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LECTURE PRESENTATIONS
For CAMPBELL BIOLOGY, NINTH EDITIONJane B. Reece, Lisa A. Urry, Micae! L. Cain, "#e$en A. %asser&an, Pe#er '. Minors(y, Ro)er# B. Jac(son
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Lectures by
Erin Barley
Kathleen Fitzpatric
Bi!techn!l!"y
Chapter #$
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O%er%ie&' The (NA T!!lb!)
• Sequencing of the genomes of more than 7,000species was under way in 2010
• DNA sequencing has depended on advances in
technology, starting with maing recom!inant DNA• "n recombinant DNA, nucleotide sequences from
two different sources, often two species, are
com!ined in vitro into the same DNA molecule
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• #ethods for maing recom!inant DNA are centralto genetic engineering, the direct manipulation of
genes for practical purposes
•
DNA technology has revolutioni$edbiotechnology, the manipulation of organisms or
their genetic components to mae useful products
• An e%ample of DNA technology is the microarray,
a measurement of gene e%pression of thousandsof different genes
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&igure 20'1
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C!ncept #$*+' (NA cl!nin" yiel,s -ultiple
c!pies !. a "ene !r !ther (NA se"-ent• (o wor directly with specific genes, scientists
prepare well)defined segments of DNA in identical
copies, a process called DNA cloning
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(NA Cl!nin" an, Its Applicati!ns'
A Preview• #ost methods for cloning pieces of DNA in the
la!oratory share general features, such as the use
of !acteria and their plasmids• Plasmids are small circular DNA molecules that
replicate separately from the !acterial
chromosome
• *loned genes are useful for maing copies of aparticular gene and producing a protein product
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• Gene cloning involves using !acteria to maemultiple copies of a gene
• &oreign DNA is inserted into a plasmid, and therecom!inant plasmid is inserted into a !acterialcell
• +eproduction in the !acterial cell results in cloningof the plasmid including the foreign DNA
•
(his results in the production of multiple copies ofa single gene
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&igure 20'2Bacterium
Bacterial
chromosome
Plasmid
2
1
3
4
Gene inserted into
plasmidCell containing gene
of interest
ecombinant
DNA !plasmid"
Gene of
interest
Plasmid put into
bacterial cell
DNA of chromosome
!#foreign$ DNA"
ecombinant
bacterium
%ost cell gro&n in culture to
form a clone of cells containing
the #cloned$ gene of interest
Gene of
interest
Protein e'pressed from
gene of interest
Protein har(estedCopies of gene
Basic research
and (arious
applications
Basic
research
on protein
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up to'ic &aste
Protein dissol(es
blood clots in heart
attac) therapy
%uman gro&th
hormone treats
stunted gro&th
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&igure 20'2a
Bacterium
Bacterial
chromosome
Plasmid
2
1 Gene inserted into
plasmidCell containing
gene of interest
ecombinant
DNA !plasmid"
Gene ofinterest
Plasmid put into
bacterial cell
DNA of
chromosome
!#foreign$ DNA"
ecombinant
bacterium
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&igure 20'2!
%ost cell gro&n in
culture to form a clone
of cells containing the
#cloned$ gene of interest
Gene ofinterest
Protein e'pressed from
gene of interest
Protein har(estedCopies of gene
Basic research
and (ariousapplications
3
4
Basicresearchon protein
Basicresearchon gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up to'ic &aste
Protein dissol(es
blood clots in heart
attac) therapy
%uman gro&th
hormone treats
stunted gro&th
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Usin" Restricti!n Enzy-es t! /ae
Rec!-binant (NA• acterial restriction en*ymes cut DNA
molecules at specific DNA sequences called
restriction sites• A restriction en$yme usually maes many cuts,
yielding restriction fragments
• (he most useful restriction en$ymes cut DNA in a
staggered way, producing fragments with -stic)yends'.
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Animation/ +estriction n$ymes
+ight)clic slide select -lay.
