Lawrence Berkeley National Lab

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Microarrays

• Lawrence Berkeley National Lab
• Center for Environmental Biotechnology
• Todd DeSantis, Sonya Murray, Jordan Moberg, Gary
  Andersen

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The ponderings of a pre-schooler
Why cant I watch Shrek 3 times per day?
Will the swings be wet at the park?
How will this lactose impact the diversity in my
lower G.I. bacterial community?
Will I inhale any archaeal microorganisms when I
visit the hot springs?
Microarrays can help answer these types of
questions.
Will I be as bald as my Dad?
Vincent DeSantis
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What can they do?

• Determine if a particular biological
  macromolecule is in a complex sample.
• Examples of biological macromolecules
• Microarrays can also quantify the abundance.

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One-of-a-kind foot

• Molecule of interest is identified by some unique
  feature.
• Cinderella is identified by her unique foot.
• The foot is the target to be sought.
• If her foot was common to all women, then would
  her foot be useful target?
• Unique molecular features (targets).
• DNA or RNA ? sequence of ACGT(U)
• Proteins ? AA, Epitopes
• Lipids ?
• Carbohydrates ? branch structure

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Need a perfect slipper

• Molecule of interest (target) must capture, or be
  captured by, a second molecule (probe).
• Many feet had to be evaluated by an easy test.
• The specific, not common, glass slipper was used
  as the test (probe).
• Unique molecular probes.
• DNA or RNA ? DNA or RNA (Oligo nt).
• Proteins ? Proteins (Antibodies)
• Lipids ?
• Carbohydrates ?
• My focus DNA targets probed with complementary
  DNA

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DNA Pairing Fundamentals

• Adenine (A) pairs with Thymine (T)
• Cytosine (C) pairs with Guanine (G)
• The two DNA strands are held together by _______
  bonds
• Complimentary Strands

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Base-pairing allows DNADNAor DNARNA
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Example application

• Do Diabetic patients have lower glycogen synthase
  levels compared to healthy individuals?
• Analyze RNA, determine specific target
• 5...UAUUAGCGCUCGAUCGCUUAGUACAGCGAGGAAAAGUCCGAUAGU
  AC...3
• Synthesize DNA probe
• 3 ATCATG 5
• Attach probe to a surface.
• Nylon sheet
• Plastic dish
• Glass slide
• Prepare samples

Label all mRNA, expose to probes
D
Extract mRNA from skeletal muscle sample
D-

• Diabetes 511913-1920, 2002Gene Expression
  Profile in Skeletal Muscle of Type 2 Diabetes and
  the Effect of Insulin Treatment, Sreekumar, et
  al.

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Hybridization

• Notice all mRNA is labeled (florescence)
• Non-binding mRNA is washed away
• If surface glows, then target was captured by
  probe.
• What does it mean if no glow is detected?

• http//www.affymetrix.com

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Example Results
Membrane with glycogen synthase probe attached



D










Labeled mRNA
Wash






D-







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Red-Green

• http//www.unc.edu

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Millions of copies per feature
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Photolithography

• Affymetrix - Photolithography
• Nimblegen microscopic independent mirrors to
  direct light.

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Coordinates of fluorescence determines test
results.
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500,000 Probe 16S array (DOE 16S Chip)
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Rapid taxonomic classification of complex
consortia of environmental rDNA using a
microarray.
What bugs are in my sample?

• Environmental Surveys
• Counter Bio-terrorism
• Bioremediation
• Clinical Investigations

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Example Microorganisms

• C. immitis
• B. anthracis
• Plus thousands more ....

Lung
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Project Overview

• Goal
• Create a single microarray capable of detecting
  and categorizing the bacteria in a complex
  sample.
• Approach
• GeneChip targeted at 16S rDNA sequence variations
  to distinguish taxa.

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The Ribosome
rDNA
rRNA (functional molecule)
LSU
SSU 16s or 18s
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• Foundations
• Maintain the largest 16S gene library (83,000).
• Cluster sequences into taxa (10,000).
• Create algorithm for picking probes for each taxa
  (500,000).

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Desulfovibrio sp. str. DMB. Desulfovibrio sp.
'Bendigo A' Desulfovibrio vulgaris DSM 644
Sequence discrepancies
Regions not unique to taxon
Regions unique to taxon
Probe set 02280401041000.2154
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General Protocol
Run Video
Air Soil Feces Blood Water
gDNA
Universal 16S rDNA PCR
rRNA
Contains probes adhered to glass surface in grid
pattern.
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Reproducibility ?
Probes for C. jejuni tiled in 4 areas
C. jejuni probe sets 45 deg C 48 deg C 50 deg C
2280503040000.010d_st 3015 2219 1927
2280503040000.010c_st 3011 2303 2126
2280503040000.010b_st 3166 2397 2548
2280503040000.010_st 3162 2320 2291
mean 3088 2309 2223
standard deviation 87 73 263
coefficient of variation 0.03 0.03 0.12
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(No Transcript)
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Counter Bio-Terrorism

