ap bio ch 13 power point

Chapter 13Chapter 13

BiotechnologyBiotechnology

Chapter 13 2 Traditional ApplicationsTraditional Applications

Biotechnology is Biotechnology is applied biologyapplied biology• Modern focus on genetic engineering, Modern focus on genetic engineering,

recombinant DNA technology, and analysis of recombinant DNA technology, and analysis of biomoleculesbiomolecules

Chapter 13 3 Traditional ApplicationsTraditional Applications

Traditional (historical) applications of Traditional (historical) applications of biotechnology date back to over 10,000 biotechnology date back to over 10,000 years agoyears ago

• Use of yeast to produce beer and wine in Egypt Use of yeast to produce beer and wine in Egypt and Near Eastand Near East

• Selective breeding of plantsSelective breeding of plants• Selective breeding of animalsSelective breeding of animals

Chapter 13 4 Genetic EngineeringGenetic Engineering

Genetic engineeringGenetic engineering refers to the modification refers to the modification of genetic material to achieve specific of genetic material to achieve specific goalsgoals

• Learn more about cellular processes, Learn more about cellular processes, including inheritance and gene expressionincluding inheritance and gene expression

• Provide better understanding and treatment Provide better understanding and treatment of diseases, particularly genetic disordersof diseases, particularly genetic disorders

• Generate economic and social benefits Generate economic and social benefits through production of valuable biomolecules through production of valuable biomolecules and improved plants and animals for and improved plants and animals for agricultureagriculture

Chapter 13 5 Recombinant DNARecombinant DNA

Genetic engineering utilizes Genetic engineering utilizes recombinant DNA recombinant DNA technologytechnology

• Splicing together of genes or portions of Splicing together of genes or portions of genes from different organismsgenes from different organisms

Recombinant DNA can be transferred to Recombinant DNA can be transferred to plants and animalsplants and animals

• Modified animals are called Modified animals are called transgenictransgenic or or genetically modified organismsgenetically modified organisms (GMOs) (GMOs)

• Most modern biotechnology includes Most modern biotechnology includes manipulation of DNAmanipulation of DNA

Chapter 13 6

Recombination in NatureRecombination in Nature

Many natural processes recombine DNA:Many natural processes recombine DNA:

Due to crossing over during meiosis, each Due to crossing over during meiosis, each chromosome in a gamete contains a chromosome in a gamete contains a mixture of alleles from the two parental mixture of alleles from the two parental chromosomeschromosomes

• Thus, eggs and sperm contain recombinant Thus, eggs and sperm contain recombinant DNADNA

Chapter 13 7

TransformationTransformation

Bacteria can naturally take up DNA from the Bacteria can naturally take up DNA from the environment (environment (transformationtransformation) and integrate ) and integrate the new genes into the genome the new genes into the genome ((recombinationrecombination))

Chapter 13 8

1 1 µµmm

RecombinationRecombinationin Bacteriain Bacteria

(a)(a) (b)(b) (c)(c)BacteriumBacterium

ChromosomeChromosomePlasmidPlasmid

Plasmid Plasmid replicates in replicates in cytoplasmcytoplasm DNA fragment DNA fragment

incorporated incorporated into into

chromosomechromosome

Plasmid Plasmid transferredtransferredto new hostto new host

DNA fragments DNA fragments transferredtransferredto new hostto new host

Chapter 13 9

TransformationTransformation

Small circular DNA molecules (Small circular DNA molecules (plasmidsplasmids) carry ) carry supplementary genessupplementary genes

• Plasmid genes may allow bacteria to grow in Plasmid genes may allow bacteria to grow in novel environmentsnovel environments

• Plasmid genes may enhance virulence of Plasmid genes may enhance virulence of bacteria in establishing an infectionbacteria in establishing an infection

• Plasmid genes may confer resistance to Plasmid genes may confer resistance to antimicrobial drugsantimicrobial drugs