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• Sticy ends can !ond with complementary sticyends of other fragments
• DNA ligase is an en$yme that seals the !onds
!etween restriction fragments
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&igure 20'3)1
estriction en*yme
cuts sugar+phosphatebac)bones,
estriction site
DNA
-′
-′
-′
-′
-′
-′
3′
3′
3′
3′
3′
3′
1
.tic)yend
GAA//CC//AAG
C / /AA
G AA/ / C
G
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&igure 20'3)3
ecombinant DNA molecule
0ne possible combinationDNA ligaseseals strands
DNA fragment addedfrom another moleculecut by same en*yme,Base pairing occurs,
estriction en*yme
cuts sugar+phosphatebac)bones,
estriction site
DNA
-′
-′
-′
-′
-′
-′
-′
-′
-′-′
-′
-′
-′-′
-′
-′
3′
3′
3′
3′
3′
3′
3′
3′
3′
3′
3′
3′
3′
3′
3′
3′
2
3
1
.tic)yend
GAA//CC//AAG
C / /AA
G AA/ / C
G
GG
AA/ / C
C/ / AA
G
G
G
G
AA// CAA// C
C //AA C //AA
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Cl!nin" a Euary!tic 0ene in a
Bacterial Plas-i,
• "n gene cloning, the original plasmid is called a
cloning (ector
•
A cloning vector is a DNA molecule that can carryforeign 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
humming!ird β)glo!in gene in a !acterial plasmid
4 (he humming!ird genomic DNA and a !acterialplasmid are isolated
4 oth are cut with the same restriction en$yme
4 (he fragments are mi%ed, and DNA ligase is
added to !ond the fragment sticy ends
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Animation/ *loning a 5ene
+ight)clic slide select -lay.
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4 Some recom!inant plasmids now containhumming!ird DNA
4 (he DNA mi%ture is added to !acteria that have
!een genetically engineered to accept it
4 (he !acteria are plated on a type of agar that
selects for the !acteria with recom!inant
plasmids
4 (his results in the cloning of many humming!irdDNA fragments, including the β)glo!in gene
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&i 20 6
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&igure 20'6
Bacterial plasmid
/C%N
.5/.
ampR gene lacZ gene
estrictionsite
%ummingbird cell
.tic)yends Gene of
interest
%umming+bird DNAfragments
ecombinant plasmids Nonrecombinantplasmid
Bacteria carryingplasmids
Colony carrying non+recombinant plasmid&ith intact lacZ gene
Colony carryingrecombinantplasmid&ith disruptedlacZ gene
0ne of manybacterialclones
&igure 20 6a 1
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&igure 20'6a)1
Bacterial plasmid
/C%N
ampR gene lacZ gene
estriction
site
%ummingbird cell
.tic)yends Gene of
interest
%umming+bird DNA
fragments
&igure 20 6a 2
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&igure 20'6a)2
Bacterial plasmid
/C%N
ampR gene lacZ gene
estriction
site
%ummingbird cell
.tic)yends Gene of
interest
%umming+bird DNA
fragments
ecombinant plasmids Nonrecombinantplasmid
&igure 20 6a 3
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&igure 20'6a)3
Bacterial plasmid
/C%N
ampR gene lacZ gene
estriction
site
%ummingbird cell
.tic)yends Gene of
interest
%umming+bird DNA
fragments
ecombinant plasmids Nonrecombinantplasmid
Bacteria carryingplasmids
&igure 20 6!
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&igure 20'6!
.5/.
Bacteria carryingplasmids
Colony carrying non+recombinant plasmid&ith intact lacZ gene
Colony carryingrecombinantplasmid&ith disruptedlacZ gene
0ne of manybacterialclones
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Storing Cloned Genes in DNA Libraries
• A genomic library that is made using !acteria isthe collection of recom!inant vector clones
produced !y cloning DNA fragments from an entire
genome
• A genomic li!rary that is made using
!acteriophages is stored as a collection of phage
clones
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&igure 20
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&igure 20'
6oreign genome
Cut &ith restriction en*ymes into either
small
fragmentslargefragments
or
ecombinantplasmids
Plasmidclone
!a" Plasmid library
!b" BAC clone
Bacterial artificialchromosome !BAC"
5argeinsert&ithmany
genes
!c" .toring genome libraries
&igure 20 a
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&igure 20'a
!c" .toring genome libraries
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• A bacterial artificial chromosome !BAC" is alarge plasmid that has !een trimmed down and
can carry a large DNA insert
• A*s are another type of vector used in DNA
li!rary construction
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• A complementary DNA !cDNA" li!rary is made !ycloning DNA made in vitro !y reverse transcription
of all the m+NA produced !y a particular cell
• A cDNA library represents only part of the
genome8only the su!set of genes transcri!ed
into m+NA in the original cells
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&igure 20'9)1
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g
DNA innucleus
mNAs incytoplasm
&igure 20'9)2
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g
