Transcript Chapt. 4 Molecular Cloning Methods Learning outcomes: • Explain major tools of recombinant DNA technology – date from 1970s • Expand understanding of new.

Chapt. 4 Molecular Cloning Methods
Learning outcomes:
• Explain major tools of recombinant DNA
technology – date from 1970s
• Expand understanding of new tools
• Impt. Figs: 1, 2, 3, 4, 5, 10,
11, 12, 15*, 16, 17, 18, 20,
23, 24, 25, 26; Table 1
• Review Q: 2, 4*, 6, 7, 8, 9*,
10, 11, 13*, 14*; AQ 1
4-1
4.1 Gene Cloning
in Bacteria
• Link prokaryotic or eukaryotic
genes to small bacterial DNAs
– (plasmids or phage)
• Insert recombinant molecules
into bacterial hosts to replicate
• Can produce large quantities of
these genes (or proteins) in
pure form
Fig. 4
4-2
Type II Restriction
Endonucleases
• Discovered late ‘60s
• Named for organism
• Recognize specific DNA
sequence, cut ONLY at
that sequence
– Recognize 4-bp to 8-bp
sequences
– Frequency of cuts less
when recognition
sequence is longer: (1/4)n
– (size of piece larger ~4n)
4-3
Restriction Endonucleases, DNA Ligase
• Many make staggered cuts in DNA strands
– Single-stranded overhangs, sticky ends, can base-pair
together (5’-PO4, 3’-OH ends)
– Makes joining 2 different DNA molecules easier
– DNA ligase needs 5-PO4, 3’-OH ends + ATP
• Recognition sequence
usually symmetrical
palindromes
EcoRI: 5’-GAATTC-3’
3’-CTTAAG-5’
4-4
Restriction-Modification
System
• Enzymes cut foreign DNA,
protect cells
• What prevents enzymes
from cutting up host DNA?
– paired methylases recognize,
methylate, same site
• Form restriction-modification
system, R-M system
• Methylation protects DNA;
after replication, parental
strand is partially methylated
Fig. 1
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Early Gene Cloning –
recombinant DNA
• Boyer & Cohen used EcoRI to
cut 2 plasmids, small circular
pieces of DNA independent of host
chromosome
• Each plasmid had site for EcoRI
– Linear DNA with same sticky ends
– Sticky ends base pair
• Some ends re-close
• Others join 2 pieces
• DNA ligase joins 2 pieces with
covalent bonds
• Transform E. coli; select drugr
Fig. 2
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Vectors
• Vectors function as DNA carriers, permit replication
of recombinant DNAs
• Typically 1 vector, 1 piece of foreign DNA
– Need vector for replication
– Foreign DNA has no origin of replication,
• (site where DNA replication begins)
• Prokaryotic vectors:
– Plasmids; Phages
• Selectable marker,
• Restriction sites for cloning
• Origin of replication
pBR322
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pBR322 Cloning
Clone foreign DNA into PstI site
of pBR322
• Cut vector to generate sticky
ends with PstI
• Cut foreign DNA with PstI
• Combine vector and foreign
DNA: DNA ligase seals
• Transform E. coli
–(CaCl2, electroporate)
• Select Tetr
Fig. 4
4-8
Screening Transformants
pBR322
• Transformation yields bacteria :
– Religated plasmid
– Religated insert
– Recombinants
• Identify transformants, recombinants
with antibiotic resistance
– In tetracycline, only cells with plasmid
grow, not just foreign DNA pieces
– Test copies of original colonies with
ampicillin (kills cells with plasmid
including foreign DNA)
– Identify desired recombinants
Fig. 4
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Screen With Replica Plating
• Transfer copies of clones
from original tetracycline
plate to plate with ampicillin
• Transfer with sterile velvet
• Desired recombinant
colonies do NOT grow on
ampicillin plate
• Pick clone from original
plate
Fig. 5
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pUC plasmid has b-galactosidase fragment
– Ampicillin resistance gene
– MCS in lacZ’ (N-terminal a-peptide of b-galactosidase)
– Foreign DNA in MCS disrupts ability to make b-gal apeptide:
– On X-gal media: clones producing functional b-gal enzyme
(from a and host b peptide) produce blue colonies; clones
with insert are white.
