MCB 317
Genetics and Genomics
Topic 11, part 2
Genomics
Need to Add to part 2 or 3
A. Chip-seq
B. Deep sequencing for expression profiling
C. Illumina? movie
Genomics Summary
A.
B.
C.
D.
E.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other
organisms
F. RNAi
G. Transgenics, gene “knock-outs” (genetics not
genomics)
H. Human Genome Project, Next Generation
Sequencing, and Comparative Genomics
Yeast “Knockout” Library
Delete YFG
Delete all genes (individually)
Disruption of “All” Yeast Genes
•
•
•
•
Approx 6000 genes
Make 6000 sets of disruption primers
Disrupt each gene in a diploid
Dissect all 6000 diploids
– Identify set of essential genes
– Identify set of non-essential genes
Yeast “Knockout” Library
• Delete one copy of each gene in diploid
– 5,916 “genes” deleted
– 5,916 diploid strains constructed
• Dissect to determine if gene is essential
– 1,105 genes = essential
– 18.7% of genes = essential
• Construct an ordered library of haploids for nonessential genes
– 4,811 mutant strains in library
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Genomics
Biochemistry
Subunits of Protein
Complex
Genomics:
High-throughput
genetics
Protein
D
Orthologs and
Paralogs
E
H
Gene
Ab
B, G
A
F
Txn
Profile
C
Mutant Gene
B, G
Protein
Profile/
Localization
Mutant Organism
Genetics
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling (and other uses)
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Genomics
Biochemistry
Subunits of Protein
Complex
Genomics:
High-throughput
genetics
Protein
D
Orthologs and
Paralogs
E
H
Gene
Ab
B, G
A
F
Txn
Profile
C
Mutant Gene
B, G
Protein
Profile/
Localization
Mutant Organism
Genetics
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Genomics
Biochemistry
Subunits of Protein
Complex
Genomics:Hi
ghthroughput
genetics
Protein
D
Orthologs and
Paralogs
E
H
Gene
Ab
B, G
A
F
Txn
Profile
C
Mutant Gene
B, G
Protein
Profile/
Localization
Mutant Organism
Genetics
8100 Human DBD-ORFs x 8100 Human AD-ORFs
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Evolution of RNAi (current model)
1. a. Viruses are bad (so are transposons).
b. Many viruses have dsRNA genomes
c. euks originally lacked dsRNAs
d. invent mechanism to kill dsRNA
2. Evolve mechanism to regulate endogenous genes
a. RNA degradation
b. inhibit translation
c. form heterochromatin
3. Use as experimental technique
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Knockout Mouse: The Goal
YFG
Marker Gene
Replace the coding region of YFG with a selectable marker
gene
Knockout Mouse
Transfected DNA can integrate at random sites
(standard transgenic organism). This is a relatively
common event.
Or the Transfected DNA can Replace the Endogenous
Copy of the Gene via Homologous Recombination.
This is a relatively rare event.
Gene Deletion Deletion by Homologous Recombination
Marker
Gene
Knockout Mouse
Knockout Mouse
How to select for the cells in which the Occludin gene is
replaced with a mutant allele (a null allele) in the face of the
fact that most of the transformed DNA will integrate at
random sites?
neor gene makes mammalian cells resistance to the drug G418
tkNSV makes mammalian cells sensitive to the drug ganciclovir
Knockout Mouse
YFG
neor
tkHSV
Red = Mouse DNA including YFG and regions upstream and downstream of YFG
Blue = neor gene, Green = tkHSV gene
Black = plasmid DNA (not homologous to any mouse DNA)
Knockout Mouse
Lodish 5-40
Knockout Mouse
Different Types of Transgenic Organisms
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Goals of Human Genome Project
1.
Generate Genetic, Physical and Sequence maps of the human
genome
2.
Sequence genomes of a variety of model organisms:
Comparative Genomics
3.
Develop improved technology for mapping and sequencing
4.
Develop computational tools for capturing, storing, analyzing,
displaying, and distributing map and sequence data
5.
Sequence ESTs and cDNAs
6.
Consider social, ethical and legal challenges posed by genetic
information
Genomicists look at two basic features of
genomes: sequence and polymorphism
• Major challenges to determine sequence of each chromosome
in genome and identify many polymorphisms
– How does one sequence a 500 Mb chromosome 600 bp at a time?
– How accurate should a genome sequence be?
• DNA sequencing error rate is about 1 per 600 bp
– How does one distinguish sequence errors from polymorphisms?
• Rate of polymorphism in diploid human genome is about 1 in 1000 bp
– Repeat sequences may be hard to place
– Unclonable DNA cannot be sequenced
• Up to 30% of genome is heterochromatic DNA that can not be cloned
Whole-genome shotgun sequencing
Private company Celera used to sequence whole human genome
• Whole genome randomly
sheared three times
– Plasmid library constructed
with ~ 2kb inserts
– Plasmid library with ~10 kb
inserts
– BAC library with ~ 200 kb
inserts
• Computer program assembles
sequences into chromosomes
• No physical map construction
• Only one BAC library
• Overcomes problems of repeat
sequences
Fig. 10.13
Pyrosequencing, pt 1
Rxn1
(DNA)n + dNTP
DNAP
(DNA)n+1 + PPi
Rxn2
Adenosine phosphosulfate = APS
APS + PPi
ATP sulfurylase
ATP
Pyrosequencing, pt 2
Luciferin
Luciferase
ATP
Oxyuciferin + Light
ADP
Apyrase: dNTP -> dNDP + Pi -> dNMP + Pi + Pi
Pyrosequencing, overview
dTTP
APS
PPi
GCTACACT
CGATGTGACTGTA
ATP
Luciferin
Oxyuciferin + Light
Luciferase
Pyrosequencing
Add one nt (A) -> detect light (yes or no)
Apyrase degrades excess nt (A)
Add next nt (C) -> detect light (yes or no)
Apyrase degrades excess nt (C)
Repeat cycle 100’s of times
Pyrosequencing
Pyrosequencing
Emulsion PCR
1. Add linkers (primers) to ends of
genomic fragments
2. Attach frags to 1000’s of beads in a
mixture
3. Add PCR reagents
4. Add oil and make an emulsion so that
each bead is in it’s own droplet (it’s own
PCR reaction)
5. Amplify DNA to make millions of
identical copies. Each bead has millions of
copies of a single DNA
Pyrosequencing
Pico-titer plate 200,000-400,000 wells per
plate
1. Add beads to picotiter plate, only one
bead fits in each well
2. Add a second type of bead, smaller, that
holds the DNA bead in the wells and
delivers enzymes to the wells
3. Flow the nts into the wells one at a time
and record the light emitted from each
well using a CCD camera
Pyrosequencing
Currently the best machines can sequence 400 - 600 million base
pairs in one 10 hour run
Haploid human genome = 3,000,000,000 bp therefore sequence
haploid human genome to 1x depth in 6 days with one machine.
The current target goal for sequencing individual human genomes is
to get the cost down to $1,000 per genome. At present the cost is
around “$5,000-$10,000” per individual (last year)… Illumina claims
to have hit the $1,000 cost per genome in January of 2014
Illumina Sequencing Technology
See Movie