DNA innucleus
mNAs incytoplasm
mNA
e(ersetranscriptase Poly+A tail
DNA
strand
Primer
-′-′
3′3′
A A A A A A
/ / / / /
&igure 20'9)3
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g
DNA innucleus
mNAs incytoplasm
mNA
e(ersetranscriptase Poly+A tail
DNAstrand
Primer
-′-′
-′-′
3′3′
3′3′
A A A A A A
A A A A A A
/ / / / /
/ / / / /
&igure 20'9)6
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g
DNA innucleus
mNAs incytoplasm
mNA
e(ersetranscriptase Poly+A tail
DNAstrand
Primer
DNApolymerase
-′-′
-′-′
-′ -′
3′3′
3′3′
3′3′
A A A A A A
A A A A A A
/ / / / /
/ / / / /
&igure 20'9)
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DNA innucleus
mNAs incytoplasm
mNA
e(ersetranscriptase Poly+A tail
DNAstrand
Primer
DNApolymerase
cDNA
-′-′
-′-′
-′ -′
-′-′
3′3′
3′3′
3′3′
3′3′
A A A A A A
A A A A A A
/ / / / /
/ / / / /
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Screening a Library for Clones Carrying a
Gene of Interest • A clone carrying the gene of interest can !e
identified with a nucleic acid probe having a
sequence complementary to the gene
• (his process is called nucleic acid hybridi*ation
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• A pro!e can !e synthesi$ed that iscomplementary to the gene of interest
• &or e%ample, if the desired gene is
4 (hen we would synthesi$e this pro!e
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-′ 3′⋅⋅
C/CA/CACCGGC⋅⋅⋅
-′
3′
G A G / A G / G G C C G
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•(he DNA pro!e can !e used to screen a largenum!er of clones simultaneously for the gene of
interest
• :nce identified, the clone carrying the gene of
interest can !e cultured
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&igure 20'7
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adioacti(elylabeled probemolecules Gene of
interest
ProbeDNA
.ingle+stranded
DNA fromcell
6ilm
5ocation of DNA &ith thecomplementaryse7uence
Nylonmembrane
Nylon membrane
8ulti&ell platesholding libraryclones
/C%N -′
-′3′
3′
GAG/AG/GGCCG⋅⋅ ⋅ C/CA/CACCGGC⋅⋅ ⋅
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E)pressin" Cl!ne, Euary!tic 0enes
• After a gene has !een cloned, its protein productcan !e produced in larger amounts for research
• *loned genes can !e e%pressed as protein in
either !acterial or euaryotic cells
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Bacterial !"ression Systems
•Several technical difficulties hinder e%pression ofcloned euaryotic genes in !acterial host cells
• (o overcome differences in promoters and other
DNA control sequences, scientists usually employ
an e'pression (ector , a cloning vector that
contains a highly active !acterial promoter
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u#aryotic Cloning and !"ression Systems
•#olecular !iologists can avoid euaryote)!acterialincompati!ility issues !y using euaryotic cells,
such as yeasts, as hosts for cloning and
e%pressing genes
• ven yeasts may not possess the proteins
required to modify e%pressed mammalian proteins
properly
•
"n such cases, cultured mammalian or insect cellsmay !e used to e%press and study proteins
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•:ne method of introducing recom!inant DNA intoeuaryotic cells is electroporation, applying a
!rief electrical pulse to create temporary holes in
plasma mem!ranes
• Alternatively, scientists can in;ect DNA into cells
using microscopically thin needles
• :nce inside the cell, the DNA is incorporated into
the cell<s DNA !y natural genetic recom!ination
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Cross$S"ecies Gene !"ression and
volutionary Ancestry• (he remara!le a!ility of !acteria to e%press some
euaryotic proteins underscores the shared
evolutionary ancestry of living species
• &or e%ample, Pax-6 is a gene that directs formation
of a verte!rate eye= the same gene in flies directs
the formation of an insect eye >which is quite
different from the verte!rate eye?• (he Pax-6 genes in flies and verte!rates can
su!stitute for each other
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A-pli.yin" (NA in %itro' The P!ly-erase
Chain Reacti!n 1PCR2• (he polymerase chain reaction, PC, can
produce many copies of a specific target segment
of DNA
• A three)step cycle8heating, cooling, and
replication8!rings a!out a chain reaction that
produces an e%ponentially growing population of
identical DNA molecules• (he ey to *+ is an unusual, heat)sta!le DNA
polymerase called (aq polymerase'
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&igure 20'@ -′
3′/C%N
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Genomic DNA
/argetse7uence
Denaturation
Annealing
'tension
Primers
Ne&nucleotides
Cycle 1yields
2molecules
Cycle 2yields
4molecules
Cycle 3yields 9
molecules:2 molecules
!in &hite bo'es"match target
se7uence
-′
-′
-′
3′
3′
3′
2
3
1
&igure 20'@a
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Genomic DNA
/argetse7uence
-′
-′
3′
3′
/C%N
&igure 20'@!
