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Lecture 10

Chapter 12 (Lecture 10-12) – Review Quicksheet.docx

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Margaret Fahnestock

Biochem 2B03 Chapter 12 – Review Quicksheet Cloning Overview 1. Select Host (often E. coli) 2. Reverse Transcription (make DNA from mRNA using Reverse Transcriptase) 3. Amplify DNA (make many copies of DNA by PCR) 4. Digesting (cut DNA and cloning vector/plasmid with restriction enzyme) 5. Purification (retrieve desired DNA; DNA Separation by Electrophoresis) 6. Ligation (insert DNA into plasmid using DNA Ligase) 7. Transformation (introduce recombinant plasmid into host then screen for recombinant plasmid) 8. DNA Sequencing (confirm DNA insert has desired sequence; Sanger/Dideoxy Sequencing) 1. Select Host  E. coli often used  Well understood  Rapid reproduction (20 minutes/generation)  Contains single chromosome 2. Reverse Transcription  mRNA – single stranded and has poly-A tail  Add primer  15 deoxythymidines (dT15) OR  Designed unique primer  Reverse transcriptase – RNA-dependent DNA Polymerase  Synthesizes DNA from mRNA in same 5’3’ direction  Cant do first bond synthesis (reason for primers)  Produced by eukaryotic retroviruses (eg/ HIV, common cold)  dNTPs (dATP, dGTP, dTTP, dCTP) – added by reverse transcriptase to make double stranded RNA:DNA Duplex  NaOH/ribonuclease – degrades mRNA  Results in ssDNA  cDNA 3. Amplify DNA (PCR) i. Denature – separate dsDNA by heating (95° C) ii. Anneal – cool (70°C) and add primers  Primers can be synthesized; designed to be complementary to the 3’ ends of DNA to be amplified  Boundaries of primers determine boundaries of amplified DNA (established after multiple cycles)  Primers allow addition of extra nucleotides to 5’ end – useful sequences such as restriction sites and tags iii. Extend – add DNA Polymerase and dNTPs  DNA Polymerase must be Thermotolerant (Taq, Pfu, Vent etc)  Extension time determined by length of desired product  Repeat steps for multiple cycles; subsequent rounds use newly synthesized strands as template  Other applications of PCR  DNA Sequencing  GENE SOEing – Splicing by Overlap Extension  Site-directed Mutagenesis  Error prone due to lack of/inefficient 3’5’ exonuclease activity 4. Digestion (Restriction Enzymes)  Digest plasmid and insert DNA with same restriction enzyme – they will have compatible sticky ends 5. Purification (DNA Separation by Electrophoresis)  DNA added to wells in agarose or polyacrylamide gel near cathode (-ve); gel acts as a molecular mesh  Voltage applied across gel (from –ve to +ve; cathode to anode)  DNA migrates toward anode; larger DNA slower than smaller DNA  Visualize DNA by staining with chromosphores that bind to DNA (Eg/ ethidium bromide)  Ladder – DNA molecules of known size used as markers  Cut bands from gel 6. Ligation (DNA Ligase)  Mix digested DNA and plasmid – DNA inserted into plasmid due to compatible ends  DNA ligase seals gaps in plasmid  Incubate at lower temperature  Single Restriction Enzyme (in digestion)  DNA can be inserted into plasmid in two directions  DNA can ligate to other copies of DNA multiple times  Vector can religate  Two Restriction Enzymes (in digestion)  Directional ligation/cloning 7. Transformation (introduce recombination plasmid into host)  Create competent cells – treat with 0.1 M CaC2  Heat shock at 42°C  Plate on selective medium; 37°C, 1 hour  Screen for Recombinant Plasmids  Replica Plating r r  Eg/ Plasmid containing 2 resistance genes (amp and tet )  DNA inserted into tet of plasmid – put into E. coli Biochem 2B03  Transformed E. coli plated on ampicillin containing medium  Colonies lifted off plate using velvet covered disk  Colonies on disk pressed onto new tetracycline containing medium  Colonies that appear on tetracycline containing medium do not contain desired DNA r  DNA insert in tet would disrupt resistance gene function; would not allow cell to survive on tetracycline  Blue/White Screening  Colony PCR  E-lyse (in-well lysis procedure; uncommon) 8. DNA Sequencing (Sanger/Dideoxy Sequencing)  dNTPs – added to ssDNA by DNA Polymerase (primer start)  Fluorescently labeled ddNTPs – eventually added to DNA strand; causes chain termination  ~10x lower concentration than dNTPs  Newly synthesiz
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