Chapter 20

Chapter 20

DNA Technology and Genomics

Overview: Understanding and Manipulating Genomes

• Sequencing of the human genome was largely completed by 2003

• DNA sequencing has depended on advances in technology, starting with making recombinant DNA

• In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

• Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

• DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

Concept 20.1: DNA cloning permits production of multiple copies of a

specific gene or other DNA segment

• To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning

DNA Cloning and Its Applications: A Preview

• Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids

• Cloned genes are useful for making copies of a particular gene and producing a gene product

LE 20-2Bacterium

Bacterialchromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

Using Restriction Enzymes to Make Recombinant DNA

• Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites

• A restriction enzyme usually makes many cuts, yielding restriction fragments

• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments

• DNA ligase is an enzyme that seals the bonds between restriction fragments

LE 20-3Restriction site

DNA 53

35

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow.

One possible combination

DNA fragment from anothersource is added. Base pairingof sticky ends producesvarious combinations.

Fragment from differentDNA molecule cut by thesame restriction enzyme

DNA ligaseseals the strands.

Recombinant DNA molecule

Sticky end

Animation: Restriction Enzymes

Cloning a Eukaryotic Gene in a Bacterial Plasmid

• In gene cloning, the original plasmid is called a cloning vector

• A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

Producing Clones of Cells• Cloning a human gene in a bacterial plasmid

can be divided into six steps:1. Vector and gene-source DNA are isolated2. DNA is inserted into the vector3. Human DNA fragments are mixed with cut

plasmids, and base-pairing takes place4. Recombinant plasmids are mixed with bacteria5. The bacteria are plated and incubated6. Cell clones with the right gene are identified

Animation: Cloning a Gene

LE 20-4_1

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

LE 20-4_2

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

LE 20-4_3

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carryingrecombinantplasmid withdisrupted lacZ gene

Bacterialclone

Identifying Clones Carrying a Gene of Interest

• A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene

• This process is called nucleic acid hybridization

• An essential step in this process is denaturation of the cells’ DNA, separation of its two strands

LE 20-5

Master plate

Filter

Solutioncontainingprobe

Filter liftedand flipped over

Radioactivesingle-strandedDNA

ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Film

Hybridizationon filter

Master plate

Coloniescontaininggene ofinterest

A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.

The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).

After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

Bacterial Expression Systems• Several technical difficulties hinder expression of

cloned eukaryotic genes in bacterial host cells• To overcome differences in promoters and other

DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active prokaryotic promoter

• One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes

• Alternatively, scientists can inject DNA into cells using microscopic needles

• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

Amplifying DNA in Vitro: The Polymerase Chain Reaction

(PCR)

• The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA

• A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

LE 20-7

Genomic DNA

Targetsequence

5

3

3

5

5

3

3

5

Primers

Denaturation:Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3 end of eachprimer

Cycle 1yields

2molecules

Newnucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction

sites• Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules• Such analysis can rapidly provide comparative information about DNA sequences

Gel Electrophoresis and Southern Blotting

• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis

• This technique uses a gel as a molecular sieve to separate nuclei acids or proteins by size

Video: Biotechnology Lab

LE 20-8

Cathode

Powersource

Anode

Mixtureof DNAmoleculesof differ-ent sizes

Gel

Glassplates

Longermolecules

Shortermolecules

• In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis

• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

LE 20-9Normal -globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant -globin allele

376 bp Large fragment

Ddel Ddel Ddel Ddel

Ddel Ddel Ddel

Ddel restriction sites in normal and sickle-cell alleles of-globin gene

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles

• A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization

• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

LE 20-10

DNA + restriction enzyme Restrictionfragments

Normal-globinallele

Sickle-cellallele

Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

Radioactivelylabeled probefor -globingene is addedto solution ina plastic bag

Paper blot

Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globinallele

Autoradiography.

Film overpaper blot

Restriction Fragment Length Differences as Genetic Markers

• Restriction fragment length polymorphisms (RFLPs, or Rif-lips) are differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths

• A RFLP can serve as a genetic marker for a particular location (locus) in the genome

• RFLPs are detected by Southern blotting

Concept 20.3: Entire genomes can be mapped at the DNA level

• The most ambitious mapping project to date has been the sequencing of the human genome

• Officially begun as the Human Genome Project in 1990, the sequencing was largely completed by 2003

• Scientists have also sequenced genomes of other organisms, providing insights of general biological significance

• Go to video

Genetic (Linkage) Mapping: Relative Ordering of Markers

• The first stage in mapping a large genome is constructing a linkage map of several thousand genetic markers throughout each chromosome

• The order of markers and relative distances between them are based on recombination frequencies

LE 20-11

Cytogenetic map

Genes locatedby FISH

Chromosomebands

Geneticmarkers

Genetic (linkage)mapping

Physical mapping

Overlappingfragments

DNA sequencing

Physical Mapping: Ordering DNA Fragments

• A physical map is constructed by cutting a DNA molecule into many short fragments and arranging them in order by identifying overlaps

