Chapter 20Chapter 20 DNA TechnologyDNA Technology

DNA CloningDNA Cloning Gene cloning allows scientists to work Gene cloning allows scientists to work

with small sections of DNA (single with small sections of DNA (single genes) in isolation.genes) in isolation.

– Exactly Exactly what does the gene code for?what does the gene code for? Much of a DNA molecule is noncoding, Much of a DNA molecule is noncoding,

and scientists are mostly interested in and scientists are mostly interested in the genes.the genes.

Cloning makes identical copies of the Cloning makes identical copies of the same gene (or genes)same gene (or genes)

Figure 20.1 An overview of how bacterial plasmids are used to clone genesFigure 20.1 An overview of how bacterial plasmids are used to clone genes

Bacterial PlasmidsBacterial Plasmids Plasmids are small, circular DNA molecules Plasmids are small, circular DNA molecules

in bacteria.in bacteria. By inserting genes into plasmids, scientists By inserting genes into plasmids, scientists

can combine eukaryotic and prokaryotic can combine eukaryotic and prokaryotic DNA. (Recombinant DNA)DNA. (Recombinant DNA)

Bacterial cells continually replicate the Bacterial cells continually replicate the foreign gene along with their DNA.foreign gene along with their DNA.

Cloning using plasmids can be used to:Cloning using plasmids can be used to:– Identify a particular protein a gene makes Identify a particular protein a gene makes

(ie: for study)(ie: for study)– Produce large amounts of a particular Produce large amounts of a particular

protein/gene (ie: for use in medicine)protein/gene (ie: for use in medicine)

Restriction EnzymesRestriction Enzymes Also used Also used

to make to make recombinanrecombinant DNA.t DNA.

Specifically Specifically cut DNA cut DNA molecules molecules at precise at precise base base locations.locations.

(restriction)(restriction)

Making Recombinant DNA (Fig 20.3)Making Recombinant DNA (Fig 20.3)

Making Recombinant DNA (Fig 20.3)

……Still Making Recombinant DNAStill Making Recombinant DNA

……Almost RecombinantAlmost Recombinant

DNA Technology FilesDNA Technology Files

Restriction Enzyme and Cloning Movies

Why Use Bacteria as vectors?Why Use Bacteria as vectors?

1.1. Plasmids are easy to use to Plasmids are easy to use to manipulate which genes are manipulate which genes are expressed in clones.expressed in clones.

2.2. Bacteria replicate very quickly and Bacteria replicate very quickly and allow you to produce a large number allow you to produce a large number of a desired gene.of a desired gene.

Identifying ClonesIdentifying Clones

Not all of the reproduced bacteria are Not all of the reproduced bacteria are clones carrying the desired gene.clones carrying the desired gene.

Two ways to identify which are clones:Two ways to identify which are clones:

–Look for the geneLook for the gene

–Look for the protein the gene codes Look for the protein the gene codes forfor

Nucleic Acid Hybridization (find gene)Nucleic Acid Hybridization (find gene) If you know the sequence of the cloned gene If you know the sequence of the cloned gene

you are looking for, you can make a nucleic you are looking for, you can make a nucleic acid probe with a complementary sequence.acid probe with a complementary sequence.

The probe is radioactively labeled and The probe is radioactively labeled and allowed to base pair with the denatured allowed to base pair with the denatured (separated strands) DNA.(separated strands) DNA.

The probes H-bond with their complement The probes H-bond with their complement (cloned gene), thus identifying the cloned (cloned gene), thus identifying the cloned cells.cells.

Identified cells are cultured to produce more.Identified cells are cultured to produce more.

Figure 20.4 Using a nucleic acid probe to identify a cloned geneFigure 20.4 Using a nucleic acid probe to identify a cloned gene

Expressing Euk. Proteins in BacteriaExpressing Euk. Proteins in Bacteria It is more difficult to get the bacteria to It is more difficult to get the bacteria to

translate the proteins because of translate the proteins because of differences in promotor sequences b/t differences in promotor sequences b/t prokaryotes and eukaryotes.prokaryotes and eukaryotes.

