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Cloning lab results Cloning the human genome Physical map of the chromosomes Genome sequencing

Nov. 9, 2009. Cloning lab results Cloning the human genome Physical map of the chromosomes Genome sequencing Integrating physical and recombination maps Polymorphic DNA markers CF jumping Example of a molecular forensics study. Research paper outlines: you can still email me your outline.

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Cloning lab results Cloning the human genome Physical map of the chromosomes Genome sequencing

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  1. Nov. 9, 2009 Cloning lab results Cloning the human genome Physical map of the chromosomes Genome sequencing Integrating physical and recombination maps Polymorphic DNA markers CF jumping Example of a molecular forensics study Research paper outlines: you can still email me your outline

  2. Cloning a gene into a plasmid for replication in E. coli

  3. HindIII BamH1 CAT

  4. Restriction enzymes used in cloning experiments Blunt ends are compatible with any other Blunt end Sticky ends are only compatible with an end that leaves the complementary single- stranded overhang. Because sites are palindromic, sites cut with the same enzyme are compatible. Single stranded overhang aids ligation preligation complex lasts longer.

  5. Tuesday class gel results pBKS Un pCAT BH pCAT Un pBKS BH Open circle supercoil

  6. Libraries of genome sequences or transcripts (cDNA)

  7. Random pieces of DNA are cloned into replication vectors where they can be accessed whenever needed. Cut DNA into fragments, Ligate to replication vectors

  8. Generate contigs that replicate DNA sequence of entire chromosomes

  9. To make overlapping contigs, DNA is partially digested with restriction enzymes or physically sheared

  10. Different kinds of replication vectors are available for maintaining cloned DNA fragments in bacteria or yeast

  11. Choice of vectors depends in part on the desired size of the cloned DNA inserts

  12. High copy number plasmids for inserts the size of single genes Low copy number plasmids for larger inserts - genome fragments

  13. Use end probes and fingerprinting to generate contigs

  14. Fig. 10.8

  15. Fig. 10.11 Combination of mapped polymorphic sequences and genomic DNA clones enables reconstruction of chromosome sequence STS are polymorphic DNA sequences BACS are cloning vectors with genomic DNA inserts

  16. Polymorphic DNA markers allow association of Phenotype with position on DNA map Linkage with a family, high lod score of DNA markers With disease. Find contigs that span markers Compare DNA sequence.

  17. Forensics: Using DNA polymorphism to distinguish individuals Need: Rapid and reliable markers Sufficient numbers of polymorphic markers to be sure that no other person could be a match DNA amplification methods to obtain information from samples that are: very small have DNA degradation

  18. Example of paternity test With this information, we can only exclude the accused from being a parent. With DNA technology, we have so many markers that the probability of mistaken identity becomes very small

  19. Different types of DNA markers Insertion-deletion polymorphism Variable number tandem repeats- Minisatellites (10-100 bp repeats) Short tandem repeats- Microsatellites (1-3 bp repeats) Single nucleotide polymorphisms

  20. Insertion-deletion polymorphism Transposon, retrovirus, etc Allele 1 Allele 2 Point of reference Restriction site PCR primer Sobrino et al 2005 Forensics Sci. Int. 154: 181-194

  21. VNTR Variable Nucleotide Repeats

  22. VNTR or Up to 100bp repeats

  23. Fig. 11.3 Microsatelites 1-3 bp repeats Polymerase pauses, replication continues out of register

  24. microarrays Some single nucleotide polymorphisms (SNP) can be detected as RFLPs if they affect a restriction enzyme sequences. Any single nucleotide polymorphism can be detected by modern technologies such as oligonucleotide arrays

  25. PCR can ease identification of polymorphic sequences and can be used to analyse very small amounts of DNA

  26. PCR copies template exponentially

  27. Microsatellites or minisatellites can be detected by PCR Use PCR to amplify sequences starting with primers designed from single copy sequence that flanks repeats

  28. Genetic variation in a sequence with a minisatellite used in DNA typing

  29. Combining the information about the alleles identified in a person for many loci (or many polymorphic markers) can generate a unique pattern for every person.

  30. Thomas Jefferson paternity investigation Who’s Y chromosome is in the male offspring of Sally Hemings children? Sally Hemings was Jefferson’s slave and cared for him late in his life. She had 5 children. After he died, she claimed Jefferson was the father of her children. Jefferson was almost impeached, charged with being the father of Sally’s first son, Thomas Woodson. Jefferson denied that he was the father. Forster et al 1988 used Y chromosome DNA to see who was telling the truth.

  31. Y chromosome sequences are highly conserved. Comparing the pattern of many polymorphic markers on the Y chromosomes makes it possible to identify the lineage of Y chromosomes in modern descendents of Sally Hemings Thomas Woodson was Sally Hemings first son. Eston Jefferson was her youngest son. John Carr was the brother in-law of Thomas Jefferson. Which lineage matches the Y chromosomes of Eston Jefferson? Thomas Woodson?

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