• Like
08.13.08: DNA Sequence Variation
Upcoming SlideShare
Loading in...5
×

08.13.08: DNA Sequence Variation

  • 989 views
Uploaded on

Slideshow is from the University of Michigan Medical School's M1 Patients and Populations: Medical Genetics Sequence. …

Slideshow is from the University of Michigan Medical School's M1 Patients and Populations: Medical Genetics Sequence.

View additional course materials on Open.Michigan:
openmi.ch/med-M1PatientsPopulations

More in: Education , Technology
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads

Views

Total Views
989
On Slideshare
0
From Embeds
0
Number of Embeds
0

Actions

Shares
Downloads
22
Comments
0
Likes
0

Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
    No notes for slide

Transcript

  • 1. Author(s): David Ginsburg, 2009License: Unless otherwise noted, this material is made available under the terms ofthe Creative Commons Attribution–Non-commercial–Share Alike 3.0 License:http://creativecommons.org/licenses/by-nc-sa/3.0/ We have reviewed this material in accordance with U.S. Copyright Law and have tried to maximize your ability to use, share, and adapt it. The citation key on the following slide provides information about how you may share and adapt this material. Copyright holders of content included in this material should contact open.michigan@umich.edu with any questions, corrections, or clarification regarding the use of content. For more information about how to cite these materials visit http://open.umich.edu/education/about/terms-of-use. Any medical information in this material is intended to inform and educate and is not a tool for self-diagnosis or a replacement for medical evaluation, advice, diagnosis or treatment by a healthcare professional. Please speak to your physician if you have questions about your medical condition. Viewer discretion is advised: Some medical content is graphic and may not be suitable for all viewers.
  • 2. Citation Key for more information see: http://open.umich.edu/wiki/CitationPolicyUse + Share + Adapt { Content the copyright holder, author, or law permits you to use, share and adapt. } Public Domain – Government: Works that are produced by the U.S. Government. (USC 17 § 105) Public Domain – Expired: Works that are no longer protected due to an expired copyright term. Public Domain – Self Dedicated: Works that a copyright holder has dedicated to the public domain. Creative Commons – Zero Waiver Creative Commons – Attribution License Creative Commons – Attribution Share Alike License Creative Commons – Attribution Noncommercial License Creative Commons – Attribution Noncommercial Share Alike License GNU – Free Documentation LicenseMake Your Own Assessment { Content Open.Michigan believes can be used, shared, and adapted because it is ineligible for copyright. } Public Domain – Ineligible: Works that are ineligible for copyright protection in the U.S. (USC 17 § 102(b)) *laws in your jurisdiction may differ { Content Open.Michigan has used under a Fair Use determination. } Fair Use: Use of works that is determined to be Fair consistent with the U.S. Copyright Act. (USC 17 § 107) *laws in your jurisdiction may differ Our determination DOES NOT mean that all uses of this 3rd-party content are Fair Uses and we DO NOT guarantee that your use of the content is Fair. To use this content you should do your own independent analysis to determine whether or not your use will be Fair.
  • 3. DNA Sequence Variation M1 Patients and Populations David Ginsburg, MD August 13, 2008Fall 2008
  • 4. Learning Objectives•  Understand the meaning of DNA sequence and amino acid polymorphisms.•  Recognize the different types of DNA sequence polymorphisms: –  RFLP, VNTR, STR, SNP, CNV•  Know the different classes of DNA mutation: –  Point mutations (silent, missense, nonsense, frameshift, splicing, regulatory) insertion/deletions, rearrangements•  Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
  • 5. Chromosomes, DNA, and GenesProteins Jfdwolff (wikimedia) Wikimedia Commons
  • 6. TRANSCRIPTIONGelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.1
  • 7. 1983 2001 Huntington Draft human Disease genome gene 1989 sequence 1956 1981mapped Positional cloning Glu 6 Val in Transgenic mice without deletion sickle hemoglobin (CF) 1944 1970 1975 1995 1985 1st complete DNA is the 1953 First restriction Southern PCR genetic Double enzyme blotting bacterial material helix genome sequence 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 1972 1986 1990 1996 1949 1966 Recombinant Complete yeast Positional First NIH-Abnl Hemoglobin Completion of the genome plasmids cloning (CGD, approved in sickle cell genetic code sequence muscular gene anemia dystrophy, therapy retinoblastoma experimentSince 2001: 1987 KnockoutComplete human genome complete; human micehapmap;Other genomes: mouse, rat, dog, chimp, chicken, Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.4zebrafish…18 vertebrates, 8 invertebrates,>500 microbial genomes.