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Denaturation
Annealing
'tension
Primers
Ne&nucleo+tides
Cycle 1yields
2molecules
-′
-′
3′
3′
2
3
1
&igure 20'@c
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Cycle 2
yields4molecules
&igure 20'@d
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Cycle 3yields 9
molecules:2 molecules
!in &hite bo'es"match target
se7uence
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C!ncept #$*#' (NA techn!l!"y all!&s us t!
stu,y the se3uence4 e)pressi!n4 an, .uncti!n!. a "ene
• DNA cloning allows researchers to
4 *ompare genes and alleles !etween individuals 4 ocate gene e%pression in a !ody
4 Determine the role of a gene in an organism
• Several techniques are used to analy$e the DNA
of genes
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0el Electr!ph!resis an, S!uthern Bl!ttin"
•
:ne indirect method of rapidly analy$ing andcomparing genomes is gel electrophoresis
• (his technique uses a gel as a molecular sieve to
separate nucleic acids or proteins !y si$e,
electrical charge, and other properties
• A current is applied that causes charged
molecules to move through the gel
• #olecules are sorted into -!ands. !y their si$e
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Animation/ iotechnology a!
+ight)clic slide select -lay.
&igure 20'B /C%N
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8i'ture of
DNA mol+
ecules of
different
si*es
Po&er
source
Po&er
source
5onger
molecules
Cathode Anode
;ells
Gel
.horter
molecules
.5/.
1
2
&igure 20'Ba/C%N
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8i'ture of
DNA mol+
ecules of different
si*es
Po&er source
Po&er source
5onger
molecules
Cathode Anode
;ells
Gel
.horter
molecules
2
1
&igure 20'B!
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.5/.
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•
"n restriction fragment analysis, DNA fragmentsproduced !y restriction en$yme digestion of a
DNA molecule are sorted !y gel electrophoresis
• +estriction fragment analysis can !e used to
compare two different DNA molecules, such astwo alleles for a gene, if the nucleotide difference
alters a restriction site
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•
Cariations in DNA sequence are calledpolymorphisms
• Sequence changes that alter restriction sites are
called 65Ps >restriction fragment length
polymorphisms?
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&igure 20'10
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Normal β+globin allele
.ic)le+cell mutantβ
+globin allele
5arge
fragment
Normal
allele
.ic)le+cell
allele
2<1 bp1=- bp
3=> bp
!a" DdeI restriction sites in normal and
sic)le+cell alleles of theβ
+globin gene
!b"lectrophoresis of restriction
fragments from normal andsic)le+cell alleles
2<1 bp1=- bp
3=> bp
5arge fragment
5arge fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
&igure 20'10a
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Normal β+globin allele
.ic)le+cell mutantβ
+globin allele
!a" DdeI restriction sites in normal and
sic)le+cell alleles of the β+globin gene
2<1 bp1=- bp
3=> bp
5arge fragment
5arge fragment
DdeI DdeI DdeI DdeI
DdeI
DdeI
DdeI
&igure 20'10!
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5arge
fragment
Normal
allele
.ic)le+cell
allele
2<1 bp1=- bp
3=> bp
!b" lectrophoresis of restrictionfragments from normal andsic)le+cell alleles
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•
A technique called .outhern blotting com!inesgel electrophoresis of DNA fragments with nucleic
acid hy!ridi$ation
• Specific DNA fragments can !e identified !y
Southern !lotting, using la!eled pro!es thathy!ridi$e to the DNA immo!ili$ed on a -!lot. of gel
* +-- Pearson E/ca#ion, Inc.
&igure 20'11
/C%N
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DNA restriction en*yme
321
4
/C%N
I Normal
β+globin
allele
II .ic)le+cell
alleleIII %etero*ygote
estriction
fragmentsNitrocellulose
membrane !blot"
%ea(y
&eight
Gel
.ponge
Al)aline
solution Paper
to&els
III III
III III III III
Preparation of
restriction fragments
Gel electrophoresis DNA transfer !blotting"
adioacti(ely labeled
probe for β+globin
gene
Nitrocellulose blot
Probe base+pairs
&ith fragments
6ragment from
sic)le+cell β+globin allele
6ragment from
normal β+ globin
allele
6ilm
o(er
blot
%ybridi*ation &ith labeled probe Probe detection-
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(NA Se3uencin"
•+elatively short DNA fragments can !e sequenced!y the dideo%y chain termination method, the firstautomated method to !e employed
• #odified nucleotides called dideo%yri!onucleotides>ddN(? attach to synthesi$ed DNA strands ofdifferent lengths
• ach type of ddN( is tagged with a distinct
fluorescent la!el that identifies the nucleotide atthe end of each DNA fragment
• (he DNA sequence can !e read from the resultingspectrogram
* +-- Pearson E/ca#ion, Inc.
