Application of Recombinant

DNA Technology
Chapter 13
Mapping mutations in eukaryotes

Cloning eukaryotic genes

Eukaryotic vectors

Introducing foreign DNA into cells

Mouse genetics - transgenics, knockouts

Human gene therapy

Cloning
Mapping Mutations in Eukaryotes
DNA Markers

RFLPs (Restriction Fragment Length
Polymorphisms)

VNTRs (Variable Number Tandem Repeats,
minisatellites)

Microsatellites

SNPs (Single-Nucleotide Polymorphisms)
RFLP
A nucleotide change that results in either elimination
or creation of a restriction enzyme site

technique to detect :
Southern Blot
digest genomic DNA
electrophorese resulting DNA fragments
hybridize using radiolabeled DNA probe that
overlaps restriction site(s)

Molecular Characterization of a
RFLP












RFLPs: Applications
Used to directly diagnose an inherited disease
Sickle cell anemia:
Change in gene sequence of -globin gene
(change of an A to a T in the DNA)
Alters restriction site
Probe hybridizes to DNA region where
restriction site (MstII) is found
In sickle cell anemia, restriction site is
missing due to change in -globin gene
sequence
RFLPs can distinguish -globin in
wild type and sickle cell anemia
VNTRs (Minisatellites):
Techniques
Southern Blot:
1. Digest genomic DNA
2. Electrophorese resulting DNA
fragments
3. Hybridize using radiolabeled DNA
probe that contains VNTR sequence
4. Expose to X-ray film

Molecular Characterization of a
VNTR
allele A
allele B
locus 1A, 5 copies
locus 1B, 4 copies
locus 3B, 4 copies
locus 2A, 3 copies
locus 3A, 3 copies
locus 2B, 2 copies
Microsatellites
Tandem Repeats, 2-5 base pairs
(smaller than VNTRs)
Total size of microsatellite:100-1000
base pairs
Use PCR to detect using primers that
span tandem repeats

Advantages of Microsatellites
Used to detect triplet repeat diseases:
Huntingtons disease
Fragile X
Used to map genes through
recombination
Scattered throughout genome
Large number of alleles in a population
Molecular Characterization of
Microsatellites
Microsatellites and Triplet Repeat
Diseases
Age of onset of disease and severity of
disease is related to number of triplet repeats
Huntingtons disease-causes
neurodegeneration, due to expansion of
triplet repeats (CAG) in ORF
Fragile X-causes mental impairment, due to
expansion of triplet repeats in front of ORF

Clinical Diagnosis of Huntingtons
Using Microsatellite Analysis
The higher the number of triplet repeats in the
microsatellite, the more severe the disease
Single-Nucleotide
Polymorphisms (SNPs)
changes in a single nucleotide
SNPs are more randomly and densely
distributed throughout the genome
frequency : ~1 out of 1000 base pairs
1.8 million SNPs identified in human
genome
Difference between SNPs and
RFLPs
* SNP does not have to be in a site of a restriction endonuclease
Molecular Characterization of
SNPs by S1 nuclease mapping
Digest DNA into small
fragments

Anneal single-stranded
probe to denatured DNA
Treat with S1 nuclease
(digests single-stranded
DNA)
Electrophorese S1
products
1. If probe and target
DNA sequence are
different at SNP site,
shortened probe
because S1 cleaves
both strands

2. If probe and target
DNA sequence are
same at SNP site,
probe will be full length
SNP
Recombination Mapping with
Microsatellites
Follow pattern of inheritance of SNP through
generations on pedigree
Identify SNP that is associated with trait
(Which SNP is always seen in individuals with
disease trait?)
Determine which individuals have disease but
do not have SNP associated with disease:
those individuals had recombination event
between the disease gene and the SNP
Cloning Eukaryotic Genes
Genes associated with a mutant phenotype can be
localized to a chromosomal region by
recombination mapping or by characterizing
chromosomal rearrangements (insertions,
deletions, translocations), then identifying
mutation and corresponding gene

