The Human Genome The Human Genome ProjectProjectAnd how we got there…Sequencing technologiesSequencing strategiesSo what?What’s next

But before that…But before that…How do you find out the

sequence of DNA?◦Sanger’s dideoxy sequencing

method

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Frederick SangerFrederick Sanger

Won the Nobel Prize in Chemistry twice

1958 – for sequencing insulin

1980 – for inventing a method for sequencing DNA (together with Gilbert)

All the high-throughput sequencing methods in use today are based on the Sanger dideoxy method

http://www.nlm.nih.gov/visibleproofs/media/detailed/vi_a_208b.jpg

Sanger’s recipe: Sanger’s recipe: IngredientsIngredientsThe DNA of interest - templateAn oligonucleotide primer to get

the ball rollingA DNA polymerasedNTPs (deoxyribonucleotide

triphosphates) – dATP, dCTP, dGTP, dTTPThe special ingredient: ddNTPs

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Revision from ATBMS: Nucleosides Triphosphates/deoxyribonucleotide triphosphates

Concepts of Genetics 7th Ed, Klug and Cummings

Concepts of Genetics 7th Ed, Klug and Cummings

• Phosphodiester bonds are formed between the 3’ carbon of one nucleotide and the 5’ carbon of the next nucleotide

Revision from ATBMS: Nucleosides Triphosphates/deoxyribonucleotide triphosphates

Linkage of two nucleotides

Concepts of Genetics 7th Ed, Klug and Cummings

Revision from ATBMS: Nucleosides Triphosphates/deoxyribonucleotide triphosphates

Concepts of Genetics 7th Ed, Klug and Cummings

What’s special about What’s special about ddNTP?ddNTP?

This method is also known as the chain termination method

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What’s special about What’s special about ddNTP?ddNTP?

Fluorescent dye coupled to N-base

Each ddNTP - ddATP, ddCTP, ddGTP, ddTTP– is coupled to a different type of fluorescent dye – each ddNTP will absorb a characteristic laser wavelength and emit a characteristic colour

Sanger recipe: MethodSanger recipe: MethodDivide DNA into 4 tubes with dNTPs

and a different ddNTP in each tube and incubate

Polymerase catalyses addition of dNTPs

ddNTPs will terminate reactions Form oligonucleotides of varying

lengths terminated by fluorescent ddNTPs

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Denature DNA to produce single stranded oligonucleotidesLoad single stranded oligonucleotides and separate by electrophoresis – usually by capillary electrophoresis‘Read’ DNA sequence

What would an agarose gel look like?

Advances in technology…Advances in technology…The use of fluorescently labelled ddNTPs

(previously radioactive isotopes were used)◦Each ddNTP could be labelled with a different

flurochrome◦Sequencing could be done in a single tube◦Capillaries replaced large sheet gels◦Fluorescence could be read by a laser,

leading to:AutomationThe human genome was sequenced

using Sanger’s dideoxy method

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Capillary electrophoresis (from wikipedia)

Capillary tube filled with agarose and bufferElectrical voltage applied across the capillaryOligonucleotides move across capillary, according to size

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Typical Electropherogram

But usually first 10-20 bp are not reliable, also limited to about 600-800 bp- Peaks get broader and smaller

THE HUMAN GENOMETHE HUMAN GENOME

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What’s in a genome?What’s in a genome?Genes that code for proteins – 2-3% - contain Open Reading Frame (ORF) beginning

with start and stop codons

Many genes have multiple copies or have several closely related ‘family’ members

Regions coding for structural RNA (not proteins)– eg ribosomal RNA, tRNA

Regulatory regions – binding regions for regulatory proteins, transcription factors

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Moderately Repetitive Moderately Repetitive DNADNAFunctional

◦Gene families eg globin, actin◦Gene family arrays eg histone genes,

rRNA genes (250 copies), tRNA genesWithout known function

◦Short interspersed elements (SINES) eg Alu 200-300 bp long, 100,000s of copies, 13%

◦Long interspersed elements (LINES) 1-5 kb long 10-10000 copies per genome, 21%

◦Pseudogenes

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Highly repetitive DNAHighly repetitive DNA About 15% of genome Minisatellites (Variable number tandem repeats

(VNTR)◦ Repeats of 14-500 bp segments◦ scattered throughout genome, number of repeats

varies on different chromosomes Microsatellites (Short tandem repeat

polymorphisms (STRP)◦ Regions up to 2-5 bp repeated many times 10-30

copies◦ Hundreds of kb long◦ Eg heterochromatin

Telomeres◦ 6 bp repeat◦ 250 – 1000 repeats at the end of each chromosome

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The race to sequence the The race to sequence the human genomehuman genome3 billion bases in the human

genomeIn 22 pairs of chromosomes + 2

sex chromosomesOnly about 30,000 genes

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2 competing approaches2 competing approachesHierarchical method

◦Adopted by the publicly funded Human Genome Project

◦Sequence of 12 individualsWhole genome shotgun (WGS)

method◦Adopted by Celera, a for-profit

company◦Sequence of 1 individual

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Craig VenterCraig Venter

Founder of Celera

Applied whole genome shotgun sequencing method to human genome

Made the first synthetic chromosome

Assignment for next weekAssignment for next weekYou will be working in 4 groups of 4-5.

Explain to the class (10-15 min):Group 1and 2 – explain the following

terms related to genome sequencing: 1: mapping, STSs and ESTs, coverage, contigs, golden tiling path, 2: library, BACs, finishing, annotation

Group 3 – explain the hierarchical approach

Group 4 – explain the whole genome shotgun approach

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