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Biotechnology and Human Disease
(Molecular Basis of Inherited Diseases)
1
Outline
Restriction endonucleases
DNA cloning
Probes
Southern Blotting
Restriction Fragment Length Polymorphism
Polymerase chain reaction
Analysis of gene expression
Gene therapy
Transgenic animals
Biopharmaceuticals
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OVERVIEW
• The entire sequence of the human genome is now
known. This effort is called the Human Genome
Project, with the help of:.
• Restriction endonucleases: this permits the
dissection of huge DNA molecules into defined
fragments.
• Cloning techniques: for amplification of specific
nucleotide sequences.
• Synthesis of specific probes: for identification and
manipulation of the sequence of interest.
• These techniques allow the identification of both
normal and mutant sequences in DNA, leading to
the development of methods for: Prenatal diagnosis
of genetic diseases and Treatment of patients by
gene therapy.
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RESTRICTION ENDONUCLEASES
• Restriction endonucleases (restriction enzymes): Bacterial
enzymes that cleave double-stranded DNA into smaller, more
manageable fragments
• Each enzyme cleaves DNA at a specific palindromic nucleotide
sequence (4-6bp), producing restriction fragments.
• DNA sequence that is recognized by a restriction enzyme is called
restriction site
• These enzymes form either staggered cuts (sticky or cohesive ends)
or blunt end cuts on the DNA
• Bacterial DNA ligases can anneal two DNA fragments from
different sources if they have been cut by the same restriction
endonucleasethe hybrid combination of two fragments is called a
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recombinant DNA molecule
Specificity of restriction endonucleases
• This means, within a short
region of the double helix, the
sequence on the "top" strand,
read 5  3, is identical to that
of the "bottom" strand, also
read in the 5  3 direction.
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Nomenclature
According to the organism from which the enzyme was isolated:
•
The 1st letter of the name from the genus of the
bacterium.
•
The next 2 letters (from the type or strain).
•
A final number: to indicate the order in which the
enzyme was discovered in that organism.
Ex.: HaeIII is the third restriction endonuclease isolated
from the bacterium Haemophilus aegyptius.
TaqI is the first restriction endonuclease isolated from
the bacterium Thermus aquaticus.
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•
"Sticky" and "blunt" ends:
– These enzymes cleave DNA to
produce a 3-OH group on one end
and a 5-phosphate group on the other.
– Some of them (ex: TaqI)  form
staggered cuts that produce "sticky" or
cohesive ends (gives DNA fragments
with complementary single-stranded
sequences).
– Others, (ex: Haelll) cleave in the middle
of their recognition sequence (i.e., at
the axis of symmetry) to produce
fragments with "blunt" ends that do
not form H-bonds with each other.
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• "Sticky" and "blunt" ends:
- Using DNA Ligase, sticky ends of a
DNA fragment can be covalently
jointed with other DNA fragments
with sticky ends produced by the
same restriction enzyme.
- The hybrid combination of the 2
fragments is a recombinant DNA
molecule.
- Another Ligase encoded by
bacteriophage T4 can covalently join
blunt-ended fragments.
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DNA CLONING
The introduction of a foreign DNA molecule into a
replicating cell for amplification (production of many copies)
of the DNA
1.
2.
3.
4.
5.
The total cellular DNA is cleaved with a specific restriction
enzyme to give 100,000s of fragments
Each fragment is ligated to a DNA vector (cloning vector) to
form a hybrid molecule.
Hybrid recombinant DNA molecule transfers its DNA
fragment into a single host cell (ex: bacterium) for replication
(amplification).
Then the host cell multiplies forming a clone in which each
cell carries copies of the same inserted DNA fragment
(cloning).
The cloned DNA is released from its vector by cleavage using
the appropriate restriction endonuclease, then isolation
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Vectors:



A vector is a molecule of DNA to which the fragment of DNA
to be cloned is joined.
Commonly used vectors: plasmids. and bacterial and animal
viruses
Essential properties of a vector include:
 It must be capable of autonomous (independent)
replication within a host cell.
 It must contain at least one specific nucleotide sequence
recognized by a restriction endonuclease.
 It must carry at least one gene that confers the ability to
select for the vector such as an antibiotic resistance gene.
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Prokaryotic plasmids:
•
•
The small, circular, extra chromosome
DNA.
