1. Genomics and
Proteomics
Brian Strahl
2013
1

2. Outline
I.
Background and history of Genomics
• How did the genomics field come about?
• Key molecular biology advances
• Brief review of Epigenetics
I.
Genomic methods and their applications in Medicine
• Basic techniques used in genomics methods
• Microarray technologies
• Genomic tests currently used in the clinical setting
• High-throughput genomic sequencing
I.
Proteomics and and their applications in Medicine
• Proteomic platforms and clinical uses
• Mass spectrometry and biomarker discovery in human diseases
2

3. What is Genomics?
“The study of DNA sequences, genes, and genome
organization and function”
The word “Genomics” is most commonly used to
describe any form of high-throughput and/or largescale approach towards understanding gene and
genome function
3

4. Why should I care to learn about genomics and proteomics?
How will this knowledge help me in the clinic someday?
Biomedical researchers use genomics to:
•Determine the extent of genetic variation between individuals
•Understand disease etiology (causes)
•Distinguish disease/cancer subtypes and categories
•Identify new diagnostic biomarkers
•Facilitate faster drug development (pharmacogenomics)*discussed by Dr. Mcleod
•Reveal new therapeutic targets
The outcome of this biomedical research will allow the average
physician to:
•Determine an individual’s predispositions to certain diseases or illnesses
•Diagnose disease and cancer subtypes
•Predict patient outcome, disease recurrence and treatment strategies
•Individualize (or tailor) patient treatments
4

5. The Genomics Revolution
How did it begin?
• DNA structure and
the genetic code
1950’s
• Recombinant DNA technology
• Polymerase Chain Reaction 1970’s/1980’s
• Automated DNA sequencing
•The Human Genome Project
1990’s
5

6. Epigenetics
However, genetic diversity is also regulated by events
outside of the DNA!
1.
DNA information
•
The genetic code - i.e. triplicate base pairs in genes that codes for a particular
amino acid.
2.
Epigenetic information
•
Effects on gene or genome function that are not specified in the DNA sequence
itself (e.g. histone post-translational modifications).
6

7. Organization of Eukaryotic Chromatin
DNA double helix
Histone
H3
H2B
H2A
H4
Nucleosomes
Solenoid
Chromatin loop:
Chromatin
~100,000 bp DNA
7

8. Epigenetic regulators
1. Chromatin remodeling complexes (e.g. Swi/Snf)
2. Histone modifications
Acetylation
Phosphorylation
Methylation
Ubiquitylation
Sumoylation
3. Histone variants (e.g. H2A.Z, CENP-A, etc.)
4. DNA methylation
8

9. Outline
I.
Background and history of Genomics
• How did the genomics field come about?
• Key molecular biology advances
• Brief review of Epigenetics
I.
Genomic methods and their applications in Medicine
• Basic techniques used in genomics methods
• Microarray technologies
• Genomic tests currently used in the clinical setting
• High-throughput genomic sequencing
I.
Proteomics and and their applications in Medicine
• Proteomic platforms and clinical uses
• Mass spectrometry and biomarker discovery in human diseases
9

10. Basic techniques used
in genomics methods
10

11. Key methods in Genomics: I. Blotting and Hybridization
Southern
blots
Northern
blots
western
blots
DNA
RNA
Protein
1. Separate molecules by electrophoresis according to size
2. Transfer to solid support (membrane)
3. Probe for specific sequence (hybridization for nucleic acid;
antibodies for proteins)
11

12. Key methods in Genomics: I. Blotting and Hybridization
Southern
blots
Target: Genomic DNA
Goal: to examine gene structure, organization, size and copy number
Northern
blots
Target: RNA
Goal: to examine gene expression
western
blots
Target: Protein
Goal: to examine protein levels and their post-translational
modifications (e.g. phosphorylation)
12

13. Southern blot procedure:
Probing the membrane
Blot after transfer, UV light, 80oC
X-ray film
after exposure
to blot
Blot washed
to remove
excess, probe
Blot with
radioactive
probe
Hot probe!
Complementary
base pairing
13

14. Southern blot Application
Example showing increased numbers (amplification) of the
Her2/neu gene in breast cancers
2-5 Single
copies copy
5-20
copies
>20 copies
14

15. Western blots:
General procedure
Electroblotting tank
Power supply
_
+
Proteins
transferred
to membrane
Gel after
electrophoresis
Visualize
using X-ray
film
Incubate membrane
with antibody against
protein of interest
Detect bound
antibody
15

