PCR for Aquatic Animal Health
Web Training Module V1; August 2011
Created by Maureen Purcell, Ph.D.
Goal

To provide an overview of PCR-based diagnostic
assays with an emphasis on basic theory
•
Want to learn more?
•
Click on the reference links located at the bottom of certain
slides
Content Overview
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PCR basics
Commonly used PCR assays
Advantages and disadvantages of PCR
Good laboratory practices
Analytical validation
Sampling and template preparation
Primers
Standards, controls and normalization
Quantitative PCR – in depth
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
PCR Basics

Polymerase chain reaction (PCR) is a method to
amplify a target sequence from background
nucleic acid
PCR uses synthetic
oligonucleotide primers
that flank target
sequence
Forward Primer
T
DNA synthesis is
catalyzed in vitro by a
heat stable DNA
polymerase
Target Sequence
5’
Forward Primer
3’
Reverse Primer
Reverse Primer
Taq
Polymerase
Lodish H, A. et al.. (2000) Polymerase chain reaction, an alternative to cloning. In Molecular Cell Biology. 4th edition. W.H.
Freeman, NY. Section 7.7. http://www.ncbi.nlm.nih.gov/books/NBK21541/
PCR Basics

PCR basic steps
Denature DNA
(94°C)
Anneal primer
(~50 = 65°C)
5’
3’
3’
5’
5’
3’
Forward Primer
Reverse Primer
3’
T
Extension
(72°C)
5’
Forward Primer
Reverse Primer
http://www.idtdna.com/pages/docs/educational-resources/the-polymerase-chain-reaction.pdf
PCR Basics
Stages of PCR
Plateau Phase
Linear Phase
Log Target

Exponential (Geometric) Phase
Stochastic/ ‘lag’ phase
Cycle Number
http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
PCR Basics

Theoretically the target sequence is doubled every
PCR cycle
This doubling each cycle equates to 100% PCR
efficiency or an efficiency (E) of 2
Theoretical
Log Target

Cycle Number
PCR Basics
In practice, PCR efficiency will vary depending
on a range of factors
Theoretical Efficiency (E) = 2
Log Target DNA

Actual Efficiency (E) < 2
Cycle Number
Commonly Used PCR Assays
Conventional PCR utilizes two primers and
products are detected by gel electrophoresis
“cPCR”
Agarose gel
electrophoresis
following PCR
Log Target

Cycle Number
http://www.idtdna.com/pages/docs/educational-resources/gel-electrophoresis.pdf
Commonly Used PCR Assays

A reverse-transcriptase step can be added to
the PCR when the starting template is RNA
“RT-PCR”
The RT reaction can be primed by a:
target specific primer (i.e. primer targeting VHSV nucleocapsid (N) gene)
oligo dT primer (a primer consisting of a run of T’s that targets the mRNA
poly A tail)
random primers (a mix of 6 base primers consisting of random nucleotides)
All messenger RNAs (mRNA) have a poly A tail
RNA
AAAAAAAAAAAA
TTTTTT
oligo dT primer
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-andExpression-Profiling/Reverse-Transcription-and-cDNA-Synthesis/RNA-Priming-Strategies.html
Commonly Used PCR Assays

A reverse-transcriptase step can be added to
the PCR when the starting template is RNA
“RT-PCR”
RNA is copied into
complementary DNA
(cDNA) by the reverse
transcriptase enzyme
RNA
cDNA
AAAAAAAAAAAA
TTTTTT
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-andExpression-Profiling/Reverse-Transcription-and-cDNA-Synthesis/RNA-Priming-Strategies.html
Commonly Used PCR Assays

Some vendors sell “one-step RT-PCR” master mixes
 This is a misnomer and should be called onetube RT-PCR
 RT-PCR always involves two steps
1. Reverse-transcriptase
2. PCR
 These steps can be performed in the same
reaction tube (aka one-step) or in separate
reaction tubes
Commonly Used PCR Assays

