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Introduction to DNA Sequencing Technology

Introduction to DNA Sequencing Technology. Dideoxy Sequencing (Sanger Sequencing, Chain Terminator method). Clone the fragments to be sequenced into the virus M13. Why M13? The clones that are isolated are single-stranded DNA. . Primer. ^^^. . . . . . TGATGTCGAGCGAGTCGTACGGT-----.

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Introduction to DNA Sequencing Technology

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  1. Introduction to DNA Sequencing Technology

  2. Dideoxy Sequencing (Sanger Sequencing, Chain Terminator method). • Clone the fragments to be sequenced into the virus M13. • Why M13? • The clones that are isolated are single-stranded DNA.

  3. Primer ^^^ . . . . . TGATGTCGAGCGAGTCGTACGGT----- Fragment to be deciphered M13

  4. DNA sequencing reaction: 1) DNA fragment to be sequenced cloned into the vector M13 2) DNA polymerase 3) “Universal” primer 4) All 4 DNA building blocks 5) One ddNTP tagged with a radioactive tracer

  5. The most popular technique is based on the dideoxynucleotide.

  6. Pyrimidine Purine

  7. Set up 4 separate reactions. Each reaction contians one of the 4 ddNTPs. Each ddNTP is tagged with a radioactive tracer.

  8. A reaction (with ddA)  21, 26, 29, . . . .T reaction (with ddT)  25, 31, 35, . . . . .C reaction (with ddC)  22, 23, 27, . . . . G reaction (with ddG)  ?? (3’ end of primer) Primer (20 nt.) ^^^ . . . . . TGATGTCGAGCGAGTCGTACGGT----- M13

  9. Each reaction generates a set of unique fragment lengths. • All fragment lengths are represented (from 21 - > 1,000 nucleotides). • None of the fragments are present in more than one reaction.

  10. DNA sequencing technology requires gel electrophoresis system with the ability to separate DNA fragments that separate by one b.p.

  11. DNA sequencing, as performed in the 1980s (manually) is slow and labor intensive. • NCBI HomePage

  12. ~1988- First big change in DNA sequencing technology: • Introduction of ‘automated DNA sequencing’: • This technique uses 4 fluorescent labels (red, yellow, blue, green) rather than one radioactive tag. • The bases are read by a laser/detector rather than by humans.

  13. York University

  14. ? Questions ?

  15. Newest Innovations in DNA Sequencing Technology • 1) Capillary Electrophoresis • 2) Robotics

  16. CE Theory

  17. Capillary Gel Electrophoresis: “The capillaries we typically use in CE are inexpensive and commercially available. We use capillaries that range about 30 to 50 centimeters in length, 0.150 to 0.375 millimeters in outer diameter, and a 0.010 to 0.075 millimeter diameter channel down the center. “

  18. DNA sequencing with CE # of capillary tubes/machine: Initally- one (Introduced ~ 1998) State of the Art- 2000: 96 tube CE (cost $300k) Today- 384 tube CE (cost of one unit- $500k)

  19. DOE Joint Genome Institute

  20. HUMAN GENOME PROJECT (HGP) • The ultimate goal of the HGP is to decipher the 3.3 billion b.p. of the human genome. • When the project was initiated, its was technologically unfeasible.

  21. Genomic Sequencing Organisms sequenced • Year # genomes sequenced • 1994 0 • 1995 2 • 1996 4 • 1997 8 (est.) • 1998 30 (est.) • 2001 ~75

  22. Genomics Research Funding(selected programs; $ millions)

  23. Why such a sudden increase in funding?? • It became apparent that if the public agencies didn’t get their act together, an upstart organization might sequence the HG before they did (despite their ~ 8 year head start).

  24. Sequencing the human genome suddenly had become a race. • The competitors: • Publicly funded genome centers, scattered throughout the U.S., Europe, and Japan. • Celera, the private company directed by J. C raig Venter.

  25. The story of how J. Craig Venter brought about a paradigm shift in genomic sequencing has now entered the mythology of science.

  26. Craig VenterScientist of the Year • from Time Magazine: What was perhaps the most important scientific event of the past century occurred this year when scientists announced the cracking of the human genetic code. And what everyone, including his numerous critics, acknowledges is that the brash and impatient Venter is the man who made it happen years before it would have otherwise by throwing computing power at the traditional, laborious process of manually examining every bit of human DNA to find the genes within.

  27. Why did Craig Venter and his new company Celera threaten the established genome sequencers? • Venter’s new company had 300 $300k state-of-the-art sequencing machines and an $80 million dollar supercomputer. • Venter suggested Celera could sequence the genome in but 3 years at a cost of $300 million.

  28. Venter’s first company, TIGR, pioneered the ‘shotgun sequencing’ approach to sequencing a genome: • 1) Shear the DNA into thousands of random pieces. • 2) Sequence the DNA of each fragment. • 3) Use a computer to align the overlapping fragments to produce a single, contiguous DNA sequence of the entire organism.

  29. Advantages/Disadvantages of the ‘shotgun approach’: Disadvantages- Requires significant over-sequencing Requires powerful alignment software There may be problems ‘finishing’ certain regions Advantages- Eliminates the needing for mapping

  30. Sequencing of Archaeoglobus fulgidus: • 29,000 sequencing reactions • 500 bp. Average ‘read’ • 14,500,000 bases aligned  2,178,400 bp. • 6.7- fold sequence coverage (14,500,000 / 2,178,400 = 6.7)

  31. Even with remarkable success sequencing bacterial genomes, skeptics doubted a whole genome random sequencing approach would work with a eukaryotic genome. Why? 2 Reasons- • Eukaryotic genomes are much larger. • Eukaryotic genomes carry significant amounts of repetetive DNA.

  32. Who won the race? • With much fanfare, the rough draft of the human genome was ‘declared’ a draw. Both Celera and the various public agencies shared credit for the rough draft of the human genome (‘announced Feb. 2000).

  33. Insert Video (10’)

  34. What is meant by the term mapping? • Mapping to a geneticist means the same as it does to a non-scientist: • A drawing showing the spatial relationship between a series of points.

  35. Western U.S.- Seattle- Portland- S.F. - L. A. - Human Chromosome # 11 Traditional map: Gene Map: Hemoglobin-b Insulin Parathyroid Hormone Albinism

  36. Mouse Clickable Cytogenetic MapChromosome X is selected

  37. Restriction Enzyme Map HinDIII EcoRI HinDIII HinDIII • ____|__________|________|_________|_

  38. Construction of various maps has been a major goal of genetic research. Why? • Maps serve as navigational tools. They are useful in finding genes or other genetic features and ordering fragments of DNA. • There is a direct correlation between the usefulness of a map, and the number of points on the map. Analogy??

  39. The STS map: • STS = sequence-tagged site. • STS are short, unique fragments of DNA generated by PCR. • Verification of a human STS: PCR amplification of the human genome generates one small fragment  unique lanckmark

  40. Usefulness of STSs • STSs are used to find overlaps between fragments of genomic DNA. • Finding overlaps  ordering of fragments (see handout).

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