Lecture 2


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Lecture 2

  1. 1. Genetic and Physical Mapping<br />Lecture 2<br />
  2. 2. Why map genes?<br /> Many diseases are partially genetic<br />– Also: environmental factors, randomness<br />2. We want to identify these genes<br />– Early diagnosis for abortion or regular checks<br /> – First step towards developing treatment<br />3. Individual sequencing is too costly (today)<br />– Sequence a small number of markers<br /> – Analyze statistically via biological principles<br />
  3. 3. <ul><li>Genetic mapping:
  4. 4. based on the use of genetic techniques to construct linkage maps showing the positions of genes and other sequence features in a genome
  5. 5. Physical mapping:
  6. 6. uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the exact position of genes and other sequences</li></li></ul><li>
  7. 7.
  8. 8.
  9. 9.
  10. 10. Genetic Linkage Maps<br />A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker<br />Markers can be expressed DNA regions (genes) or DNA segments that have no known coding function but whose inheritance pattern can be followed. DNA sequence differences are especially useful markers because they are plentiful and easy to characterize precisely<br />Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist among individuals so that they are detectable among different members in family studies<br />Polymorphisms are variations in DNA sequence that occur on average once every 300 to 500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility<br />
  11. 11. Stages of Mapping a Gene<br />• Demonstrate disease is hereditary<br /> – Show it runs in families<br />• Linkage analysis to identify region<br /> – Widely-spaced markers, e.g. RFLPs<br />• Association analysis to narrow region<br /> – Closely-spaced markers, usually SNPs<br />• Clone the gene within found region<br /> – Investigate its metabolic relevance<br />
  12. 12.
  13. 13.
  14. 14. Summary<br />A genetic linkage map shows the relative locations of specific DNA markers along the chromosome<br />2. Five major DNA markers for genetic mapping: <br /><ul><li>RFLP
  15. 15. RAPD
  16. 16. SNP
  17. 17. AFLP
  18. 18. Microsatellites</li></ul>3. A physical map shows the exact position of genes and other sequences on the chromosome<br />4. Five physical mapping methods: <br /><ul><li>optical mapping
  19. 19. fingerprinting
  20. 20. chromosome walking,
  21. 21. STS and FISH </li></li></ul><li>
  22. 22.
  23. 23.
  24. 24. Types of DNA Markers<br />1. Restriction fragment length polymorphisms (RFLP)<br />2. RAPD-Random amplified polymorphic DNA (RAPD)<br />3. Microsatellite, simple sequence repeat (SSR)<br />markers or short tandem repeat (STR)<br />4. Single nucleotide polymorphisms (SNPs)<br />5. Amplified fragment length polymorphism (AFLP)<br />
  25. 25.
  26. 26. <ul><li>Restriction Fragment Length Polymorhisms = RFLP
  27. 27. Minisatellites
  28. 28. Variable number of tandem repeats = VNTR
  29. 29. High number of alleles
  30. 30. High level of heterozygocity
  31. 31. Problem: southern blots and radioactive probes, minisatellites are to long to be amplified by PCR
  32. 32. Not evenly spread over the genome
  33. 33. Microsatellites
  34. 34. Single Nucleotide Polymorphisms (SNP)</li></li></ul><li><ul><li>Restriction Fragment Length Polymorhisms = RFLP
  35. 35. Minisatellites
  36. 36. Microsatellites
  37. 37. Standard tools for mapping studies and linkega analysis
  38. 38. Mostly (CA)n repeats
  39. 39. Tri-en tetranucleotide repeats replace more and more the dinucleotide repeats, because of the better results
  40. 40. Dinucleotide-repeats are volnurable to “replication slippage” during PCR as such that each allel can give a small ladder on a gel
  41. 41. Development of multiplex PCR reactions
  42. 42. Single Nucleotide Polymorphisms (SNP)</li></li></ul><li>
  43. 43.
  44. 44.
  45. 45.
  46. 46.
  47. 47.
  48. 48. Physical Mapping<br /><ul><li>Construction of a physical map which consists of continuous overlapping fragments of cloned DNA that has the same linear order as found on the chromosomes from which they were derived</li></li></ul><li>Physical Map vs Genetic Map<br />Limitations of genetic maps<br />1. The resolution of a genetic map depends on the number of crossovers that have been scored. Limited resolution (1cM is approx. 1 Mb)<br />2. Genetic maps have limited accuracy. Certain genomic regions more sensitive to recombination<br />3. Markers must be polymorphic for genetic mapping<br />
  49. 49. Physical Map of a Chromosome<br />Contig: <br /><ul><li>A series of overlapping clones or sequences that collectively span a particular chromosomal region</li></ul>Depicts genetic markers and DNA sequences between the markers measured in base pairs (High Resolution)<br />
  50. 50. Physical Mapping Methods<br />Optical mapping<br />2. Restriction fragment fingerprinting<br />3. Chromosome walking<br />4. Sequence tagged site (STS) mapping<br />5. Fluorescent in situ hybridization (FISH)<br />
  51. 51.
