Your Genes and Your Health

http://bio84.stanford.edu/ The Human Genome Project

Doug Brutlag Professor Emeritus Biochemistry and Medicine (by courtesy) brutlag@stanford.edu Doug Brutlag 2011

Thanks
Pat Muller Antacids versus Histamine-2 Blockers Melissa Del Sol Genetics for Dummies and 23andMe Discount Dan Feldman Pointer to Epigenetics John Stark and Talia Kostick to futher epigenetic references including NOVA show

Doug Brutlag 2011

The Human Genome Project: Should we do it?

Not hypothesis driven. Fishing expedition or stamp collecting. Eliminate funds from investigator initiated science.

Service, R. F. (2001). The human genome: Objection #1: big biology is b Science, 291(5507), 1182.

Vogel, G. (2001). The human genome: Objection #2: why sequence the j Science, 291(5507), 1184.
Limit sequencing to 1.5% of genome that codes proteins. Do not sequence intergenic regions genetic wastelands. Do not sequence repeated regions (telomeres and heterochromatin).

Service, R. F. (2001). The human genome: Objection #3: impossible to d Science, 291(5507), 1186.
Technology of the time permitted 500 bp per day per person. Move from radioactively labeled sequencing to fuorescent sequencing permitted complete automation up to 1 gigabyte per year.
Doug Brutlag 2011

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Hierarchical Sequencing Vs. Whole Genome Shotgun Sequencing

(from Gibson & Muse, A Primer of Genome Science)

Gibson & Muse A Primer of Genome Brutlag 2011 Doug Science

Whole Genome Shotgun versus BAC Sequencing

1997
Lets sequence the human genome with the shotgun strategy

That is impossible, and a bad idea anyway

Phil Green
Thanks to Seraf m Batzoglou i Doug Brutlag 2011

Gene Myers

Public Human Genome Project Strategy

http://www.nhgri.nih.gov/

Doug Brutlag 2011

Doug Brutlag 2011

Subclone Construction for Sequencing

Doug Brutlag 2011

M13mp18 Sequencing Vector

http://www.mikeblaber.org/oldwine/bch5425/lect33/lect33.htm

Michael Blaber@sb.fsu.edu Doug Brutlag 2011

Sanger Sequencing Technology

(from Gibson & Muse, A Primer of Genome Science)

ABI Sequence Trace

Doug Brutlag 2011

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Sequence Assembly Sequence Assembly

Contig

Doug Brutlag 2011

Shotgun Sequencing Strategy

Doug Brutlag 2011

Polymerase Chain Reaction Overview: Exponential Amplification of DNA

Doug Brutlag 2011

PCR Requirements
DNA
Need to know at least the beginning and end of DNA sequence These flanking regions have to be unique to strand interested in amplifying Region of interest can be present in as little as one copy Enough DNA in 0.1 microliter of human saliva to use PCR

DNA Polymerase Enzyme

DNA polymerase from Thermus aquaticus--Yellowstone Alternatives: Thermococcus litoralis, Pyrococcus furiosus

Thermocycler

Doug Brutlag 2011

PCR Applications
Forensics assessment/reassessment of crimes Archaeology determine gene sequences of ancient organisms rethinking the past, human origins Molecular Biology Cloning genes Sequencing genes Finishing genome sequences Amplification of DNA or RNA Medicine Diagnostics for inherited disease Diagnostics for gene expression Diagnostics for gene methylation
Doug Brutlag 2011

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Single Nucleotide Polymorphisms (SNPs) and Short Insertion/Deletions in The Human Genome

Doug Brutlag 2011

Duplications and Deletions in the Human Genome

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Illumina Solexa Sequencing Technology

Doug Brutlag 2011

Pacific Biosciences Sequencing

Doug Brutlag 2011

Phospholinked Fluorophores

Doug Brutlag 2011

Circular Templates Gives Redundant Sequencing and Accuracy

Doug Brutlag 2011

The Human Genome How fast is the cost going down?

2006: $10 million 2008: $100,000 2009: $10,000 2010: $1,000 ??? $100

Thanks to Seraf m Batzoglou i Doug Brutlag 2011

Archon X-Prize

Doug Brutlag 2011

Components of a Typical Human Gene

TFBS Promoter Exon

Intron

Exon

Intron

Exon

Terminator Gene (DNA)

Doug Brutlag 2011

Active Genes are Transcribed into RNA

Promoter

Exon

Intron

Exon

Intron

Exon

Terminator
Gene (DNA)

Transcription

Primary Transcript (RNA)

Doug Brutlag 2011

Splicing Transcript Yields Mature mRNA

Promoter Exon

Intron

Exon

Intron

Exon

Terminator
Gene (DNA) Transcript (RNA)

Transcription

Splicing

mRNA (RNA)

Doug Brutlag 2011

Alternative Splicing Generates Distinct Proteins in Different Tissues

Promoter Exon

Intron

Exon

Intron

Exon

Terminator Gene (DNA)

Transcript (RNA)

Transcription

Splicing

5
Alternate Splicing

mRNA-1 (RNA) Transcript (RNA)

mRNA-2 (RNA)
Doug Brutlag 2011

Mature mRNA contains Coding Region and 5 and 3 Untranslated Regions

Promoter Exon
5UTR

Intron

Exon

Intron

Exon
3UTR 3UTR

Terminator Gene (DNA) Transcript (RNA)

Transcription

Splicing

5
5UTR

Coding Region

3
3UTR 3UTR

mRNA (RNA)

Translation
N-terminus C-terminus

Protein
Doug Brutlag 2011

The Genetic Code

Doug Brutlag 2011

Messenger RNA (mRNA) Databases ESTs UniGene & Full Length cDNA RefSeq
Promoter Exon

Intron

Exon

Intron
Transcription

Exon

Terminator
Gene (DNA) Transcript (RNA)

Splicing

5
5 ESTs

mRNA (RNA)

3 ESTs Full Length cDNA

Doug Brutlag 2011

ESTs, Full Length cDNA UniGene & RefSeq Databases

Promoter Exon
5 UTR

Intron

Exon

Intron

Exon
3 UTR

Terminator Gene (DNA) Transcript (RNA) mRNA (RNA) 3 ESTs

Transcription

Splicing

5
5 ESTs
5 UTR 3 UTR

Full Length cDNA Proteins

Protein

Doug Brutlag 2011

NCBI Genomes
http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome

Doug Brutlag 2011

Human Genome Resources

http://www.ncbi.nlm.nih.gov/genome/guide/human/

Doug Brutlag 2011

NCBI Entrez Gene

http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene

Doug Brutlag 2011

Human Beta-Hemoglobin Gene Entry

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Gene Entry

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Hemoglobin Gene Entry

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Phenotypes

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Hemoglobin Phenotypes

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Gene Entry

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Biosystems

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Conserved Domain

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Homologene

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Genome Map Viewer

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

Human Beta-Hemoglobin Sequence Vari

http://www.ncbi.nlm.nih.gov/gene/3043

Doug Brutlag 2011

EBI Genomes Home Page

http://www.ensembl.org/

Doug Brutlag 2011

Ensembl Human Genome

http://www.ensembl.org/Homo_sapiens/

Doug Brutlag 2011

UCSC Genome Browser

http://genome.ucsc.edu/

Doug Brutlag 2011

Doug Brutlag 2011

Doug Brutlag 2011