DNA TechnologyAsilomar 1973. Singer,Zinder, Brenner, BergDNA TechnologyThe following are some of the most important molecular methods we willbe using in this course. They will be used, among other things, for• Sequencing genes and genomes• PCR• Making recombinant DNA• Detecting mutations at the molecular level• Studying the control of gene actionDNA SequencingReminder: DNA polymerase adds nucleotides, one at atime, to a single nucleotide strand, in the 5' to 3' direction,using a DNA template and RNA or DNA primers.• Polymerase + DNA template + pair of DNA primers -->copy DNA sequence between the primers.• Interrupt reaction by dideoxynucloside triphosphates whichcan have labels attached to them. You don’t need to knowthe method; sequencing is done for you at facilities at UAand elsewhere.• Sequence both strands.• Proofread sequences.Cambridge biochemist FredSanger won Nobel Prizes forsequencing proteins andDNA.DNA Denaturation and Renaturation(Melting and Annealing)DNA Denaturation and Renaturation(Melting and Annealing)Molecule A has 30% GC.Molecule B has 45% GC.Which one will melt at a lower temperature?Polymerase Chain Reaction (PCR)-1A pre-defined DNAsequence in thegenome can be greatlyamplified by repeatedPolymerization cycles using 2primers whichhybridize to the endsof the target DNA. Ineach cycle, the amount oftarget DNAis doubled. After 10, 20 and 30cycles, there is a 1000-, million-and billion-fold amplificationrespectively.Polymerase Chain Reaction (PCR)-2Each PCR cycle has 3 steps-a. Melting of DNA(e.g. 94°)b. Hybridization ofprimer (e.g. 40°(ca. 20 bp long)c. DNA synthesis (e.g.68°)PCR Requires Thermostable PolymeraseThermus aquaticus: bacterium found in hotsprings of Yellowstone National Park in1969. Survives at up to 80° C.DNA polymerase from Thermus aquaticus =Taq DNA Polymerase has optimumtemperature 80°.Monetary value of natural habitats andorganisms. If we don’t conserve them, wewon’t be able to exploit them.Size separation of DNA fragmentsby electrophoresis in agarose gelsDNA is negatively charged due to phosphates on its surface.As a result, it moves towards the positive pole.Distance migrated by a DNA fragmentin a gel is related to log10 of its sizeGel Electrophoresis to Verify AmplificationPCR very powerful,amplifies tinyamounts ofcontaminating DNA.Should have negativecontrol = all reagentsbut no added DNA.If we amplified thecorrect DNAsegment, we shouldget a product of thesize of that segment.SizeStandardQ & AQuestion: A pair of PCR primers are designed to be complementary to sites 120-135 and440-465 of the human β-globin gene. What is the expected size of the amplification productif these primers are used with a human DNA template?Q & AQuestion: A pair of PCR primers are designed to be complementary to sites 120-135 and440-465 of the human β-globin gene. What is the expected size of the amplification productif these primers are used with a human DNA template?Answer: 120 - 465 +1 = 346 bpQuestion: If the template is DNA from a baboon, do we expect to get the same size?Q & AQuestion: A pair of PCR primers are designed to be complementary to sites 120-135 and440-465 of the human β-globin gene. What is the expected size of the amplification productif these primers are used with a human DNA template?Answer: 120 - 465 = 345 bpQuestion: If the template is DNA from a baboon, what size product do we expect?Answer: Similar but not necessarily identical, because the genes of these closely-relatedanimals are similar but not identical and can differ in length as well as in sequence.Powers of 2Memorize andunderstand:21 = 222 = 423 = 824 = 1625 = 3226 = 6427 = 12828 = 25629 = 512210 = 1024 ≈ 103211 ≈ 2 x103212 ≈ 4 x103220 ≈ 106Useful in thinking about:• Anything that doubles innumber: PCR, increase in cellnumber during binary fission•Dilution series in the lab•Genetic ratios 1:1, 3:1, 9:3:3:1etc., etc.Engineering DNAThe same enzymes that are used in DNA replication can be used to rearrange DNAmolecules at will.Endonuclease: breaks 5'-3' phosphodiester bonds in backbone of DNA; "nicks"DNA.Restriction endonucleases are enzymes made by bacteria. They recognize andcleave specific sequences. These sequences are usually palindromes. E.g.:EcoRI 5' G|A A T T C 3'3' C T T A A|G 5'6-cutter, staggeredAvaII 5' G|G W C C 3' W = A or T3' C C W G|G 5'5-cutter, staggered, degenerateSau3A 5'|G A T C 3'3' C T A G|5'4-cutter, staggeredAluI 5' A G|C T 3'3' T C|G A 5'4-cutter, blunt endEngineering DNAHow EcoRI cutsReminderLigase: makes 5'-3' phosphodiester bonds in backbone ofDNA; "ligates" DNA.Blunt-end ligation:Recombinant DNACloning DNA: Making Cells Amplify DNA for USPlasmid = small DNA molecule that can replicate insidebacterial cell independently of cell chromosome to achieve highcopy number.Cloning DNASelecting cells with vector + insert:• Join DNA cut with restriction enzyme to plasmid cut with same enzyme, e.g.BamH1• Transform bacteria with DNA -> cells cells + plasmid cells + plasmid + insertAntibiotic sensitive/resistant phenotypes?Cloning vector needs:• Replication origin• Selectable genes, e.g. antibiotic resistance• Restriction sites in genesCloning DNASelecting cells with vector + insert:• Join DNA cut with restriction enzyme to plasmid cut with same enzyme, e.g.BamH1• Transform bacteria with DNA -> cells ampS tetS cells + plasmid ampR tetR cells + plasmid + insert ampR tetS•How would I select cells + plasmid + insert?Cloning vector needs:• Replication origin• Selectable genes, e.g. antibiotic resistance• Restriction sites in