# Presentation: 429824

## Lawrence Berkeley National Lab
Center for Environmental Biotechnology

Todd DeSantis, Sonya Murray, Jordan Moberg, Gary Andersen

- Center for Environmental Biotechnology
- Todd DeSantis, Sonya Murray, Jordan Moberg, Gary Andersen

- Microarrays

## The ponderings of a pre-schooler

**The ponderings of a pre-schooler**

- Why can’t 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?

- Vincent DeSantis

- Microarrays can help answer these types of questions.

- Will I be as bald as my Dad?

## 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.

## 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

## 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

## 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”

## Base-pairing allows DNA:DNAor       DNA:RNA

## Example application

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

*Diabetes* 51:1913-1920, 2002**Gene Expression Profile in Skeletal Muscle of Type 2 Diabetes and the Effect of Insulin Treatment, Sreekumar, *****et al*****.**

- Extract mRNA from skeletal muscle sample

- D+

- D-

- Label all mRNA, expose to probes

## 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

## Example Results

- D-

- D+

- Membrane with glycogen synthase probe attached

- Labeled mRNA*

- Wash

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## Red-Green

- http://www.unc.edu

## Millions of copies per feature

## Photolithography

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

## Coordinates of fluorescence determines test results.

## 500,000 Probe 16S array

**500,000 Probe 16S array**
**(DOE 16S Chip)**

## 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

## Example Microorganisms

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

- Lung

## 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.

## The Ribosome

**The Ribosome**

- rDNA

- rRNA (functional molecule)

- LSU

- SSU
- 16s or 18s

## 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).

**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).**

## Probe set: 02280401041000.2154

- Desulfovibrio sp. str. DMB.
- Desulfovibrio sp. 'Bendigo A'
- Desulfovibrio vulgaris DSM 644

- Regions unique to taxon

- Regions not unique to taxon

- Sequence discrepancies

## General Protocol

**Air**
**Soil**
**Feces**
**Blood**
**Water**

- rRNA

- gDNA

- Universal 16S rDNA PCR

- Contains probes adhered to glass surface in grid pattern.

- Run Video

## Reproducibility 

| 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 |

- Reproducibility 

- Probes for C. jejuni tiled in 4 areas

## Slide 24

## Counter Bio-Terrorism

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

## 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

## PCR

- Polymerase Chain Reaction
- Makes many DNA copies from few

## Hybridization Occurs Between Complimentary Strands

- Separate when heated

- Join properly when cooled

## Probes Search for Targets

- Probes

## Probes Search for Targets

## 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

- 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”

- Polymerase Chain Reaction (PCR)

- Let’s do it...

## Polymerase Chain Reaction (PCR)

- - 5’

- 3’-

- Single stranded template to be “xeroxed”

- 95°

- Hot temperature ensures template is single stranded.

## Polymerase Chain Reaction (PCR)

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- 5’-

- - 3’

- - 5’

- 3’-

- Primer designed by lab

- 95°

## Polymerase Chain Reaction (PCR)

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- 5’ -

- - 3’

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- 55°

- Lowering temperature allows hydrogen bonding to form

## Polymerase Chain Reaction (PCR)

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- - 5’

- 3’ -

- - 3’

- 72°

- Polymerase can act upon free 3’ end when it has bound to the template.

- Temperature raised to optimize polymerase activity

## Polymerase Chain Reaction (PCR)

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- Temperature raised to optimize polymerase activity

- Polymerase can act upon free 3’ end when it has bound to the template.

## 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

## Attractive Perl Properties:

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

## 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

## 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  ...TTGTACACACCGCCCGTCACACCATGGGAGTGGNCTGCAAAAGA-GCAGGTAGTTTAACC...**
**V.mimicus   ...TTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCAAAAGAAGCAGGTAGTTTAACC...**

## 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

## Start Here

## Answers (Targets)

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

## 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?*