• Sampling the Air
• Extracting DNA
• Hybridization
• PCR
• Single Organism Detection
• Detection Arrays for Multiple Organisms

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Traditional Detection

• Collect air sample
• Swab sample onto petri dish
• Wait 2 days to 4 weeks for organisms to grow
• Isolate
• Biochemical tests
• Visual Inspection

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PCR

• Polymerase Chain Reaction
• Makes many DNA copies from few

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Hybridization Occurs Between Complimentary Strands



Separate when heated
Join properly when cooled






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Probes Search for Targets
Probes
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Probes Search for Targets
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DNA Replication Foundations

• DNA Polymerase
• enzyme which xeroxes a single strand in to a
  double strand
• assembles dNTPs monomers into a polymeric strand
• adds dNTPs to 3 end of polymer

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Polymerase Chain Reaction (PCR)

• An in vitro technique for creating many copies of
  a gene segment
• Components
• polymerase
• template DNA
• dNTPs (individual A, C, G, or T)
• Small probes called primers

Lets do it...
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Polymerase Chain Reaction (PCR)
95
Hot temperature ensures template is single
stranded.
- 5
3-
Single stranded template to be xeroxed
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Polymerase Chain Reaction (PCR)
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Primer designed by lab
G
C
C
A
A
T
G
- 3
A
T
G
G
A
T
5-
A
C
G
A
- 5
3-
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Polymerase Chain Reaction (PCR)
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Lowering temperature allows hydrogen bonding to
form
G
C
C
A
A
T
G
- 3
A
T
G
G
A
T
5 -
A
C
G
A
3 -
- 5
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Polymerase Chain Reaction (PCR)
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Polymerase can act upon free 3 end when it has
bound to the template.
5 -
A
A
A
- 3
G
A
T
A
C
G
G
C
T
T
A
G
C
A
G
G
T
A
A
T
T
T
G
T
G
G
C
T
3 -
A
A
A
G
G
G
T
T
C
C
C
C
C
C
T
T
- 5
G
G
A
A
G
G
A
A
Temperature raised to optimize polymerase activity
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Polymerase Chain Reaction (PCR)
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Polymerase can act upon free 3 end when it has
bound to the template.
5 -
A
A
A
T
C
A
G
A
C
A
A
T
A
C
G
G
C
T
T
A
G
C
A
G
G
T
G
G
- 3
A
A
T
T
T
G
C
C
T
T
G
G
C
T
3 -
C
T
C
T
T
A
A
A
G
G
G
T
T
C
C
C
C
C
C
T
T
- 5
G
G
A
A
G
G
A
A
Temperature raised to optimize polymerase activity
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career preparation

• Do a Senior Project that involves both CS and BIO
  students
• Find Mentor
• Interview, Interview, Interview (on campus,
  off-campus, maintain contacts)
• Learn HTML/Perl/Java/CGI

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Attractive Perl Properties

• Forgiving syntax
• Interpreted, not compiled
• Platform independent
• Text manipulation
• Libraries, modules, etc.
• Object oriented optional

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Why Perl is the leading language of
Bioinformatics?

• Perl easy to learn
• Perl is powerful
• Perl is free
• Large support group www.bioPerl.org
• Interfaces easily to relational databases
• In fact, it has been claimed that Perl saved the
  Human Genome Project

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Examples

• Find all the genomic 20-mers in common between
  Vibrio cholera str.14 and Vibrio mimicus
• This could take a long time by hand.

V.choler14 ...TTGTACACACCGCCCGTCACACCATGGGAGTGGNC
TGCAAAAGA-GCAGGTAGTTTAACC... V.mimicus
...TTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCAAAAGAAGC
AGGTAGTTTAACC...
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Probe Finding Project

• Given
• one microbial taxon
• Purpose
• Describe its taxonomic placement.
• Find two interesting things about the organisms
  in that taxa.
• Find a probe that is specific for a group of
  organisms.
• Method
• Obtain 16S Sequences.
• Align them to each other (MSA)
• Determine best target from a short list
  (provided).
• Verify that probe exists in all/most orgs of taxa
• Check for X-hybe (non-specificity)
• Check w/in sub-division, not all sequences

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Start Here
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Answers (Targets)

• Oceanospirillum
• TGCTACTTCGCCGGCGAGCGGCGGA
• Streptococcus
• CTTGACATCCTTCTGACCGGCCTAG
• Rhodococcus
• CGGGTCTCTGGGAAACAACTGACGC
• TGGGAAACAACTGACGCTGAGGAAC
• Methanocorpusculum
• TGGAGAATACTCCCGGGAAACTGGG
• Desulfovibrio
• GCGTGAAAGGACTTCGGTCCGAGTA

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Multi Microarray Analysis

• Track a bio-marker over many experiments.
• Download file
• Fluorescence intensity for118 taxa reported for 5
  experimental conditions, and a negative control.
• Find
• Taxa with highest intensity overall.
• Possible PCR contaminants
• Taxa with greatest intensity fluctuation overall
• Condition producing the brightest signal for
  Paracoccus yeeii?