Chapter 13 10 Viral Transfer of DNAViral Transfer of DNA

Viral life cycleViral life cycle1.1. Viral particle invades host cellViral particle invades host cell2.2. Viral DNA is replicatedViral DNA is replicated3.3. Viral protein molecules are synthesizedViral protein molecules are synthesized4.4. Offspring viruses are assembled and break Offspring viruses are assembled and break

out of the host cellout of the host cell

Chapter 13 11 Viral Transfer of DNAViral Transfer of DNA

Viral transfer of DNAViral transfer of DNA• Viruses may package some genes from host Viruses may package some genes from host

cell into viral particles during assemblycell into viral particles during assembly• Infection of new host cell injects genes from Infection of new host cell injects genes from

previous host, allowing for recombinationprevious host, allowing for recombination

Chapter 13 12 Viruses May Transfer GenesViruses May Transfer Genes

Chapter 13 13 Biotechnology and ForensicsBiotechnology and Forensics

ForensicsForensics is the science of criminal and victim is the science of criminal and victim identificationidentification

DNA technology has allowed forensic science DNA technology has allowed forensic science to identify victims and criminals from trace to identify victims and criminals from trace biological samplesbiological samples

• Genetic sequences of any human individual are Genetic sequences of any human individual are uniqueunique

• DNA analysis reveals patterns that identify DNA analysis reveals patterns that identify people with a high degree of accuracypeople with a high degree of accuracy

Chapter 13 14 Polymerase Chain ReactionPolymerase Chain Reaction

Forensic technicians typically have very little Forensic technicians typically have very little DNA with which to perform analysesDNA with which to perform analyses

Polymerase Chain ReactionPolymerase Chain Reaction (PCR) produces (PCR) produces virtually unlimited copies of a very small virtually unlimited copies of a very small DNA sampleDNA sample


PCR requires small pieces of DNA (called PCR requires small pieces of DNA (called primersprimers) that are complementary to the ) that are complementary to the gene sequences targeted for copyinggene sequences targeted for copying

A PCR “run” is basically DNA replication in a A PCR “run” is basically DNA replication in a tiny test tubetiny test tube

• Template DNA, primer, nucleotides, and DNA Template DNA, primer, nucleotides, and DNA polymerase are all in the reaction mixpolymerase are all in the reaction mix


Four steps of a PCR cycleFour steps of a PCR cycle1.1. Template strand separationTemplate strand separation

– The test tube is heated to 90-95The test tube is heated to 90-95ooC to cause the C to cause the double stranded template DNA to separate into double stranded template DNA to separate into single strands…single strands…

2.2. Binding of the primersBinding of the primers– The temperature is lowered to 50The temperature is lowered to 50ooC to allow the C to allow the

primer DNA segments to bind to the targeted gene primer DNA segments to bind to the targeted gene sequences through hydrogen bonding…sequences through hydrogen bonding…


3.3. New DNA synthesis at targeted sequencesNew DNA synthesis at targeted sequencesThe temperature is raised to 70-72The temperature is raised to 70-72ooC where the C where the

heat-stable DNA polymerase synthesizes new heat-stable DNA polymerase synthesizes new DNA of the sequences targeted by the DNA of the sequences targeted by the primers…primers…

4.4. Repetition of the cycleRepetition of the cycleThe cycle is repeated automatically (by a The cycle is repeated automatically (by a

thermocycler machine) for 20-30 cycles, thermocycler machine) for 20-30 cycles, producing up to 1 billion copies of the original producing up to 1 billion copies of the original targeted DNA sequencetargeted DNA sequence

Chapter 13 18 Polymerase Chain Reaction:Polymerase Chain Reaction:

(a) One PCR Cycle(a) One PCR Cycle

OriginalOriginalDouble-Double-helixhelixDNADNA

SeparateSeparateDNADNAStrandsStrands

90 °C90 °C

Primers &Primers &DNADNApolymerasepolymerasebindbind

50 °C50 °C

DNA PolymeraseDNA Polymerase PrimerPrimerDNADNA

72 °C72 °C

DNADNAsynthesizedsynthesized

Chapter 13 19 Polymerase Chain Reaction:Polymerase Chain Reaction:

