biotechnology - ocw.nthu.edu.tw
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Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
PowerPoint® Lecture Presentations for
BiologyEighth Edition
Neil Campbell and Jane Reece
Lectures by Chris Romero, updated by Erin Barley with contributions from Joan Sharp
Chapter 20Chapter 20
Biotechnology
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Overview: The DNA Toolbox
• One of the greatest achievements of modern science has been the sequencing of the human genome (2003)--- depended on advances in recombinant DNA technology
• In recombinant DNA , nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule
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• 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
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Fig. 20-1
An example of DNA technology is the microarray , a measurement of gene expression of tens of thousands of different genes
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Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment
• The first step in recombinant DNA technology
– DNA cloning
• It is the production of multiple identical copiesof a specific gene (gene cloning ) or other DNA segment
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DNA Cloning and Its Applications: A Preview
• How to do DNA cloning:
– (1) 目標:DNA of interest
– (2) 載體: Plasmids (cloning vector) : replication origin, multiple cloning sites, selection marker (ampicillin resistance gene; LacZ gene : b-galactosidae digests X-gal into blue product)
– (3) 宿主:Host: bacteria (E. Coli), yeast
• Cloned genes are useful for making copies of a particular gene and producing a protein product
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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
cut, ligate
A preview of gene cloning and some uses of cloned genes
Easily isolated
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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
Multiple copies
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How to Make Recombinant DNA: Restriction Enzymes
(1) Bacterial restriction enzymes -- cut DNA molecules at a limited number of specific DNA sequences (4-8nucleotides), called restriction sites – GAATTC (EcoRI),
EcoRIHindIII HindIII
Restriction fragments
single cut
Double cuts/digests
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Using Restriction Enzymes to Make Recombinant DNA
• 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
Animation: Restriction EnzymesAnimation: Restriction Enzymes
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• DNA ligase is an enzyme that seals the bonds between restriction fragments
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Fig. 20-3-1Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
5′′′′3′′′′
3′′′′5′′′′
1
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Fig. 20-3-2Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
5′′′′3′′′′
3′′′′5′′′′
1
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
One possible combination
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Fig. 20-3-3Restriction site
DNA
Sticky end (overhang)
Restriction enzymecuts sugar-phosphatebackbones .
5′′′′3′′′′
3′′′′5′′′′
1
One possible combination
Recombinant DNA molecule
DNA ligaseseals strands.
3
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
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Intermission 12102009
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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
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Producing Clones of Cells Carrying Recombinant Plasmids
• Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid:
– The hummingbird genomic DNA and a bacterial plasmid are isolated
– Both are digested with the same restriction enzyme
– The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends
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– Some recombinant plasmids now contain hummingbird DNA
– The DNA mixture is added to bacteria that have been genetically engineered to accept it
– The bacteria are plated on a specific type of agar that selects for the bacteria with recombinant plasmids
– This results in the cloning of many hummingbird DNA fragments, including the β-globin gene
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Fig. 20-4-1
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
ampR gene: makes E. coli cells resistant to the antibiotic ampicillin.
lacZ gene: encodes β-galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. (藍白篩)
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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
self-ligation (intact lacZ )
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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
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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
White is good!
Screen for right insert
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Summay of cloning a gene
Animation: Cloning a GeneAnimation: Cloning a Gene
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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 - complete sequence with regulatory region (enhancers), exon, introns, nonconding region, etc.