Fig. 6 Fig. 6
4-11
Summary Basic Plasmid Cloning Vectors
• pBR322 and the pUC plasmids
• pBR322 has
– 2 antibiotic resistance genes
– Many unique restriction sites for inserting foreign DNA
– Most of these sites interrupt antibiotic resistance, making
screening straightforward
• pUC has
– Ampicillin resistance gene
– MCS that interrupts the b-galactosidase gene a-peptide
• MCS also facilitates directional cloning, using pieces cut
with 2 different restriction enzymes
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Phages As Vectors
• Bacteriophages: natural vectors transduce bacterial
DNA from one cell to another
• Phage vectors infect cells much more
efficiently than plasmids transform cells
lambda
• Clones are not colonies of cells, but rather plaques,
clearing of bacterial lawn due to
phage infect, kill bacteria in area
4-13
Lambda (l) Phage Vectors
• Replacement vectors:
– Removed middle region (needed only for lysogeny)
– Retained genes for phage replication
– Replace removed phage genes with foreign DNA
• Phage vectors hold larger amounts of foreign DNA
– Accept up to 20-kb of DNA
– Traditional plasmid vectors usually less
• Lambda phage vectors require minimum size DNA
(12 kb) insert to package into phage particle
4-14
Cloning Using
a Lambda
Phage Vector
• Join DNA in vitro
• Add packaging
proteins from cell
extract
• Infect cells
Fig.4-15
8
Genomic Libraries
• Large number of clones: all genes from species’
genome
• Restriction fragments can be packaged into phage,
about 16 – 20 kb per fragment (often partial digests)
• Need thousands of clones for library:
ex. Yeast genome 107 bp/ 104 bp/clone -> ____ clones
ex. Human genome 3 x 109 bp /104 bp/clone -> ___
• Once library is established, search for gene of
interest (BL 415 experiment with plasmid library)
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Plaque Hybridization
• Search genomic library
with probe to identify
clone containing desired
gene
• Ideal probe – labeled
nucleic acid with
sequence matching the
gene of interest
Fig. 9 4-17
Cosmids
Cosmids designed to clone large DNA fragments
– Behave as plasmid and phage
– Contain
• cos sites, cohesive ends of l phage DNA, allow DNA
to be packaged into l phage head
• Plasmid origin of replication permits replication as a
plasmid in bacteria
– Most l genome removed so hold large inserts (40-50 kb)
– So little phage DNA, can’t replicate like phage; infectious,
carry recombinant DNA into bacterial cell, then plasmid
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M13 Phage Vectors
• Long, thin, filamentous phage M13
– Normal size 6.4 kb (10 genes)
• Modified vector contains:
– Gene fragment with b-galactosidase
– Multiple cloning site like pUC plasmid
• Advantages
– Can hold large DNA; length of phage increases
– Phage genome is single-stranded DNA
– Fragments cloned (into ds intracellular form)
are recovered in single-stranded form
– Phage does not kill cells; extrudes particles
– Useful for sequencing DNA, mutagenesis
4-19
M13 Cloning permits recovery of Singlestranded DNA
• After infecting E. coli, singlestranded phage DNA was converted
to double-stranded replicative form
(RF, plasmid-like)
• Purify replicative form to clone
foreign DNA into MCS; ligate vitro
• Transform recombinant DNA into
host cells, results in plasmid-like RF
molecule in cells
• Phage particles, contain one singlestranded DNA (+ strand), secreted
from transformed cells, can be
collected from media
Fig. 10
4-20
Phagemids are
versatile Vectors:
pBluescript
(pBSK)
Fig. 11
Aspects of both phages and plasmids:
– Ampr for selection of clones
– MCS inserted into lacZ’ gene permit blue/white screening
– Origin of replication of single-stranded phage f1 permits
recovery of single-stranded recombinant DNA (like M13)
– MCS has phage RNA polymerase promoters, 1 on each
side of MCS; permits specific RNA transcripts in vitro
4-21
Eukaryotic Vectors,
High Capacity vectors
• Plasmids and Viruses
clone genes in eukaryotic cells
– Plasmid shuttle vectors (origins of replication for both
bacterial and eukaryotic host cell; ex. Yeast YEP24
– Selectable markers in both hosts; MCS sites
• Yeast artificial chromosomes (YAC), bacterial
artificial chromosomes (BAC) clone huge pieces
• Vectors based on Ti plasmid for plant cells
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Identifying specific clone with specific probe
• Probes can identify a desired clone from among
