Ch. 9: Frontiers of Biotechnology€¦ · 1 _ x 1 x 1_ 500 90 120 1 chance in 5.4 million people! 9.3: DNA Fingerprinting Uses: To determine guilt ... Implant nucleus from cell of

Ch. 9: Frontiers of BiotechnologyCh. 9: Frontiers of Biotechnology

9.1: Manipulating DNA9.1: Manipulating DNA

Restriction EnzymesRestriction EnzymesAKA AKA endonucleasesCut DNA at precise locations (Cut DNA at precise locations (restriction sites) based on nucleotide sequence) based on nucleotide sequenceRecognize sequences 4 Recognize sequences 4 –– 8 base pairs 8 base pairs longlongObtained from bacteriaObtained from bacteria

Used by bacteria to restrict effect of a virusUsed by bacteria to restrict effect of a virus


Two types of Restriction Enzyme:Two types of Restriction Enzyme:Yield different types of cuts:Yield different types of cuts:

Blunt ends: straight cutBlunt ends: straight cut

Sticky ends: staggered cutSticky ends: staggered cutOften used in biotechOften used in biotechComplimentary BPComplimentary BP’’s will hydrogen bonds will hydrogen bond


Sticky EndsSticky Ends


Once cut, DNA sequence can be studiedOnce cut, DNA sequence can be studiede.g. gene sequencinge.g. gene sequencinge.g. gene can be cut out e.g. gene can be cut out from DNA and placed into from DNA and placed into the DNA of another the DNA of another organismorganism


Gel Electrophoresis Gel Electrophoresis (Fig. 9.3, p. 267)Method used to separate DNA fragments Method used to separate DNA fragments by size (length)by size (length)

DNA is negatively chargedDNA is negatively chargedPlace DNA sample on gel and pass electricity Place DNA sample on gel and pass electricity through gelthrough gelDNA migrates toward positive electrodeDNA migrates toward positive electrodeSmaller fragments move faster (farther)Smaller fragments move faster (farther)Fragments appear as bands (lines) on the gel, Fragments appear as bands (lines) on the gel, forming a forming a restriction map..


Gel Electrophoresis Gel Electrophoresis (Fig. 9.3, p. 267)Can be used to diagnose genetic disease by Can be used to diagnose genetic disease by comparing length of fragment with normal comparing length of fragment with normal DNADNA

9.2: Copying DNA9.2: Copying DNAAmt. of DNA from individual sample is Amt. of DNA from individual sample is insufficient for most studies, so . . . insufficient for most studies, so . . .

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)Process that produces multiple (millions or Process that produces multiple (millions or billions) copies of DNA billions) copies of DNA sequence rapidlysequence rapidlyDeveloped by Developed by KaryKary MullisMullis

9.2: Copying DNA9.2: Copying DNA1. Separating: Heat DNA (90Heat DNA (90°°C/194C/194°°F) F) to to

separate the two strandsseparate the two strandsa.a. Also breaks down DNA polymerases, so . . . Also breaks down DNA polymerases, so . . . b.b. Use polymerases from heat tolerant Use polymerases from heat tolerant

organism, organism, Thermus aquaticus, found in , found in Yellowstone hot springs: Yellowstone hot springs: Taq polymerase

Note: Patent for PCR Note: Patent for PCR process sold for $300 process sold for $300 million. NPS never million. NPS never received any royaltiesreceived any royalties

9.2: Copying DNA9.2: Copying DNA2. Binding: Cool DNA (55Cool DNA (55°°C/131C/131°°F)F) so so

primer can bind to each DNA strandprimer can bind to each DNA strand

9.2: Copying DNA9.2: Copying DNA3. Copying: Heat sample again Heat sample again

(72(72°°C/152C/152°°F)F)a.a. DNA polymerases build new complimentary DNA polymerases build new complimentary

strands of DNAstrands of DNA

9.2: Copying DNA9.2: Copying DNARun cycle 30 times Run cycle 30 times over 1 billion copiesover 1 billion copies

Makes 1 million copies/hrMakes 1 million copies/hr

9.3: DNA Fingerprinting9.3: DNA Fingerprinting

DNA Fingerprint: DNA Fingerprint: a representation of parts a representation of parts of an individualof an individual’’s DNAs DNAA specific type of restriction mapA specific type of restriction mapUsed to identify a person at the molecular Used to identify a person at the molecular levellevel


DNA Fingerprint:DNA Fingerprint:OneOne’’s DNA fingerprint is a combination of s DNA fingerprint is a combination of oneone’’s parentss parents’’ fingerprints (fingerprints (Fig. 9.6, p. 272))Greatest differences are in Greatest differences are in noncodingnoncodingsections of DNA sections of DNA –– repeating sequences

