WHODUNNIT?WHODUNNIT?

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DNA Technology- What is it?DNA Technology- What is it?► Making recombinant Making recombinant

DNA (DNA in which DNA (DNA in which genes from 2 different genes from 2 different sources are combined)sources are combined)

► Biotechnology-Biotechnology-manipulation of genes manipulation of genes of organisms for our of organisms for our benefitbenefit

► Genetic engineering-Genetic engineering-directly changing directly changing genes of an organismgenes of an organism

► Examples:Examples: CloningCloning Gel electrophoresisGel electrophoresis Gene therapyGene therapy Test-tube babiesTest-tube babies Sequence DNA/genesSequence DNA/genes Stem cell researchStem cell research

What can we do with it?What can we do with it?The Big PictureThe Big Picture

► Diagnose diseasesDiagnose diseases► Cure diseasesCure diseases► Produce better Produce better

medicines/vaccines medicines/vaccines more efficientlymore efficiently

► Solve crimesSolve crimes► Paternity testsPaternity tests► Produce more nutritious Produce more nutritious

foodsfoods► Produce food in Produce food in

countries with little or countries with little or no arable landno arable land

► More efficiently Dispose More efficiently Dispose of wasteof waste

Important Techniques/Tools Used in DNA Important Techniques/Tools Used in DNA Technology Technology

► Restriction EnzymesRestriction Enzymes► DNA librariesDNA libraries► Gel electrophoresisGel electrophoresis► Bacterial transformation, plasmidsBacterial transformation, plasmids► cDNAcDNA► Polymerase chain reaction (PCR)Polymerase chain reaction (PCR)► probing/DNA hybridizationprobing/DNA hybridization► DNA sequencingDNA sequencing► BioinformaticsBioinformatics► Southern blottingSouthern blotting► Microarray analysisMicroarray analysis► RFLPsRFLPs► vectorsvectors

A few words about bacterial genetics….A few words about bacterial genetics….► ProkaryotesProkaryotes► Nucleoid regionNucleoid region► Reproduce by binary fission-Reproduce by binary fission-

10108 8 in 12 hrs!!in 12 hrs!!► GenomeGenome

1 DS circular chromosome1 DS circular chromosome Plasmids-smaller circles of DNAPlasmids-smaller circles of DNA

► Self replicatingSelf replicating► Confer new characteristicsConfer new characteristics

R plasmids, F plasmidsR plasmids, F plasmids► Can be genetically engineeredCan be genetically engineered

► Genetic recombination in Genetic recombination in bacteriabacteria Transformation-uptake of Transformation-uptake of

naked foreign DNAnaked foreign DNA Transduction-virusesTransduction-viruses Conjugation using pilliConjugation using pilli

Restriction EnzymesRestriction Enzymes

►Used in almost areas of recombinant Used in almost areas of recombinant DNA technologyDNA technology

►Create DNA “libraries”- collections of Create DNA “libraries”- collections of bacteria each having a random bacteria each having a random fragment of DNAfragment of DNA

Restriction Enzymes/Restriction Enzymes/EndonucleasesEndonucleases

► Enzymes able to cleave Enzymes able to cleave DNA at specific DNA at specific sequences 4-8 bps in sequences 4-8 bps in lengthlength

► Originally found in Originally found in bacteria, used as a bacteria, used as a defense against viral defense against viral infectioninfection

► Presently over 200 Presently over 200 known REs, each known REs, each recognizing a different recognizing a different DNA sequenceDNA sequence

► May make blunt (linear) cuts or staggered May make blunt (linear) cuts or staggered (zig zag) cuts(zig zag) cuts

► Staggered cuts leave “sticky” Staggered cuts leave “sticky” complementary ends-this allows DNA from complementary ends-this allows DNA from 2 sources to join together (recombinant 2 sources to join together (recombinant DNA)DNA)

► Not every sequence is Not every sequence is found in every found in every genome/DNA fragmentgenome/DNA fragment

► Enzymes recognizing Enzymes recognizing smaller sequences smaller sequences usually cut more often usually cut more often than enzymes than enzymes recognizing larger recognizing larger sequencessequences

► Since every organisms Since every organisms DNA is different, DNA is different, cutting 2 or more cutting 2 or more samples w/ the same samples w/ the same RE will yield different RE will yield different sizes & numbers of sizes & numbers of fragmentsfragments

► Cutting one DNA Cutting one DNA sample w/ the SAME RE sample w/ the SAME RE will yield fragments will yield fragments which can be used to which can be used to create a restriction create a restriction mapmap

Gel ElectrophoresisGel Electrophoresis

►UsesUses Paternity testingPaternity testing Solving crimesSolving crimes Diagnosing diseasesDiagnosing diseases Finding a particular gene or DNA Finding a particular gene or DNA

sequence in a DNA librarysequence in a DNA library Taxonomical studiesTaxonomical studies

The basics…….The basics…….http://gslc.genetics.utah.edu/units/biotech/gelhttp://gslc.genetics.utah.edu/units/biotech/gel

A few more points…..A few more points…..

► Can be used to separate proteins, lipids, nucleic acidsCan be used to separate proteins, lipids, nucleic acids► Gel is porous agarose (starch or cellulose) and consistency Gel is porous agarose (starch or cellulose) and consistency

can varycan vary► Rate of movement is a function of fragment length/agarose Rate of movement is a function of fragment length/agarose

consistency/time/ and voltageconsistency/time/ and voltage► Gels/DNA have dyes added to increase visibility of Gels/DNA have dyes added to increase visibility of

fragments both during & after electrophoresisfragments both during & after electrophoresis

Whodunnit?Whodunnit?Crime ScenesCrime Scenes

Who’s my Baby Daddy?Who’s my Baby Daddy?Aka paternity casesAka paternity cases

Size DOES Matter!Size DOES Matter!Determining the size (in bps) of DNA fragments of Determining the size (in bps) of DNA fragments of

unknown lengthsunknown lengths

► Generate a Generate a STANDARD CURVE STANDARD CURVE using known using known samplesample

► Compare distance Compare distance migrated of migrated of unknown fragments unknown fragments to standard curveto standard curve

► Estimate unknown Estimate unknown fragment sizesfragment sizes