&igure 20'12
DNA
!t l t t d"
/C%N
/
Primer 3
Deo'yribonucleotides Dideo'yribonucleotides
!fluorescently tagged"
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!template strand"-′
3′
C
C
C
C
/
//
G
G
A
AAA
G//
/
DNA
polymerase
-′
3′
P P P
0%
G
dA/P
dC/P
d//P
dG/P
!fluorescently tagged"
P P P
%
G
ddA/P
ddC/P
dd//P
ddG/P
-′
3′
C
C
C
C
/
//
G
G
A
AAA
DNA !template
strand"5abeled strands
.hortest 5ongest-′
3′
ddCddG
ddAddA
ddA
ddG
ddG
dd/ddC
G//
/ G//
/
C
G//
/
C
/ /
G
G//
/
C
/
GA
G//
/
C
/
GAA
G//
/
C
/
GAAG
G//
/
C
/
GAAG/
G//
/
C
/
GAAG/C
G//
/
C
/
GAAG/CA
Direction
of mo(ement
of strands
5ongest labeled strand
Detector
5aser .hortest labeled strand
.5/.
5ast nucleotide
of longest
labeled strand
5ast nucleotide
of shortest
labeled strand
G
G
G
A
AA
C
C
/
&igure 20'12a
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DNA
!template strand"
/C%NPrimer Deo'yribonucleotides Dideo'yribonucleotides
!fluorescently tagged"
DNA
polymerase
-′
-′
3′
3′
0% %
GG
dA/P
dC/P
d//P
dG/P
P P P P P P
ddA/P
ddC/P
dd//PddG/P
/
//
G
G
G
C
C
C
C/
//
A
A
AA
&igure 20'12!
/C%N !continued"
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DNA !template
strand"5abeled strands
.hortest 5ongest
Direction
of mo(ement
of strands5ongest labeled strand
Detector
5aser .hortest labeled strand
/C%N !continued"
-′
3′
G
G
C
C
C
C/
//
A
A
A
A
/
/
/
G
ddC
ddC
ddG
ddG
ddG
ddAddA
ddA
dd/
3′
-′
/
/
/
G
C/
/
/
G
CG
/
/
/
G
CGA
/
/
/
G
CGAA
/
/
/
G
CGAAG
/
/
/
CGAAG/
/
/
/
CGAAG/
CA
/
/
/
CGAAG/
C
G G G
&igure 20'12c
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.5/.
5ast nucleotide
of longest
labeled strand
5ast nucleotide
of shortest
labeled strand
G
G
G
A
AA
C
C
/
Direction
of mo(ement
of strands5ongest labeled strand
Detector
5aser .hortest labeled strand
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Analyzin" 0ene E)pressi!n
•
Nucleic acid pro!es can hy!ridi$e with m+NAstranscri!ed from a gene
• ro!es can !e used to identify where or when a
gene is transcri!ed in an organism
* +-- Pearson E/ca#ion, Inc.
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Studying t&e !"ression of Single Genes
•
*hanges in the e%pression of a gene duringem!ryonic development can !e tested using
4 Northern !lotting
4 +everse transcriptase)polymerase chain reaction
• oth methods are used to compare m+NA from
different developmental stages
* +-- Pearson E/ca#ion, Inc.
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•
Northern blotting com!ines gel electrophoresisof m+NA followed !y hy!ridi$ation with a pro!e on
a mem!rane
• "dentification of m+NA at a particular
developmental stage suggests protein function atthat stage
* +-- Pearson E/ca#ion, Inc.
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•
e(erse transcriptase+polymerase chainreaction !/+PC" is quicer and more sensitive
!ecause it requires less m+NA than Northern
!lotting
• +everse transcriptase is added to m+NA to maecDNA, which serves as a template for *+
amplification of the gene of interest
•
(he products are run on a gel and the m+NA ofinterest is identified
* +-- Pearson E/ca#ion, Inc.
&igure 20'13
cDNA synthesis NA1
/C%N
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cDNA synthesis
PC amplification
Gel electrophoresis
mNAs
cDNAs
Primers
β
+globingene
mbryonic stages1 2 3 4 - >
2
3
1
.5/.
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•
In situ hybridi*ation uses fluorescent dyesattached to pro!es to identify the location of
specific m+NAs in place in the intact organism
* +-- Pearson E/ca#ion, Inc.