Number of repeats differs from one person to Number of repeats differs from one person to anotheranother


DNA Fingerprinting Method:DNA Fingerprinting Method:1.1. Cut DNA in known locations with restriction Cut DNA in known locations with restriction

enzymesenzymes2.2. Separate fragments with gel electrophoresisSeparate fragments with gel electrophoresis3.3. The more repeats The more repeats the larger the DNA the larger the DNA

fragmentfragment1.1. Unlikely more than one individual will have multiple Unlikely more than one individual will have multiple

fragments in common fragments in common –– even among brothers and even among brothers and sisterssisters

2.2. Usually compare five (5) regions of the genomeUsually compare five (5) regions of the genome

9.3: DNA Fingerprinting9.3: DNA FingerprintingProbabilityProbability

Suppose:Suppose:1 in 500 people have matching DNA in region A,1 in 500 people have matching DNA in region A,1 in 90 have matching DNA in region B1 in 90 have matching DNA in region B1 in 120 have matching DNA in region C1 in 120 have matching DNA in region C

What is the chance that two people will match in all three regions?1 _ x 1 x 1_

500 90 120

1 chance in 5.4 million people!


Uses:Uses:To determine guilt or innocenceTo determine guilt or innocence

Easier to prove innocence than guiltEasier to prove innocence than guiltPossible contamination of samplePossible contamination of sampleNo legal standard for probability of random matchNo legal standard for probability of random match

To determine family relationshipsTo determine family relationshipsTo study biodiversityTo study biodiversity

e.g. ID native Galapagos tortoises or locate e.g. ID native Galapagos tortoises or locate genetically engineered cropsgenetically engineered crops

9.4: Genetic Engineering9.4: Genetic EngineeringExamples:Examples:

Glowing plants used to track genetically Glowing plants used to track genetically modified cropsmodified cropsGlowing mice used in cancer Glowing mice used in cancer researchresearchGlowing yeast used to Glowing yeast used to locate water pollutionlocate water pollution

9.4: Genetic Engineering9.4: Genetic Engineering

Clone: Clone: a genetically identical copy of a a genetically identical copy of a single gene or entire organismsingle gene or entire organismAsexual reproduction in plantsAsexual reproduction in plantsBacterial reproductionBacterial reproductionIdentical twinsIdentical twinsRegeneration in sea starsRegeneration in sea stars


To clone a mammal:To clone a mammal:Swap DNA between cells (Swap DNA between cells (nuclear transfernuclear transfer))1.1. Obtain unfertilized egg and remove nucleusObtain unfertilized egg and remove nucleus2.2. Implant nucleus from cell of Implant nucleus from cell of

animal to be clonedanimal to be cloned3.3. Stimulate egg to begin cell Stimulate egg to begin cell

divisiondivision4.4. Transplant embryo into Transplant embryo into

femalefemale

Cows, pigs, mice, Cows, pigs, mice, your pet!!!your pet!!!


Clone may not look like or act like Clone may not look like or act like originaloriginalEnvironment plays a role in gene Environment plays a role in gene expressionexpression


Uses:Uses:Clone organs from mammals for transplant Clone organs from mammals for transplant into humansinto humansSave endangered speciesSave endangered species

Concerns:Concerns:Health of cloneHealth of clone

Dolly aged quickly Dolly aged quickly –– possibly possibly from having from having ““oldold”” DNADNA

Ecological biodiversity reducedEcological biodiversity reducedPopulation more susceptible to disease, etc.Population more susceptible to disease, etc.


Genetic Engineering: Genetic Engineering: changing an changing an organismorganism’’s DNA to give it new traitss DNA to give it new traitsUses DNA with genes from more than Uses DNA with genes from more than one organism (one organism (recombinant DNA))Possible uses:Possible uses:

Produce crops that make medicines or Produce crops that make medicines or vitamins vitamins (“pharming”)Make vaccines that protect against HIVMake vaccines that protect against HIV

9.4: Genetic Engineering9.4: Genetic EngineeringGenetic Engineering:Genetic Engineering:

Bacterial Bacterial plasmids used to manufacture large amounts used to manufacture large amounts of targeted geneof targeted geneSee Fig. 9.11, p. 277 See Fig. 9.11, p. 277 for procedurefor procedure

Bacteria become Bacteria become ““gene factoriesgene factories””Bacteria are Bacteria are transgenic –an organism that has one an organism that has one or more genes from another or more genes from another organismorganism

9.4: Genetic 9.4: Genetic EngineeringEngineering

Genetic Engineering in Plants:Genetic Engineering in Plants:Allow transgenic bacteria to infect plant Allow transgenic bacteria to infect plant new gene becomes part of plantnew gene becomes part of plant’’s DNAs DNAGGenetically enetically mmodified plants (odified plants (GM plants))Scientists can give plants new traits, e.g.Scientists can give plants new traits, e.g.