Plasmid may:
– carry antibiotic resistance genes
– facilitate the transfer of genetic
information from one bacterium to
another.
– be isolated from bacterial
cells and cleaved at specific sites by
restriction endonucleases, and foreign DNA is inserted.
- The hybrid plasmid can be reintroduced into a bacterium for
amplification (large number of copies of the plasmid containing
the foreign DNA)
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DNA libraries:
 A DNA library is a collection of cloned restriction fragments
of the DNA of an organism.
 There are two kinds of libraries: genomic libraries an cDNA
libraries.
 Genomic libraries contain a copy of every DNA nucleotide
sequence in the genome, while cDNA libraries contain DNA
sequences that appear as mRNA molecules and these differ
from one cell type to another.
 Cloned cDNAs lack introns and the control regions of the
genes, whereas these are present in genomic libraries.
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Genomic DNA libraries:
1.
It is the collection of fragments of double-stranded DNA
obtained by digestion of the total DNA of the organism
with a restriction enzyme.
2.
Ligation of the fragments to a vector.
3.
The recombinant DNA molecules are replicated within
host bacteria.
4.
The amplified DNA fragments represented the entire
genome of the organism and are called a genomic library.
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Complementary DNA (cDNA)
libraries:
• Using mRNA as a template to make a
complementary double-stranded DNA
(cDNA) using the enzyme reverse
transcriptase.
• The resulting cDNA is a double-stranded
copy of mRNA.
• cDNA can be amplified by cloning or by
polymerase chain reaction.
• It can be used as a probe to locate the
gene that coded for the original mRNA
(or fragments of the gene) in mixtures
containing many unrelated DNA
fragments.
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cDNA libraries
• If the mRNA used as a template is a
mixture of different species, the
resulting cDNAs are heterogenous
and cloned to form a cDNA library.
• cDNA has no intervening sequences
(introns and control regions), it can
be cloned into an expression vector
for the synthesis of eukaryotic
proteins by bacteria.
• Expression vectors are plasmids containing a bacterial
promoter for transcription of the cDNA, and a ShineDalgarno sequence for translation of the resulting mRNA
by bacterial ribosome.
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Sequencing of cloned DNA fragments:
•
•
•
Sanger dideoxy method: is a procedure used to determine the
base sequence of DNA fragments that have been cloned and
purified.
The Human Genome Project used highly automat variations of
this technique to determine the base sequence of the entire human
genome
Procedure:
1. The single-stranded DNA to be sequenced is used as the
template for DNA synthesis by DNA polymerase.
2. A radioactive primer complementary to the 3-end of the target
DNA is added along with the four deoxyribonucleoside
triphosphates (dNTPs).
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Sequencing of cloned DNA fragments
3. Divide the sample into 4 rxn tubes and add to each one a
small amount of one of the 4 dideoxy ribonucleoside
triphosphate (ddNTPs). Because ddNTPs contain no 3OH group, incorporation of a ddNTPs into a newly
synthesized strand terminates its elongation.
4. The products of this reaction is a mixture of DNA strands
of different lengths, each terminating at a specific base.
5. Separation of DNA products by size using polyacrylamide
gel electrophoresis
6. Autoradiography; yields a pattern of bands from which the
DNA sequence can be read.
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Dideoxynucleotides
2’-dideoxynucleotide
monophosphate
• DNA Sequencing using the Sanger
method involves the use of 2’3’OH
dideoxynucleotide triphosphates Phosphate
HO P
O
in addition to regular 2’-deoxy
nucleotide triphosphates
O
• 2’3’-dideoxynucleotide triphosphates
lack a 3’ OH group, and DNA
polymerization occurs only in the 3’
direction, once 2’3’-dideoxynucleotide
triphosphates are incorporated, primer
extension stops
3’
N
N
5’CH2
4’
Base
NH2
N
N
O
Sugar 2’
H
OH
1’
H
2’3’-dideoxynucleotide
monophosphate
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OH
P
HO
O
N
O
CH2
N
N
O
OH
O
N
CH2
O
H
O
HO
P
O
O
N
O
CH2
OH
H
H
NH
N
O
OH
P
O
NH2
N
O
CH2
O
H
O
H
H2O
N
O
CH2
N
O
O
O
CH2
O
P
HO
H
O
OH
HO
P
NH2
HO
P
O
H
O
HO
O
2’3’
dideoxynucleotides
Terminate
DNA
Replication
H
NH2
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DNA Sequencing
• In DNA sequencing reactions all the basic components needed to
replicate DNA are used
• 4 reactions are set up, each containing:
– DNA Polymerase
– Primer
– Template to be sequenced
– dNTPs
– A small amount of one ddNTP
ddATP, ddCTP, ddGTP, ddTTP
• As incorporation of ddNTPs terminates DNA replication, a series
of fragments is produced all terminating with the ddNTP that was
added to each reaction
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DNA Sequencing
Cloned
fragment
Primer
Primer Binding sites
Plasmid (or phage)
with cloned DNA
fragment
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The ddATP Reaction
Pol.
