Chapter 4. Recombinant DNA Technology
I. Molecular Cloning
II. Restriction Endonucleases
III. Cloning Vectors
IV. Creating and Screening Gene Libraries
V. Cloning Eukaryotic DNA
VI. Transformation, Transfection, Transduction and Conjugation

I. Molecular Cloning

· Molecular cloning involves isolation of a cell line, derived from a single cell, that contains a gene or DNA fragment from another organism (foreign DNA) inserted into a independently-replicating cloning vector.
See Fig. 4.1
· 5 Main steps
1. Extract total genomic DNA from cells containing target DNA
2. Use a restriction enzyme to cut the DNA into smller fragments and to linearize the cloning vector.
3. Join (ligate) the DNA fragments to the cloning vector to produce recombinant DNA molecules
4. Introduce the recombinant vector into a host cell.
5. Identify and isolate a strain containg the cloned target DNA.
 
· Most of the discussion in this chapter assumes that you know that a prokaryotic organism has a potentially useful gene (the target) that you would like to isolate by cloning. But, you do not know its location in the genome or its nucleotide sequence, preventing you from specifically cloning just the target gene. Therfore, you must create a gene library of the entire genome that you then search for the target gene. Eukaryotic genes can also be cloned with this approach but a somewhat different strategy is often used for biotechnology applications and is described at the end of this web page.

II. Restriction Enzymes (Restriction Endonucleases)
 
Natural Function
Protection of cell from infection by foreign DNA (bacteriophage viruses)
Component of restriction-modification system (see below)
 
Three types (I, II, III)
Type II for Recombinant DNA Methods
-Specific for particular nucleotide sequence
-Cleave DNA in a reproducible manner --always the same for a particular enzyme
Only Mg⁺⁺ is required for activity
-Found in many species of bacteria
Named after species in which they were first discovered
Ex. EcoR I from Escherichia coli
-Over 200 different restriction enzymes are commercially available
 
Restriction-Modification Systems
Two components
1. Restriction enzyme. Cleaves foreign (bacteriophage) DNA to protect cell from infection.
EcoRI. Recognizes 6 base-pair palindromic sequence of DNA.
 
---GAATTC---
---CTTAAG---
 
---G............AATTC---
---CTTAA............G---
 
Cleaves DNA backbone of each strand by catalyzing hydrolysis of phosphodiester bonds and produces a staggered cut
 
 
2. Modification enzyme. Modifies (methylates) restriction sites present in the cell's DNA to protect them from cleavage by the restriction enzyme.
CH₃ is added to the red A bases of the top and bottom strands which inhibits cleavage by EcoR I
---GAATTC---
---CTTAAG---
 

Restriction Enzymes (cont.)
Some produce staggered ("sticky") ends, others blunt ends.
See Figs. 4.2 and 4.3
 
Some recognize short sequences (4 nucleotides long) , others longer sequences (6 to 8 nucleotides long)
See Table 4.1
 
Use of a Restriction Enzyme to Make a Recombinant DNA Molecule
See Fig. 4.6
Ex. BamHI from Bacillus amyloliquefaciens
Recognizes and cuts the sequence (between the two adjacent Gs of each strand)
-GGATCC-
-CCTAGG-
Steps:
1. Digest the DNA from two different sources with the enzyme
2. Mix the fragments produced from both sources together
The fragments will recombine, in a variety of different combinations, because of complimentary base pairing of the sticky ends.
 
3. Use a DNA ligase to seal the nicks (reform the phosphodiester bonds) in the backbone of the DNA strands.
See Fig. 4.7
ATP is required for the reaction to occur
Ligation of DNA fragments with sticky ends is more efficient than ligation of blunt ends
 

III. Cloning Vectors
1. Plasmids.
Efficient for cloning fragments up to ~ 10 kbp
(kbp = kilobase pairs )
1 kbp = 1000 base pairs = double stranded DNA 1000 nucleotides long
 
2. Bacteriophage lambda.
~ 9 to 23 kbp fragments
 
3. Cosmids.
~ 40 kbp fragments
 
4. Yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and bacteriophage P1 artificial chromosomes (PACS).
~ 100 to >2,000 kbp

Plasmids as Cloning Vectors
Plasmids
· Found in most bacteria (also yeast and plants) as extrachromosomal DNA
· Circular, double stranded DNA (dsDNA)
· Size of different plasmids ranges from ~1 to 500 kbp
· Replicates in host cell, usually more than one copy produced.
· Low copy number or high copy number
low : ~1 to 5 copies per cell.
high ~10 to 100's of copies per cell
 
