Chapter 13

Application of Recombinant DNA Technology

 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
SNP

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

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
Once general location of mutation in genome is found:

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

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

if we want to clone a gene we know is mapped between 2 markers in genome :
gene may be 100s 1000s of nucleotides away from markers ! - use unique sequences next to VNTR and microsatellite markers as probes

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

(recombination mapping showed it was between XV-2c and KM-19

CTFR gene identified

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)

Cloning Eukaryotic Genes: Using a Haplotype Map

HapMap (Haplotype Map) project : analyze 1 SNP every 5kb across the entire human genome in individuals from different geographic populations

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
can be used to introduce recombinant DNA into eukaryotic cells, including human cells

Yeast vectors - 2 micron plasmid Plant vectors - Ti (tumor inducing) plasmid Transposable elements - P elements in Drosophila Viral vectors - SV40 (Simian virus 40)

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

Introducing foreign DNA into cells

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

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
(smaller ; missing introns)

Southern Blot
(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)

Snuppy and surrogate mom Somatic nucleus donor and Snuppy (2005)

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