710.LC GRADUATE MOLECULAR BIOLOGY

9/15/2010

Lecture 4 Competency Test.

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

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) Template2) Buffer3) Primers (two of them)4) Taq Polymerase5) 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 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’

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

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

2) Name two ways to synthesize a gene.

1) Recombinant PCRAlso: Polymerase cycle assembly2) Assembly PCR

Polymerase cycle assembly

Assembly PCR

What is Nested PCR?

3) What is the purpose of codon optimizing genes?

To maximize the translationto the host tRNA population

You must know single letter codes

What doesDegree of DegeneracyReflect?

http://www.encorbio.com/protocols/Codon.htm

eGFP

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*

eGFP (eucaryotic vs for bacterial expression)

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]

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

6) Are oligonucleotides and primers single stranded?

Yes. We use them to anneal toother 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 DNAMake very stronger hybrids.

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

9) What does DNA ligase do?

DNA ligase catalyzes the Phosphodiester bond formationbetween two nucleotides.ATP is used in the reaction to donatea phosphate.

DNA Ligase Covalently Closes Nicks in DNA

DNA ligase forms a high energy intermediate that

Calf Intestinal Phosphotase?

GAATTCCTTAAG

G-OH p-AATTCCTTAA-p HO-G

Cut with EcoR1

Aside:

Calf Intestinal Phosphotase?

G-OH p-AATTCCTTAA-p HO-G

Cut with EcoR1

G-OH HO-AATTCCTTAA-OH HO-G

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