Download Cloning Restriction Fragments of Cellular DNA

Recombinant DNA
OVERVIEW OF RECOMBINANT DNA TECHNOLOGY
• Recombinant DNA technology allows a DNA
fragment from any source to be joined in vitro
with a nucleic acid vector that can replicate
autonomously in microorganisms.
• This provides a means of analyzing and altering
genes and proteins. It provides the reagents
necessary for genetic testing for carrier detection
and prenatal diagnosis of genetic diseases and for
gene therapy.
• Additionally, this technology can provide a source
of a specific protein, such as recombinant human
insulin, in almost unlimited quantities.
Two approaches to producing recombinant DNA for
cloning have been developed for use with somewhat
different applications:
• Cloning restriction fragments of cellular DNA
• Cloning cDNA produced by reverse transcription of
cellular mRNA
Cloning Restriction Fragments of Cellular DNA
• Cloning DNA restriction fragments is useful in
the following applications:
• Sequencing DNA (Human Genome Project,
Genetic diagnosis)
• Producing restriction maps for gene mapping
• Studies involving non-expressed DNA sequences
The first step in this procedure is to produce restriction
fragments of DNA using restriction endonucleases.
Restriction Endonucleases
• These enzymes are isolated from bacteria, their
natural source.
• There are many different restriction endonucleases
isolated from a variety of bacteria that are now
readily available commercially.
• In bacteria they act as part of a restriction/
modification system that protects the bacteria from
infection by DNA viruses.
• Restriction endonucleases recognize double-stranded
DNA sequences called palindromes (inverted repeats)
usually of four to eight base pairs in length. For example,
Figure 1-6-1 shows the recognition site for EcoRI, a
restriction endonuclease isolated from Escherichia coli.
• A palindrome can be identified by examining the
sequence of only one strand. Draw a line through the
center of the sequence (through the central base for
palindromes with an odd number of nucleotides). If the
sequence is folded along this line the bases should pair.
Prokaryotic Restriction Modification Systems
• Provide defense against infecting DNA
viruses
• Methylase enzyme modifies and protects
palindromes in bacterial DNA.
• Unmethylated palindromes of infecting viral
DNA are recognized by restriction
endonuclease.
• Viral DNA is fragmented and destroyed.
EcoRI Recognition Sequence
• DNA from a source to be cloned is mixed with a
particular restriction endonuclease such as EcoRI,
producing DNA restriction fragments.
• Some restriction endonucleases such as EcoRI produce
asymmetric cuts within the palindrome yielding
"sticky ends" on the fragments.
• Sticky ends are advantageous in facilitating the
recombination of a restriction fragment with the vector
DNA.
• Others, like HaeIII, cut both strands in the same
location yielding "blunt ends" on the restriction
fragments.
Examples of Restriction Endonucleases
Cloning Restriction Fragments Using Vectors
• To clone the restriction fragments, they must each be inserted
into a vector.
• A vector is a piece of DNA (plasmid, viral chromosome,
yeast chromosome) capable of autonomous replication in a
host cell, for instance, plasmid pBR322.
• The DNA used as a vector usually has. At least one type of
palindrome recognized by a restriction endonuclease
• An origin for autonomous replication
• At least one gene for resistance to an antibiotic
Cloning vectors
• Common properties
– origin of DNA replication
– unique restriction sites for insertion of DNA
• multiple cloning sites containing many restriction sites engineered into many
plasmid vectors
– Easy identification and recovery of clones
• Types of vectors
– plasmids containing drug resistance gene (up to 10 kb)
• many commercially available plasmids
–
–
–
–
bacteriophage, e.g., lambda (~ 15-20 kb)
cosmids for larger DNA molecules (~ 40-50 kb)
BAC: bacterial artificial chromosome (~ 100-300 kb)
YAC: yeast artificial chromosome (~ 1000 kb)
• The vector is cut with the restriction endonuclease
and mixed with the DNA restriction fragments to be
cloned. As shown in Figure 1-6-3, once the vectors
have combined with one of the restriction fragments,
DNA ligase is used to form permanent PDE bonds
between the fragment and the vector. This produces
recombinant DNA.
• Once the recombinant vectors have been
produced, they are used to transform host cells.
• In the example of the plasmid pBR322, the host
cells are bacteria. Once transformed, the bacteria
are plated on selective media so that bacteria
transformed with a recombinant plasmid can be
easily identified.
• In the case of plasmid pBR322, bacteria with
recombinant plasmids would be resistant to
ampicillin but sensitive to tetracycline.
