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Eukaryotic vectors
Introducing foreign DNA into cells Mouse genetics - transgenics, knockouts Human gene therapy Cloning
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)
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
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
The higher the number of triplet repeats in the microsatellite, the more severe the disease
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
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
goal is to isolate a clone of DNA, between original markers, that contains the gene of interest
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)
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
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)
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
Transgenic Mouse
Transgenic Mouse
Analysis of genomic DNA of transgenic mouse
PCR amplify
(smaller ; missing introns)
Southern Blot
(2 differently-sized EcoRI fragments)
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)
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
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?