• Physical mapping gives the actual distance in base pairs between markers

DNA Sequencing

• Relatively short DNA fragments can be sequenced by the dideoxy chain-termination method

• Inclusion of special dideoxyribonucleotides in the reaction mix ensures that fragments of various lengths will be synthesized

LE 20-12DNA(template strand)

5

3

Primer3

5

DNApolymerase

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

3

5DNA (templatestrand)

Labeled strands3

Directionof movementof strands

Laser Detector

• Linkage mapping, physical mapping, and DNA sequencing represent the overarching strategy of the Human Genome Project

• An alternative approach to sequencing genomes starts with sequencing random DNA fragments

• Computer programs then assemble overlapping short sequences into one continuous sequence

LE 20-13Cut the DNA from many copies of an entire chromosome into overlapping frag-ments short enough for sequencing

Clone the fragments in plasmid or phagevectors

Sequence each fragment

Order the sequences into one overall sequence with computer software

Concept 20.4: Genome sequences provide clues to

important biological questions

• In genomics, scientists study whole sets of genes and their interactions

• Genomics is yielding new insights into genome organization, regulation of gene expression, growth and development, and evolution

Identifying Protein-Coding Genes in DNA Sequences

• Computer analysis of genome sequences helps identify sequences likely to encode proteins

• The human genome contains about 25,000 genes, but the number of human proteins is much larger

• Comparison of sequences of “new” genes with those of known genes in other species may help identify new genes

Determining Gene Function

• One way to determine function is to disable the gene and observe the consequences

• Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function

• When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype

• In nonmammalian organisms, a simpler and faster method, RNA interference (RNAi), has been used to silence expression of selected genes

Studying Expression of Interacting Groups of Genes

• Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays

• DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions

LE 20-14

Make cDNA by reverse transcription, using fluorescently labeled nucleotides.

Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism’s genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.

Rinse off excess cDNA; scan microarray for fluorescent. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample.

Isolate mRNA.Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNAmicroarray

Size of an actualDNA microarraywith all the genesof yeast (6,400 spots)

Comparing Genomes of Different Species

• Comparative studies of genomes from related and widely divergent species provide information in many fields of biology

• The more similar the nucleotide sequences between two species, the more closely related these species are in their evolutionary history

• Comparative genome studies confirm the relevance of research on simpler organisms to understanding human biology

Future Directions in Genomics• Genomics is the study of entire genomes• Proteomics is the systematic study of all proteins

encoded by a genome• Single nucleotide polymorphisms (SNPs) provide

markers for studying human genetic variation

Concept 20.5: The practical applications of DNA technology

affect our lives in many ways

• Many fields benefit from DNA technology and genetic engineering

Medical Applications

• One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

Diagnosis of Diseases• Scientists can diagnose many human genetic

disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation

• Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

LE 20-15

DNA

RFLP marker

Disease-causingallele

Normal allele

Restrictionsites

Human Gene Therapy• Gene therapy is the alteration of an afflicted

individual’s genes• Gene therapy holds great potential for treating

disorders traceable to a single defective gene• Vectors are used for delivery of genes into cells• Gene therapy raises ethical questions, such as

whether human germ-line cells should be treated to correct the defect in future generations

LE 20-16

Cloned gene

Retroviruscapsid

Bonemarrowcell frompatient

Inject engineeredcells into patient.

Insert RNA version of normal alleleinto retrovirus.

Viral RNA

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Bonemarrow

Pharmaceutical Products

• Some pharmaceutical applications of DNA technology:– Large-scale production of human hormones

and other proteins with therapeutic uses– Production of safer vaccines

Forensic Evidence• DNA “fingerprints” obtained by analysis of

tissue or body fluids can provide evidence in criminal and paternity cases

• A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel

• The probability that two people who are not identical twins have the same DNA fingerprint is very small

• Exact probability depends on the number of markers and their frequency in the population

LE 20-17Defendant’sblood (D)

Blood from defendant’sclothes

Victim’sblood (V)

Environmental Cleanup

• Genetic engineering can be used to modify the metabolism of microorganisms

• Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

Agricultural Applications• DNA technology is being used to improve

agricultural productivity and food quality

Animal Husbandry and “Pharm” Animals

• Transgenic organisms are made by introducing genes from one species into the genome of another organism

• Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles)

• Other transgenic organisms are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use

Genetic Engineering in Plants

• Agricultural scientists have endowed a number of crop plants with genes for desirable traits

• The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

LE 20-19Agrobacterium tumefaciens

Tiplasmid

Site whererestrictionenzyme cuts

DNA withthe geneof interest

T DNA

RecombinantTi plasmid

Plant withnew trait

Safety and Ethical Questions Raised by DNA Technology

• Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures

• Most public concern about possible hazards centers on genetically modified (GM) organisms used as food