• The collection of colonies produced is referred to
as a genomic DNA library. The library must be
screened with a radioactive probe to identify the
colony with the desired restriction fragment.
Uses of Genomic Libraries
• Large quantities of each clone can be grown for DNA
sequencing studies, similar to what is being done in the
Human Genome Project.
• By producing genomic libraries using different restriction
endonucleases (or allowing one type of restriction
endonuclease to digest a DNA sample for different
times), regions of overlap can be identified and the
fragments ordered, producing DNA restriction maps
useful for genetic testing and sequencing.
• Other genetic markers may be identified in this way, such
as minisatellite and microsatellite sequences.
• Genomic libraries are also useful to clone and study
DNA sequences that are not expressed in cells (response
elements, introns, promoters).
Restriction Maps
• A restriction map is a linear (or circular)
map of the order and distances of restriction
endonuclease cut sites in a segment of DNA
• Each DNA fragment, no matter what size,
has its own unique restriction map
• Restriction maps are useful in comparing
DNA fragments to look for regions of identity
Restriction Maps
• Line drawings of DNA identifying sites cut by
restriction endonucleases.
• Identify potential RFLP markers for genetic
diagnosis.
• Example: Restriction site polymorphism for Mstll
may be used to identify individuals with the sickle cell
mutation
Cloning cDNA Produced by Reverse Transcription
of Cellular mRNA
• If the end goal of cloning is to have a cloned gene
expressed in a cell, the entire coding sequence must
be cloned intact. Furthermore, if a cloned eukaryotic
gene is to be expressed in bacteria (to make
recombinant proteins), the gene must not contain
introns, which could not be processed in a
prokaryotic cell. In these cases it is more convenient
to clone cDNA rather than DNA restriction
fragments.
Producing cDNA by Reverse Transcription of mRNA
• Cytoplasmic mRNA is isolated from a cell known to express the
desired gene. Reverse transcriptase, along with other components
(Figure 1-6-4), is used in vitro to produce double stranded cDNA
that is subsequently recombined with a chosen vector to produce the
recombinant DNA for cloning. In this approach:
• All genes expressed will be cloned along with the desired gene.
• None of the non-expressed DNA in the cell will be cloned.
• Each cDNA represents the complete coding sequence of a gene.
• The cDNAs have no introns.
• An expression library is produced at the end of the cloning
procedure.
Synthesis of cDNA In Vitro
Expression Vectors
• If the goal of the cloning procedure is to obtain a
recombinant protein, appropriate sequences required
for transcription and translation in the cloning host
cell must be provided because they will not be part of
the cDNA.
• For instance, to produce recombinant human insulin
in bacteria, a bacterial promoter and a ShineDalgarno sequence must be included in the cloning
plasmid near the insertion site for the cDNA.
• Figure 1-6-5 shows an example of an expression
vector, pUC. In some expression vectors, other
regulatory sequences such as operators are added to
allow expression of the cloned gene to be controlled.
An Expression Vector (pUC)
Uses of cDNA (Expression) Libraries
• Once the recombinant expression vectors containing the
cDNA inserts are produced, they are used to transform
bacteria (or other host cells) and produce cDNA
(expression) libraries. Expression libraries may be used
to produce recombinant proteins that in some cases have
significant advantages over isolating them from natural
sources:
• Larger quantities may be produced. Recombinant human
insulin used to treat diabetics.
• Natural source may carry risk of infection. Recombinant
Factor VIII used to treat hemophilia A has helped reduce
the incidence of HIV infection in hemophiliacs.
Recombinant HbsAg is now used to immunize against
hepatitis B, eliminating the risk of introducing a viral
infection during vaccination.
• Genes cloned as cDNA are used for
gene therapy protocols and for
producing transgenic animals.
• cDNA probes, for many of the
blotting techniques, are produced
by cloning.
Screening libraries for a Specific DNA Sequence
• Figure 1-6-6 shows how libraries are screened to
identify a desired DNA sequence. The top circle
represents either a genomic library or an
expression library on a growth plate.
• A blot is made from the plate.
• Colonies on the blot are lysed and treated with a
radioactive probe specific for the DNA sequence
32P-DNA) or recombinant protein (125I-antibody
reactive with the recombinant protein).
• An autoradiogram of the probed blot is produced and
the radioactive colony identified.
• Once the corresponding colony has been identified, a
sample can be used to inoculate a large broth culture
from which one can isolate the cloned DNA or the
recombinant protein.