16. Western blots:
Clinical applications
Clinical Example of an HIV test using a Western blot
Band pattern Interpretation
Lane 1, HIV+ serum (positive control)
Lane 2, HIV- serum (negative control)
Lane A, Patient A
Lane B, Patient B
Lane C, Patient C
16

17. Northern blots:
Applications
o Transcript size
o Gene regulation
o Alternate transcripts
o Transcript (expression) levels
o Related transcripts
Ex: induction of Fos mRNA after growth stimulation
RNA from unstimulated cells
RNA from cells
stimulated for:
RNA from
tumor cells
15” 30” 60”
Fos mRNA
control mRNA
X-ray
film
17

18. The Polymerase Chain Reaction
1 copy
Double-stranded DNA
3’
5’
5’
3’
Primers complimentary
to ends of target region
35 cycles yields 235
= 34.4 billion copies
Repeat denature,
anneal, & extend
+
5’
3’
Heat denature
strands, cool to
anneal primers
5’
3’
3’
4 copies
5’
Thermo-stable
DNA polymerase
3’
5’
Extend
extend primers
5’
5’
3’
2 copies
3’
5’
3’
3’
5’
Denature, anneal
18

19. Reverse Transcriptase (RT)-PCR:
Detecting Specific mRNAs
PCR can also be used to detect specific mRNAs via amplification
of Reverse Transcriptase-derived cDNA (RT-PCR)
RT-PCR
mRNA:
TTTTTTTTTT 5’ dT primer
AAAAAAAAAA
HO
+ 1. Reverse Transcriptase (RT)
cDNA:
PCR
DNA:
etc
19

20. Real-time PCR:
Towards High-throughput Quantitation of DNA & RNA
oligonucleotide
complimentary
to target
Quencher
Fluorescent
Reporter Dye
+ target DNA + primers
heat and anneal
3’
5’
3’
5’
3’
5’
Taq
polymerase
3’
5’
Fig from:
www.hgbiochip.com/images/QuantitativePCR.gif
Repeat, measure fluorescence at each cycle
3’
5’
3’
5’
Fluorescence no
longer quenched!
20

21. Some Clinical Applications of Real-time PCR:
Genomic PCR
o Determining the number of gene copies (e.g. HER2)
RT-PCR
o Quantitating the level of RNA viral infection (e.g. HIV)
o Comparing expression of suspect genes in normal versus
diseased tissues or tumors (e.g. p53, RB, ER)
21

22. High-through put genomic
technologies
22

23. Microarrays: gene expression profiling
Genome wide expression (“Transcriptome”) analysis
o Emphasis on global changes instead of single genes
o Towards compiling atlases of genome expression
Important clinical impact, including:
o Understanding disease etiology
o Distinguishing disease subtypes (molecular signatures)
o Identifying new diagnostic markers
o Revealing new therapeutic targets
23

24. Two-color cDNA Microarrays:
A Comparative Analysis
Approach: compare levels of expression of individual genes in
a large population between test and reference samples . . .
Cancer cell
Make cDNA pools
using nucleotides
tagged with
red (Cy5) or
green (Cy3)
fluorescent dye
Normal cell
Here:
Test = cancer cell
Ref = normal cell
mRNAs
cDNAs
24

25. Outline of the Two Color –
cDNA Microarray Approach
Cancer cell
Normal cell
Combine
cDNAs
Hybridize
to array
Wash, excite with
lasers
Cy5/Cy3
0.03
50
Record emissions
w/ detectors
1
Gene exp higher in cancer cell
Gene exp lower in cancer cell
Gene exp similar in cancer cell
25

26. Microarray Technology
26

27. Data Selection & Clustering:
Making Sense Out of Arrays
Original data
Clustered data
Genes inhibited
quickly
Inhibited after
lag, then induced
Induced
after lag
123456
Time
123456
Induced after
longer lag
Inhibited
Induced
Time
27

28. Gene Arrays:
Distinguishing Different Cancers
Mesenchymal
Leukemia
Epithelial
Distinct gene
expression profiles
mark different
cancers, help
distinguish primary
tumor
Melanoma
28

29. Gene Arrays: Distinguishing Different Disease Subtypes
Tumor groupings
Probability
The 5 tumor types:
HER2+
Basal tumors
Survival months
29

30. Gene Arrays: Distinguishing Different Disease Subtypes
Example 2: Distinguishing acute myeloid leukemia (AML) from acute lymphoblastoid
leukemia (ALL)
These two cancer types are difficult to distinguish using traditional cytological, cytogenetic and
biochemical assays. This is relevant as while daunorubicin and cytarabine work best for AML,
vincristine and methotrexate works better for ALL
30

