concept 20.1: dna cloning yields multiple copies of a gene or other dna segment to work directly...
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Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment
• To work directly with specific genes, scientists prepare well-defined segments of DNA in identical copies, a process called DNA cloning
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DNA Cloning and Its Applications: A Preview
• Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids
• Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
• Cloned genes are useful for making copies of a particular gene and producing a protein product
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• Gene cloning involves using 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 in the bacterial cell results in cloning of the plasmid including the foreign DNA
• This results in the production of multiple copies of a single gene
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Figure 20.2 Bacterium
Bacterialchromosome
Plasmid
2
1
3
4
Gene inserted intoplasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene of interest
Plasmid put intobacterial cell
DNA ofchromosome(“foreign” DNA)
Recombinantbacterium
Host cell grown in culture toform a clone of cells containingthe “cloned” gene of interest
Gene of interest
Protein expressed fromgene of interest
Protein harvestedCopies of gene
Basic researchand variousapplications
Basicresearchon protein
Basic research on gene
Gene for pestresistance insertedinto plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growthhormone treatsstunted growth
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Figure 20.2a
Bacterium
Bacterialchromosome
Plasmid
2
1 Gene inserted intoplasmid
Cell containing gene of interest
RecombinantDNA (plasmid)
Gene of interest
Plasmid put intobacterial cell
DNA ofchromosome(“foreign” DNA)
Recombinantbacterium
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Figure 20.2b
Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
Gene of interest
Protein expressed fromgene of interest
Protein harvestedCopies of gene
Basic researchand variousapplications
3
4Basicresearchon protein
Basic research on gene
Gene for pestresistance insertedinto plants
Gene used to alterbacteria for cleaningup toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growthhormone treatsstunted growth
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Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
• A restriction enzyme usually makes many cuts, yielding restriction fragments
• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends.”
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© 2011 Pearson Education, Inc.
Animation: Restriction Enzymes
Right-click slide / select “Play”
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• Sticky ends can bond with complementary sticky ends of other fragments
• DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 20.3-1
Restriction enzymecuts sugar-phosphatebackbones.
Restriction site
DNA5
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1
Sticky end
GAATTCCTTAAG
CTTAAG AATTC
G
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Figure 20.3-2
One possible combination
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
Restriction enzymecuts sugar-phosphatebackbones.
Restriction site
DNA5
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Sticky end
GAATTCCTTAAG
CTTAAG AATTC
G
GGAATTC
CTTAA
GG
GG
AATT CAATT CC TTAA C TTAA
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Figure 20.3-3
Recombinant DNA molecule
One possible combinationDNA ligaseseals strands
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
Restriction enzymecuts sugar-phosphatebackbones.
Restriction site
DNA5
5
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55
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Sticky end
GAATTCCTTAAG
CTTAAG AATTC
G
GGAATTC
CTTAA
GG
GG
AATT CAATT CC TTAA C TTAA
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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 cut with the same restriction enzyme
– The fragments are mixed, and DNA ligase is added to bond the fragment sticky ends
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Animation: Cloning a Gene
Right-click slide / select “Play”
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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 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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Figure 20.4
Bacterial plasmidTECHNIQUE
RESULTS
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
Recombinant plasmids Nonrecombinant plasmid
Bacteria carryingplasmids
Colony carrying non-recombinant plasmidwith intact lacZ gene
Colony carrying recombinantplasmidwith disruptedlacZ gene
One of manybacterialclones
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Figure 20.4a-1
Bacterial plasmidTECHNIQUE
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
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Figure 20.4a-2
Bacterial plasmidTECHNIQUE
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
Recombinant plasmids Nonrecombinant plasmid
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Figure 20.4a-3
Bacterial plasmidTECHNIQUE
ampR gene lacZ gene
Restrictionsite
Hummingbird cell
Sticky ends Gene of
interest
Humming-bird DNAfragments
Recombinant plasmids Nonrecombinant plasmid
Bacteria carryingplasmids
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Figure 20.4b
RESULTS
Bacteria carryingplasmids
Colony carrying non-recombinant plasmidwith intact lacZ gene
Colony carrying recombinantplasmidwith disruptedlacZ gene
One of manybacterialclones
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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
• A genomic library that is made using bacteriophages is stored as a collection of phage clones
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Figure 20.5
Foreign genome
Cut with restriction enzymes into eithersmallfragments
largefragments
or
Recombinantplasmids
Plasmidclone
(a) Plasmid library
(b) BAC clone
Bacterial artificialchromosome (BAC)
Largeinsertwithmanygenes
(c) Storing genome libraries
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Figure 20.5a
(c) Storing genome libraries
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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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• A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell
• A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells
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Figure 20.6-1
DNA innucleus
mRNAs incytoplasm
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Figure 20.6-2
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
55
33
A A A A A AT T T T T
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Figure 20.6-3
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
55
55
33
33
A A A A A A
A A A A A A
T T T T T
T T T T T
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Figure 20.6-4
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
DNA polymerase
55
55
55
33
33
33
A A A A A A
A A A A A A
T T T T T
T T T T T
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Figure 20.6-5
DNA innucleus
mRNAs incytoplasm
mRNA
Reversetranscriptase Poly-A tail
DNAstrand
Primer
DNA polymerase
cDNA
55
55
55
55
33
33
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33
A A A A A A
A A A A A A
T T T T T
T T T T T
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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
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5 3 CTCAT CACCGGC
53 G A G T A G T G G C C G
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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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Figure 20.7
Radioactivelylabeled probemolecules Gene of
interestProbeDNA
Single-strandedDNA fromcell
Film
Location ofDNA with thecomplementarysequence
Nylonmembrane
Nylon membrane
Multiwell platesholding libraryclones
TECHNIQUE 5
53
3
GAGTAGTGGCCG CTCATCACCGGC
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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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