Screening a DNA Library
Common Sources of 32P-DNA probes
• cDNA can be used as a probe to locate a
genomic clone
• A cloned gene from another species can be
used to locate the human homolog.
• An approximate DNA sequence can be
deduced and synthesized in the laboratory
provided the amino acid sequence of the
protein is known.
Gene Therapy and Transgenic Animals
• Gene therapy now offers potential cures for individuals with
inherited diseases. The initial goal is to introduce a normal
copy of the gene that is defective into the tissues that give rise
to the pathology of the genetic disease. For instance, about
50% of the children with severe combined immunodeficiency
have a mutation in the gene encoding the "γ chain common to
several of the interleukin receptors. cDNA from a normal "γchain gene was used to transduce autologous cells from infants
with X-linked SCID with subsequent correction of the defects
in their T-cells and natural killer cells.
• Gene transfer requires a delivery vector (retrovirus;
adenovirus, liposome).
• Only tissues giving rise to the disease pathology are targeted
for gene therapy.
• Normal gene is not inherited by offspring.
Transgenic Animals
• Transgenic animals are produced by transferring cDNA into
the pronucleus of a fertilized ovum. The resultant transgenic
animal has the new gene (transgene) in all of its cells including
its reproductive tissues. Transgenic animals are now widely
used as experimental models in which to study human diseases.
• A variation of this technique produces a knockout animal, in
which a normal gene has been functionally eliminated. This
may be done by site-specific mutagenesis.
• Transgenic animals have a new gene (transgene) introduced
into their germline.
• All cells of a transgenic animal contain the transgene.
• Transgene is inherited by offspring.
• Knockout animals have a normal gene intentionally
inactivated/destroyed.
• Transgenic and knockout animals are used as models of
human disease.
Incorporation of Cloned DNA Into Humans and Other Animals
Gene Therapy
• When using gene therapy to correct genetic deficiencies in
humans, the cloned normal gene is targeted only to the tissues
giving rise to the major symptoms.
• For instance about 50% of the cases of severe combined
immunodeficiency (SCID) are caused by mutations in the gene
for a subunit common to several interleukin receptors.
• The approach to gene therapy has been to introduce a normal
cloned gene into the patient's bone marrow cells that will
subsequently divide and differentiate to produce T and B
lymphocytes expressing the gene.
• These patients subsequently have improved immune function.
Because the cloned normal gene has not been introduced into
reproductive tissues any children of the patient would inherit
the defective allele.
Review Questions
Select the ONE best answer.
1. If a cystic fibrosis patient were to be treated by
gene therapy, which type of cells should be
targeted as host cells?
A. Germ cells
B. Epithelial cells
C. T cells
D. Hemopoietic stem cells
2. A pharmaceutical firm is interested in the bacterial
production of thymidylate synthase in large quantities for
drug-targeting studies. An important step in the overall
cloning strategy involves the ligation of synthase cDNA
into a plasmid vector containing a replication origin, an
antibiotic resistance gene, and a promoter sequence.
Which additional nucleotide sequence should be included
in this vector to ensure optimal production of the
thymidylate synthase?
A. Operator sequence
B. PolyA sequence
C. Shine-Dalgarno sequence
D. Attenuator sequence
E. 3' -splice acceptor sequence
3. Restriction fragment length polymorphisms may be
produced by mutations in the sites for restriction
endonucleases. For instance, a single base change in the
site for the nuclease SalI produces the sequence
GTGGAC, which can no longer be recognized by the
enzyme. The original sequence recognized by SalI was:
A. GTAGAC
B. GCGGAC
C. CTGGAC
D. GTCGAC
E. GTGTAC
4. Scientists studying a common mutation in the LDL
receptor gene have inserted the defective gene into
fertilized murine ova. The altered ova are implanted
in a foster mother and the progeny are used to study
the effects of the mutant allele. The mice produced
in this procedure would be referred to as:
A. Knockout mice
B. Transgenic mice
C. Allogenic mice
D. Cloned mice
E. Somatic-cell engineered mice
5. A linear DNA fragment is cleaved with the individual
restriction enzymes HindIII and SmaI and then with a
combination of the two enzymes. The fragments
obtained are:
HindIII
2.5 kb; 5.0 kb
SmaI
2.0 kb; 5.5 kb
HindIII + SmaI 2.5 kb; 3.0 kb; 2.0 kb
- Draw the restriction map.
- The mixture of fragments produced by the combined
enzymes is cleaved with EcoRI, resulting in the loss of
the 3.0 kb band and the appearance of a band at 1.5 kb.
Mark the EcoRI restriction site on the map.