&igure 20'16
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-<µ
m
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Studying t&e !"ression of Interacting
Grou"s of Genes
• Automation has allowed scientists to measure
the e%pression of thousands of genes at one
time using DNA microarray assays
• DNA microarray assays compare patterns of
gene e%pression in different tissues, at different
times, or under different conditions
* +-- Pearson E/ca#ion, Inc.
solate mNA1
/C%N&igure 20'1
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solate mNA,
2
1
3
4
8a)e cDNA by re(ersetranscription? usingfluorescently labelednucleotides,
Apply the cDNA mi'ture to amicroarray? a different genein each spot, /he cDNA hybridi*es
&ith any complementary DNA onthe microarray,
inse off e'cess cDNA: scan microarrayfor fluorescence, ach fluorescent spot!yello&" represents a gene e'pressed
in the tissue sample,
/issue sample
mNA molecules
5abeled cDNA molecules!single strands"
DNA fragmentsrepresenting aspecific gene
DNA microarray
DNA microarray&ith 2?4<<human genes
&igure 20'1a
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DNA microarray
&ith 2?4<<human genes
( t i i 0 F ti
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(eter-inin" 0ene Functi!n
•
:ne way to determine function is to disa!le thegene and o!serve the consequences
• sing in vitro mutagenesis, mutations are
introduced into a cloned gene, altering or
destroying its function
• Ehen the mutated gene is returned to the cell, the
normal gene<s function might !e determined !y
e%amining the mutant<s phenotype
* +-- Pearson E/ca#ion, Inc.
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•
5ene e%pression can also !e silenced using NAinterference !NAi"
• Synthetic dou!le)stranded +NA molecules
matching the sequence of a particular gene are
used to !rea down or !loc the gene<s m+NA
* +-- Pearson E/ca#ion, Inc.
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•
"n humans, researchers analy$e the genomes ofmany people with a certain genetic condition to try
to find nucleotide changes specific to the condition
• 5enetic marers called .NPs >single nucleotide
polymorphisms? occur on average every 1004300 !ase pairs
• SNs can !e detected !y *+, and any SN
shared !y people affected with a disorder !ut notamong unaffected people may pinpoint the
location of the disease)causing gene
* +-- Pearson E/ca#ion, Inc.
&igure 20'19
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DNA
.NP
Normal allele
Disease+causing
allele
/
C
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• :rganismal cloning produces one or more
organisms genetically identical to the -parent. that
donated the single cell
C!ncept #$*5' Cl!nin" !r"anis-s -ay
lea, t! pr!,ucti!n !. ste- cells .!rresearch an, !ther applicati!ns
* +-- Pearson E/ca#ion, Inc.
Cl i Pl t Si l C ll C lt
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Cl!nin" Plants' Sin"le6Cell Cultures
•
:ne e%perimental approach for testing genomicequivalence is to see whether a differentiated cell
can generate a whole organism
• A totipotent cell is one that can generate a
complete new organism
• lant cloning is used e%tensively in agriculture
* +-- Pearson E/ca#ion, Inc.
&igure 20'17
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Crosssection of carrot root
2+mgfragments
6ragments &erecultured in nu+
trient medium:stirring causedsingle cells toshear off intothe li7uid,
.ingle cellsfree in
suspensionbegan todi(ide,
mbryonicplant de(eloped
from a culturedsingle cell,
Plantlet &ascultured on
agar medium,5ater it &asplanted in soil,
Adultplant
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Cl!nin" Ani-als' Nuclear Transplantati!n
•
"n nuclear transplantation, the nucleus of anunfertili$ed egg cell or $ygote is replaced with the
nucleus of a differentiated cell
• %periments with frog em!ryos have shown that a
transplanted nucleus can often support normaldevelopment of the egg
• Fowever, the older the donor nucleus, the lower
the percentage of normally developing tadpoles
* +-- Pearson E/ca#ion, Inc.
6rog embryo 6rog egg cell 6rog tadpoleP8N/&igure 20'1@
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@
5ess differ+entiated cell
Donor
nucleus
trans+
planted
nucleated
egg cell
6ully differ+
entiated!intestinal" cell
Donor
nucleus
trans+
plantedgg &ith donor nucleus
acti(ated to begin
de(elopment
8ost de(elop
into tadpoles,
8ost stop de(eloping
before tadpole stage,
.5/.
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&igure 20'1B
8ammary
/C%N
gg cell
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cell donor
21
3
4
-
>
.5/.
Culturedmammarycells
ggcell from
o(ary
donor
Nucleusremo(edCells fused
Gro&n in culture
mplanted in uterusof a third sheep
mbryonicde(elopment
Nucleus frommammary cell
arly embryo
.urrogatemother
5amb !#Dolly$" geneticallyidentical to mammary cell donor
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Nucleus from
&igure 20'1B!
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4
-
>
.5/.
Gro&n in culture
mplanted in uterusof a third sheep
mbryonicde(elopment
mammary cell
arly embryo
.urrogatemother
5amb !#Dolly$" geneticallyidentical to mammary cell donor
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•
Since 1BB7, cloning has !een demonstrated inmany mammals, including mice, cats, cows,horses, mules, pigs, and dogs
• ** >for *ar!on *opy? was the first cat cloned=
however, ** differed somewhat from her female-parent.