(b) Multiple PCR Cycles(b) Multiple PCR Cycles

DNADNAfragmentfragment

to beto beamplifiedamplified

2 copies2 copies 4 copies4 copies 8 copies8 copies


Choice of primers determines which Choice of primers determines which sequences are amplified (copied)sequences are amplified (copied)

Forensic scientists focus on Forensic scientists focus on short tandem short tandem repeatsrepeats (STRs) found within the human (STRs) found within the human genomegenome


STRs are repeated sequences of DNA within STRs are repeated sequences of DNA within the chromosomes that do not code for the chromosomes that do not code for proteinsproteins

STRs vary greatly between different human STRs vary greatly between different human individualsindividuals

A match of 10 different STRs between A match of 10 different STRs between suspect and crime scene DNA virtually suspect and crime scene DNA virtually proves the suspect was at the crime sceneproves the suspect was at the crime scene

Chapter 13 22

Chapter 13 23 Gel ElectrophoresisGel Electrophoresis

Mixtures of DNA fragments can be separated Mixtures of DNA fragments can be separated on the basis of sizeon the basis of size

Gel electrophoresisGel electrophoresis is a technique used to is a technique used to spread out different-length DNA fragments spread out different-length DNA fragments in a mixturein a mixture


Four steps of gel electrophoresisFour steps of gel electrophoresis1.1. DNA mixtures are placed into wells at one DNA mixtures are placed into wells at one

end of a slab of end of a slab of agarose gelagarose gel

2.2. An electric current introduced through the An electric current introduced through the gel causes the negatively-charged DNA gel causes the negatively-charged DNA fragments to migrate towards the positive fragments to migrate towards the positive electrodeelectrode


Four steps of gel electrophoresisFour steps of gel electrophoresis3.3. Short DNA fragments move more easily Short DNA fragments move more easily

through the three-dimensional meshwork of through the three-dimensional meshwork of fibers between the gelfibers between the gel

Short DNA fragments migrate farther than Short DNA fragments migrate farther than long DNA fragments so the mixture is long DNA fragments so the mixture is separated into bands of DNA of specific separated into bands of DNA of specific lengthslengths

4.4. The invisible bands of DNA are made visible The invisible bands of DNA are made visible using stains or DNA probesusing stains or DNA probes

Chapter 13 26

Chapter 13 27

Chapter 13 28 RFLP: Gel ElectrophoresisRFLP: Gel Electrophoresis

DirectionDirectionof Migrationof Migration

Larger fragmentsLarger fragmentsmove more slowly;move more slowly;smaller fragmentssmaller fragmentsmove more rapidlymove more rapidly

Chapter 13 29 DNA FingerprintingDNA Fingerprinting

DNA from a crime scene sample can be DNA from a crime scene sample can be amplified by PCR and run on a gel with amplified by PCR and run on a gel with suspect DNAssuspect DNAs

Short tandem repeats (STRs) in the gel DNA Short tandem repeats (STRs) in the gel DNA can be identified by DNA probescan be identified by DNA probes

Distinctive pattern of STR numbers and Distinctive pattern of STR numbers and lengths are fairly unique to a specific lengths are fairly unique to a specific individual (forming a individual (forming a DNA fingerprintDNA fingerprint))

DNA fingerprint from crime scene can be DNA fingerprint from crime scene can be matched with DNA fingerprint of suspectmatched with DNA fingerprint of suspect

Chapter 13 30

CC SSRR CCII EE

MM NNEE EE

DNA Fingerprint in ForensicsDNA Fingerprint in Forensics

11 22 33 44 55 66 77

SuspectsSuspects SuspectsSuspects

Q: Which suspectQ: Which suspectshould beshould beindicted?indicted?