Resistance to frost, insects, diseaseResistance to frost, insects, diseaseBtBt gene = a natural pesticidegene = a natural pesticideModify to increase crop yield, Modify to increase crop yield, e.g. potatoes and corne.g. potatoes and corn

9.4: Genetic Engineering9.4: Genetic EngineeringGenetic Engineering in Animals:Genetic Engineering in Animals:

More difficultMore difficultAnimals are more resistant to genetic Animals are more resistant to genetic modificationmodification

Only small percentage mature normallyOnly small percentage mature normallyOf those that do, only a few develop into a Of those that do, only a few develop into a transgenic animaltransgenic animal


Genetic Engineering in Animals:Genetic Engineering in Animals:Process:Process:

1.1. Obtain fertilized egg cellObtain fertilized egg cell2.2. Insert foreign DNA into nucleusInsert foreign DNA into nucleus3.3. Implant egg back into femaleImplant egg back into female

9.4: Genetic Engineering9.4: Genetic EngineeringGenetic Engineering in Animals:Genetic Engineering in Animals:

Uses:Uses:Transgenic mice Transgenic mice to study cancer and to study cancer and cancer drugs (cancer drugs (oncomouse), diabetes, brain ), diabetes, brain function & development, sex determinationfunction & development, sex determinationGene knockout: genes : genes purposely purposely ““turned offturned off”” in in mice to study gene function, mice to study gene function, genetic diseases, obesity genetic diseases, obesity (gene for (gene for leptinleptin, which , which controls hunger)controls hunger)


Concerns:Concerns:Insufficient research done so far?Insufficient research done so far?Possible sidePossible side--effects of GE organismseffects of GE organisms

Allergic reactions?Allergic reactions?Environmental decrease in genetic diversityEnvironmental decrease in genetic diversity

9.5: Genomics and Bioinformatics9.5: Genomics and BioinformaticsGenomics: Genomics: study of genomesstudy of genomes

Genome: Genome: sequence ofsequence of an organisman organism’’s genetic s genetic information, for one gene or for all its DNAinformation, for one gene or for all its DNAComparing DNA of many different people canComparing DNA of many different people can

Find genes that cause diseaseFind genes that cause diseaseUnderstand how medicines Understand how medicines work in different individualswork in different individualsLearn evolutionary Learn evolutionary relationshipsrelationshipsLearn how genes interactLearn how genes interactDetermine how oneDetermine how one’’s s genome makes that genome makes that individual uniqueindividual unique

9.5: Genomics and Bioinformatics9.5: Genomics and Bioinformatics

Comparing DNA of many different people Comparing DNA of many different people cancan

Find genes that cause diseaseFind genes that cause diseaseUnderstand how medicines work in different Understand how medicines work in different individuals, e.g. an organismindividuals, e.g. an organism’’s DNA is used s DNA is used as a model in medical researchas a model in medical researchLearn evolutionary relationshipsLearn evolutionary relationshipsLearn how genes interactLearn how genes interactDetermine how oneDetermine how one’’s genome makes that s genome makes that individual uniqueindividual unique


Gene sequencing: Determining the order : Determining the order of DNA nucleotides in genes or entire of DNA nucleotides in genes or entire genomesgenomes

Frederic Sanger (1970Frederic Sanger (1970’’s) s) developed techniquedeveloped techniqueSee See Fig. 9.13, p. 281Fig. 9.13, p. 281for comparison of selected for comparison of selected genomesgenomes

9.5: Genomics and Bioinformatics9.5: Genomics and BioinformaticsHuman Genome Project:Human Genome Project:

Goals:Goals:Map and sequence all of DNA Map and sequence all of DNA base pairs of all 24 human base pairs of all 24 human chromosomes (finished in 2003)chromosomes (finished in 2003)Identify all of the genes within the sequenceIdentify all of the genes within the sequence

Current work:Current work:Identifying genesIdentifying genesLocating genes on Locating genes on chromosomeschromosomesDetermining gene functionDetermining gene function

9.5: Genomics and Bioinformatics9.5: Genomics and BioinformaticsBioinformatics: Bioinformatics: Use of computer Use of computer

databases to organize and analyze databases to organize and analyze biological databiological dataStore,Store, share, find share, find datadataCompare genomes Compare genomes of various individuals of various individuals or organismsor organismsPredict and model Predict and model gene and protein gene and protein functionfunction