3’AATAGCATGGTACTGATCTTACGCTAT5’
Pol.
Pol.
Pol.
5’TTATCG
5’TTATCGTA
5’TTATCGTACCATGA
5’TTATCGTACCATGACTAGA
5’TTATCGTACCATGACTAGATGCGATA
Let me
Through!
Oh come
on!
Not
Again!
Agggg….
5’TTATCGTA
5’TTATCGTACCA
5’TTATCGTACCATGA
5’TTATCGTACCATGACTA
5’TTATCGTACCATGACTAGA
5’TTATCGTACCATGACTAGATGCGA
5’TTATCGTACCATGACTAGATGCGATA
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DNA Sequencing
ddCTP
ddGTP
ddTTP
Read 5’ to 3’ from bottom to top
• Products from 4 reactions each
containing a small amount of a
dideoxynucleotide are loaded onto a
gel
ddATP
• Polyacrlyamide gels capable of
separating fragments differing in size
by only one base
• High concentrations of urea are used
to prevent formation of double
stranded DNA or secondary
structures
• Because polymerization goes 5’ to 3’
shortest fragments are 5’ compared to
longer fragments which are in the 3’
direction
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A C G T
DNA Sequencing
What A Sequencing
Autorad Actually
Looks Like
• To read the autorad it is important to start at the
bottom and work up so that it is read in the 5’ to
3’ direction
5’CTAGAGGATCCCCGGGTACCGAGCT...3’
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PROBES
•
•
•
Probe: is a single-stranded piece of DNA, labeled with a
radioisotope (e.g. 32P) or with a non-radioactive probe, (e.g.
biotin).
The nucleotide sequence of a probe is complementary to the
target DNA.
Probes are used to identify which clone of a library or which
band on a gel contains the target DNA.
Hybridization of a probe to DNA fragments:
•
•
The utility of probes depends on the phenomenon of
hybridization.
Hybridization: is a process in which a single-stranded sequence
of a target DNA binds to a probe of a complementary
nucleotide sequence (DNA probe-target DNA hybrid duplex)
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Hybridization of a probe to DNA fragments
1) ssDNA is produced by alkaline denaturation of
dsDNA.
2) This ssDNA is first bound to a nitrocellulose
membrane to prevent self-annealing. (DNA
immobilization).
3) The immobilized DNA strands are available for
hybridization to an exogenous, single-stranded
complementary, radio labeled DNA probe.
4) The extent of hybridization is measured by the
retention of radioactivity on the membrane.
5) Excess probe molecules that do not hybridize are
removed by washing the filter and so do not interfere.
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Synthetic oligonucleotide probes
• If the sequence of all or part of the target DNA is known,
single stranded oligonucleotide probes of 20-30 nucleotides can
be synthesized that are complementary to a small region of the
gene of interest.
• If the sequence of the gene is unknown, the amino acid
sequence of the protein-that is the gene product-may be used to
construct a probe. Short, single-stranded DNA sequences (15-30
nucleotides) are synthesized, using the genetic code as a guide.
• Because of the degeneracy of the genetic code  synthesize
several oligonucleotides.
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Detecting the ßS-globin mutation
Allele-specific oligonucleotide (ASO) probe can be used to detect the
presence of the sickle cell mutation in the ß-globin gene.
1.
DNA, isolated from white blood cells, is denatured into single
strands.
2.
An oligonucleotide is constructed that is complementary to the
portion of the mutant globin gene coding for the aminoterminal sequence of the ß-globin protein.