thousands of irrelevant ones:
• Two types widely used:
Recognize gene or mRNA with Oligonucleotides:
Short RNA or DNA sequences that– hybridize
– Can use homologous gene from another organism
– If amino acid sequence is known, deduce possible
nucleotide sequences to code for these amino acids
Recognize specific protein product with Antibodies
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cDNA Cloning from Eukaryotes into Bacteria
• cDNA: complementary DNA or copy DNA
– made from mRNA
• A cDNA library: set of clones representing many
mRNAs from cell type or tissue or time
– Library can contain thousands of different clones
– * Need cDNA clones because genomic clones
have introns, aren’t expressed in bacteria
4-24
Making cDNA Library
from eukaryotic mRNA
Reverse Transcriptase (RT)
• mRNA -> DNA
Primer
• oligo(dT) to polyA at 3’ end
RNAse H degrades RNA
DNA polymerase Pol I finishes
Terminal transferase to add C
tails and G tails for cloning
Fig. 12
4-25
Vector choice for cDNA cloning
• Purpose and method to detect positive clones
• If cloning gene and small fragment, plasmid or
phagemid like pUC or pBS used, with colony
hybridization and labeled DNA probe
• If large fragments, use cosmids, or l phage or YAC,
BAC
• For expression of gene product, use cDNA of
eukaryote cloned in pUC, phagemids of lambda
– under control of lac promoter for transcription and translation
of gene; use hybridization, antibody screening (more later)
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Summary clone cDNA into Bacteria
• Make cDNA library with synthesis of cDNAs one
strand at a time
– Use mRNAs from a cell as templates for 1st strands, then
1st strand as template for 2nd
– Reverse transcriptase generates 1st strand
– DNA polymerase I generates 2nd strand
• Give cDNAs oligonucleotide tails that base-pair with
complementary tails on a cloning vector
• Use recombinant DNAs to transform bacteria
• Detect clones with:
– Colony hybridization using labeled probes
– Antibodies if gene product is translated
4-27
Polymerase chain reaction (PCR):
amplifies DNA exponentially;
yields DNA fragments for cloning, forensics, diagnostics
Fig. 15
4-28
Standard PCR
• Invented by Kary Mullis in 1980s
• DNA polymerase copies selected region of DNA
– Short DNA primers hybridize to DNA on either side of piece
of interest – initiate DNA synthesis through that area, X
– Selected region DNA doubles in amount each cycle
• Special heat-stable polymerases work after high
temperatures that separate (denature) strands
simplifies process – ex. Taq polymerase
• Real-time PCR uses fluorescent dyes to quantify
amplification, reflect amount of mRNA or DNA in cells
– (Fig. 17)
4-29
Reverse transcriptase PCR
(RT-PCR) to clone specific
cDNA
Start with mRNA, not dsDNA
Convert mRNA to DNA with
primer specific to gene
Use forward primer to convert
ssDNA to dsDNA
Then standard PCR
• With primer choice, restriction
enzyme sites added to cDNA
• 2 sticky ends permit directional
cloning – good for protein expression
Fig. 16
4-30
4.3 Expressing Cloned Genes
• Cloned gene permits production of large quantities of
particular DNA sequence
• Expression vectors yield protein products
– Strong promoter, ribosome binding site near ATG codon
– Make as pure protein, or as fusion to bacterial peptide
• Regulated expression:
Inducible expression vectors keep cloned gene
repressed until time to express:
– Large quantities of eukaryotic protein can be toxic
4-31
Fusion proteins:
Oligo-histidine
(His-tagged)
Expression Vector
Sequence upstream of MCS encodes
6 His which get added to protein:
• Oligo-histidine high affinity for
divalent metal ions (Ni2+)
• Purify protein by nickel affinity
chromatography
• His tag removed using enzyme
enterokinase if needed
Fig. 20
4-32
Fusion Proteins
with lacZ’
• Cloning vectors (pUC and pBS)
are expression vectors using lac
promoter with lacZ’ a-peptide
• If inserted DNA in same reading
frame as interrupted gene,
fusion protein results
–
–
–
–
Partial b-galactosidase at N- end
Inserted protein at C-end
Fusions often more stable
Fusions also assist detection,
purification
Fig.4-33
18
Regulation: lac Promoter/ operator
• lac promoter
inducible with
lactose or
synthetic
IPTG; binds
repressor and
removes it
from O
• Transcription
stays off until
induced
4-34
Fusion Proteins also in lgt11 vector
• Phage contains lac control
region and lacZ’ gene (apeptide)
• Products of gene correctly