DNA Microarrays: DNA Microarrays: Tools for studying many Tools for studying many genes at oncegenes at onceSmall chips dotted with multiple genes in a Small chips dotted with multiple genes in a grid patterngrid pattern


DNA Microarrays: DNA Microarrays: 1.1. Make fluorescently labeled singleMake fluorescently labeled single--stranded stranded cDNAcDNA from from

mRNAmRNA2.2. Add labeled Add labeled cDNAcDNA to microarrayto microarray3.3. cDNAcDNA binds to genes in array; gene glowsbinds to genes in array; gene glows4.4. Glowing indicates which genes were expressed Glowing indicates which genes were expressed

(mRNA was synthesized) in that (mRNA was synthesized) in that cellcell

Can compare gene expression in Can compare gene expression in cancer vs. healthy cells, for cancer vs. healthy cells, for instanceinstance


Proteomics: Proteomics: Comparison and study of all Comparison and study of all proteins resulting from oneproteins resulting from one’’s genome.s genome.Studies protein function and interactionStudies protein function and interactionMore difficult than studying genesMore difficult than studying genes

A single gene can code for >1 polypeptideA single gene can code for >1 polypeptideDifferent cells/tissues have different proteinsDifferent cells/tissues have different proteinsProtein function must often be studied in Protein function must often be studied in living, functional organismliving, functional organism


Potential benefits:Potential benefits:Study evolutionary relatedness Study evolutionary relatedness ((phylogenies))Learn how proteins are involved in diseaseLearn how proteins are involved in disease

Cancer, heart disease, arthritisCancer, heart disease, arthritisDevelop new treatments that target proteins Develop new treatments that target proteins involvedinvolvedMay be able to match medical treatment to May be able to match medical treatment to individual patient, based on their unique body individual patient, based on their unique body chemistrychemistry

9.6: Genetic Screening & Gene 9.6: Genetic Screening & Gene TherapyTherapy

Genetic Screening: Genetic Screening: Process of testing DNA to Process of testing DNA to determine a persondetermine a person’’s risk of having, or passing s risk of having, or passing on, a genetic disorderon, a genetic disorderScreens for specific genes known to be Screens for specific genes known to be associated with certain disorders, e.g. certain associated with certain disorders, e.g. certain cancers (e.g. cancers (e.g. BRCA1BRCA1 gene gene –– breast cancer), breast cancer), cystic fibrosis, cystic fibrosis, DuchenneDuchenne’’ss muscular dystrophymuscular dystrophyNot all defective genes have been identified yetNot all defective genes have been identified yet


Genetic Screening:Genetic Screening:Can save livesCan save livesCan Can difficult choicesdifficult choices

How would knowledge you had a gene for a How would knowledge you had a gene for a terminal illness affect your daily living?terminal illness affect your daily living?

Ethical dilemmas:Ethical dilemmas:Should screening be mandatory?Should screening be mandatory?Should employers/health insurance providers Should employers/health insurance providers have access to this information?have access to this information?


Gene Therapy: Gene Therapy: Replacement of a defective Replacement of a defective or missing gene; addition of a new geneor missing gene; addition of a new geneStill largely experimentalStill largely experimentalMust get new gene Must get new gene into proper cellsinto proper cellsCells must Cells must incorporate new incorporate new gene into their DNAgene into their DNA


One method:One method:1.1. Obtain sample of bone marrow stem Obtain sample of bone marrow stem

cellscells2.2. ““InfectInfect”” cells with GE virus with new genecells with GE virus with new gene3.3. Replace stem cells in personReplace stem cells in person’’ bone bone

marrowmarrow4.4. Stem cells divide and make more blood Stem cells divide and make more blood

cells with the new genecells with the new gene



1990: First successful trial1990: First successful trialTwo children with genetic autoimmune Two children with genetic autoimmune disorder disorder Now living healthy adult livesNow living healthy adult lives

““SuicideSuicide”” genes may genes may be inserted into cancer be inserted into cancer cells cells Kill only Kill only cancer cellscancer cells


Challenges:Challenges:Must get correct gene into correct cellsMust get correct gene into correct cellsGene expression must be regulatedGene expression must be regulated

Avoid too much or too little of the geneAvoid too much or too little of the gene’’s s proteinprotein

Will new gene affect other geneWill new gene affect other gene’’s s activities?activities?

So far, few positive longSo far, few positive long--term resultsterm results

Documents

Ch. 9: Frontiers of Biotechnology€¦ · 1 _ x 1 x 1_ 500 90 120 1 chance in 5.4 million people! 9.3: DNA Fingerprinting Uses: To determine guilt ... Implant nucleus from cell of