• *loned animals do not always loo or !ehavee%actly the same
* +-- Pearson E/ca#ion, Inc.
&igure 20'20
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Problems Associated wit& Animal Cloning
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Problems Associated wit& Animal Cloning
•
"n most nuclear transplantation studies, only asmall percentage of cloned em!ryos have
developed normally to !irth, and many cloned
animals e%hi!it defects
• #any epigenetic changes, such as acetylation ofhistones or methylation of DNA, must !e reversed
in the nucleus from a donor animal in order for
genes to !e e%pressed or repressed appropriately
for early stages of development
* +-- Pearson E/ca#ion, Inc.
Ste- Cells !. Ani-als
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Ste- Cells !. Ani-als
•
A stem cell is a relatively unspeciali$ed cell thatcan reproduce itself indefinitely and differentiate
into speciali$ed cells of one or more types
• Stem cells isolated from early em!ryos at the
!lastocyst stage are called em!ryonic stem >S?cells= these are a!le to differentiate into all cell
types
•
(he adult !ody also has stem cells, which replacenonreproducing speciali$ed cells
* +-- Pearson E/ca#ion, Inc.
&igure 20'21mbryonicstem cells
Adultstem cells
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Cultured
stem cells
Differentcultureconditions
Differenttypes of differentiatedcells
Cells generatingall embryoniccell types
Cells generatingsome cell types
5i(er cells
Ner(ecells
Bloodcells
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•
+esearchers can transform sin cells into S cells!y using viruses to introduce stem cell master
regulatory genes
• (hese transformed cells are called iS cells
>induced pluripotent cells?• (hese cells can !e used to treat some diseases
and to replace nonfunctional tissues
* +-- Pearson E/ca#ion, Inc.
&igure 20'22
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emo(e s)in cells
from patient, 2
1
3
4
eprogram s)in cells
so the cells become
induced pluripotentstem !iP." cells,
Patient &ith
damaged heart
tissue or other
disease
eturn cells to
patient? &here
they can repair
damaged tissue,
/reat iP. cells so
that they differentiate
into a specific
cell type,
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C!ncept #$*7' The practical applicati!ns !.
(NA techn!l!"y a..ect !ur li%es in -any&ays
• #any fields !enefit from DNA technology and
genetic engineering
* +-- Pearson E/ca#ion, Inc.
/e,ical Applicati!ns
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/e,ical Applicati!ns
•
:ne !enefit of DNA technology is identification ofhuman genes in which mutation plays a role in
genetic diseases
* +-- Pearson E/ca#ion, Inc.
Diagnosis and (reatment of Diseases
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Diagnosis and (reatment of Diseases
•
Scientists can diagnose many human geneticdisorders using *+ and sequence)specific
primers, then sequencing the amplified product to
loo for the disease)causing mutation
• SNs may !e associated with a disease)causingmutation
• SNs may also !e correlated with increased riss
for conditions such as heart disease or certaintypes of cancer
* +-- Pearson E/ca#ion, Inc.
)uman Gene (&era"y
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)uman Gene (&era"y
•
Gene therapy is the alteration of an afflictedindividual<s genes
• 5ene therapy holds great potential for treatingdisorders tracea!le to a single defective gene
• Cectors are used for delivery of genes intospecific types of cells, for e%ample !one marrow
• 5ene therapy provoes !oth technical and ethicalquestions
* +-- Pearson E/ca#ion, Inc.
&igure 20'23 Cloned gene
1 nsert NA (ersion of normal allele
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2
1
3
4
etro(irus
capsid
Bone
marro&
cell from
patient
@iral NA
Bone
marro&
nsert NA (ersion of normal allele
into retro(irus,
5et retro(irus infect bone marro& cells
that ha(e been remo(ed from the
patient and cultured,
@iral DNA carrying the normal
allele inserts into chromosome,
nect engineered
cells into patient,
P&armaceutical Products
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P&armaceutical Products
•
Advances in DNA technology and geneticresearch are important to the development of new
drugs to treat diseases
* +-- Pearson E/ca#ion, Inc.
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• (he drug imatini! is a small molecule that inhi!its
overe%pression of a specific leuemia)causing
receptor • harmaceutical products that are proteins can !e
synthesi$ed on a large scale
.ynthesis of .mall 8olecules for se asDrugs
* +-- Pearson E/ca#ion, Inc.
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• Fost cells in culture can !e engineered to secrete
a protein as it is made, simplifying the tas of
purifying it
• (his is useful for the production of insulin, human
growth hormones, and vaccines
Protein Production in Cell Cultures
* +-- Pearson E/ca#ion, Inc.