· Carry nonessential genes but they may provide selective advantage to host cell under certain conditions. (e.g. antibiotic resistance genes)
· May have a narrow or broad range of species that can serve as hosts
· Some plasmids are incompatible with other plasmids
Cannot reside in the same cell together
 
Desirable Features of a Plasmid Cloning Vector
Unique restriction site (occurs only once): for introducing foreign DNA
Small size: for efficient introduction into the host cell
Replicates in host: often want high copy number and broad host range
Selectable marker and/or reporter genes that help to:

1. Maintain plasmid in host cell
2. Identify cells that contain the plasmid
 
Naturally occurring plasmids have been genetically engineered to posses these features for use as cloning vectors

Cloning with the plasmid cloning vector pBR322
See Figs. 4.8 and 4.9
Size 4.4 kbp
Unique restriction sites: PstI, EcoRI, HindIII, BamHI and SalI
Origin of replication: ori
Two selectable markers: resistance genes for the antibiotics tetracycline and ampicillin
Steps:
1. In separate reactions, cut pBR322 and source DNA with the same restriction enzyme (e.g. Pst I)
Pst I cuts within the Amp resistance gene.
Foreign DNA inserted into this site disrupts the gene so that it cannot protect cells from exposure to ampicillin
2. Remove the 5' phosphate groups from the vector with alkaline phosphatase
This prevents recircularization of the vectors lacking an insert
3. Mix the two restricted DNA samples together and allow the sticky ends to anneal to each other
4. Seal nicks with a DNA ligase (e.g. T4 DNA ligase)
One of the knicks in each strand cannot be sealed because the vector was dephosphorylated
5. Introduce the DNA into a host by transformation and plate onto a selective medium ( containing tetracycline in this example)
Cells containing a plasmid will grow, the nicks in the plasmid will be sealed and the plasmid will be replicated
6. Transfer colonies to a plate containing ampicillin
Only cells that contain a recircularized plasmid without a DNA insert in the ampicillin resistance gene will grow
7. Isolate colonies that grew on the tetracycline-containing plate but not on the ampicilling-containing plate.
8. Screen for the target gene (see below).

The lac operon
Normal function: allows E. coli to utilize lactose
lacZ gene encodes b-galactosidase
Hydrolyzes lactose or X-gal --artificial substrate, turns blue
Operon is under the transcriptional control of lac promoter/operator
lacI gene encodes a repressor protein (LacI)
LacI binds to lac promoter/operator and blocks transcription
Natural effector is a form of lactose (allolactose) that induces expression
IPTG (isopropylthiogalactopyranoside): chemical that is an artificial inducer
Binds to LacI and prevents it from binding to lac operator
Lac operon is transcribed

pUC19 Cloning Vector
See Fig. 4.10
Smaller than pBR322, can clone larger DNA fragments
Selectable marker:
Ampr selects for cells containing a plasmid
Origin of replication
Promoter/operator from lac operon
Multiple cloning site allows choice of more restriction enzymes
Under transcriptional control of lac promoter/operator
Located between lac promoter/operator and within lacZ'
lacZ', to identify cells containing a recombinant plasmid by colony color
Encodes only the amino terminal end of b-galactosidase (the a-peptide)
 
pUC19 is used with an E. coli host that contains lacZDM15 on the chromosome
lacZDM15 contains a deletion mutation of lacZ and is missing the sequences that encode the amino-terminal end (i.e. lacking the lacZ' sequence) of b-galactosidase.
Neither lacZ' or lacZDM15 alone codes for an acitve b-galactosidase
Expression of lacZ' and lacZDM15 in the same cell produces an active form of b-galactosidase
DNA inserted into the multiple cloning site disrupts lacZ', preventing expression of active b-galactosidase
 
Detection of E. coli cells containing pUC19 with cloned DNA
· After transformation of E. coli with pUC19, plate cells onto a solid medium containing IPTG, X-gal and ampicillin
· As stated above, pUC19 has lac Z' which encodes the amino terminus of b-galactosidase
· The rest of the gene (lacZDM15) is present on the chromosome of E. coli
· When pUC19 is inside E. coli, expression of both lacZ' and lacZDM15 produces a functional b-galactosidase enzyme which hydrolyzes X-gal, producing a blue product.
These cells contain pUC19 without cloned DNA (lacZ' was not disrupted)
· DNA cloned into the multiple cloning site disrupts lacZ', preventing expression of the a-peptide
Cells containing pUC19 with cloned DNA are white
 
Cells w/o a plasmid will not grow because _________ ?

