1 / 30

Sanger Dideoxy sequencing

Sanger Dideoxy sequencing. Purpose: to determine the primary sequence of a DNA molecule. Interactive Demo for Cycle sequencing http://www.dnalc.org/view/15923-Cycle-sequencing.html. Terms: Polymerase Primer Template DNA strand PCR Deoxynucleotide Dideoxynucleotide

kail
Télécharger la présentation

Sanger Dideoxy sequencing

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Sanger Dideoxy sequencing Purpose: to determine the primary sequence of a DNA molecule. Interactive Demo for Cycle sequencing http://www.dnalc.org/view/15923-Cycle-sequencing.html Terms: Polymerase Primer Template DNA strand PCR Deoxynucleotide Dideoxynucleotide Polyacrylamide gel electrophoresis http://www.dnalc.org/view/15479-Sanger-method-of-DNA-sequencing-3D-animation-with-narration.html

  2. Length of DNA sequence in one run: 500-800 bp Length of a chromosome: 10-200 million bp • How can we sequence something so large? • Sequences have to be separated from each other to use Sanger sequencing. • Then we need to assemble the sequence data of ~750 bp lengths back together. (3,000,000,000 ÷ 750 = 4,000,000 pieces… • How?

  3. Hierarchical versus Shotgun sequencing

  4. DNA vectors for Sequencing

  5. Chapter 1 Genome Projects Objectives • To assemble physical and genetic maps of the genome (today) • To establish an integrated Web-based database and research interface (done) • To generate and order genomic and expressed gene sequences (this week and next) • To identify and annotate the complete set of genes in genome (this week and next)

  6. Chapter 1 Genome Projects Objectives • To characterize DNA sequence diversity(Chapter 3) • To compile atlases of gene expression(Chapter 4) • To accumulate functional data (biochemical and phenotypic properties of genes) (Chapter 4,5,6) • To provide the resources for comparison with other genome (Chapter 3,4,5,6)

  7. Genetic Linkage Mapping describes the order of variablemarkers* segregating in an informative population Unit of distance- centiMorgan (cM) What can we use a linkage map for? Limitations of linkage mapping *What can “markers” consist of?

  8. Physical Mapping- advantages versus Linkage Mapping for Genome Analysis • A physical map shows the molecular distance between specific locations on a single physical molecule or a set of overlapping molecules • No polymorphism needed- important when assembling conserved sequences or sequence of limited number of individuals • Map resolution can be very high with relatively minimal effort (Radiation mapping sequencing) • Alternative to linkage mapping which can be confirmatory or corrective

  9. Resolution of Various Physical Techniques ResolutionSize of DNA Fragment (bp) • Low: (Chromosomal painting; SCHP) 25-250,000,000 • Middle-low:5-25,000,000 (Chr Regions; SCHP, painting, Chr scrapes) • Middle-high: (RH maps, Gene-FISH*) 100,000-5,000,000 • High: Cosmids1,000-10,000 Plasmids 100-1,000 Sequencing1 *Fluorescent In Situ Hybridization How do you increase resolution of a genetic linkage map?

  10. Figure 1.4 Chromosome painting

  11. Figure 1.5 Chromosome painting shows conserved syntenybetween genomes

  12. Bidirectional ZOO-FISH between SSC12 and HSA 17 HSA17 labeled by SSC12 probe SSC12 labeled by HSA17 probe FISH= Fluorescent In Situ Hybridization Pictures courtesy of M. Yerle, INRA

  13. Schematic of Human:Pig Zoo- FISH Human 17 Pig 12

  14. Physical Mapping Techniques • Gene-specific FISH • Somatic Cell Hybrid Panels • Radiation Hybrid Panels

  15. Fluorescent In Situ Hybridization(FISH) Mapping on Pig Chromosome 13

  16. Single-gene FISH for SSC11 (Dual-color) HTR2A RB1

  17. Physical Mapping Techniques • Gene-specific FISH • Somatic Cell Hybrid Panels • Radiation Hybrid Panels

  18. Steps in Developing a Somatic Cell Hybrid Panel 1. Cellfusion 2. Selection 3. Cytogeneticanalysis. 4. Moleculargeneticanalysis

  19. Somatic Cell Hybrid Panel Characterization 1. Cytogeneticanalysis to determine which chromosomes or chromosome fragments are retained in each hybrid ‘clone’ in panel. 2. Moleculargeneticanalysis: Presence or absence of gene sequence is correlated with chromosome presence/absence in each hybrid. • Requires species specific hybridization or PCR An informative panel requires: -specific pattern for each region -100% coverage of the genome

  20. Example: SCHP Mapping to Chromosome13 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F SSC 13 D=F

  21. Example: Human 3 and 21 Paints Overlap on Pig 13 QTL HSA3 SSC13 HSA21

  22. Physical Mapping Techniques • Gene-specific FISH • Somatic Cell Hybrid Panels • Radiation Hybrid Panels

  23. Approach to Improve Physical Map Resolution Radiation Hybrids (RH) • Resolution of RH Mapping is HIGH- between current • linkage maps and physically cloned DNA • No polymorphism needed, highly useful in • comparative mapping of Type I loci • • Can be used to make fast progress in • detailed comparative mapping • • RH mapping of microsatellite loci allows linkage • map connection

  24. X-rayIrradiation breaks chromosomes into fragments Steps in the Development of Radiation Hybrid Panels What process is conceptually similar to recombination here? Donor DNA fragments, not chromosomes, are retained Much higher resolution possible

  25. “Two-point linkage” calculations for RH mapping a) Frequency of breakage (R = Rays (equivalent to Morgans) • = (A+B - + A-B+) T( Ra + Rb - 2RaRb) Ra fraction of hybrids analyzed for marker a that contains a Rb fraction of hybrids analyzed for marker b that contains b T total number of hybrids analyzed for both markers b) Distance between A and B in R: D = -ln (1-)

  26. PRSS7 APP IFNAR MX ITGB2 PRSS7 APP IFNAR PIT1,PRSS7 MX, ITGB2 APP, IFNAR MX HSA21 Gene Mapping in Pig SCHP Results FISH Results RH Results PIT1 208 cR PRSS7 83 cR APP 181 cR IFNAR 124 cR HSA21 MX SSC13 SSC13

  27. Transition to molecular maps • Methods already discussed don’t require a global effort in cloning large regions of the genome of interest. • Rest of the physical mapping discussion will involve the idea of cloning all parts of the genome and fitting those parts together. • Chapter 2- Genome sequencing

  28. How to put pieces together? • “chromosome walking” to sequence a region of a chromosome: • Example; sequence region between two genes in the pig HTR2A RB1

  29. Human Genome Project • Details of how the sequence was created will be described in Chapter 2 • HGP Goals: • Creation of genetic and physical maps as tools to find disease genes • Develop high-throughput, cheap means to sequence lots of DNA • Use resulting sequence to find and characterize every gene in the genome- computation, aligning many expressed sequences, collecting functional data from experimental models • Compile exhaustive polymorphisms data on human populations

  30. From Mendel to the Human Genome Project

More Related