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• /ransgenic animals are made !y introducinggenes from one species into the genome ofanother animal
• (ransgenic animals are pharmaceutical -factories,.producers of large amounts of otherwise raresu!stances for medical use
Protein Production by #Pharm$ Animals
* +-- Pearson E/ca#ion, Inc.
&igure 20'26
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&igure 20'26a
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&igure 20'26!
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F!rensic E%i,ence an, 0enetic Pr!.iles
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•
An individual<s unique DNA sequence, or geneticprofile, can !e o!tained !y analysis of tissue or
!ody fluids
• DNA testing can identify individuals with a high
degree of certainty• 5enetic profiles can !e analy$ed using +&
analysis !y Southern !lotting
* +-- Pearson E/ca#ion, Inc.
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•
ven more sensitive is the use of genetic marerscalled short tandem repeats !./s", which arevariations in the num!er of repeats of specificDNA sequences
•
*+ and gel electrophoresis are used to amplifyand then identify S(+s of different lengths
• (he pro!a!ility that two people who are notidentical twins have the same S(+ marers is
e%ceptionally small
* +-- Pearson E/ca#ion, Inc.
&igure 20'2 /his photo sho&s;ashington ust beforehis release in 2<<1?
!a"
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after 1= years in prison,
!b"/hese and other ./ data e'onerated ;ashingtonand led /insley to plead guilty to the murder,
.emen on (ictim
arl ;ashington
enneth /insley
1=?1
1>?19
1=?1
13?1>
14?1-
13?1>
12?12
11?12
12?12
.ource of sample
./mar)er 1
./mar)er 2
./mar)er 3
&igure 20'2a
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/his photo sho&s
;ashington ust beforehis release in 2<<1?after 1= years in prison,
!a"
En%ir!n-ental Cleanup
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p
•
5enetic engineering can !e used to modify themeta!olism of microorganisms
• Some modified microorganisms can !e used to
e%tract minerals from the environment or degrade
potentially to%ic waste materials
* +-- Pearson E/ca#ion, Inc.
A"ricultural Applicati!ns
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" pp
•
DNA technology is !eing used to improveagricultural productivity and food quality
• 5enetic engineering of transgenic animals speeds
up the selective !reeding process
• eneficial genes can !e transferred !etweenvarieties or species
* +-- Pearson E/ca#ion, Inc.
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•
Agricultural scientists have endowed a num!er ofcrop plants with genes for desira!le traits
• (he /i plasmid is the most commonly used vector
for introducing new genes into plant cells
• 5enetic engineering in plants has !een used totransfer many useful genes including those for
her!icide resistance, increased resistance to
pests, increased resistance to salinity, and
improved nutritional value of crops
* +-- Pearson E/ca#ion, Inc.
&igure 20'29 /C%N Agrobacterium tumefaciens
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Plant &ith ne& trait
.5/.
/iplasmid
.ite &hererestrictionen*yme cuts
DNA &iththe geneof interest
ecombinant
/i plasmid
/ DNA
Sa.ety an, Ethical 8uesti!ns Raise, by
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Sa.ety an, Ethical 8uesti!ns Raise, by
(NA Techn!l!"y
• otential !enefits of genetic engineering must
!e weighed against potential ha$ards of
creating harmful products or procedures
• 5uidelines are in place in the nited States
and other countries to ensure safe practices for
recom!inant DNA technology
* +-- Pearson E/ca#ion, Inc.
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•
#ost pu!lic concern a!out possi!le ha$ardscenters on genetically modified !G8" organisms
used as food
• Some are concerned a!out the creation of -super
weeds. from the transfer of genes from 5# cropsto their wild relatives
• :ther worries include the possi!ility that
transgenic protein products might cause allergic
reactions
* +-- Pearson E/ca#ion, Inc.
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• As !iotechnology continues to change, so does its
use in agriculture, industry, and medicine
• National agencies and international organi$ations
strive to set guidelines for safe and ethical
practices in the use of !iotechnology
* +-- Pearson E/ca#ion, Inc.
&igure 20'N03
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3′
3′ 3′
3′
-′
-′
-′
-′C//AA
AA//CG
G
.tic)y end
&igure 20'N06
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DNA fragments from genomic DNA
or cDNA or copy of DNA obtainedby PC
Cloning
(ector
8i' and ligate
ecombinant DNA plasmids
&igure 20'N0
- 3/CCA/GAA//C/AAAGCGC//A/GAA//CACGGC
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Aard(ar) DNA
Plasmid
-′
3′ -′
3′/CCA/GAA//C/AAAGCGC//A/GAA//CACGGC
AGG/AC//AAGA///CGCGAA/AC//AAG/GCCG
G
G
A A
AA / /
/ / C C
&igure 20'N09
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&igure 20'N07
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&igure 20'N0@
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