide and a primer?
Nothing. It is the usage whichdiffers. A primer is always usedwith a polymerase. An oligois simply a chain of nucleotides
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6) Are oligonucleotides and primers single stranded?
Yes. We use them to anneal toother single stranded templates.
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7) Do oligonucleotides and primers have to be DNA?
No. They can be RNA.Why do we use RNA sometimes:Because annealing RNA to DNAMake very stronger hybrids.
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8) Name 4 parameters that affect annealing of two single stranded
DNA chains?
1)Temperature2) Salt concentration3) DNA concentration4) Length of complementarity5) Time of re-annealing
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9) What does DNA ligase do?
DNA ligase catalyzes the Phosphodiester bond formationbetween two nucleotides.ATP is used in the reaction to donatea phosphate.
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DNA Ligase Covalently Closes Nicks in DNA
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DNA ligase forms a high energy intermediate that
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Calf Intestinal Phosphotase?
GAATTCCTTAAG
G-OH p-AATTCCTTAA-p HO-G
Cut with EcoR1
Aside:
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Calf Intestinal Phosphotase?
G-OH p-AATTCCTTAA-p HO-G
Cut with EcoR1
G-OH HO-AATTCCTTAA-OH HO-G
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Calf Intestinal Phosphotase?
p-AATTCgatacagagagactcatgacgG-OH
HO-GctatgtctctctgagtactgcCTTAA-p
Cut with EcoR1
G-OH HO-AATTCCTTAA-OH HO-G
Vector won’t religate,But will take in insert
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