  • 8. DNA Samples and PCR DNA for analysis cheek swabBlood sample urine sample Forensic specimens Source: Dennis Myts (wikimedia) Repeat DNA cycles primers Target sequence Amplified product D. Ginsburg
  • 9. SINGLE-STRANDED DNA PROBES FOR GENE A C D B MIXTURE OF + SINGLE-STRANDED DNA MOLECULES E A F C C DB B D A A E E F F ONLY A FORMS A STABLE A, C, E ALL FORMDOUBLE-STRANDED COMPLEXES STABLE COMPLEXES STRINGENT HYBRIDIZATION REDUCED-STRINGENCY HYBRIDIZATION Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.8
  • 10. Hybridization array: gene chip DNA chip v6.0: ~1 million SNPs RNA expression: All ~ 20,000 genes CGH: Survey whole genome Ricardipus (wikipedia) for large deletions, insertions, rearrangements
  • 11. Image of DNA sequencingprocess removed
  • 12. DNA SequencingSource Undetermined
  • 13. New Sequencing TechnologiesSource Undetermined
  • 14. Learning Objectives•  Understand the meaning of DNA sequence and amino acid polymorphisms.•  Recognize the different types of DNA sequence polymorphisms: –  RFLP, VNTR, STR, SNP, CNV•  Know the different classes of DNA mutation: –  Point mutations (silent, missense, nonsense, frameshift, splicing, regulatory) insertion/deletions, rearrangements•  Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
  • 15. DNA Sequence Variation•  DNA Sequence Variation: –  Human to human: ~0.1% (1:1000 bp) •  Human genome = 3X109 bp X 0.1% =~3X106 DNA common variants –  Human to chimp: ~1-2% –  More common in junk DNA: introns, intergenic regions•  poly·mor·phism Pronunciation: "päl-i-mor-"fiz-&m Function: noun : the quality or state of existing in or assuming different forms: as a (1) : existence of a species in several forms independent of the variations of sex (2) : existence of a gene in several allelic forms (3) : existence of a molecule (as an enzyme) in several forms in a single species
  • 16. MutationsA mutation is a change in the normal base pair sequence Dennis Myts (wikimedia) •  Can be: –  a single base pair substitution –  a deletion or insertions of 1 or more base pairs (indel) –  a larger deletion/insertion or rearrangement
  • 17. Polymorphisms and Mutations•  Genetic polymorphism: –  Common variation in the population: •  Phenotype (eye color, height, etc) •  genotype (DNA sequence polymorphism) –  Minor allele frequency > 1%•  DNA (and amino acid) sequence variation: –  Most common allele < 0.99 = polymorphism (minor allele > 1%) –  Variant alleles < 0.01 = rare variant•  Mutation-- any change in DNA sequence –  Silent vs. amino acid substitution vs. other –  neutral vs. disease-causing•  Common but incorrect usage: –  mutation vs. polymorphism•  balanced polymorphism= disease + polymorphism
  • 18. All DNA sequence variation arises via mutation of an ancestral sequence < 1% > 1% Rare variant orNormal private polymorphism polymorphism Example: Factor V LeidenDisease Disease mutation (thrombosis) 5% allele frequency Common but incorrect usage: a disease-causing mutation OR a polymorphism
  • 19. Learning Objectives•  Understand the meaning of DNA sequence and amino acid polymorphisms.•  Recognize the different types of DNA sequence polymorphisms: –  RFLP, VNTR, STR, SNP, CNV•  Know the different classes of DNA mutation: –  Point mutations (silent, missense, nonsense, frameshift, splicing, regulatory) insertion/deletions, rearrangements•  Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
  • 20. Types of DNA Sequence Variation•  RFLP: Restriction Fragment Length Polymorphism•  VNTR: Variable Number of Tandem Repeats –  or minisatellite –  ~10-100 bp core unit•  SSR : Simple Sequence Repeat –  or STR (simple tandem repeat) –  or microsatellite –  ~1-5 bp core unit•  SNP: Single Nucleotide Polymorphism –  Commonly used to also include rare variants•  Insertions or deletions –  INDEL – small (few nucleotides) insertion or deletion•  Rearrangement (inversion, duplication, complex rearrangement)•  CNV: Copy Number Variation
  • 21. VNTRGelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.19
  • 22. STR Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E; Figure 5.22
  • 23. SNPAllele 1 A U G A A G U U U G G C G C A U U G C A AAllele 2 A U G A A G U U U G G U G C A U U G C A A Adapted from ASCO teaching slides •  Most are silent •  Intragenic •  Promoters and other regulatory sequences •  Introns •  Exons –  5 and 3 untranslated regions –  Coding sequence (~1-2% of genome)
