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Chapter 5

Chapter 5. Chemical Synthesis, Sequencing, and Amplification of DNA. Chemical Synthesis of DNA. The Phosphoramidite method Uses of synthesized oligonucleotides. Fig 5.1. Fig 5.2. Fig 5.3. Fig 5.4. Fig 5.5. Fig 5.6. Fig 5.7. Fig 5.8. Fig 5.9. Fig 5.10. Fig 5.11. Fig 5.12.

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Chapter 5

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  1. Chapter 5 Chemical Synthesis, Sequencing, and Amplification of DNA

  2. Chemical Synthesis of DNA • The Phosphoramidite method • Uses of synthesized oligonucleotides

  3. Fig 5.1

  4. Fig 5.2

  5. Fig 5.3

  6. Fig 5.4

  7. Fig 5.5

  8. Fig 5.6

  9. Fig 5.7

  10. Fig 5.8

  11. Fig 5.9

  12. Fig 5.10

  13. Fig 5.11

  14. Fig 5.12

  15. Fig 5.13

  16. table 5.1

  17. DNA Sequencing Techniques • Dideoxynucleotide procedure for sequencing DNA • Automated DNA sequencing • Using bacteriophage M13 as a DNA sequencing vector • Primer walking

  18. Dideoxynucleotide

  19. Cycle Sequencing Reaction * Templates * Primers * AmpliTaq DNA Polymerase, FS * BigDye Terminators * Others (Buffer, Mg2+ , dNTP…)

  20. Templates • Double Stranded Plasmid DNA • PCR Product • Single Stranded DNA

  21. Templates: Quality Requirement Plasmid DNA: • RNA, Genomic DNA and Protein free • Recommended Purification Method • QIAGEN DNA Isolation Products • Alkaline lysis with PEG PPT PCR Product: • if single band produced: remove excess primers, dNTP • if multiple band produced: gel purification followed by extraction protocol is required

  22. Primers Length: • generally 18-24 bases • Specific Hybridization Site • Avoid Secondary Structure • Avoid Strings of 3 or More of One Base in the Primer Sequence • High Purity

  23. Sequencing Reaction ( standard protocol) Component Quantity Plasmid DNA 0.2-0.5 mg Primer 3.2 pmole BigDye Terminator Kit 8 mL Deionized water q.s. Total Volume 20 mL *Adjust the concentration of template and primer to make the total volume of template and primer < 12 mL

  24. PCR Product Required for Cycle Sequencing Reaction Size(bp) Template quantity(ng) 100-200 1-3 200-500 3-10 500-1000 5-20 1000-2000 10-40 >2000 40-100

  25. Cycle Sequencing Program • 96 ℃, 2 min • 96 ℃, 10 sec • 50 ℃, 5 sec 25 cycle • 60 ℃, 4 min

  26. Recommended Alcohol Precipitation Method Protocol • Add the ethanol/EDTA solution to each well: • 5.0 uL of 125mM EDTA • 60 uL of 100% ethanol • Mix by inverting 4 times than leave the tubes at room temperature for • 15 minutes to precipitate the extension products • Spin the tubes for 20 minutes at maximum speed in a microcentrifuge • ( Proceed to the next step immediately) • Carefully aspirate the supernatants completely • Add 60 uL of 70% ethanol to the tubes and mix briefly • Spin the tubes for 5 minutes at maximum speed (at 4ºC) • Aspirate the supernatants completely • Dry the samples in a vacuum centrifuge for 5-10 minutes

  27. Next-Generation Sequencing • 高通量定序(High -Throughput Sequencing, HTS) • 合成定序法(Sequencing by Synthesis, SBS) • 大量同步定序法(Massive Parallel Sequencing, MPS)

  28. Next Generation Sequencing • Applied Biosystems/Agencourt (SOLiD)

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