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Transcript of Chapter 20 Biotechnology. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin...
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Overview: The DNA Toolbox
• Sequencing of the human genome was completed by 2007
• 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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• Methods for making recombinant DNA are central to genetic engineering (direct manipulation of genes for practical purposes)
• DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products
• An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment
• To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
I. DNA Cloning
• Use bacteria and their plasmids
– Remember: Plasmids = small circular DNA molecules that replicate separately from the bacterial chromosome
• Cloning is used for making copies of gene and producing a protein product
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• Gene cloning = uses bacteria to make multiple copies of a gene
– Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell
• Reproduction of bacterial cell results in cloning of the recombinant plasmid
• Results = production of multiple copies of gene
Fig. 20-2a
DNA of chromosome
Cell containing geneof interest
Gene inserted intoplasmid
Plasmid put intobacterial cell
RecombinantDNA (plasmid)
Recombinantbacterium
Bacterialchromosome
Bacterium
Gene ofinterest
Plasmid
2
1
2
Fig. 20-2b
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Gene ofInterest
Protein expressedby gene of interest
Basic research andvarious applications
Copies of gene Protein harvested
Basicresearchon gene
Basicresearchon protein
4
Recombinantbacterium
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
3
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
II. Restriction Enzymes
• Bacterial restriction enzymes (protects bacteria from phages) cut DNA molecules at specific DNA sequences (restriction sites)
– Restriction enz makes many cuts, yielding restriction fragments
– Rest enz cut DNA in a staggered way, makes fragments with “sticky ends”, that bond with complementary sticky ends of other fragments
– DNA ligase = seals bonds between fragments
Fig. 20-3-3Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
One possible combination
Recombinant DNA molecule
DNA ligaseseals strands.
3
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
III. Cloning a Euk Gene in a Bacterial Plasmid
• cloning vector = the original plasmid that can carry foreign DNA into a host cell and replicate there
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A. Producing Cells w/ Recombinant Plasmids
• Steps to clone the hummingbird β-globin gene in a bacterial plasmid:
– Isolate genomic DNA and a bacterial plasmid
– Digest both with SAME rest enz
– Fragments are mixed, DNA ligase is added to bond the fragment sticky ends
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– Some recombinant plasmids now contain hummingbird DNA
– DNA mixture is added to bacteria
– Bacteria are plated on agar that selects for the bacteria with recombinant plasmids
– Results in the cloning of many hummingbird DNA fragments, including the β-globin gene
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
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C3 5C CG G GA AATT T
B. Screening Clones for a Gene
• Nucleic acid probe = identifies clone by using a sequence complementary to the gene
• This process is called nucleic acid hybridization
• For example, if the desired gene is
– Then we would synthesize this probe
G5 3… …G GC C CT TTAA A
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• DNA probe is used to screen a large number of clones at same time for gene of interest
• Once identified, the clone can be cultured
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
•
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IV. Expressing Cloned Euk Genes
• Protein products of cloned genes can be produced in larger amounts for research
• Cloned genes can be expressed in either bacterial or eukaryotic cells
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A. Bacterial Expression
• Scientists use an expression vector (a cloning vector that contains a highly active prokaryotic promoter) to overcome differences in promoters and other DNA control sequences
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
B. Eukaryotic Expression
• Using eukaryotic cells as hosts and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems
• YACs can carry more DNA than a plasmid
• Euk hosts can provide the post-translational modifications that many proteins require
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• Electroporation = applying an electrical pulse to create temp holes in plasma membranes introducing recombinant DNA
• Can inject DNA into cells using microscopically thin needles
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
V. Amplifying DNA in Vitro
• 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
3
3
3
3
5
5
51
2
3
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
• DNA Cloning - Andersen
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
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
