Nanopore for dna sequencing by shreya


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Nanopore for dna sequencing by shreya

  1. 1. A NANOPORE FOR DNA SEQUENCING Guided and Checked By: Dr. Prakash C. Jha Sir Presented BY: Shreya M .Modi M.Phil. in Nano science Central University of Gujarat. 1
  2. 2. OUTLINE Introduction Types & Application DNA Sequencing Different methods -Using α-Hemolysin -Using MspA -Using Graphene etc…… Conclusion References 2
  3. 3. INTRODUCTION• Nanoporous materials consist of a regular organic or inorganic framework supporting a regular, porous structure.• The size of the pores is generally 100 nanometers or smaller.• Classified as Bulk• Examples – Zeolites, A.Carbon, etc… 3
  4. 4. Types Microporous Mesoporous Macroporous materials materials materials 0.2-2.0 nm 2-50 nm 50-1000 nm Images/Nanoporous%20materials- t/journal/v11/n11/carousel/n 4-400-FOR-TRIDION_tcm18-139492.jpg 2s.jpg mat3404-f4.jpg
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  7. 7. DNA SEQUENCING• DNA sequencing is the process of determining the precise order of Nucleotides within a DNA molecule.• DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions, full chromosomes or entire genomes. 7Radioactive_Fluorescent_Seq.jpg
  8. 8. DNA SEQUENCING GENERATIONS Then + Now Now Now + anticipated Anticipated 1st Gen 2nd Gen 2nd Gen Next Sanger -parallised -single mol or electronic -single mol AND electronic •Optical •Optical •Low •Single-molecule •Direct electrical (no optics) •Amplification needed throughput •Highly parallel •Single-molecule, highly parallel •Highly parallel •High cost •Cost similar •Transformation of workflow •Improved cost and •Accurate •New applications •Designed to broaden user base, Throughput •Broad user deliver step change in cost, power •More centralised base •Or electronic, •New applications users clonal Helicos GAII (Solexa/Illumina) Pacific Biosciences Sanger SOLiD (Agencourt/LIFE) Nanopores Ion Torrent FLX (454/Roche) (LIFE Starlight)Estimated cost of a human genome using these technologies$70M $200k --- $50k ---- $20k --- 15k--- ?$5k - $? 8
  9. 9. De NOVO • DNA from individual bacterial clones is sequenced and the sequence is assembled by using overlapping DNA regions. 9
  11. 11. 454: Pyrosequencing 11
  12. 12. WHAT IS A NANOPORE? Nanopore = „very small hole‟ Electrical current flows through the hole Introduce analyte of interest into the hole  identify “analyte” by the disruption or block toCurrent electrical current theflow 12
  13. 13. APPLICATION OF NANOPORE Adaptable protein nanopore:Application Specific DNA Sequencing Proteins Polymers Small Molecules Sensor array chip: many nanopores in parallel Generic Platform Electronic read-out system 13
  14. 14. NANO PORE FOR DNA SEQUENCING• A nanopore is simply a small hole, of the order of 1 nanometer in internal diameter.• Certain porous transmembrane cellular proteins act as nanopores, and nanopores have also been made by etching a somewhat larger hole• α-Hemolysin Nanopore• MspA Nanopore• Graphene Nanopore 14
  15. 15. α-HEMOLYSIN • TOXIN • DNA or RNA strands can translocate through the pore of alpha-hemolysin, producing the ionic current blockades that reflect the chemical structure of individual strands 15The_process_of_hemolysis.png
  16. 16. ALPHA HEMOLYSIN• A single nucleotide resolution has been demonstrated for DNA hairpins raising the prospect of creating a nanopore sensor capable of reading the nucleotide 16
  17. 17. DNA SEQUENCING BY MSPA Mycobacterium smegmatis 17
  18. 18. Conti….It resolve single-nucleotides in single-stranded DNAwhen double-stranded DNA temporarily holds thenucleotides in the pore constriction.Passing DNA with a series of double-strandedsections through MspA provides proof of principle ofa simple DNA sequencing method using a nanopore. 18
  19. 19. • (A) The positive voltage attracts the negatively charged hairpin DNA into the pore.• (B) The DNA threads through the pore until the wider hairpin duplex prevents further translocation.• (C) After a few milliseconds the hairpin dissociates allowing for complete translocation. 19
  20. 20. GRAPHENE FOR DNA SEQUENCING Each nucleotide interacts with the nanopore to a varying degree, resulting in a characteristic electronic signal for each of the 4 nucleotides. 20
  21. 21. ADVANTAGES• Direct• Fast• InexpensiveDisadvantage- Reduced Conduction- Smaller Coupling 21
  22. 22. EDGE HYDROGENATIONWhen two H-bonds (dotted yellow lines) are formed simultaneouslybetween the nitrogen atom of a DNA nucleobase and two H atomsattached to the graphene-edge.For the sake of clarity, only relevant atoms from the edge hydrogenatedgraphene electrodes and the DNA molecule have been visualized,omitting water molecules, counter ions, and the silicon-nitridemembrane. 22
  23. 23. DETECTING A-T AND G-C BASE PAIRS A-T base pairs to stretch more readily. It can also discriminate between A-T and G-C base pairs which is the first step towards sequencing DNA. 23
  24. 24. Voltage-dependent Kinetics of DNA Transport• The blocking is more effective at lower bias voltages.• At low bias hydrophobic interaction is strong so DNA stick to graphene membrane. 24
  25. 25. ULTIMATELY: WILL WE SEQUENCE EVERY SPECIES? 1995 2002 2005 20002002 2009 25
  26. 26. CONCLUSION• According to advancement and applications of nanopores, it indicates that DNA sequencing carried out by Nanopores are best Methods than others. 26
  27. 27. REFERENCES••••• Faller M, et al. (2004) "The structure of a mycobacterial outer- membrane channel." Science.• Butler TZ, Pavlenok M, Derrington I, Niederweis M, Gundlach J (2008). "Single-molecule DNA detection with an engineered MspA protein nanopore." Proc. Natl. Acad. Sci. 106 (9): 20647-20652.• Purnell R, Mehta K, Schmidt J (2008). Nucleotide identification and orientation discrimination of DNA homopolymers immobilized in a protein nanopore. Nano Letters 8 (9): 3029-3034 27
  28. 28. Conti…• Church, G.M.; Deamer, D.W., Branton, D., Baldarelli, R., Kasianowicz, J. (1998) "US patent # 5,795,782 (filed March 1995) Characterization of individual polymer molecules based on monomer-interface interaction".• Kasianowicz, JJ; Brandin E, Branton D, Deamer DW (1996-11-26). "Characterization of individual polynucleotide molecules using a membrane channel.". Proc Natl Acad Sci USA 93 (24): 13770–3. doi:10.1073/pnas.93.24.13770. PMC 19421. PMID 8943010.• Manrao E, Derrington I, Pavlenok M, Niederweis M, Gundlach J (2011). "Nucleotide discrimination with DNA immobilized in the MspA nanopore." PLoS ONE 6• Stoddart D, Heron A, Mikhailova E, Maglia G, Bayley H (2009). "Single-nucleotide discrimination in immobilized DNA oglionucleoties with a biological nanopore". Proc. Natl. Acad. Sci. USA 106: 7702– 28 7707.
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