lecture 1,2
DESCRIPTION
Genomics Part 1 and Part 2TRANSCRIPT
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Sucheta TripathyGenomics
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• Introduction.• History of Genome Sequencing.• How genomes are sequenced.
• Packaging• Transfection• Recovery of clones• Strategies of genome sequencing
• Application of genome sequencing.
Topics to be covered
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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 (?)
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• Gene + Chromosome -> Genome
What is a Genome?
A/T/G/C
A/U/G/C
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• 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!!!!
Why determine the order of nucleotides?
5-100 million living species???
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Genome Sequencing HistoryOrganism Year Institute Genome Size
Bacteriophage MS2
1976 Walter Fiers at the University of Ghent
3569 bp
Phage Φ-X174 1977 Fred Sanger Cambridge
5386 bp
Haemophilus influenzae
1995 TIGR 1,830,138 bp
Saccharomyces cerevisiae
1996 European Effort
12,495,682(16 chromosomes)
Human Genome Project
2000 Multiple Organizations
3.3 x 109
(3 billion letters)
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• Eukaryotes [2231] – Animal– Fungi– Plants– Protists– Others
• Prokaryotes [14268] • Viruses [3219]Ref:
http://www.ncbi.nlm.nih.gov/genome/browse/
Genomes Sequenced so far…19987 – 19718 (26th Sept 2012)
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Genomic Libraries
Cell
DNA Extraction and Purification
Restriction Digestion
Size Selection
3 KB
End sealingBlunt End
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Types of LibrariesGenomic Libraries
Plasmids (2-10 KB)Bacteriophage (9-23 kb)
Cosmid libraries (30 – 40 kb)BAC libraries (125 – 200 kb)YAC libraries
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Restriction Enzymes4 cutters6 cutters8 cutters
¼ * ¼ * ¼ * ¼ = 1/256; 1/4096; 1/65536
Small 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
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Genomic LibrariesB-Glucuronidase
Glucuronides
Blue
Antibiotics Resistant Genes
DNA to be cloned
One in thousand plasmid will get foreign DNA
Electroporate
Enzymes
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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 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 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
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Bacteriophage librariesInsert size is larger -> Number of clones needed is
smallerLytic and LysogenicHead, tailRecombinant DNAAssembly ProteinCos site (200 bp long, nicked 12 bp overhang :
terminase)Organism Genome size is 50 KBCritical KB is required for PackagingVectors are of size 25KBUpto 25 KB external DNA can be added
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Infect Bacteria with Mutant phage•Lacking critical size•Lacking Assembly protein
Large Number of Empty heads and tails
Step - 1
Extract Empty Head and Tails
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Step - 2
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Step -3
Mix Empty heads + tails + Recombinant DNA
Add Packaging enzyme Packaged viral
Particles
Made to Infect Bacterial cells
Transfection
Grow infected and non-infected cells
Transparent plaques:Each one contains a fragment multiplied
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Cosmid LibrariesTakes larger insert sizesCan grow in bacteria or any other hostNeeds an origin of replication
SV40 ori can grow in mammalsColE1 in E.coli
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BAC LibrariesCan take even larger insert sizesHas origin of replicationMust have less copy numbers per cell.
• Partially digest chromosome• Fraction select• Clone it to a specialized plasmid
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Various uses of BAC librariesPhysical mapping of genesCloning of valuable genesChromosome walkingBAC end sequencing
For gap filling in genome sequencing projects.Powerful tools when used with genome
sequencing data.
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A B
BAC End Sequencing
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• 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 sequences4. Hitachi Laboratory developed High throughput capillary array sequencer, 1996.1991, A patent filed by EMBL on media less, solid support based sequencing.
How Genomes are sequenced?
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• 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 sequences4. Hitachi Laboratory developed High throughput capillary array sequencer, 1996.1991, A patent filed by EMBL on media less, solid support based sequencing.
How Genomes are sequenced?
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Sanger Di-deoxy method
Figures taken from http://www.bio.davidson.edu/courses/bio111/seq.html
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Maxam-Gilbert’s chemical cleavage method
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Application of Genome SequencingPrediction of novel genes/transcriptsStudy of genome organizationStudy of genome evolutionRelationship between organismsGenetic basis of complex diseaseLinkage analysisEvolution of genes