inserted are fusion
proteins with bgalactosidase peptide
• Use blue-white screening
on host with b-peptide
• Fusion reduces
degradation of foreign
protein
Fig. 21
4-35
Antibody screening with lgt11 (or plasmids)
• Lambda phages with
cDNA inserts on E. coli
• Proteins released are
blotted onto membrane
• Probe with specific 1o
antibody to protein
• Antibody bound to
protein from plaque is
detected with labeled 2o
antibody
Fig. 22
4-36
Summary – Expression in Bacteria
• Expression vectors can produce fusion proteins
– One part from coding sequences in vector
– Other part from sequences in cloned gene
• Many fusion proteins can be simply purified by
affinity chromatography (His-tag; Flag-tag; TAP-tag)
• Fusion proteins can be detected with antibodies
after blotting of colonies or plaques:
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Bacterial Expression System Shortcomings
• Bacteria recognize proteins as foreign and destroy
• Posttranslational modifications (glycosylation,
phosphorylation) are not done correctly
• Bacterial cytoplasm not permit correct protein folding
• High levels of cloned eukaryotic proteins can be
expressed in useless, insoluble (inclusion) form
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Use Eukaryotic Expression Systems
• Initial cloning in E. coli using shuttle vector, that
replicates in both bacterial and eukaryotic cells
• Ex. Yeast Saccharomyces cerevisiae well-suited :
– Rapid growth, ease of culture
– Multicopy plasmids (from natural 2 m plasmids)
– Eukaryote, so more appropriate posttranslational
modifications to expressed proteins
– Can secrete protein for easy purification
– Ex. Plasmid YEP24 shown earlier
4-39
Ex. Baculovirus expression vectors;
transform (transfect)
insect cells in culture
Large circular DNA genome;
Major viral structural protein
made in huge amounts
– Strong promoter for
polyhedrin protein
– Produce lots of recombinant
protein per liter of medium
– Non-recombinant viral DNA
do not replicate because
lack essential gene
Fig. 23
4-40
Methods for Transfection of animal cells with
vectors for clones or protein production
[Transformation for animal cells used for cancer cells]
• Calcium phosphate
– Mix cells with DNA in phosphate buffer
– Solution of calcium salt added to form precipitate
– Cells take up calcium phosphate crystals, includes DNA
• Electroporation
– Electric shock for DNA to enter cells
• Liposomes
– DNA mixed with lipid to form liposomes, small vesicles
– Liposomes fuse with cell membrane, carry DNA inside
4-41
Summary
• Foreign genes can be cloned and expressed
in eukaryotic cells
• Eukaryotic systems have some advantages:
– Proteins tend to fold properly, are soluble rather
than aggregated into insoluble inclusion bodies
– Posttranslational modifications (phosphorylation,
glycosylation) made in eukaryotic manner
– Can use transient clones for tests gene function
– Select stable clones expressing transgene (as for
construction transgenic organisms)
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Review problems
2. Why does one need to attach DNAs to vectors to
clone them?
4. Diagram the process of cloning a DNA fragment
into BamHI and PstI sites of vector pUC18. How
would you screen for clones that contain an insert?
Explain the blue/white lacZ’ detection system
7. You want to make a genomic library with DNA
fragments averaging about 45 kb in length. Which
vector discussed in this chapter would be most
4-43
appropriate? Why?
Review problems
11. Diagram a method for creating a cDNA library.
13.Outline the polymerase chain reaction (PCR)
method for amplifying a given stretch of DNA.
14.What is the difference between reverse
transcriptase PCR (RT-PCR) and standard PCR?
For what purpose would you use RT-PCR?
For what purpose might you want cDNA vs genomic
4-44
library?
Review problems
AQ 1. Given an amino acid sequence of part of a
protein from a hypothetical gene you want to clone:
Pro-arg-tyr-met-cys-trp-ile-leu-met-ser
Look at the code chart
a. What sequence of 5 aa would give a 14-mer probe with the
least degeneracy for probing a library to find gene of
interest?
b. How many different 14-mers would you need to make to be
sure probe matches sequence in cloned gene perfectly?
c. lf you started your probe one aa to the left of the one you
chose in (a), how many different 14-mers would you have to
make?
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