Expression vectors are plasmids that Expression vectors are plasmids that contain the promotor sequence just before contain the promotor sequence just before the restriction site.the restriction site.

This allows the insertion of a eukaryotic This allows the insertion of a eukaryotic gene right next to the prokaryotic gene right next to the prokaryotic promotor.promotor.

Expressing Euk. Proteins in BacteriaExpressing Euk. Proteins in Bacteria

Bacteria also lack the enzymes needed to Bacteria also lack the enzymes needed to remove introns from DNA.remove introns from DNA.

Therefore, cDNA (no introns) is inserted Therefore, cDNA (no introns) is inserted into plasmids to allow expression of the into plasmids to allow expression of the eukaryotic gene.eukaryotic gene.

Reverse transcriptase is the enzyme used Reverse transcriptase is the enzyme used to make cDNA from a fully processed to make cDNA from a fully processed mRNA strand.mRNA strand.

Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene

Another Solution: Use Yeast (eukaryotic)Another Solution: Use Yeast (eukaryotic)

Why? Why?

–They grow quickly like bacteriaThey grow quickly like bacteria

–They are eukaryotes (They are eukaryotes (similarsimilar enzymes, enzymes, metabolic mechanisms, protein mods)metabolic mechanisms, protein mods)

–They have plasmids (rare for They have plasmids (rare for eukaryotes)eukaryotes)

–Can replicate artificial chromosomes as Can replicate artificial chromosomes as well as DNA in plasmidswell as DNA in plasmids

Genomic LibrariesGenomic Libraries Plasmids and phages used to store copies of specific Plasmids and phages used to store copies of specific

genes.genes.

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)

PCRPCR FasterFaster and and more specificmore specific method for method for

amplifying short DNA sequencesamplifying short DNA sequences After DNA is denatured (split), primers start After DNA is denatured (split), primers start

new complementary strands with each new complementary strands with each strand producing more molecules of the strand producing more molecules of the sequence.sequence.

In vitro = doesn’t require living cells In vitro = doesn’t require living cells – In test tube: denatured DNA, free In test tube: denatured DNA, free

nucleotides, DNA primers (specific to gene nucleotides, DNA primers (specific to gene desired), “special” DNA polymerase (can desired), “special” DNA polymerase (can withstand high heat w/o denaturing)withstand high heat w/o denaturing)

PCRPCR

Analyzing DNAAnalyzing DNA Gel electrophoresis separates molecules Gel electrophoresis separates molecules

based on size, charge, density, etc.based on size, charge, density, etc.

Linear DNA – mainly separated by Linear DNA – mainly separated by fragment length (size)fragment length (size)

Molecules of DNA are separated into Molecules of DNA are separated into bands of molecules of the same length.bands of molecules of the same length.

Gel ElectrophoresisGel Electrophoresis

Restriction Fragment AnalysisRestriction Fragment Analysis

Southern BlottingSouthern Blotting

Southern BlottingSouthern Blotting1.1. Produce restriction fragments of DNA Produce restriction fragments of DNA

(rest. enzyme used)(rest. enzyme used)

2.2. Separate fragments (gel electrophoresis)Separate fragments (gel electrophoresis)

3.3. Blotting Blotting Transfer DNA to nitrocellulose paper Transfer DNA to nitrocellulose paper

via cap. actionvia cap. action

4.4. Hybridize with radioactive probes (know Hybridize with radioactive probes (know seq.)seq.)

5.5. Autoradiography to identify which have Autoradiography to identify which have probes. probes.

RFLPs (rif-lips)RFLPs (rif-lips) Polymorphisms that result from differences in Polymorphisms that result from differences in

noncoding regions of DNA.noncoding regions of DNA. Restriction enzymes cut DNA into different Restriction enzymes cut DNA into different

fragments in each variant.fragments in each variant. RFLP markers allowed scientists to more RFLP markers allowed scientists to more

accurately map the human genome.accurately map the human genome. Genetic studies do not have to rely on Genetic studies do not have to rely on

phenotypic (appearance/proteins) differences to phenotypic (appearance/proteins) differences to guide them anymore.guide them anymore.

