Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Genomics
Topics to be covered• Introduction.• History of Genome Sequencing.• How genomes are sequenced.   • Packaging   • Transfect...
Period of time between first man-powered flight and landing on the moon (1902-1969):                                      ...
What is a Genome?• Gene + Chromosome -> Genome                                A/T/G/C                                A/U/G/C
Why determine the order of                nucleotides?• Determining the order of billions of  chemical units that builds t...
Genome Sequencing HistoryOrganism        Year   Institute         Genome SizeBacteriophage   1976   Walter Fiers at   3569...
Genomes Sequenced so far…                 19987 – 19718 (26th Sept 2012)• Eukaryotes [2231]  –   Animal  –   Fungi  –   Pl...
Genomic Libraries                             DNA                             Extraction and          Restriction        C...
Types of Libraries Genomic Libraries    Plasmids (2-10 KB)    Bacteriophage (9-23 kb) Cosmid libraries (30 – 40 kb) B...
Restriction Enzymes 4 cutters 6 cutters 8 cutters¼ * ¼ * ¼ * ¼ = 1/256; 1/4096;1/65536Small Problem: Human genome size:...
BlueGlucuronides       Genomic LibrariesB-Glucuronidase                   Antibiotics                   Resistant Genes   ...
The exact probability of having any given DNA sequence in the library can be calculatedfrom the equationN = ln(1 -P)/ln(1 ...
Bacteriophage libraries  Insert size is larger -> Number of clones needed is smaller     Lytic and Lysogenic     Head, ...
Step - 1                                                 Large                                                 Number     ...
Step - 2
Step -3                         Add Packaging                         enzymeMix Empty heads +                            P...
Cosmid Libraries Takes larger insert sizes Can grow in bacteria or any other host Needs an origin of replication   SV4...
BAC Libraries Can take even larger insert sizes Has origin of replication Must have less copy numbers per cell.     •Pa...
Various uses of BAC libraries Physical mapping of genes Cloning of valuable genes Chromosome walking BAC end sequencin...
A                B    BAC End    Sequencing
How Genomes are sequenced?• Sanger Dideoxy Sequencing methods(1977)• Maxam Gilberts Chemical degradation methods(1977)• Tw...
How Genomes are sequenced?• Sanger Dideoxy Sequencing methods(1977)• Maxam Gilberts Chemical degradation methods(1977)• Tw...
Sanger Di-deoxy method    Figures taken from    http://www.bio.davidson.edu/courses/bio111/seq.html
Maxam-Gilbert’s chemical cleavagemethod
Application of Genome Sequencing Prediction of novel genes/transcripts Study of genome organization Study of genome evo...
Upcoming SlideShare
Loading in …5
×