A: #3 is primeA: #3 is primesuspectsuspect

Chapter 13 31

You can read the stuff on DNA probes and You can read the stuff on DNA probes and Biotech in Agriculture on your own…Biotech in Agriculture on your own…

Chapter 13 32 Restriction Enzymes Cut DNARestriction Enzymes Cut DNA

A DNA sequence (e.g. a gene) can be removed A DNA sequence (e.g. a gene) can be removed from a chromosome using special enzymesfrom a chromosome using special enzymes

Restriction enzymesRestriction enzymes are nucleases that cut DNA are nucleases that cut DNA at specific nucleotide sequencesat specific nucleotide sequences

Chapter 13 33

Chapter 13 34 Restriction Enzymes Cut DNARestriction Enzymes Cut DNA

Enzymes that create staggered cuts with “Enzymes that create staggered cuts with “sticky sticky endsends” are the most useful in gene cloning” are the most useful in gene cloning

Chapter 13 35 Splicing of DNA FragmentsSplicing of DNA Fragments

Sticky ends allow for splicing of a DNA Sticky ends allow for splicing of a DNA fragment with another complementary fragment with another complementary fragmentfragment

• Bt gene can be cut out of the Bt gene can be cut out of the BacillusBacillus chromosome with the same enzyme used to chromosome with the same enzyme used to cut open the plasmidcut open the plasmid

• Bt gene fragment ends can base-pair with Bt gene fragment ends can base-pair with sticky ends of the opened plasmid, adding sticky ends of the opened plasmid, adding gene to the plasmid circlegene to the plasmid circle

Chapter 13 36 Splicing of DNA FragmentsSplicing of DNA Fragments

DNA ligase enzyme used next to permanently DNA ligase enzyme used next to permanently bond gene into plasmidbond gene into plasmid

Chapter 13 37

Chapter 13 38

Chapter 13 39

Chapter 13 40

Chapter 13 41

Chapter 13 42

Chapter 13 43 Plasmids Are Used to Insert GenesPlasmids Are Used to Insert Genes

The Ti plasmid from The Ti plasmid from Agrobacterium Agrobacterium tumefacienstumefaciens is ideal for transferring genes is ideal for transferring genes into plant chromosomesinto plant chromosomes

Chapter 13 44 Plasmids Are Used to Insert GenesPlasmids Are Used to Insert Genes

Agrobacterium infects plant cells and inserts Agrobacterium infects plant cells and inserts its small Ti plasmid into a plant its small Ti plasmid into a plant chromosome in the nucleuschromosome in the nucleus

• Pathogenic effects of certain tumor-causing Ti Pathogenic effects of certain tumor-causing Ti plasmid genes can be disabledplasmid genes can be disabled

• A gene inserted into a Ti plasmid is therefore A gene inserted into a Ti plasmid is therefore carried into the plant cell chromosomes by a carried into the plant cell chromosomes by a natural processnatural process

Chapter 13 45

Chapter 13 46 The Human Genome ProjectThe Human Genome Project

FindingsFindings• Human genome contains ~25,000 genes Human genome contains ~25,000 genes • New genes, including many disease-associated New genes, including many disease-associated

genes have been discoveredgenes have been discovered• Has determined the nucleotide sequence of all Has determined the nucleotide sequence of all

the DNA in our entire set of genes, called the the DNA in our entire set of genes, called the human genome human genome

• The genes comprise 2% of all the DNAThe genes comprise 2% of all the DNA

Chapter 13 47 The Human Genome ProjectThe Human Genome Project

ApplicationsApplications• Improved diagnosis, treatment and cures of Improved diagnosis, treatment and cures of

genetic disorders or predispositionsgenetic disorders or predispositions• Comparison of our genome to those of other Comparison of our genome to those of other

species will clarify the genetic differences that species will clarify the genetic differences that help to make us human help to make us human

Chapter 13 48 DNA ProbesDNA Probes

Defective alleles can also be identified using Defective alleles can also be identified using DNA probesDNA probes

DNA probing is especially useful where there DNA probing is especially useful where there are many different alleles at a single gene are many different alleles at a single gene locuslocus

• Cystic fibrosis is a disease caused by any of Cystic fibrosis is a disease caused by any of 32 alleles out of 1000 total possible alleles32 alleles out of 1000 total possible alleles


Arrays of single-stranded DNA complementary Arrays of single-stranded DNA complementary to each of the defective alleles can be bound to each of the defective alleles can be bound to filter paperto filter paper

A person’s DNA sample is cut up and separated into A person’s DNA sample is cut up and separated into single-strands single-strands

