Recombinant DNA technology•Genetic Engineering•DNA cloning•Modifying DNA sequence called22 Methods in GeneticsMonday, March 17, 201410:09 AM Exam 4 Page 1Exam 4 Page 2Exam 4 Page 3Site-directed mutagenesis by PCR Exam 4 Page 4Characterizing DNA Exam 4 Page 5Ethidium Bromide intercalates DNA and fluoresces under ultraviolet light. Exam 4 Page 6The pairing of DNA is called DNA hybridization. It is highly specific and this is how nucleic acids are detected in complex mixtures. Exam 4 Page 7South - DNANorth - RNAWest - ProteinThe Sanger sequencing method relies on ddNTPS terminating DNA synthesis. Exam 4 Page 8Sanger inserts dideoxyribonucleoside triphosphates into the DNA synthesis steps which prevents further phosphodiester bond linkage, terminating the sequence. Natural phosphodiester bond formation is shown below. Exam 4 Page 9Sanger disrupts continual synthesis by adding a small amount of one ddNTP to the mix of dNTPs during synthesis. By doing this with all four bases, the terminations happen randomly Exam 4 Page 10By doing this with all four bases, the terminations happen randomly and then are all put into gel electrophoresis which then can sequence DNA by reading from bottom up. Exam 4 Page 11Gene Cloning - isolate, manipulate, and modify DNA sequences by inserting them into specially designed plasmids called "vectors". Then put them into host cells. Transgene - sequence that has been moved from one species to anotherTransgenic - organism carrying a transgeneBiotechnology: ex. creating quantities of human insulin from bacteria.1.Basic Science: mutate proteins to investigate function; fuse proteins or domains to investigate function or location.2.Applications of Genetic EngineeringHow to clone a gene:A cloning vector must contain three thingsRestriction endonuclease/enzymes cut plasmids at restriction sites. 23 Methods in GeneticsThursday, March 20, 20149:44 PM Exam 4 Page 12Restriction endonuclease/enzymes cut plasmids at restriction sites.Restriction sites are sequences of palindromes in genes that get cut very specifically. This allows two ends to be ligated together for recombinant DNA.Overexpressing Protein Genes Exam 4 Page 13Misfoldi.Cannot undergo processing/modificationsii.E. coli overexpression is the most common but because eukaryotic proteins usuallyYeasti.Cultured insect cellsii.Cultured mammalian cellsiii.We are forced to use Exam 4 Page 14Forward genetics example: Exam 4 Page 15Exam 4 Page 16Instead of using genomic DNA to template PCR we can use mRNAs to template reverse transcriptase to make a "complementary DNA" or cDNA Exam 4 Page 17Exam 4 Page 18The Limit of Resolution is set by the wavelength of irradiation applied to the sample: light, electrons, X-rays24 Visualizing the CellSunday, March 23, 20141:21 PM Exam 4 Page 19Exam 4 Page 20Sample Consideration for Light MicroscopyNot a problem with single cellsa.Tissues needed to be thinly sliced with a tool called a microtome (razor)b.Is the sample thin enough to transmit light?1.Fixation - preserve the overall integrity of samples. Fixation kills cells and is accomplished using formaldehyde which chemically crosslinks molecules together.a.Will the sample degrade before it can be examined?2.Light must be somewhat perturbeda.Samples can be stained. Chemical dyes that bind tightly to certain biomoleculesb.Does the sample have enough contrast? 3. Exam 4 Page 21At edges of cover slips, phase-contrast microscopy is a benefit of constructive light which increases contrast of a sample. • Exam 4 Page 22Exam 4 Page 23Benefits of fluorescence microscopyLOCATION - Highly selective fluorescence tags allow one to determine the location of specific molecules in a cell1.COMBINATION OF TAGS - Combining different fluorescent tags can allow you to show two proteins are located in the same place and might work together2.CONCENTRATION - Fluorescent intensity reflects the concentration 3. Exam 4 Page 24CONCENTRATION - Fluorescent intensity reflects the concentration of tagged molecules3.INTERNALLY EXPRESSIVE - Fluorescently tagged molecules can be introduced into cell and their behaviors can be viewed in a live cell4.Fluorescent microscopy can be used to monitor the dynamics of the movement of molecules in the cellFRAP - fluorescent recovery from photo bleachingClean up out - of - focus fluorescence with math and software. Exam 4 Page 25Clean up out - of - focus fluorescence with math and software.Confocal fluorescence microscopy - hardware focusing Exam 4 Page 26Lets us make 3D z-stacks•TEM Exam 4 Page 27Radiation bean is electrons which have very short wavelengths. Practical resolution for biological samples is 2nm - about 1/10th the diameter of a ribosome•Lenses are magnets that focus electron beam•Image is created by components scattering electrons•Samples must be very thin - 50 to 100 nm•The samples are dead and fixed•Samples are stained with heavy metals: uranium, lead, platinum, osmium, gold since organic elements do not scatter electrons well•Scanning Electron Microscopy (SEM) Exam 4 Page 28Exam 4 Page 29Allows organelles to execute specialized functions by keeping the contents of the organelle separate from the rest of the cell•Provides boundaries that establish electrochemical gradients. These are used to make ATP and to generate nerve impulses •Has proteins embedded in them to adhere to surfaces and other cells•Biologicals membranes are thin films composed mainly of amphilic lipids and proteins. Most of the molecules are held together by noncovalent interactions. This model of the membrane is known as the Fluid Mosaic ModelLipid molecules organized into a bilayer approximately 5 nm think○Each layer is called a leaflet○Bilayers are self-sealing○LipidsPlasma membrane separates cellular components from the environment.25 Membrane StructureSunday, March 30, 20148:14 PM Exam 4 Page 30Lipids diffuse rapidly in the plane of the bilayer○Lipids flex and rotate but DO NOT FLIP FLOT (without help)○Membranes of animal cells are composed primarily of 3 types of lipids.Phospholipids - most abundant1.Cholesterol2.Glycolipids3.Phospholipids - spontaneously form bilayers by exclusion of hydrophobic side chains from the aqueous phase Exam 4 Page 31Trans-unsaturated chains cause linear tails○Cis-unsaturated chains cause kinks○ Exam 4 Page 32Cholesterol - increases membrane packing decreasing fluidity and permeability.Glycolipids - common in neuronal membranes3. Exam 4 Page 33Exam 4 Page