3.
Thus, a double-stranded hybrid forms that can be detected by
electrophoresis.
.
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Detecting the ßS-globin mutation
•
DNA isolated from a heterozygous
individual (sickle cell trait) or a
homozygous patient (sickle cell disease)
contains a nucleotide sequence that is
complementary to the probe
•
DNA obtained from normal individuals
is not complementary at the sixth
codon (coding for glutamate in normal
individuals but for valine in patients with
the ßS-gene) and, therefore, does not
form a hybrid
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• Use of a pair of such ASOs
(one specific for the normal
allele and one specific for the
mutant allele) allows one to
distinguish the DNA from all
three possible genotypeshomozygous normal,
heterozygous, and
homozygous mutant.
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Biotinylated probes
• Because the expensive disposal of radioactive waste, nonradioactive probes have been developed. The vitamin biotin, can
be chemically coupled to the nucleotides used to synthesize the
probe. Biotin binds very tenaciously to avidin-a readily available
protein contained in chicken egg whites
• Avidin can be attached to a fluorescent dye detectable optically
with great sensitivity.
• Thus, a DNA fragment (displayed by gel electrophoresis) that
hybridizes with the biotinylated probe is made visible by
immersing the gel in a solution of dye-coupled avidin. After
washing away the excess avidin, the DNA fragment that binds
the probe is fluorescent.
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SOUTHERN BLOTTING
•
•
•
A technique that detects mutations in DNA. It combines the
use of restriction enzymes and DNA probes.
Named after its inventor Edwards Southern
Procedure (fragmentation electrophoresis detection)
1. DNA (entire genomic) is extracted from leukocytes and
cleaved using a specific restriction enzymemillion of
fragments
2. Resulting fragments are separated on the basis of size by
electrophoresis (larger is slower, smaller is faster)and their
lengths are determined in bp
3. DNA fragments in gel are denatured and transferred to
nitrocellulose membrane
4. A specific probe hybridization helps to visualize DNA
fragments of interest
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RESTRICTION FRAGMENT LENGTH
POLYMORPHISM
• Genome variations are differences in the sequence of DNA among
individuals. They include both polymorphisms and mutations.
• A polymorphism is a clinically harmless DNA variation. It often
occurs in the intervening sequences that do not code for proteins
• Mutation refers to an infrequent potentially harmful genome variation
that is associated with a specific human disease.
• A restriction fragment length polymorphism (RFLP) is a genetic
variant that can be examined by cleaving the DNA into fragments
(restriction fragments) with a restriction enzyme. The length of the
restriction fragments is altered if the genetic variant alters the DNA so
as to create or abolish a restriction site In either case, cleavage with
an endonuclease results in fragments of lengths differing from the
normal ( more or fewer), which can be detected by DNA hybridization
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•
Two DNA variations commonly resulting in RFLPs:
1. Single base changes in DNA:
•
About 90% of human genome variation comes in the
form of single nucleotide polymorphisms, or SNPs
(pronounced "snips"), that is, variations that involve just
one base.
• The alteration of one or more nucleotides at a restriction
site can render the site unrecognizable by a particular
restriction endonuclease. A new restriction site can also be
created by the same mechanism.
• In either case, cleavage with an endonuclease results in
fragments of lengths differing from the normal, which can
be detected by DNA hybridization
2. Tandem repeats:
Polymorphism in chromosomal DNA
can arise from the presence of a
variable number of tandem
repeats. These are short sequences
of DNA at scattered locations in
the genome, repeated in tandem
(like freight cars of a train).
• The number of these repeat units
varies from person to person, but is
unique for any given individual and,
therefore, serves as a molecular
fingerprint.
• Cleavage by restriction enzymes
yields fragments that vary in length
depending on how many repeated
segments are contained in the
fragment.
• Variations in the number of tandem
repeats can lead to polymorphism.
Prenatal diagnosis
Families with a history of severe genetic disease, may
wish to determine the presence of the disorder in a
developing fetus by prenatal diagnosis.
Many methods are available but molecular analysis of
fetal DNA promises to provide the most detailed
genetic picture.
