Sucheta TripathyGenomics

• 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

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 (?)

• Gene + Chromosome -> Genome

What is a Genome?

A/T/G/C

A/U/G/C

• 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???

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)

• 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)

Genomic Libraries

Cell

DNA Extraction and Purification

Restriction Digestion

Size Selection

3 KB

End sealingBlunt End

Types of LibrariesGenomic Libraries

Plasmids (2-10 KB)Bacteriophage (9-23 kb)

Cosmid libraries (30 – 40 kb)BAC libraries (125 – 200 kb)YAC libraries

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

Genomic LibrariesB-Glucuronidase

Glucuronides

Blue

Antibiotics Resistant Genes

DNA to be cloned

One in thousand plasmid will get foreign DNA

Electroporate

Enzymes

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

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

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

Step - 2

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

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

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

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.

A B

BAC End Sequencing

• 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?

• 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?

Sanger Di-deoxy method

Figures taken from http://www.bio.davidson.edu/courses/bio111/seq.html

Maxam-Gilbert’s chemical cleavage method

Application of Genome SequencingPrediction of novel genes/transcriptsStudy of genome organizationStudy of genome evolutionRelationship between organismsGenetic basis of complex diseaseLinkage analysisEvolution of genes