In Situ (on a slide) HybridizationIn Situ (on a slide) Hybridization

Radioactively (or fluorescently) labeled Radioactively (or fluorescently) labeled probes base pair with complementary probes base pair with complementary denatured DNA on a microscope slide.denatured DNA on a microscope slide.

Autoradiography and staining identify the Autoradiography and staining identify the location of the bound probe.location of the bound probe.

Human Genome ProjectHuman Genome Project Attempt to map the genes on every Attempt to map the genes on every

human chromosome as well as noncoding human chromosome as well as noncoding information.information.

Three stagesThree stages

–Genetic Mapping (linkage)Genetic Mapping (linkage)

–Physical MappingPhysical Mapping

–Gene (DNA) SequencingGene (DNA) Sequencing Genomes of species that give insight to Genomes of species that give insight to

human codes are also being done (fruit human codes are also being done (fruit fly, fly, E coliE coli, yeast), yeast)

Genetic Mapping (Stage 1)Genetic Mapping (Stage 1) Linkage maps based on recombination Linkage maps based on recombination

frequencies created.frequencies created.

Linkage maps portray gene sequences as Linkage maps portray gene sequences as you physically move along a you physically move along a chromosome.chromosome.

Genetic markers along the chromosome Genetic markers along the chromosome allow researchers to use them as allow researchers to use them as reference points while studying other reference points while studying other genes.genes.

Physical Mapping (Stage 2)Physical Mapping (Stage 2) Determines the actual distance between Determines the actual distance between

the markers along a chromosome (# of the markers along a chromosome (# of bases)bases)

Utilizes chromosome walking to identify Utilizes chromosome walking to identify the distance between.the distance between.

–Use a series of probes to identify the Use a series of probes to identify the DNA sequence of various restriction DNA sequence of various restriction fragments, and ultimately the entire fragments, and ultimately the entire length of DNA sample.length of DNA sample.

Chromosome WalkingChromosome Walking

DNA Sequencing (Stage 3)DNA Sequencing (Stage 3) As of 1998, 3% of the human genome had As of 1998, 3% of the human genome had

been sequenced using automation. (Sanger been sequenced using automation. (Sanger Method)Method)

Once the sequences of all the genes are Once the sequences of all the genes are known, scientists can begin to study all of their known, scientists can begin to study all of their functions, and manipulate their products in functions, and manipulate their products in many ways.many ways.

Applied GeneticsApplied Genetics Diagnosis of Genetic DisordersDiagnosis of Genetic Disorders

–Sequence individuals before birth to Sequence individuals before birth to know if their DNA contains abnormalitiesknow if their DNA contains abnormalities

Human Gene TherapyHuman Gene Therapy

–Replace missing or fix damaged genes Replace missing or fix damaged genes in affected individualsin affected individuals

Gene TherapyGene Therapy

PharmaceuticalsPharmaceuticals Hormone production (ie: Human Growth)Hormone production (ie: Human Growth) Protein supplementsProtein supplements

– HIV treatment: “decoy” receptor protein HIV treatment: “decoy” receptor protein used to inhibit HIV virus’ ability to enter cellused to inhibit HIV virus’ ability to enter cell

VaccinesVaccines– Proteins that stimulate immune response Proteins that stimulate immune response

can be used instead of traditional vaccinescan be used instead of traditional vaccines Antisense Nucleic AcidsAntisense Nucleic Acids

– Block translation of certain proteinsBlock translation of certain proteins

Other Uses of DNA TechOther Uses of DNA Tech DNA Fingerprinting for forensic casesDNA Fingerprinting for forensic cases

Environmental cleanupEnvironmental cleanup

AgricultureAgriculture

–Animal HusbandryAnimal Husbandry

–Genetic Engineering of PlantsGenetic Engineering of Plants

The Future of GeneticsThe Future of Genetics

The future of science lies in genetics.The future of science lies in genetics.

The question is not whether or not we The question is not whether or not we cancan do the things discussed in this chapter, do the things discussed in this chapter, but whether or not we but whether or not we shouldshould. This is a . This is a question you will ultimately have to help question you will ultimately have to help answer.answer.