Positional Gene Cloning identifying the actual
gene based on its location in the genome
Chromosome walking - sequencing
overlapping clones to determine position of gene
Expression patterns to identify candidate genes
Cloning using haplotype maps
Once general location of mutation in genome is found:
Cloning Eukaryotic Genes:
Chromosome Walking
1. Identify molecular markers near gene
2. Generate unique sequence probe
3. Probe genomic library to isolate clones
4. Generate restriction map of clones to identify
ends of clones that extend the farthest toward
gene of interest
6. Use those end sequences as new probes
walk closer to gene of interest using
probe to isolate new clones

Cloning Eukaryotic Genes:
1. Chromosome Walking
gene may be 100s 1000s of nucleotides
away from markers !
- use unique sequences next to VNTR
and microsatellite markers as probes
if we want to clone a gene we know is mapped
between 2 markers in genome :
generate new probes extending the
furthest in both directions
goal is to isolate a clone
of DNA, between original
markers, that contains the
gene of interest
Cloning Eukaryotic Genes:
2. Identifying Candidate Genes,
Expression Patterns
Example: cystic fibrosis (autosomal recessive)
Mapped gene to small region on chromosome 7
containing 4 genes
Isolated mRNAs from tissue where cystic fibrosis is
expressed (lungs, pancreas, sweat glands)
Northern blots with probes for 4 genes identified
only 1 gene (cystic fibrosis transmembrane
conductance regulator, or CFTR) expressed in all
of the expected tissues
affected patients had mutations in CFTR gene
Cloning Eukaryotic Genes:
Identifying Candidate Genes:
Expression Patterns
CTFR gene identified
(recombination mapping showed it was
between XV-2c and KM-19
Cloning Eukaryotic Genes:
Identifying Candidate Genes,
Expression Patterns
Cystic fibrosis
Confirmation: clone and sequence CFTR
gene from affected and unaffected
individuals
Affected individuals had mutations in both
copies of CFTR gene
Cloning Eukaryotic Genes: Using
a Haplotype Map
haplotype : Haploid genotype
-specific combinations of markers (SNPs or
PCR fragments) on the alleles of a chromosome
for a given individual

within a population, clusters of SNPs do not exhibit
recombination (always found together in genome)

HapMap (Haplotype Map) project :
analyze 1 SNP every 5kb across the entire human
genome in individuals from different geographic populations
Cloning Eukaryotic Genes: Using
a Haplotype Map
Tag SNPs : because of the recombination-free regions, a subset
of SNP alleles can uniquely identify a specific haplotype
- for example, Tag SNPs ATC correspond to haplotype 1
Cloning Eukaryotic Genes: Using
Association Mapping
association mapping:
identifying Tag SNPs that are associated with disease
a gene that may be involved with heart disease is associated
with the Tag SNP in red ; C at this location suggests disease allele
Eukaryotic Vectors
Yeast vectors
- 2 micron plasmid
Plant vectors
- Ti (tumor inducing) plasmid
Transposable elements
- P elements in Drosophila
Viral vectors
- SV40 (Simian virus 40)




can be used to introduce recombinant DNA into
eukaryotic cells, including human cells
Use of Vectors to Express
Foreign Genes
Requires appropriate vector
Requires appropriate promoter elements
(so gene is expressed in correct tissue)
Requires appropriate posttranscriptional
processing signals
Requires appropriate translational signals

need to express the gene in the correct
cell at the proper time in the proper amount
Introducing foreign DNA into cells
Transformation (in prokaryotes)
Treat E. coli cells to make them more
permeable to plasmid DNA
1. Chemical Transformation - expose
E. coli cells to salt (calcium chloride)
2. Electroporation - expose E. coli cells to
electrical current
both allow E.coli to take in DNA
Transfection (in eukaryotes)
1. Chemical (calcium phosphate)
2. Electroporation
3. Liposomes
- DNA carried in to cell in membrane
bound vesicles)
4. Injection
5. Biolistic projectiles
- introduce DNA into mitochondria and
chloroplasts on tungsten bullets