Direct diagnosis of sickle cell disease is preformed
using RFLPs
The Sickle Cell Anemia Mutation
Normal b-globin DNA
C
Mutant b-globin DNA
T
T
C
G A
A
G U A
mRNA
mRNA
Normal b-globin
Mutant b-globin
Glu
H2 N
C
C
A T
Val
O
OH
H
CH2
H2C
C OH
O Acid
H2 N
C
C
O
OH
H
CH
CH3
H3C
Neutral
Non-polar
Direct diagnosis of sickle cell disease using RFLPs:
• The genetic disorders of hemoglobin are the most common
genetic diseases in humans.
• In the case of sickle cell disease, the mutation that gives rise to
the disease is actually one and the same as the mutation that
gives rise to the polymorphism. Direct detection by RFLPs of
diseases that result from point mutations is at present limited
to only a few genetic diseases.
• Sickle cell anemia is caused by a point mutation. The sequence
altered by the mutation abolishes the recognition site of the
restriction endonuclease MstII that recognizes the nucleotide
sequence CCTNAGG (where N is any nucleotide).
• Thus, the A to T mutation within a codon of the bs-globin
gene eliminates a cleavage site for the enzyme.
RFLP analysis
• Sickle cell anemia is caused by a point
mutation (A to T mutation (base
substitution) within a codon of the bsglobin gene). The sequence altered by the
mutation abolishes the recognition site
CCTNAGG (where N is any nucleotide).
of the MstII restriction endonuclease
• Normal DNA digested with MstII yields a
1.15 kb fragment, whereas a 1.35 kb
fragment is generated from the ßs gene as a
result of the loss of one MstII cleavage
site.
• Diagnostic techniques for analyzing fetal
DNA provide safe, early detection of
sickle cell anemia, as well as other genetic
diseases.
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POLYMERASE CHAIN REACTION(PCR)
• PCR is a test tube method for amplifying a selected DNA
sequence that does not rely on the biologic cloning method.
• PCR permits the synthesis of millions of identical copies of a
specific nucleotide sequence in a few hours.
• The method can be used to amplify DNA sequences from any
source-bacterial, viral, plant, or animal.
• PCR is an artificial way of doing DNA replication. PCR uses DNA
polymerase to repetitively amplify target DNA
• Each cycle of amplification doubles the amount of DNA in the
sample, leading to an exponential increase in DNA with repeated
cycles of amplification
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Components of a PCR Reaction
•
•
•
•
•
Buffer (containing Mg++)
Template DNA
2 Primers that flank the fragment of DNA to be amplified
dNTPs
Taq DNA Polymerase (or another thermally stable DNA
polymerase)
How The Functions Of Replication Are Achieved
During PCR
Function
• Melting DNA
• Polymerizing DNA
• Providing primer
• Joining nicks
PCR
Heat
Taq DNA Polymerase
Primers are added to the
reaction mix
N/A as fragments are short 4343
PCR
Temperature
100
Melting
94 oC
Extension
Annealing
Primers
50 oC
50
0
94 oC
72 oC
T i m e
3’
3’
5’
5’
3’
3’
5’
5’
5’
3’
5’
5’
5’
3’
5’
5’
5’
3’
5’
5’
5’
30x
Melting
5’
3’
44
3’
5’
3’
Temperature
100
Melting
94 oC
PCR
50
0
T i m e
3’
5’
5’
3’
45
Temperature
100
50
0
3’
5’
5’
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
30x
T i m e
5’
5’
5’
Melting
94 oC
PCR
5’
3’
5’
5’
5’
5’
5’
5’
46
Temperature
100
Melting
94 oC
50
0
3’
5’
5’
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
30x
T i m e
5’
5’
5’
PCR
5’
3’
Fragments of
defined length
5’
5’
5’
5’
5’
5’
47
DNA Between The Primers Doubles With Each
Thermal Cycle
Number
1
2
0
1
Cycles
4
8
16
32
64
2
3
4
5
6
48
Steps of a PCR
1.
2.
3.
4.
Primer construction: synthetic oligonucleotide (20-35 NMP)
single stranded and complementary to the Flanking
sequences -nucleotide sequence on each side of the target
DNA .The 3'-hydroxyl end of each primer points toward the
target sequence.
Denature the DNA: The DNA to be amplified is heated to
separate the double-stranded target DNA into single strands.
