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710.LC GRADUATE MOLECULAR BIOLOGY 9/15/2010 Lecture 4 Competency Test. 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 10) What does a Kinase do? 11) What are Restriction Enzymes? 12) Given one 4-cutter restriction enzyme, how many times might it cut a 1000bp dsDNA molecule? 13) Given one 6-cutter restriction enzyme, how many times might it cut a 1000bp dsDNA molecule? 14) What is the most common type of DNA sequencing? 15) What is NextGen Sequencing? 16) What is a transcriptome? 17) Name two ways to make mutations in plasmid. 18) What is the yeast two-hybrid system? 19) What is the one-hybrid system? 20) Name the protein that binds to the TetO sequence? 21) What is GFP? 22) What is Cherry (protein)? 23) What is Venus (protein) ? 24) What is Cerulean (protein) ? 25) What are molecular beacons? 26) Define I.R.E.S. 27) What does the T2A fragment do? 28) What is Translational Frameshifting? 29) What are CRE and Flp? 30) Cre works with LoxP or FRT? Flp works with LoxP or FRT? 31) What is a Bacterial artificial chromosome? 32) How are Transgenic animals made (in general)? Be brief and specific. 33) What are Zn finger nucleases and what are they good for? 34) What do Double Strand DNA breaks promote? 35) What are ES cells? 36) How were mouse ES cells derived? 37) Why do we use ES cells to make Gene-targeted mice? 38) What is meant by Structure/Function analysis?