Lecture 1,2

995 views

Published on

Genomics Part 1 and Part 2

Published in: Education
  • Be the first to comment

Lecture 1,2

  1. 1. Genomics
  2. 2. Topics to be covered• Introduction.• History of Genome Sequencing.• How genomes are sequenced. • Packaging • Transfection • Recovery of clones • Strategies of genome sequencing• Application of genome sequencing.
  3. 3. Period of time between first man-powered flight and landing on the moon (1902-1969): 67 yearsPeriod of time between discovery of structure of DNA and determination of the sequence of the entire human genome (1953-2010?) 57 years (?)
  4. 4. What is a Genome?• Gene + Chromosome -> Genome A/T/G/C A/U/G/C
  5. 5. Why determine the order of nucleotides?• Determining the order of billions of chemical units that builds the genetic material. – Secrets of life is locked up in the order of the 4 letters!!!! 5-100 million living species???
  6. 6. Genome Sequencing HistoryOrganism Year Institute Genome SizeBacteriophage 1976 Walter Fiers at 3569 bpMS2 the University of GhentPhage Φ-X174 1977 Fred Sanger 5386 bp CambridgeHaemophilus 1995 TIGR 1,830,138 bpinfluenzaeSaccharomyces 1996 European Effort 12,495,682cerevisiae (16 chromosomes)Human Genome 2000 Multiple 3.3 x 109Project Organizations (3 billion letters)
  7. 7. Genomes Sequenced so far… 19987 – 19718 (26th Sept 2012)• Eukaryotes [2231] – Animal – Fungi – Plants – Protists – Others• Prokaryotes [14268]• Viruses [3219]Ref: http://www.ncbi.nlm.nih.gov/genome/browse/
  8. 8. Genomic Libraries DNA Extraction and Restriction Cell Purification Digestion Size Blunt End End sealing 3 KB Selection
  9. 9. Types of Libraries Genomic Libraries  Plasmids (2-10 KB)  Bacteriophage (9-23 kb) Cosmid libraries (30 – 40 kb) BAC libraries (125 – 200 kb) YAC libraries
  10. 10. Restriction Enzymes 4 cutters 6 cutters 8 cutters¼ * ¼ * ¼ * ¼ = 1/256; 1/4096;1/65536Small Problem: Human genome size: 3 billion base pairsHow many fragments can be generated using a 4 cutter, 6 cutter and 8 cutter?16 million for 4 cutters1*10^6 = 1 million for 6 cutters1/16 million for 8 cutters
  11. 11. BlueGlucuronides Genomic LibrariesB-Glucuronidase Antibiotics Resistant Genes One in thousand plasmid Enzymes will get foreign DNA DNA to be cloned Electroporate
  12. 12. The exact probability of having any given DNA sequence in the library can be calculatedfrom the equationN = ln(1 -P)/ln(1 - f)P is the desired probabilityf is the fractional proportion of the genome in a single recombinant[Ex. For 4 cutter for human genome would be 256 * 3 X 10^9]N is the necessary number of recombinantsFor example, how large a library (i.e. how many clones) would you need in order to havea 99% probability of finding a desired sequence represented in a library created bydigestion with a 6-cutter? N = ln(1 - 0.99)/ln(1 - (4096/3x109)) N = 3.37 x 106 clones
  13. 13. Bacteriophage libraries  Insert size is larger -> Number of clones needed is smaller  Lytic and Lysogenic  Head, tail  Recombinant DNA  Assembly Protein  Cos site (200 bp long, nicked 12 bp overhang : terminase)Organism Genome size is 50 KBCritical KB is required for PackagingVectors are of size 25KBUpto 25 KB external DNA can be added
  14. 14. Step - 1 Large Number of Empty heads and tailsInfect Bacteria with Mutant phage•Lacking critical size•Lacking Assembly protein Extract Empty Head and Tails
  15. 15. Step - 2
  16. 16. Step -3 Add Packaging enzymeMix Empty heads + Packaged viraltails + Recombinant ParticlesDNATransparent plaques: Made to InfectEach one contains a Bacterial cellsfragment multiplied Grow infected and non-infected cells Transfection
  17. 17. Cosmid Libraries Takes larger insert sizes Can grow in bacteria or any other host Needs an origin of replication  SV40 ori can grow in mammals  ColE1 in E.coli
  18. 18. BAC Libraries Can take even larger insert sizes Has origin of replication Must have less copy numbers per cell. •Partially digest chromosome •Fraction select •Clone it to a specialized plasmid
  19. 19. Various uses of BAC libraries Physical mapping of genes Cloning of valuable genes Chromosome walking BAC end sequencing  For gap filling in genome sequencing projects.  Powerful tools when used with genome sequencing data.
  20. 20. A B BAC End Sequencing
  21. 21. How Genomes are sequenced?• Sanger Dideoxy Sequencing methods(1977)• Maxam Gilberts Chemical degradation methods(1977)• Two Labs that owned automated sequencers: 1. Leroy Hood at Caltech, 1986(commercialized by AB) 2. Wilhelm Ansorge at EMBL, 1986(commercialized by Pharmacia-Amersham and GE healthcare) 3.Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)Alu sequences 4. Hitachi Laboratory developed High throughput capillary array sequencer, 1996.1991, A patent filed by EMBL on media less, solid support based sequencing.
  22. 22. How Genomes are sequenced?• Sanger Dideoxy Sequencing methods(1977)• Maxam Gilberts Chemical degradation methods(1977)• Two Labs that owned automated sequencers: 1. Leroy Hood at Caltech, 1986(commercialized by AB) 2. Wilhelm Ansorge at EMBL, 1986(commercialized by Pharmacia-Amersham and GE healthcare) 3.Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)Alu sequences 4. Hitachi Laboratory developed High throughput capillary array sequencer, 1996.1991, A patent filed by EMBL on media less, solid support based sequencing.
  23. 23. Sanger Di-deoxy method Figures taken from http://www.bio.davidson.edu/courses/bio111/seq.html
  24. 24. Maxam-Gilbert’s chemical cleavagemethod
  25. 25. Application of Genome Sequencing Prediction of novel genes/transcripts Study of genome organization Study of genome evolution Relationship between organisms Genetic basis of complex disease Linkage analysis Evolution of genes

×