The array is bathed in the DNA sampleThe array is bathed in the DNA sampleStrands of DNA binding to complementary sequence Strands of DNA binding to complementary sequence

on the paper indicate presence of a defective on the paper indicate presence of a defective allele in person’s genomeallele in person’s genome

Chapter 13 50


An expanded version of this type of DNA analysis is An expanded version of this type of DNA analysis is known as a known as a microarray microarray

A microarray contains up to thousands of probes for A microarray contains up to thousands of probes for a variety of disease-related alleles a variety of disease-related alleles

Microarray analysis has the potential to Microarray analysis has the potential to comprehensively identify disease susceptibilitycomprehensively identify disease susceptibility

Chapter 13 52

Chapter 13 53 Scientific Objections to GMOsScientific Objections to GMOs

Safety issues from eating GMOsSafety issues from eating GMOs• Could ingestion of Bt protein in insect-Could ingestion of Bt protein in insect-

resistant plants be dangerous to humans?resistant plants be dangerous to humans?• Are transgenic fish producing extra growth Are transgenic fish producing extra growth

hormone dangerous to eat?hormone dangerous to eat?• Could GM crops cause allergic reactions?Could GM crops cause allergic reactions?

– USDA now monitors GM foods for allergic USDA now monitors GM foods for allergic potentialpotential

• Toxicology study of GM plants (2003) Toxicology study of GM plants (2003) concluded that ingestion of current transgenic concluded that ingestion of current transgenic crops pose no significant health dangerscrops pose no significant health dangers


Environmental hazards posed by GMOsEnvironmental hazards posed by GMOs• Pollen from modified plants can carry GM Pollen from modified plants can carry GM

genes to the wild plant populationgenes to the wild plant population– Could herbicide resistance genes be transferred Could herbicide resistance genes be transferred

to weed species, creating superweeds?to weed species, creating superweeds?

• Could GM fish reduce biodiversity in the wild Could GM fish reduce biodiversity in the wild population if they escape?population if they escape?

– Reduced diversity in wild fish makes them more Reduced diversity in wild fish makes them more susceptible to catastrophic disease outbreakssusceptible to catastrophic disease outbreaks


Environmental hazards posed by GMOsEnvironmental hazards posed by GMOs• US found to lack adequate system to monitor US found to lack adequate system to monitor

changes in ecosystem wrought by GMOs changes in ecosystem wrought by GMOs (National Academy of Science Study 2003)(National Academy of Science Study 2003)

Chapter 13 56 The Human GenomeThe Human Genome

Should parents be given information about the Should parents be given information about the genetic health of an unborn fetus?genetic health of an unborn fetus?

Should parents be allowed to select the Should parents be allowed to select the genomes of their offspring?genomes of their offspring?

• Embryos from Embryos from in vitroin vitro fertilization are fertilization are currently tested before implantationcurrently tested before implantation

• Many unused embryos are discardedMany unused embryos are discarded

Should parents be allowed to design or Should parents be allowed to design or correct the genomes of their offspring?correct the genomes of their offspring?

Chapter 13 57 Hope through Gene TherapyHope through Gene Therapy

Chapter 13 58 Human Cloning:Human Cloning:

Permanent Genetic Correction?Permanent Genetic Correction?Parents withParents with

genetic diseasegenetic disease Zygote withZygote withdefective genedefective gene

Embryo withEmbryo withdefective genedefective gene

Baby withBaby withgenetic disordergenetic disorder

Cell CultureCell Culture

ViralViralvectorvectorwithwith

therapeutictherapeuticgenegene

Treated CultureTreated Culture

Genetically correctedGenetically correctedcell from culturecell from culture

EnucleatedEnucleatedegg cellegg cell GeneticallyGenetically

correctedcorrectedegg cellegg cell

GeneticallyGeneticallycorrectedcorrected

embryo cloneembryo clone

Healthy babyHealthy baby

Chapter 13Chapter 13

The endThe end

ap bio ch 13 power point

Technology

ti plasmid

dna fingerprint

sticky ends

human genome

gel

dna

genes

chromosome