Genomics Part 1 and Part 2

1. Genomics

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. Period of time between first man-powered flight and landing on the moon (1902-1969):
67 years
Period of time between discovery of structure of DNA and determination of the sequence of the
entire human genome (1953-2010?)
57 years (?)

4. What is a Genome?
• Gene + Chromosome -> Genome
A/T/G/C
A/U/G/C

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. Genome Sequencing History
Organism Year Institute Genome Size
Bacteriophage 1976 Walter Fiers at 3569 bp
MS2 the University
of Ghent
Phage Φ-X174 1977 Fred Sanger 5386 bp
Cambridge
Haemophilus 1995 TIGR 1,830,138 bp
influenzae
Saccharomyces 1996 European Effort 12,495,682
cerevisiae (16
chromosomes)
Human Genome 2000 Multiple 3.3 x 109
Project Organizations (3 billion letters)

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. Genomic Libraries
DNA
Extraction and Restriction
Cell Purification Digestion
Size
Blunt End End sealing 3 KB Selection

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. Restriction Enzymes
 4 cutters
 6 cutters
 8 cutters
¼ * ¼ * ¼ * ¼ = 1/256; 1/4096;
1/65536
Small Problem: Human genome size: 3 billion base pairs
How many fragments can be generated using a 4 cutter, 6 cutter and 8 cutter?
16 million for 4 cutters
1*10^6 = 1 million for 6 cutters
1/16 million for 8 cutters

11. Blue
Glucuronides Genomic Libraries
B-Glucuronidase
Antibiotics
Resistant Genes One in
thousand
plasmid
Enzymes will get
foreign
DNA
DNA to be
cloned
Electroporate

12. The exact probability of having any given DNA sequence in the library can be calculated
from the equation
N = ln(1 -P)/ln(1 - f)
P is the desired probability
f 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 recombinants
For example, how large a library (i.e. how many clones) would you need in order to have
a 99% probability of finding a desired sequence represented in a library created by
digestion with a 6-cutter?
N = ln(1 - 0.99)/ln(1 - (4096/3x109))
N = 3.37 x 106 clones

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. Step - 1
Large
Number
of Empty
heads and
tails
Infect Bacteria with Mutant phage
•Lacking critical size
•Lacking Assembly protein
Extract Empty Head and
Tails

15. Step - 2

16. Step -3
Add Packaging
enzyme
Mix Empty heads + Packaged viral
tails + Recombinant Particles
DNA
Transparent plaques: Made to Infect
Each one contains a Bacterial cells
fragment multiplied
Grow infected and
non-infected cells Transfection

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. 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. 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. A B
BAC End
Sequencing

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. 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. Sanger Di-deoxy method
Figures taken from
http://www.bio.davidson.edu/courses/bio111/seq.html

24. Maxam-Gilbert’s chemical cleavage
method

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