• A genomic library that is made using bacteriophages is stored as a collection of phage clones
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Organization of eukaryotic genes: with multiple control elements
complementary DNA (cDNA)
noncoding control elements coding region
Reverse transcriptase
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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
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Fig. 20-5a
Bacterial clones
Recombinantplasmids
Recombinantphage DNA
or
Foreign genomecut up withrestrictionenzyme
(a) Plasmid library (b) Phage library
Phageclones
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• 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
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Fig. 20-5b
(c) A library of bacterial artificialchromosome (BAC) clones
Large plasmidLarge insertwith many genes
BACclone
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• 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 or tissue
• A cDNA library represents only part of the genome — only the subset of genestranscribed into mRNA in the original cells
cDNA library
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Fig. 20-6-1
DNA innucleus
mRNAs in cytoplasm
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Fig. 20-6-2
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase Poly-A tail
DNAstrand
Primer
mRNA
Oligo dT or Random Primers
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Fig. 20-6-3
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase Poly-A tail
DNAstrand
Primer
mRNA
DegradedmRNA
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Fig. 20-6-4
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase Poly-A tail
DNAstrand
Primer
mRNA
DegradedmRNA
DNA polymerase
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Fig. 20-6-5
DNA innucleus
mRNAs in cytoplasm
Reversetranscriptase Poly-A tail
DNAstrand
Primer
mRNA
DegradedmRNA
DNA polymerase
cDNA
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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
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• 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′′′′ 5′′′′C CG G GA AATT T
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Template 5’…GGCTAACTTAGC….3’probe 3’…CCGATTGAATCG…5’
3’…CCGATTGAATCG…5’
DNA denature and probe hybridization (colony hybridization)
Template 5’…CCATGGATACCG….3’
probe
3’…GGTACCTATGGC…5’
5’…CCGATTGAATCG…3’
complementary
Sense vs. Anti-sense strand
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• The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest
• Once identified, the clone carrying the gene of interest can be cultured
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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
•
Radioactivesingle-stranded DNA in reaction solution
replica Hybridization
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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
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Bacterial Expression Systems
• Most popular methods to deliver plamids into E.coli – heat shock (42℃ for 45 seconds)
• Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells: no expression, not soluble, not proper folded, etc.
• 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
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Eukaryotic Cloning and Expression Systems
• 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
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• 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 microscopically thin needles (microinjection)
• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination
Delivery gene into the eukaryotic cells
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Microinjector
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Suction
Glass needle
Cell
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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
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With polymerase chain reaction (PCR), any specific segment (with too many or no restriction enzymes available) of the target sequence within a DNA sample can be copied many times (amplified) completely in vitro.
Key components in a PCR reaction:
--Uses primers (with unique restriction site) that bracket the desired sequence
--Uses a heat-resistant DNA polymerase (active at high temperature) isolated from prokaryotes living
in hot spring: extend primers in 5’→ 3’ direction
EcoRI No EcoRI site
BamHI
BamHI
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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′′′′
5′′′′1
2
3
Only tiny amount of DNA template is required
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Fig. 20-8a
5′′′′
Genomic DNA
TECHNIQUETargetsequence
3′′′′
3′′′′ 5′′′′
The polymerase chain reaction (PCR)
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Fig. 20-8b
Cycle 1yields
2molecules
Denaturation
Annealing
Extension
Primers
Newnucleo-tides
3′′′′ 5′′′′
3
2
5′′′′ 3′′′′1
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Fig. 20-8c
Cycle 2yields
4molecules
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Fig. 20-8d
Cycle 3yields 8
molecules;2 molecules
(in whiteboxes)
match targetsequence
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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
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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 nucleic acids or proteins by size
• A electrical current is applied that causes charged molecules to move through the gel