Bacteriophage lambda (l) as a cloning vector
See Figs. 4.24 and 4.25
A virus that infects E. coli cells
Three main components
1. Head: contains the viral DNA
2. Tail fiber: used to inject viral DNA into the host cell
3. DNA: encodes genes necessary for infection of host cells, replication and production of new viral particles
 
lambda DNA is linear and about 50 kbp long
Cos sites. Single stranded extensions present on each end
They are cohesive (complimentary to each other)
Allow l DNA to form a circle inside cells during replication
 
Stuffer DNA. About 20 kbp in the center
Not required by the virus to infect cells
Flanked by BamH I restriction sites
Can be replaced with foreign BamH I DNA fragments of ~9 to 23 kbp
(Smaller or larger fragments do not produce infectious phage)
 
In vitro packaging
Laboratory procedure that produces infective l phage particles
Mix together recombinant l DNA, empty phage heads and phage tails
Use to infect E. coli and grow as a lawn of cells on a solid medium
Clear plaques on the plate are areas where recombinant phage have lysed the
host's cells
Screen for the target gene (see below)
Inoculate fresh cells with phage picked from the plaques

Vectors for Cloning Larger DNA Fragments (40-2000 kbp)
Ex. Large multigene prokaryotic operons and eukaryotic genes with introns
1. Cosmid. ~40 kbp of insert DNA
See Fig. 4.26
Combines plasmid cloning vector with phage cos sites
Plasmid ori maintains cosmid as a circular plasmid in host cells (cells aren't lysed)
Cos sites allow in vitro packaging and introduction of DNA into host cells via infectious l phage
Selectable markers (e.g resistance to tetracycline)
 
2. Artificial chromosomes. ~ 100 to > 2,000 kbp of insert DNA
Ex. Yeast artificail chromosomes
Maintained as a chromosome in yeast host cells
Multiple cloning site
Yeast origin of replication
Centromere for partitioning to daughter cells during mitosis
Telomeres at ends for chromosome stability
Selectable marker

IV. Creating a Gene Library
Goal: Produce a population of host cells containining a recombinant vector carrying DNA fragments of a target organism that represents its entire genome AND that contains an intact copy of the gene of interest.
1. Isolate genomic DNA from an organism that contains the target gene(s).
2. Partially digest the DNA with a restriction enzyme.
See Fig. 4.12
Use a restriction enzyme that cuts frequently
e.g. Sau3A I. A 4 base pair cutter
GATC
CTAG
4⁴ = 256 different 4 base sequences. Theoretically restriction site would occur
every 256 bases.
 
Partial digestion results in less frequent cutting, producing fragements > 256 bp
Limit time of digestion or amount of enzyme used.
3. Insert (ligate) fragments into cloning vector.
4. Introduce the recombinant vector into host cells..

Screening a DNA Library for Clones Containing the Target DNA
 
1. DNA hybridization. Detects target DNA with a labeled DNA probe .
Requires:
1. Knowledge of the DNA sequence of the target DNA, or
2. DNA previously cloned from another organism that has a nucleotide sequence closely related to that of the target DNA
**Does not require expression (transcription/translation) of the gene
 
2. Immunoassay. Detects the gene product (protein) using antibodies

Requires:
1. Expression
2. Purified protein to produce antibodies
 
3. Detection of enzymatic activity of the gene product.
Requires:
1. Expression
2. Gene product is an enzyme that catalyzes formation of a detectable compound
 
 
4. Complementation of a mutation in the gene you wish to clone
Requires:
1. Expression
2. Availability of a strain containing the mutated gene

Detection of Target DNA by Hybridization with Labeled DNA Probes
See Fig. 4.14 and Fig. 4.17
Steps
1. Make the target DNA single stranded (denatured).
2. Immobilize ssDNA by binding to a solid support (Ex. nitrocellulose membrane).
3. Add a labeled ssDNA probe with a nucleotide sequence that is the same as that of target DNA.
4. Allow the probe to bind (hybridize) to the complimentary target DNA sequence.
5. Detection of labeled DNA on the membrane shows the location of the target DNA.

