chapter 20 biotechnology
DESCRIPTION
Fig. 20-23. Chapter 20 Biotechnology. Fig. 20-2. Cell containing gene of interest. Bacterium. 1. Gene inserted into plasmid. Bacterial chromosome. Plasmid. Gene of interest. Recombinant DNA ( plasmid ). DNA of chromosome. genetic engineering. 2. Plasmid put into bacterial cell. - PowerPoint PPT PresentationTRANSCRIPT
Fig. 20-23
Chapter 20 Biotechnology
Fig. 20-2
DNA of chromosome
Cell containing geneof interest
Gene inserted intoplasmid
Plasmid put intobacterial cell
RecombinantDNA (plasmid)
Recombinantbacterium
Bacterialchromosome
Bacterium
Gene ofinterest
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Plasmid
Gene ofInterest
Protein expressedby gene of interest
Basic research andvarious applications
Copies of gene Protein harvested
Basicresearchon gene
Basicresearchon protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growth hor-mone treats stuntedgrowth
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genetic engineering
---recombinant DNA---
Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene
• DNA cloning allows researchers to
– Compare genes and alleles between individuals
– Locate gene expression in a body
– Determine the role of a gene in an organism
• Several techniques are used to analyze the DNA of genes
Fig. 20-25
Site whererestrictionenzyme cuts
T DNA
Plant with new trait
Tiplasmid
Agrobacterium tumefaciens
DNA withthe geneof interest
RecombinantTi plasmid
TECHNIQUE
RESULTS
Fig. 20-22
Bonemarrow
Clonedgene
Bonemarrowcell frompatient
Insert RNA version of normal alleleinto retrovirus.
Retroviruscapsid
Viral RNA
Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.
Viral DNA carrying the normalallele inserts into chromosome.
Inject engineeredcells into patient.
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2
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RFLP (restriction fragment length polymorphism
Normalallele
Sickle-cellallele
Largefragment
(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene
Normal -globin allele
Sickle-cell mutant -globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
Fig. 20-10
Fig. 20-14
50 µm
•In situ hybridization
uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
Fig. 20-21
Disease-causingallele
DNA
SNP
Normal alleleT
C
•Short tandem repeats (STRs),
•which are variations in the number of repeats of specific DNA sequences
Fig. 20-24This photo shows EarlWashington just before his release in 2001,after 17 years in prison.
These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.
(a)
Semen on victim
Earl Washington
Source of sample
Kenneth Tinsley
STRmarker 1
STRmarker 2
STRmarker 3
(b)
17, 19
16, 18
17, 19
13, 16 12, 12
14, 15 11, 12
13, 16 12, 12
Fig. 20-1
Fig. 20-3-3Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
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35
1
One possible combination
Recombinant DNA molecule
DNA ligaseseals strands.
3
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
DNA cloning
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 host cell and replicate there
Fig. 20-4-1
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
DNA cloning
Fig. 20-4-2
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Fig. 20-4-3
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
Fig. 20-4-4
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
RESULTS
Colony carrying non-recombinant plasmidwith intact lacZ gene
One of manybacterialclones
Colony carrying recombinant plasmid with disrupted lacZ gene
Storing Cloned Genes in DNA Libraries
• A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome
• A genomic library that is made using bacteriophages is stored as a collection of phage clones
Fig. 20-5
Bacterial clones
Recombinantplasmids
Recombinantphage DNA
or
Foreign genomecut up withrestrictionenzyme
(a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones
Phageclones
Large plasmidLarge insertwith many genes
BACclone
Fig. 20-5a
Bacterial clones
Recombinantplasmids
Recombinantphage DNA
or
Foreign genomecut up withrestrictionenzyme
(a) Plasmid library (b) Phage library
Phageclones
• A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert
• BACs are another type of vector used in DNA library construction
Fig. 20-5b
(c) A library of bacterial artificial chromosome (BAC) clones
Large plasmidLarge insertwith many genes
BACclone
• A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell
• A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells
Fig. 20-6-5
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase Poly-A tail
DNAstrand
Primer
mRNA
DegradedmRNA
DNA polymerase
cDNA
Screening a Library for 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
• A probe can be synthesized that is complementary to the gene of interest
• For example, if the desired gene is
– Then we would synthesize this probe
G5 3… …G GC C CT TTAA A
C3 5C CG G GA AATT T
Fig. 20-7
ProbeDNA
Radioactivelylabeled probe
molecules
Film
Nylon membrane
Multiwell platesholding libraryclones
Location ofDNA with thecomplementarysequence
Gene ofinterest
Single-strandedDNA from cell
Nylonmembrane
TECHNIQUE
•
Expressing Cloned Eukaryotic Genes
• After a gene has been cloned, its protein product can be produced in larger amounts for research
• Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
• The use of cultured eukaryotic cells as host cells and 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 the post-translational modifications that many proteins require