• Molecules are sorted into “bands” by their size
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Fig. 20-9
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
RESULTS
1
2
+
+
–
–
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Fig. 20-9a
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
1
2
Powersource
– +
+–
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Fig. 20-9b
RESULTSGel electrophoresis
Visualization by EtBr staining
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• In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis
– A restriction enzyme will usually make many cuts in a DNA molecule
• Yielding a set of restriction fragments
– Gel electrophoresis to Separates DNA restriction fragments of different lengths:
• Small DNA molecules from virus,plasmid– discrete bands
• Large DNA molecules from eukaryotic chromosome --- smear
• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene
• The procedure is also used to prepare pure samples of individualfragments
Restriction fragment analysis
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Fig. 20-10
Normalallele
Sickle-cellallele
Largefragment
(b) Electrophoresis of restriction fragmentsfrom normal and sickle-cell alleles
201 bp175 bp
376 bp
(a) DdeI restriction sites in normal andsickle-cell alleles of ββββ-globin gene
Normal ββββ-globin allele
Sickle-cell mutant ββββ-globin allele
DdeI
Large fragment
Large fragment
376 bp
201 bp175 bp
DdeIDdeI
DdeI DdeI DdeI DdeI
Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene
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Fig. 17-22
Wild-type hemoglobin DNA
mRNA
Mutant hemoglobin DNA
mRNA
3′′′′3′′′′
3′′′′
3′′′′
3′′′′
3′′′′
5′′′′5′′′′
5′′′′
5′′′′5′′′′
5′′′′
C CT T TTG GA A AA
A A AGG U
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
transcription
translation
DdeI cutting site lost
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• 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 (DNA – DNA)
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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 Normalββββ-globinallele
1 32
Southern blotting of DNA fragments
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Fig. 20-11b
I II IIII II III
Film overblot
Probe detectionHybridization with radioactive probe
Fragment fromsickle-cellββββ-globin allele
Fragment fromnormal ββββ-globin allele
Probe base-pairswith fragments
Nitrocellulose blot
4 5
Radioactively labeledprobe for ββββ-globin gene
Southern blotting of DNA fragments
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DNA Sequencing
• Relatively short DNA fragments can be sequenced by the dideoxy chain termination method
• 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
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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
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Fig. 20-12a
DNA(template strand)
TECHNIQUE
DNA polymerase
Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)
dATP
dCTP
dTTP
dGTP
ddATP
ddCTP
ddTTP
ddGTP
Chain reaction terminated
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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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Latest Development on
Massive & Rapidwhole genome DNA Sequencing
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NGS
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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
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Studying the Expression of Single Genes
• Changes in the expression of a gene during embryonic development can be tested using
– Northern blotting
– Reverse transcriptase-polymerase chain reaction
• Both methods are used to compare mRNA from different developmental stages
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• Northern blotting combines gel electrophoresis of mRNA followed by hybridization with a probe on a membrane
• Identification of mRNA at a particular developmental stage suggests protein function at that stage
Northern blotting (DNA-RNA)
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http://ww
w.accessexcellence.org/R
C/V
L/GG
/nucleic.html
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• Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive
• Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest
• The products are run on a gel and the mRNA of interest identified
RT-PCR
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Fig. 20-13
TECHNIQUE
RESULTS
Gel electrophoresis
cDNAs
ββββ-globingene
PCR amplification
Embryonic stages
Primers
1 2 3 4 5 6
mRNAscDNA synthesis1
2
3
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• In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
In situ hybridization
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Fig. 20-14
50 µm
Determining where genes are expressed by in situ hybridization analysis
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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
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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
1
2
3
4
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Fluor DyesCy3 and Cy5
In a typical cDNA microarray experiments, the samples will be visibly pink (Cy3) or blue (Cy5) in color after successful labeling.
If one looks at the visible light spectrum, the Cy3 emission filter (550-605 nM) covers the blue-green region, while the Cy5 emission filter (650-725 nM) covers the orange-red region.
Note: – Cy3: Three-Tree- Tree is Green!– Cy5: Five-Fire- Fire is Red!
