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Biotech 2012 spring-7_-rflp_0

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  • 1. RFLP DNA molecular testing and DNA Typing
  • 2. Genetic testing  An individual has symptoms or  An individual is at risk of developing a disease with a family history.  DNA molecular testing:  A type of testing that focuses on the molecular nature of mutations associated with the disease.
  • 3. Complications  Many different mutations can cause symptoms of a single disease.  BRCA1 and BRCA2 are implicated in the development of breast and ovarian cancer.  Hundreds of mutations can be found in these genes; the risk of cancer varies among the mutations.  General screening and genetic testing are different (mammograms vs. testing for specific mutations in the gene).
  • 4. Genetic testing:  Prenatal diagnosis: is the fetus at risk? (amniocentesis or chorionic villus samples analyzed).  Newborns can be tested for PKU, sickle cell anemia, Tay-Sachs.
  • 5. Methods of Genetic Testing  Restriction Fragment Length Polymorphism analysis:  Loss or addition of a RE site is analyzed.  RFLP is a DNA marker.  RFLPs are useful for:  Mapping the chromosomes.  Finding out different forms of genes/sequences.
  • 6. RFLPs  RFLP’s may be changes in the gene of interest (such as with sickle cell).  Often, RFLP’s are associated with, but not in, the gene of interest. A RFLP which is very near the allele of interest will usually indicate the presence of the allele of interest.  RFLP’s can be used to follow a genetic lineage (in essence, a specific chromosome) in a population, and is a useful tool in population biology.
  • 7. Different alleles of Hb
  • 8. Microsatellites and VNTRs as DNA Markers  Analysis of “microsatellites” ( short tandem repeats or STR’s, 2-4 bases repeat), and VNTR’s (Variable number of tandem repeats, 5- 10’s of bases repeat) sequences is used in many genetic approaches.  Repeated sequences are often more variable (due to replication errors and errors in crossing over) than non repeating sequences, therefore lots of alleles are generally present in a population.  In other words, two individuals have a higher chance of genetic differences at STR’s and VNTR’s than at most sequences in the DNA.
  • 9. Microsatellites and VNTRs as DNA Markers
  • 10. Analysis of Microsatellites and VNTR’s  One way of thinking about these analyses is that this is a specialized RFLP analysis, the power is that there are lots of alleles in a population, so there is a high chance that two individuals will be different in their genotypes (informative).  Two techniques are common in these analyses:  Southern blot followed by hybridization with a probe that will detect the sequence (as in RFLP analysis).  PCR with a pair of primers which flank the variable sequence.
  • 11. Applications  Population studies: finding differences in allele frequencies can identify separate populations (not interbreeding).  Locating specific genes: associating a specific VNTR allele with a genetic disease can help localize the gene to a region of the chromosome, or trace the allele through a pedigree.  DNA typing: paternity testing (also useful in population studies, in animal breeding etc.) and in forensic analysis.
  • 12. DNA Typing in Paternity Testing • Comparison of VNTR’s can definitely exclude an individual from being the parent of a child (neither allele the child has is present in the alleged father).
  • 13. DNA Typing in Paternity Testing • Proving paternity is more difficult, and relies on statistical arguments of the probability that the child and the alleged father are related. Multiple loci (different VNTR’s) must be examined to provide convincing evidence that the alleged father is the true father. The same statements (exclusion versus proof of identity) are true for forensic arguments. Ethnicity of the accused is a factor: allele frequencies for VNTR’s are different in different population, be they elk or human., and often the frequencies (which are the basis of the statistical arguments) are not known for a specific group.
  • 14. Finding a Gene: Chromosome Walking  Identifying the gene associated with a specific disease requires years of work.  The first step is to identify the region of the chromosome the gene is in (pedigree analysis, identifying breaks in chromosomes which cause the disease, etc.)  Once the gene has been localized to a region of a chromosome, is to “walk” along the chromosome.  The walk starts at a sequence known to be nearby, and continues until the gene of interest is located.
  • 15. Isolation of Human Genes  Positional cloning: Isolation of a gene associated with a genetic disease on the basis of its approximate chromosomal position.
  • 16. Cystic Fibrosis Gene  Cystic fibrosis disease is a common lethal disease inherited as an autosomal recessive manner.  Identify RFLP markers linked to the CF gene.  Identify the chromosome on which the CF gene is located.  Identify the chromosome region on which the CF gene is located (finer mapping).  Clone the CF gene between the flanking markers.  Identify the CF gene in the cloned DNA.  Identify the defects in the CF gene.
  • 17. RFLP markers linked to the CF gene (linkage studies)  Screen many individuals in CF pedigrees with a large number of RFLPs.  Use Southern blot analysis and hybridize with probes to identify different forms.  Establish a linkage between the markers and the occurrence of the disease.
  • 18. Which chromosome?  Use in situ hybridization, where chromosomes are spread on a microscope slide, and hybridized with a labeled probe, results are analyzed by autoradiography.  A 3H-labeled RFLP probe showed that CF gene is located on chromosome 7.
  • 19. Which chromosomal region?  Search other RFLPs located on the chr. 7, to find ones that are linked to the CF gene.  Again, use the pedigrees and test the DNA for associated RFLP markers.  Two closely linked flanking markers (one marker on each side of the CF gene) were found that are 0.5 map units apart (~500.000 bp).  Their locations were 7q31-q32.