chapter 20
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Chapter 20. DNA Technology & Genomics. Biotechnology Today. Genetic Engineering manipulation of DNA if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with this unit is a survey of those tools…. Our tool kit…. - PowerPoint PPT PresentationTRANSCRIPT
Chapter 20DNA Technology & Genomics
• Genetic Engineering– manipulation of DNA– if you are going to engineer DNA & genes &
organisms, then you need a set of tools to work with
– this unit is a survey of those tools…
Biotechnology Today
Our tool kit…
Understanding and Manipulating Genomes
• DNA sequencing has depended on advances in technology, starting with making recombinant DNA
• Recombinant DNA - nucleotide sequences from 2 different sources are combined in vitro into same DNA molecule
• Methods for making recombinant DNA are central to genetic engineering, direct manipulation of genes for practical purposes
20.1: DNA cloning permits production of multiple copies of a specific gene
To work with specific genes, scientists prepare gene-sized pieces of DNA in identical copies - gene cloning
Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes - cut DNA molecules at DNA sequences called restriction sites
• Restriction enzyme usually makes many cuts, yielding restriction fragments
• Most useful restriction enzymes cut DNA in a staggered way - producing fragments with “sticky ends” - bond with complementary “sticky ends” of other fragments
• DNA ligase - enzyme that seals bonds between restriction fragments
LE 20-3Restriction site
DNA 5¢3¢
3¢5¢
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
Cloning a Eukaryotic Gene in a Bacterial Plasmid
• In gene cloning, original plasmid is called a cloning vector
• Cloning vector - DNA molecule that can carry foreign DNA into a cell and replicate there
Producing Clones of Cells• Cloning human gene in bacterial plasmid:
1. Vector and gene-source DNA are isolated2. DNA is inserted into 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
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
• Clone carrying gene of interest can be identified with a nucleic acid probe
• Called nucleic acid hybridization
• Radioactive or fluorescent probes are engineered to be complimentary to a target sequence
• First, denature of cells’ DNA
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.
Storing Cloned Genes in DNA Libraries• Genomic library - collection of recombinant
vector clones produced by cloning DNA fragments from an entire genome
• Complementary DNA (cDNA) library - made by cloning DNA made in vitro by reverse transcription of all mRNA produced by a particular cell
• cDNA library - represents only part of genome—only subset of genes transcribed into mRNA in original cells
• Solves problem of prokaryotes not having machinery to remove introns
Bacterial Expression Systems• Several technical difficulties hinder expression
of cloned eukaryotic genes in bacterial host cells
• Have to overcome differences in promoters and other DNA control sequences
Eukaryotic Cloning and Expression Systems• Use of yeast artificial
chromosomes (YACs) as vectors helps avoid gene expression problems
• YACs - behave normally in mitosis and can carry more DNA than a plasmid
• Eukaryotic hosts can provide posttranslational modifications that many proteins require
Introducing recombinant DNA into eukaryotic cells:• electroporation, - applying a brief electrical
pulse to create temporary holes in plasma membranes
• inject DNA into cells using microscopic needles
Polymerase Chain Reaction (PCR)• Polymerase chain reaction (PCR) - produce
many copies of specific target segment of DNA• 3-step cycle: heating, cooling, and replication• chain reaction that produces an exponentially
growing population of identical DNA molecules• http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
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 nucleotide sequences of DNA molecules
• provide comparative information about DNA sequences
Gel Electrophoresis and Southern Blotting
• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis
• Uses a gel as a molecular sieve to separate nuclei acids or proteins by size
• DNA is negatively charged and moves towards a positive charge when placed in an electrical field
• restriction fragment analysis - fragments of DNA molecule are sorted by gel electrophoresis
• Useful for comparing two different DNA molecules, such as two alleles for a gene
RFLP Analysis
Restriction Fragment Length Differences as Genetic Markers
• Restriction fragment length polymorphisms (RFLPs, or Rif-lips) - differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths
• A RFLP can serve as genetic marker for a particular location (locus) in the genome
• RFLPs are detected by Southern blotting
LE 20-9Normal b-globin allele
175 bp 201 bp Large fragment
Sickle-cell mutant b-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
Uses: Evolutionary relationships• Comparing DNA samples from different
organisms to measure evolutionary relationships
–
+
DNA
1 32 4 5 1 2 3 4 5
turtle snake rat squirrel fruitfly
Uses: Medical diagnostic• Comparing normal allele to disease allele
chromosome with disease-causing allele 2
chromosomewith normal allele 1 –
+
allele 1allele 2
DNA
Example: test for Huntington’s disease
Uses: Forensics• Comparing DNA sample from crime scene
with suspects & victim
–
+
S1
DNA
S2 S3 Vsuspects crime
scene sample
DNA fingerprints• Comparing blood
samples on defendant’s clothing to determine if it belongs to victim– DNA fingerprinting– comparing DNA banding
pattern between different individuals
– ~unique patterns
• Southern blotting - combines gel electrophoresis with nucleic acid hybridization
• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to DNA immobilized on a “blot” of gel
• http://highered.mcgraw-hill.com/olc/dl/120078/bio_g.swf
LE 20-10
DNA + restriction enzyme Restrictionfragments
I Normal-globinallele
I Sickle-cellallele
I Heterozygote
Preparation of restriction fragments. Gel electrophoresis. Blotting.
I I I Nitrocellulosepaper (blot)
Gel
Sponge
Alkalinesolution
Papertowels
Heavyweight
Hybridization with radioactive probe.
I I IRadioactivelylabeled 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.
I I I
Film overpaper blot
Concept 20.3: Entire genomes can be mapped at the DNA level
Most ambitious mapping project to date has been the sequencing of the human genome
Officially begun as Human Genome Project in 1990, sequencing was largely completed by 2003
Scientists have also sequenced genomes of other organisms, providing insights of general biological significance
Genetic (Linkage) Mapping: Relative Ordering of Markers
1st stage is constructing linkage map of several thousand genetic markers throughout each chromosome
Order of markers and relative distances between them are based on recombination frequencies
LE 20-11Cytogenetic map
Genes locatedby FISH
Chromosomebands
Geneticmarkers
Genetic (linkage)mapping
Physical mapping
Overlappingfragments
DNA sequencing
Physical Mapping: Ordering DNA Fragments
Physical map - constructed by cutting DNA molecule into many short fragments and arranging them in order by identifying overlaps
Physical mapping gives actual distance in base pairs between markers
DNA Sequencing Relatively short DNA fragments can be
sequenced by dideoxy chain-termination method
Inclusion of special dideoxyribonucleotides in reaction mix ensures that fragments of various lengths will be synthesized
http://www.dnalc.org/resources/animations/cycseq.html
LE 20-12DNA(template strand)
5¢
3¢
Primer3¢
5¢
DNApolymerase
Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
3¢
5¢DNA (templatestrand)
Labeled strands3¢
Directionof movementof strands
Laser Detector
Linkage mapping, physical mapping, and DNA sequencing represent overarching strategy of 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
NOVA Science Now: Public Genomes
Determining Gene Function One way to determine function is to disable gene
and observe consequences Using in vitro mutagenesis, mutations are
introduced into cloned gene, altering or destroying its function
When mutated gene is returned to cell, 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
NOVA Science NOW: Autism Video
Future Directions in Genomics Genomics - study of entire genomes Proteomics - 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
Gene testing videohttp://www.pbs.org/wgbh/nova/body/public-genomes.html
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