Random Primer Method for Producing Labeled DNA Probes
See Fig. 4.15
Steps
1. Need template DNA for synthesis of the probe.
A. DNA with a nucleotide sequence that is the same or very similar to that of the target DNA.
e.g. DNA from a close relative.
B. DNA that is known to be located near the target.
e. g. a gene from the same operon as the target
 
2. Hybridize short oligonucleotides to act as primers for DNA synthesis.
e.g. hexamers (6 nucleotides); there are 4⁶ = 4096 different hexamers
 
3. Add a DNA polymerase and the 4 different dNTPs (dATP*, dTTP, dGTP and dCTP).
Use dATP that is labeled* with something that can be easily detected
e.g. radioactive phosphorus, ³²P
 
4. After synthesis is complete, denature the dsDNA and use in a hybridization procedure to detect the target.

Library Screening by Immunoassay
See Fig. 4.18
Procedure is similar to the one for labeled probes, except the gene product is detected rather than the gene.
A primary antibody that binds specifically to the gene product is needed in addition to a secondary antibody that is conjugated to an enzyme.
The enzyme converts a colorless substrate to a colored product.
Detection of the colored product shows the location of the colony that contains the cloned gene.
You produce the primary antibody by injecting the gene product (protein) into a rabbit.
You purchase the secondary antibody from a company that produced it by injecting rabbit antibodies into a goat. The goat produced anti-rabbit antibodies which the company isolated and then attached the enzyme. The goat anti-rabbit antibodies will bind to the rabbit antibodies which are bound to the protein.

Screening by Enzyme Activity
Ex. Identification of a clone containing the Pseudomonas xylE gene which encodes the enzyme catechol dioxygenase
Library cells expressing catechol dioxygenase (the gene product of xylE) oxidize catechol to a yellow product

Screening by Complementation of a Mutation
Ex. Identification of a clone containing xylE.
Use a strain of Pseudomonas with xylE containg a mutation that prevents expression of a functional catechol dioxygenase
This strain cannot oxidize catechol and cannot use it for growth.
Transfer vector DNA from the library into the mutant cells via transformation or conjugation
Plate on a minimal medium containing catechol as the sole source of carbon and energy.
Complementation of the mutation by a cloned xylE gene restores the ability of Pseudomonas to grow on catechol.

V. Cloning Eukaryotic Genes
See Fig. 4.20 and 4.21
· Eukaryotic genes are usually not cloned directly.
· Cloning eukaryotic genes is different from prokaryotes because of introns and mRNA processing in eukaryotes.
· The processed mRNA transcript is isolated and reverse transcribed into cDNA (complementary DNA) which is then cloned.
cDNA contains only the coding parts of the gene (exons).
1.) Total RNA (rRNA, tRNA and mRNA) is isolated from cells or tissues expressing the target gene
2.) The processed mRNA transcript is isolated by binding the polyA tail to a chromatography column containing polyT.
3.) The enzyme reverse transcriptase uses the processed mRNA as a template to
produce a strand of DNA with a sequence that is complementary to that of the mRNA.
4.) Next DNA polymerase catalyzes synthesis of the second strand of DNA.
5.) The ends of the cDNA are modified so that they can be inserted into the restriction enzyme cloning site of a vector.
Blunt ends are produced by treatment with RNase H and S1 nuclease
Linker sequnces (containing restriction enzyme sites) are ligated to the blunt ends
Digestion of the linker-modified ends with the restriction enzyme allows the cDNA to be cloned into the corresponding restriction site of a cloning vector
6.) The recombinant vector can be introduced into a host cell and identified to complete cloning

VI. Introduction of Recombinant DNA into Host Cells
1. Transformation: Introduction of naked plasmid DNA into competent cells
Competence: the ability of a cell to take up extracellular DNA
Some bacteria are naturally competent (not E. coli).
Two methods to make E. coli competent
a.) E. coli can be made competent by chemical treatment of the cells with calcium chloride and a heat shock: 0^OC ---> 42^OC.
b.) Electroporation: makes cells competent using an electric shock
 
2. Transfection: Introduction of naked viral DNA into prokaryotic cells or naked plasmid DNA into eukaryotic cells.
3. Transduction: Introduction of DNA into prokaryotic cells via infection with bacteriophage.
4. Conjugation: Transfer of DNA directly from one cell to another

Last updated: September 15, 2009/jdh

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