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
VI. Gel Electrophoresis and Southern Blotting
• Gel electrophoresis = method of rapidly analyzing and comparing genomes
– Uses gel to separate nucleic acids or proteins by size
– Electrical current is applied that causes charged molecules to move through the gel
– Molecules form “bands” by their size
Fig. 20-9
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
RESULTS
1
2
+
+
–
–
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• Restriction fragment analysis = DNA fragments from rest enz digestion of DNA are sorted by gel electrophoresis
– Used for comparing two different DNA molecules
– Used to prepare pure samples of individual fragments
Fig. 20-10
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
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• Southern blotting combines gel electro with nucleic acid hybridization
– DNA fragments can be identified using labeled probes that bind to the DNA that is stuck on a “blot” of gel
Fig. 20-11a
TECHNIQUE
Nitrocellulosemembrane (blot)
Restrictionfragments
Alkalinesolution
DNA transfer (blotting)
Sponge
Gel
Heavyweight
Papertowels
Preparation of restriction fragments Gel electrophoresis
I II IIIDNA + restriction enzyme
III HeterozygoteII Sickle-cellallele
I Normal-globinallele
1 32
Fig. 20-11b
I II IIII II III
Film overblot
Probe detectionHybridization with radioactive probe
Fragment fromsickle-cell-globin allele
Fragment fromnormal -globin allele
Probe base-pairswith fragments
Nitrocellulose blot
4 5
Radioactively labeledprobe for -globin gene
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VII. DNA Sequencing
• Modified nucleotides called 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
• Read from bottom to top (smallest fragment on bottom, this is 5’ end, original DNA is complimentary)
Fig. 20-12a
DNA(template strand)
TECHNIQUE
DNA polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
Fig. 20-12bTECHNIQUE
RESULTS
DNA (template strand)
Shortest
Labeled strands
Longest
Shortest labeled strand
Longest labeled strand
Laser
Directionof movementof strands
Detector
Last baseof longest
labeledstrand
Last baseof shortest
labeledstrand
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VIII. Analyzing Gene Expression
• Nucleic acid probes can hybridize with mRNAs transcribed from a gene, used to identify where or when a gene is transcribed in an organism
• Reverse transcriptase-polymerase chain reaction (RT-PCR)
• Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest
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
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IX. Cloning Animals
• Nuclear transplantation = nucleus of an unfertilized egg is replaced w/ nucleus of a differentiated cell
• Experiments, w/ frog embryos, have shown 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
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
1
33
4
5
6
2
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
A. Problems with Animal Cloning
• Only a small percentage of cloned embryos have developed normally to birth
• Epigenetic changes (acetylation of histones or methylation of DNA) must be reversed in nucleus from a donor animal for genes to be expressed or repressed appropriately for development
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X. Medical Applications
• Gene therapy = alteration of an afflicted individual’s genes
• Vectors are used for delivery of genes into specific types of cells, for example bone marrow, lung tissue (CFTR protein)
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.
1
2
3
4
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
XI. Genetic Engineering in Plants
• Agricultural scientists have given a number of crop plants genes for desirable traits
• Ti plasmid = most commonly used vector for introducing new genes into plant cells
• Used to transfer herbicide resistance, inc resistance to pests, inc resistance to salinity, and improved nutritional value
Fig. 20-25
Site whererestrictionenzyme cuts
T DNA
Plant with new trait
Tiplasmid
Agrobacterium tumefaciens
DNA withthe geneof interest
RecombinantTi plasmid
TECHNIQUE
RESULTS
Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
You should now be able to:
1. Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology
2. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid
3. Define and distinguish between genomic libraries using plasmids, phages, and cDNA
4. Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure
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5. Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene
6. Describe and distinguish between the Southern blotting procedure, Northern blotting procedure, and RT-PCR
7. Distinguish between gene cloning, cell cloning, and organismal cloning
8. Describe how nuclear transplantation was used to produce Dolly, the first cloned sheep
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9. Describe the application of DNA technology to the diagnosis of genetic disease, the development of gene therapy, vaccine production, and the development of pharmaceutical products
10.Define a SNP and explain how it may produce a RFLP
11.Explain how DNA technology is used in the forensic sciences