Annealing of primers to single-stranded DNA: The
separated strands are cooled and allowed to anneal to the two
primers (one for each strand).
Chain extension: DNA polymerase adds deoxyribonucleotides
(within reaction mixture in excess) to the 3'-hydroxyl end of
the primer, and strand growth –complementary antiparallelextends across the target DNA.
49
Steps of a PCR
• At the completion of one cycle of replication, the reaction mixture
is heated again to denature the DNA strands (of which there are
now four). and the cycle of chain extension is repeated.
• Thus, each newly synthesized polynucleotide can act as a template
for the successive cycles. This leads to an exponential increase in the
amount of target DNA with each cycle, hence, the name
"polymerase chain reaction”
• By using a heat-stable DNA polymerase (for example, Taq
polymerase) from a thermophilic bacterium, the polymerase is not
denatureddoes not have to be added at each successive cycle.
• Each extension product of the primer includes a sequence
complementary to the primer at the 5' end of the target sequence.
• Advantages o PCR: sensitivity (target DNA is less than 1 part in a
106 of the initial sample) and speed ( as compared to recombinant
DNA cloning technology)
50
Applications of PCR
1. Comparison of a normal cloned gene with an uncloned
mutant form of the gene
2. Detection of low-abundance nucleic acid sequences
3. Forensic analysis of DNA samples
4. Prenatal diagnosis and carrier detection of cystic fibrosis
51
ANALYSIS OF GENE EXPRESSION
• Products of gene expression are mRNA and proteins.
A. Determination of mRNA levels
1. Northern blots (mRNAs electrophoresis transfer to a
membrane  hybridization to a radioactive probe)
2. Microarrays are used to determine the differing patterns of gene
expression in normal and cancer cells
• DNA microarrays contain thousands of immobilized DNA
sequences in tiny spots (1000s, each corresponding to a different
gene) organized in a microscope slide (gene chip)
• Used for genotyping (of genomic DNA variations or mutations)
or gene expression analysis (mRNA converted to cDNA
coupled to florescent tag)
• Fluorescence bound to each spot is a measure of the amount of
corresponding mRNA in sample
52
53
B. Analysis of proteins
• Proteomics is the study of all proteins expressed by a genome,
including their relative abundance, distribution, posttranslational
modifications, functions, and interactions with other
macromolecules.
• Enzyme linked immunosorbent assay (ELISA) uses labeled
antibodies (probe) in sandwich technique to detect and quantify
specific proteins in multiwell (microtiter) plate
– Antigen (protein) is bound to wells
– Enzyme-coupled antibodies are specific for this particular
antigen, and produce a colored product when exposed to
substrate
– Amount of color is used to determine amount of protein
(antigen) in sample
54
• Western Blots (immunoblots)
– Similar to Southern Blots
– Protein molecules in sample are separated by electrophoresis,
and blotted to a membrane
– Probe is labeled antibody, it produces a band at the location of
its antigen
55
GENE THERAPY
• Gene replacement therapy is
to insert the normal, cloned
DNA for a gene into the
somatic cells of a patient
who is defective in that gene
as a result of some diseasecausing mutation
• The DNA must become
permanently integrated into
the patient's chromosomes to
be expressed to produce the
correct protein.
56
TRANSGENIC ANIMALS
• Transgenic animals can be produced by injecting a cloned gene
into the fertilized egg.
• If the gene becomes successfully integrated into a chromosome,
it will be present in the germline of the resulting animal, and can
be passed along from generation to generation.
• Transgenic goats and cows can now be designed to produce
human hormones in their milk.
• Sometimes, rather than introducing a functional gene into a
transgenic mouse, a mutant gene is used to replace the normal
copies of that gene in the cells of the mouse. This can be used
to produce a colony of "knockout mice" that are deficient in a
particular enzyme. Such animals can then serve as models for the
study of a corresponding human disease.
57
Biopharmaceuticals
• Are recombinant therapeutic proteins, monoclonal antibodies
products used in vivo and nucleic acid-based medicinal products
• Represent 1 in every 4 new pharmaceuticals (new molecular
entities)
• Therapeutic goals of biopharmaceutical engineering
– The reduction/elimination of product immunogenecity
– The generation of products with altered pharmacokinetic
profiles
– The alteration of biological half life
– The generation of novel (hybrid) proteins
58