31. Gene Arrays: use in the clinic
Several FDA approved genetic tests that are available
to you now:
1) AmpliChip Cytochrome P450 Genotyping test
• Looks for common genetic mutations and/or variations that might occur in the
cytochrome P450 enzymes CYP2D6 and CYP2C19. These genes regulate
drug metabolism in the liver. Test indicates whether there is a likelihood of
certain drugs to be metabolized faster or slower.
1) MammaPrint® test
• Examines the gene signature of 70 cancer-related genes commonly activated
in breast tumors. The test determines the likelihood that a breast cancer will
return within 5 to 10 years. A score is given that determines whether the
patient is at “low risk” or “high risk” for tumor recurrence. Such information will
help guide doctors in the appropriate treatment and follow-up care for each
31
individual patient.

32. MamaPrint study
295 patients
32
N Engl J Med, Vol. 347, 2002 p1999

33. Next Generation DNA
Sequencing Technologies
Why bother?
Provides rapid and extensive sequence information at a very low price
What can you do with this technology?
1) Sequence a human genome in two weeks
Obtain SNPs, insertions and deletions (“Indels”)
1) Rapidly obtain the “transcriptome” (RNA-Seq)
RNA  cDNA  High-throughput sequencing
1) Obtain transcription and chromatin factor binding sites (ChIP-Seq)
Antibody immunoprecipitation, isolate DNA, sequence
33

34. Comparing Sequencers
Roche (454)
Illumina GAIIx
SOLiD 4
Chemistry
Pyrosequencing
Polymerase-based
Ligation-based
Amplification
Emulsion PCR
Bridge Amp
Emulsion PCR
Paired ends/sep
Yes/3kb
Yes/400 bp or
5000kb
Yes/200bp or 3-10
kb
Mb/run
100 Mb
48 Gb
100 Gb
Time/run
7h
8 days
12 days
Read length
450 bp
76bp
50 bp
Cost per run (total)
1/16 plate $562
(36bp) $700
N/A
The human genome contains about 3,400 megabases (3.4 GB)
Illumina has now lowered the cost of its individual sequencing service to ~$5000/sample. They are offering a
discounted price for people with serious medical conditions who could potentially benefit from having their
genomes decoded.
34

35. Reconstructing Genome Sequence
reads
Genomic DNA
35

36. Challenges with Fragment Assembly
•
Sequencing errors can happen!
~1-2% of bases are wrong
•
Repeat regions are difficult
False overlap due to repeat
36

37. Outline
I.
Background and history of Genomics
• How did the genomics field come about?
• Key molecular biology advances
• The Human Genome Project and its discoveries/impact
• Brief review of Epigenetics
I.
Genomic methods and their applications in Medicine
• Basic techniques used in genomics methods
• Microarray technologies
• Genomic tests currently used in the clinical setting
• High-throughput genomic sequencing
I.
Proteomics and and their applications in Medicine
• Proteomic platforms and clinical uses
• Mass spectrometry and biomarker discovery in human diseases
37

38. Proteomics: A More Challenging Undertaking
Very challenging:
o Proteins are inherently more diverse than DNA/RNA
o Tip of the iceberg: numerous covalent modifications
o Difficult to cleanly separate different cell compartments and cell types
Ca
nc
B
C
Phosphorylation
Gene
Expression
(mRNA)
No
rm
al
A
er
So why do we need to think about Proteomics?
Gene expression
Protein modifications
Protein abundance
38

39. Proteomics: Key Technologies and Platforms
Mass spectrometry - gets accurate mass of proteins, their peptide
fragments upon digestion and their post-translational modifications
Goal: to analyze complex sample of normal and diseased tissues to
generate a protein “fingerprint”. Peptide sequencing (MS/MS) can tell
you what protein is being affected.
Tissue microarrays- This approach uses well-established
immunohistochemistry techniques and formalin-fixed tissue samples
Protein microarrays - Approach uses spotted proteins as bait for proteinprotein interaction studies
39

40. Proteomics using Mass Spectrometry
From: Sebahat Ocak, et al. Proc Am Thorac Soc Vol 6. pp 159–170, 2009
40

41. Proteomics: A Clinical Pilot Study
Need: new technologies for detecting early-stage ovarian cancer
“Use of proteomic patterns in serum to identify ovarian cancer” by Petricoin
et al (FDA/NIH/MD Anderson)
- The Lancet 359, pg 572, 2002.
Serum from 50
normal women
Serum from 50
cancer patients
Generated >15,000 protein mass spectra
Identified 5-20 proteins that differ in normal patients and cancer patients
(potential diagnostic markers)
41