Introducing foreign DNA into cells
Transfection

Viral vector

Injections

Biolisitics
Introducing foreign DNA into cells
Mouse Genetics
Transgenic Mice random integration of
a foreign gene into the mouse genome
Introduce foreign gene into mouse egg
Implant fertilized egg into female
Analyze genomic DNA in offspring for
transgene

Knockout Mice - physical exchange of
transgene for endogenous gene
Transgenic Mouse
Inject foreign DNA into male pronucleus of newly
fertilized eggs







Transgenic Mouse
Analysis of genomic DNA of transgenic mouse













PCR amplify
Southern Blot
(smaller ;
missing introns)
(2 differently-sized EcoRI
fragments)
Transgenic Mouse: Creating a
Giant Mouse
Knock Out Mice: Target Vector and
Introduction into ES Cells
Knock Out Mice
Follow knock out gene in chimeric mice by using
reporter gene (coat color), mate chimeric mice

Human Gene Therapy
Introduce wild type copy of gene
into patients with defective gene
Severe Combined
Immunodeficiency (SCID)
Absence of adenosine deaminase
results in buildup of deoxyadenosine
Toxic to B and T lymphocytes
SCID Gene Therapy
Insert wild type ADA gene
into retrovirus
Isolate T cells from SCID
patient
Infect T cells with retrovirus
Reintroduce the T cells with
the wild type ADA gene into
patient

Disadvantages of Gene Therapy
Retrovirus insertion cannot be
controlled
- can insert near protooncogene and
cause T cell leukemia
Can cause immune response to virus
Requires helper virus which can
recombine with retrovirus vector
Human Gene Therapy: Cystic
Fibrosis
Adenovirus as a vector
Infects lung epithelial cells
Does not integrate into host
chromosome
Maintained as extrachromosomal DNA
Requires continual application for
patients
Cloned Organisms
Genetically identical
Replace nucleus of egg with nucleus
from epithelial cell
Mitochondrial genes still remain from
host cell
Large number of nuclear transfers
required (over 1000)
Cloned Organisms
Dolly (1997)
Somatic nucleus
donor and Snuppy
(2005)
Snuppy
and
surrogate
mom
Disadvantages of Cloned Organisms
Require multiple nuclear transplants

Cloned animals have shorter life spans

Cloned animals have more propensity
for disease and physical abnormalities
- at age 6 Dolly had lung cancer and
severe arthritis

Potential disruption of gene functioning
Applications of Genetic Engineering
Medicine
Basic knowledge of how genes work
Identifying genes that cause diseases
Producing large amounts of proteins and
antibodies to help fight disease
Gene therapy
Transgenic animals : potential for disease
models
Cloned animals : potential to produce organs
for human transplants
Applications of Gene Cloning
Agriculture
Transgenic crops
resistant to pests
resistant to frost
resistant to premature ripening
resistant to herbicides
Industry
Engineering bacteria to break down toxic
waste
Yeast that can convert glucose to ethyl
alcohol to replace fossil fuels

Ethical Considerations
Genetically modified organism (GMO)
(plants)
Are they safe for human and animal
consumption?
Is it ethical to plant GMOs which
encourage the use of herbicides?
Can we control the spread of GMOs
once they are planted?

Ethical Considerations
Cloning Organisms and Individuals
Preimplantation Genetic Diagnosis (PGD) to
reduce likelihood of bearing child with genetic
disease
- in vitro fertilization
- remove 1 cell from embryo at 8-cell stage,
screen it for mutation or desired trait
- implant 7-cell embryo in mothers uterus if
desired trait is present (or undesired is absent)

Is test dangerous to individuals later in life?
Is manipulation of a childs genome ethical?

Preimplantation Genetic Diagnosis