710.LC GRADUATE MOLECULAR BIOLOGY 10/31/2011

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) Template 2) Buffer 3) Primers (two of them) 4) Taq Polymerase 5) dNTPs

The PCR Reaction How does it work? Heat (94 o C) to denature DNA strands Cool (52 o C) to anneal primers to template Warm (72 o C) to activate Taq polymerase, which extends primers and replicates DNA Repeat 35 cycles

Denaturing Template DNA Heat causes DNA strands to separate 3’ 5’ 3’ Denaturation of DNA at 94 o C 5’ 3’ 5’

Annealing Primers Primers bind to the template Taq polymerase recognizes 3’ end of primer + template strand Taq polymerase recognizes 3’ end of primer + template strand Primers anneal at 52 o C 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Taq extends at 72 o C

Taq polymerase extends….. DNA is replicated DNA is replicated Repeat denaturing, annealing, and extending 35 cycles Cycle 1 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?

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

Calf Intestinal Phosphotase? GAATTC CTTAAG G -OH p- AATTC CTTAA -p HO- G Cut with EcoR1 Aside:

Calf Intestinal Phosphotase? G -OH p- AATTC CTTAA -p HO- G Cut with EcoR1 G -OH HO- AATTC CTTAA -OH HO- G

Calf Intestinal Phosphotase? p- AATTCgatacagagagactcatgacgG -OH HO- GctatgtctctctgagtactgcCTTAA -p Cut with EcoR1 G -OH HO- AATTC CTTAA -OH HO- G Vector won’t religate, But will take in insert