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Determining Gene Function
• One way to determine function is to disable the gene and observe the consequences – loss of function
• 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
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• Gene expression can also be silenced using RNA interference (RNAi)
• Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA
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• 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
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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
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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
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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
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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
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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
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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
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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”
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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
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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
• Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell types
• The adult body also has stem cells, which replace nonreproducing specialized cells
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Fig. 20-20
Culturedstem cells
Early human embryoat blastocyst stage
(mammalian equiva-lent of blastula)
Differentcultureconditions
Differenttypes ofdifferentiatedcells
Blood cellsNerve cellsLiver cells
Cells generatingall embryoniccell types
Adult stem cells
Cells generatingsome cell types
Embryonic stem cells
From bone marrowin this example
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• The aim of stem cell research is to supply cells for the repair of damaged or diseased organs
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Concept 20.4: The practical applications of DNA technology affect our lives in many ways
• Many fields benefit from DNA technology and genetic engineering
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Medical Applications
• One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
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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
• Genetic disorders can also be tested for using genetic markers that are linked to the disease-causing allele
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• Single nucleotide polymorphisms (SNPs) are useful genetic markers
• These are single base-pair sites that vary in a population
• When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphism (RFLP)
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Fig. 20-21
Disease-causingallele
DNA
SNP
Normal alleleT
C
Single nucleotide polymorphisms (SNPs) as genetic markers for disease-causing alleles
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SNPs in Coding Regions –No Changes in Protein
DNA SNP C to G
RNA CodonCUG to CUC
Protein Leucine to Leucine
No change in shape
Leucine Leucine
mRNA
G A C
C U G C U C
CUG CUC
G A G
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SNPs in Coding Regions –Subtle, Harmless Changes in Protein
DNA SNP A to C
RNA CodonGAU to GAG
Protein Aspartic acid
to Glutamic acid
Slight change in shape
Aspartic acid Glutamic acid
mRNA
C T A
G A U G A G
GAU GAG
C T C
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SNPs in Coding Regions –Harmful Changes in Protein – Mutations
DNA SNP T to A
RNA CodonGAU to GUU
Protein Aspartic acid
to Valine
Change in shape
Aspartic acid Valine
mRNA
C T
G A U G U U
GAU GUU
C AA A
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SNPs in Coding Regions –Subtle Changes in Proteins
That Only Switch on Under Certain ConditionsSmoking
Switched-on genes
Pattern AMany years later
= SNPs causing latent effects
Pattern BMany years later
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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 specific types of cells, for example bone marrow
• Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations
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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
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Pharmaceutical Products
• Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases
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• The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
• Pharmaceutical products that are proteins can be synthesized on a large scale
Synthesis of Small Molecules for Use as Drugs
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• Host cells in culture can be engineered to secrete a protein as it is made
• This is useful for the production of insulin , human growth hormones , and vaccines
Protein Production in Cell Cultures
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• Transgenic animals are made by introducing genes from one species into the genome of another animal
– Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use
• “Pharm” plants are also being developed to make human proteins for medical use
Protein Production by “Pharm” Animals and Plants
“Pharm” Animals and Plants
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Intrinsic PathwayExtrinsic Pathway(Tissue Damage)Factor XIa
Factor IX
Factor IXaFactor X
Factor XaProthrombin
ThrombinFibrinogen
Fibrin Clot
Antithrombin(+Heparin)
*Inhibition in Red
Regulation of the Blood Clotting Cascade By Antithrombin
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Fig. 20-23
Goats as “pharm” animals
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Forensic Evidence and Genetic Profiles
• An individual’s unique DNA sequence, or genetic profile , can be obtained by analysis of tissue or body fluids
• Genetic profiles can be used to provide evidence in criminal and paternity cases and to identify human remains
• Genetic profiles can be analyzed using RFLP analysis by Southern blotting
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• Even more sensitive is the use of genetic markers called short tandem repeats (STRs) ,which are variations in the number of repeats of specific DNA sequences
• PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
• The probability that two people who are not identical twins have the same STR markers is exceptionally small
Short tandem repeats (STRs)
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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
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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
• Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels
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Agricultural Applications
• DNA technology is being used to improve agricultural productivity and food quality
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Animal Husbandry
• Genetic engineering of transgenic animals speeds up the selective breeding process
• Beneficial genes can be transferred between varieties or species
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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
• Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops
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Fig. 20-25
Site whererestrictionenzyme cuts
T DNA
Plant with new trait
Tiplasmid
Agrobacterium tumefaciens
DNA withthe geneof interest
RecombinantTi plasmid
TECHNIQUE
RESULTS
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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
• Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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• Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
• Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives
GMO
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• As biotechnology continues to change, so does its use in agriculture, industry, and medicine
• National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
Ethical guidelines
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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
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12.Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry