PCR & Gel Electrophoresis

BiotechnologyAP Biology Chapter 20History of BiotechnologyBiotechnologyUtilizing living systems for technological advancement

Origins of BiotechnologyBiotechnology has been around for ~10,000 yearsDomesticated plants and animalsUsing yeast to make bread riseUsing bacteria or yeast to ferment grapes into wine

4Current BiotechnologyBiotechnology is used in three main ways:Directly using cellsUsing the proteins/enzymes made by cellsUsing the genetic material inside of cell

5Future of Biotechnologyhttps://www.youtube.com/watch?v=rD5uNAMbDaQ DNA CloningPart 1DNA CloningScientists:Only want to study specific genesGenes:Small segments of DNA strand

E. coli plasmidSmall circular DNA that replicates independently from nucleoid chromosome

DNA Cloning ProcessFind gene of interest in any organism

DNA Cloning ProcessCut gene with restriction enzymesCut bacteria plasmid w/ same enzymes

DNA Cloning ProcessAttach gene to plasmid with DNA Ligase

DNA Cloning ProcessReinsert into bacteriaAllow bacteria to replicate

1414Restriction EnzymeCut DNA at known palindromic sequencesEnzymeSourceRecognition SequenceCutEcoRIEscherichia coli5'GAATTC 3'CTTAAG 5'---G AATTC---3' 3'---CTTAA G---5' EcoRIIEscherichia coli5'CCWGG 3'GGWCC 5'--- CCWGG---3' 3'---GGWCC ---5' BamHIBacillus amyloliquefaciens5'GGATCC 3'CCTAGG 5'---G GATCC---3' 3'---CCTAG G---5' HindIIIHaemophilus influenzae5'AAGCTT 3'TTCGAA 5'---A AGCTT---3' 3'---TTCGA A---5' TaqIThermus aquaticus5'TCGA 3'AGCT 5'---T CGA---3' 3'---AGC T---5' Current UsesMedicine:Insulin, HGH, Blood clottingTreatment:Hepatitis B Vaccine, HIV identificationFood:Golden Rice, Countless Resistant Crops

Environmental ConcernsEscapees in nature!Jumping into you!Part 2PCR & Gel ElectrophoresisPolymerase Chain Reaction FunctionExponentially amplify small amounts of DNA

PCR ProcessDenaturingHeating DNA separates the H bonds

PCR ProcessAnnealingCooling DNA allows RNA primers to attach

PCR ProcessExtensionDNA polymerase adds nucleotides to 3 end of primer

PCR ResultAfter 30 rounds of PCR:Copy 1 segment of DNA into ~1,000,000,00099.9% are only target sequencehttp://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

PCR ApplicationsShare 40,000 yr old woolly mammoth DNAIdentify crime scene DNAScreen fcDNA for genetic disordersDetect hidden viral genes (HIV)

Gel Electrophoresis FunctionVisualize various sized sections of DNA

GE ProcessUse restriction enzymes to cut DNA

GE ProcessPipette DNA into the gel (well)

GE ProcessTurn on electrical currentDNA phosphates have a negative chargeDNA travels to the positive electrodeGel separates DNA segments based on size

GE ResultStain gel to show bands of DNA

GE ApplicationsCompare DNAExtract pure samples of DNA

Part 3DNA sequencingDNA Sequencing FunctionAnalyze complete genome of an organism

DNA Sequencing ProcessIsolate DNAGrind up organism cells with DNA in nucleusAdd soap break down lipid membraneAdd meat tenderizer break down proteinSpin DNA in centrifuge DNA sinks

DNA Sequencing ProcessSend DNA through PCR with normal nucleotides

DNA Sequencing ProcessSend DNA through PCR with radioactive nucleotides

DNA Sequencing ResultMany partial sequences of various lengths with a radioactive terminal nitrogen baseRun this solution through a gel or sequencer

DNA Sequencing ApplicationHas shown evolutionary relationshipsWill unlock genetic mysterieshttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter15/animation_quiz_1.html