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
Fig. 20-85
Genomic DNA
TECHNIQUE
Cycle 1yields
2molecules
Denaturation
Annealing
Extension
Cycle 2yields
4molecules
Cycle 3yields 8
molecules;2 molecules
(in whiteboxes)
match targetsequence
Targetsequence
Primers
Newnucleo-tides
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3
3
3
5
5
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2
3
Fig. 20-9
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
RESULTS
1
2
+
+
–
–
Gel Electrophoresis and Southern Blotting
• A technique called Southern blotting combines gel electrophoresis of DNA fragments 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
Southern blotting
Fig. 20-11TECHNIQUE
Nitrocellulosemembrane (blot)
Restrictionfragments
Alkalinesolution
DNA transfer (blotting)
Sponge
Gel
Heavyweight
Papertowels
Preparation of restriction fragments Gel electrophoresis
I II III
I II IIII II III
Radioactively labeledprobe for -globin gene
DNA + restriction enzyme
III HeterozygoteII Sickle-cellallele
I Normal-globinallele
Film overblot
Probe detectionHybridization with radioactive probe
Fragment fromsickle-cell-globin allele
Fragment fromnormal -globin allele
Probe base-pairswith fragments
Nitrocellulose blot
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4 5
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DNA Sequencing---dideoxy chain termination method
• Relatively short DNA fragments can be sequenced by the dideoxy chain termination method
• dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths
• Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment
• The DNA sequence can be read from the resulting spectrogram
Fig. 20-12
DNA(template strand)
TECHNIQUE
RESULTS
DNA (template strand)
DNA polymerase
Primer Deoxyribonucleotides
Shortest
Dideoxyribonucleotides(fluorescently tagged)
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Directionof movementof strands
Detector
Last baseof longest
labeledstrand
Last baseof shortest
labeledstrand
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
Analyzing Gene Expression
• Nucleic acid probes can hybridize with mRNAs transcribed from a gene
• Probes can be used to identify where or when a gene is transcribed in an organism
• mRNA
– Northern blotting
– Reverse transcriptase-polymerase chain reaction (RT-PCR)
Fig. 20-13
TECHNIQUE
RESULTS
Gel electrophoresis
cDNAs
-globingene
PCR amplification
Embryonic stages
Primers
1 2 3 4 5 6
mRNAscDNA synthesis 1
2
3
Studying the 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
Fig. 20-15
TECHNIQUE
Isolate mRNA.
Make cDNA by reversetranscription, usingfluorescently labelednucleotides.
Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray.
Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.
Tissue sample
mRNA molecules
Labeled cDNA molecules(single strands)
DNA fragmentsrepresentingspecific genes
DNA microarraywith 2,400human genes
DNA microarray
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4
Determining Gene Function in vitro mutagenesis and RNA interference (RNAi)
• 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
• Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell
Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications
Cloning Plants: Single-Cell Cultures
• One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism
• A totipotent cell is one that can generate a complete new organism
Fig. 20-16
EXPERIMENT
Transversesection ofcarrot root
2-mgfragments
Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid.
Singlecellsfree insuspensionbegan todivide.
Embryonicplant developedfrom a culturedsingle cell.
Plantlet wascultured onagar medium.Later it wasplantedin soil.
A singlesomaticcarrot celldevelopedinto a maturecarrot plant.
RESULTS
Cloning Animals: Nuclear Transplantation
• In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
• Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg
• However, the older the donor nucleus, the lower the percentage of normally developing tadpoles
Fig. 20-17
EXPERIMENT
Less differ-entiated cell
RESULTS
Frog embryo Frog egg cell
UV
Donornucleustrans-planted
Frog tadpole
Enucleated egg cell
Egg with donor nucleus activated to begin
development
Fully differ-entiated(intestinal) cell
Donor nucleus trans-planted
Most developinto tadpoles
Most stop developingbefore tadpole stage
Reproductive Cloning of Mammals
• In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
• Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus
Fig. 20-18
TECHNIQUE
Mammarycell donor
RESULTS
Surrogatemother
Nucleus frommammary cell
Culturedmammary cells
Implantedin uterusof a thirdsheep
Early embryo
Nucleusremoved
Egg celldonor
Embryonicdevelopment Lamb (“Dolly”)
genetically identical tomammary cell donor
Egg cellfrom ovary
Cells fused
Grown inculture
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• Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs
• CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”
Fig. 20-19
Problems Associated with Animal Cloning
• In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth
• Many epigenetic changes,
– such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
Stem Cells of Animals
• A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types
• embryonic stem cells :at the blastocyst stage, to differentiate into all cell types
• The aim of stem cell research is to supply cells for the repair of damaged or diseased organs