  • 24. Learning Objectives•  Understand the meaning of DNA sequence and amino acid polymorphisms.•  Recognize the different types of DNA sequence polymorphisms: –  RFLP, VNTR, STR, SNP, CNV•  Know the different classes of DNA mutation: –  Point mutations (silent, missense, nonsense, frameshift, splicing, regulatory) insertion/deletions, rearrangements•  Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
  • 25. Silent Sequence Variants (Synonymous SNP) mRNANormal A U G A A G U U U GGC GC A U UG C A A Protein Met Lys Phe Gly Ala Leu GlnSequence mRNA A U G A A G U U U GGU GC A U UG C A A variant Protein Met Lys Phe Gly Ala Leu Gln Adapted from ASCO teaching slides Changes that do not alter the encoded amino acid
  • 26. Missense Mutations mRNANormal A U G A A G U U U GGC GC A U UG C A A Protein Met Lys Phe Gly Ala Leu Gln mRNA A U G A A G U U U AGC GC A U UG C A AMissense Protein Met Lys Phe Ser Ala Leu Gln Adapted from ASCO teaching slides Missense: changes to a codon for another amino acid (can be harmful mutation or neutral variant) (nonsynonymous SNP)
  • 27. Nonsense Mutations mRNANormal A U G A A G U U U GGC GC A U UG C A A Protein Met Lys Phe Gly Ala Leu Gln mRNA A U G U A G U U U GGC GC A U UG C A ANonsense Protein Met STOP Adapted from ASCO teaching slides Nonsense: change from an amino acid codon to a stop codon, producing a shortened protein (nonsynonymous SNP)
  • 28. Frameshift Mutations mRNA Normal A U G A A G U U U GGC GC A U UG C A A Protein Met Lys Phe Gly Ala Leu Gln mRNAFrameshift A U G A A G U U G GC G C A U UGC A A Protein Met Lys Leu Ala STOP Adapted from ASCO teaching slides Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened protein
  • 29. Splice-site Mutations Exon 1 Intron Exon 2 Intron Exon 3 Exon 2 Exon 1 Exon 3Altered mRNA Adapted from ASCO teaching slidesSplice-site mutation: a change that results in altered RNA sequence
  • 30. Other Types of Mutations•  Mutations in regulatory regions of the gene•  Large deletions or insertions•  Chromosomal translocations or inversions
  • 31. Copy Number Variation (CNV)•  Kb to Mb in size (average ~250 Kb)•  >>2000 known, affect ~12% of human genome•  ? ~100 / person•  ? Role in human disease/normal traits Nature 444:444, Nov 2006.
  • 32. Learning Objectives•  Understand the meaning of DNA sequence and amino acid polymorphisms.•  Recognize the different types of DNA sequence polymorphisms: –  RFLP, VNTR, STR, SNP, CNV•  Know the different classes of DNA mutation: –  Point mutations (silent, missense, nonsense, frameshift, splicing, regulatory) insertion/deletions, rearrangements•  Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
  • 33. Tests to Detect Mutations•  Many methods/technologies•  Rapidly changing•  DNA sequencing –  Most direct and informative –  The gold standard –  Targeted region (known mutation) –  Whole gene (unknown mutation) –  … Whole genome
  • 34. How do we distinguish a disease causing mutation from a silent sequence variation?•  Obvious disruption of gene –  large deletion or rearrangement –  frameshift –  nonsense mutation•  Functional analysis of gene product –  expression of recombinant protein –  transgenic mice•  New mutation by phenotype and genotype•  Computer predictions•  Disease-specific mutation databases –  Same/similar mutation in other patients, not in controls X•  Rare disease-causing mutation vs. private polymorphism (rare variant)
  • 35. Learning Objectives•  Understand the meaning of DNA sequence and amino acid polymorphisms.•  Recognize the different types of DNA sequence polymorphisms: –  RFLP, VNTR, STR, SNP, CNV•  Know the different classes of DNA mutation: –  Point mutations (silent, missense, nonsense, frameshift, splicing, regulatory) insertion/deletions, rearrangements•  Understand how to distinguish a disease-causing mutation from a neutral DNA sequence variation
  • 36. Additional Source Information for more information see: http://open.umich.edu/wiki/CitationPolicy Slide 5: Jfdwolff (wikimedia) http://creativecommons.org/licenses/by-sa/3.0/; http://commons.wikimedia.org/wiki/File:Chromosome_zh-tw.svg Slide 6: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.1 Slide 7: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.4 Slide 8: D. Ginsburg Slide 9: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.8 Slide 10: CC: BY-SA: Ricardipus (wikipedia) http://creativecommons.org/licenses/by-sa/2.0/deed.en Slide 11: Image of DNA sequencing process removed Slide 12: Undetermined Slide 13: Undertirmined Slide 16: Dennis Myts (wikimedia) Slide 21: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.19 Slide 22: Gelehrter, Collins and Ginsburg: Principles of Medical Genetics 2E, Figure 5.22 Slide 23: Adapted from ASCO teaching slides Slide 25: Adapted from ASCO teaching slides Slide 26: Adapted from ASCO teaching slides Slide 27: Adapted from ASCO teaching slides Slide 28: Adapted from ASCO teaching slides Slide 29: Adapted from ASCO teaching slides Slide 31: Nature 444:444, Nov 2006.