  52. 52. 2 Restriction fragment fingerprinting<br /><ul><li>Individual clones are digested with different restriction enzymes
  53. 53. The digested DNA is labeled with radioactive or fluorescent dye and run on a sequencing gel
  54. 54. The fingerprint data is collected and analyzed for contigassembly</li></ul>Disadvantages: labor intensive and difficult to fill gaps.<br />
  55. 55.
  56. 56.
  57. 57.
  58. 58. 3 Chromosome walking<br /><ul><li>Markers with known map position are used as probe to screen the large insert library
  59. 59. Clones hybridizing with the same single copy marker are considered to be overlapping
  60. 60. PCR amplification of DNA pools using primers derived from DNA markers with known position was also used for physical map construction</li></ul>Disadvantage: <br /><ul><li>Laborintensive, repetitive sequence misleading, markers unevenly distributed in the genome.</li></li></ul><li>Chromosome Walking<br />
  61. 61. 4 Sequence Tagged Sites (STSs)<br /><ul><li>An STS is a short region of DNA about 200-300 bases long whose exact sequence is found nowhere else in the genome
  62. 62. Two or more clones containing the same STS must overlap and the overlap must include the STS</li></ul>Disadvantages: <br /><ul><li>still very labor intensive and
  63. 63. high expensive for primer synthesis</li></li></ul><li>STS mapping of linked markers and BAC clones<br />
  64. 64. PCR confirmation of STS markers in the genome Each STS contains a unique sequence<br />
  65. 65. 5 Fluorescence in situ hybridization (FISH)<br /><ul><li>This technique uses synthetic polynucleotide strands or short DNA fragment that bear sequences known to be complementary to specific target sequences at specific chromosomal locations.
  66. 66. The polynucleotides are bound via a series of link molecules to a fluorescent dye that can be detected by a fluorescence microscope</li></li></ul><li>
  67. 67.
  68. 68. <ul><li>Human metaphase chromosomes hybridized to fluorescent probes from two overlapping micro-dissection libraries.
  69. 69. Probes specific to chromosome regions 1p34– 35 and 1p36 were labeled using the ULYSIS Oregon Green 488 (U21659) and Alexa Fluor 594 (U21654) Nucleic Acid Labeling Kits, respectively.</li></ul>(http://www.probes.com/servlets/photohigh?fileid=g001276)<br />
  70. 70. FISH mapping of BACs (bacterial artificial chromosomes)<br />
  71. 71. Application of FISH in physical mapping<br />Jackson S. et al. 2000. Genetics, 156: 833-838<br />
  72. 72. Combination of fingerprinting, molecular linkage map, STS, end sequencing and FISH mapping<br />Combination of STS markers, BACs and FISH<br />Human genome anatomy: <br /><ul><li>BACs integrating the genetic and cytogenetic maps for bridging genome and biomedicine. Genome Res. 1999 9(10):994-1001
  73. 73. 872 unique STSs and 957 BACs were used</li></li></ul><li>
  74. 74.
  75. 75.
  76. 76.
  77. 77.
  78. 78. Gene mapping applications. <br />Useful for locating the position of genes on chromosomes, e.g. if two genes are closely linked and the position of one is known, then the other must also be nearby. <br /> Useful in estimating genetic risk, e.g. if a gene cannot be tested directly, then variation at a closely linked locus may indicate the presence or absence of a detrimental allele. <br /> Has aided in:<br /><ul><li>Mapping of all human genes (Human Genome Project)
  79. 79. Mice,
  80. 80. Drosophila,
  81. 81. Caenorabditiselegans , Arabidopsis thaliana
  82. 82. Yeast, Escherichia coli. </li></li></ul><li>By 1999:<br /><ul><li>Yeast;E. coli; C. elegans
  83. 83. A dozen other bacteria have been completely sequenced and all their genes identified, although the functions of most are unknown.
  84. 84. Major progress has been made in mapping human genes, and a "rough draft" of the human genome was read by 2000.
  85. 85. Understanding of function of the many newly discovered human genes is being greatly aided by the studies of yeast, which has many genes similar to those of humans. </li></li></ul><li>Resources<br />• Genetic Analysis Software<br />– http://linkage.rockefeller.edu/soft/list.html<br />• Introduction to Genetic Analysis<br />– http://www2.qimr.edu.au/davidD/Course/<br />• Genetic Analysis Resources<br />http://ihome.cuhk.edu.hk/~b400559/complexdismap.html<br />• NCBI Human Genes and Disease<br />– http://www.ncbi.nlm.nih.gov/disease/<br />• International Haplotype Map Project<br />– http://www.genome.gov/10001688<br />