Nested PCR (“nPCR”) involves two rounds of PCR
utilizing outer and inner primer sets to improve
sensitivity (because two rounds of PCR are
performed) and specificity (since all four primers
must match the target sequence)
Target DNA Region (i.e. Msa gene from R. salmoninarum)
Outer Forward Primer
Outer Reverse Primer
1st Round PCR Product
Inner Forward Primer
Inner Reverse Primer
2nd Round PCR Product
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/1.1.05._VALID_PCR.pdf
Commonly Used PCR Assays
Real-time PCR detects a fluorescent signal that
is increased each time a template is copied; the
fluorescent signal is monitored each cycle or in
‘real-time’
∆ Fluorescence

Threshold
CT CT
Cycle Number
CT = The cycle that a PCR reaction crosses the designated threshold
Also called cycle quantification (CQ) or crossing point (CP)
http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf
Commonly Used PCR Assays
Quantitative PCR relies on the principal that
the quantity of target at the start of the reaction
is proportional to amount of product produced
during the exponential phase
∆ Fluorescence

Greater starting target
Less starting target
CT < CT
Commonly Used PCR Assays

Real-time PCR is often used synonymously with
quantitative PCR
 Real-time PCR involves monitoring the fluorescent
signal produced during every cycle
 Real-time PCR results can be interpreted as plus
or minus (detectable / not detectable)
amplification
 Real-time PCR results can be used to estimate
starting quantity of the target sequence in a
sample = quantitative PCR
Commonly Used PCR Assays

Suggested terminology and acronyms for each assay type
Assay type
Acronym
Nucleic acid
target
Result
cPCR
DNA
Plus / Minus
RT-cPCR
RNA
Plus / Minus
nPCR
DNA
Plus / Minus
RT-nPCR
RNA
Plus / Minus
Detection by gel-based electrophoresis
Conventional PCR
Reverse transcriptase conventional PCR
Nested PCR
Reverse transcriptase nested PCR
Detection by fluorescent monitoring in a real-time PCR instrument
Quantitative PCR
Reverse transcriptase quantitative PCR
Real-time PCR
Reverse transcriptase real-time PCR
qPCR
DNA
CT / Pathogen copy
RT-qPCR
RNA
CT / Pathogen copy
rPCR
DNA
Plus / Minus
RT-rPCR
RNA
Plus / Minus
Advantages and Disadvantages of PCR

Detection of pathogens with PCR-based tests
have a number of general advantages



Assays are typically highly sensitive
Assays are typically highly specific
Assays can be run in a high through-put manner
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Advantages and Disadvantages of PCR

Detection of pathogens with PCR-based tests
have a number of general disadvantages
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Failure to detect pathogen template due to genetic variation
at primer sites leading to false-negative results
Inhibitors in samples leading to false-negative results
High risk of contamination leading to false-positive results
No indication of pathogen viability
Confirms presence of nucleic acid but not infection
Only a small proportion of the tissue is examined per
reaction
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Advantages and Disadvantages of PCR

Nested PCR for pathogen
detection

104 103 102 101
Advantages

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Bacterial Quantity
Two rounds of PCR
improves sensitivity
Two sets of primers
improves specificity
Conventional
PCR
- -
Disadvantages


Prone to contamination
from amplified PCR
products
Time consuming to perform
two PCR rounds
Nested
PCR
+ +
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Advantages and Disadvantages of PCR

Quantitative PCR has several advantages over
conventional and nested PCR assays
 Obtain quantitative estimate of target
 Semi-automated
 Rapid results
 No handling of amplified DNA which limits potential
laboratory contamination
 Some assays use an internal probe that provides added
specificity
 Good assay parameters
 Large dynamic range
 Low inter-assay variation
 Highly reliable
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Good Laboratory Practices

All PCR-based assays are prone to contamination

Need dedicated spaces for different activities:
Clean Room:
storage and
preparation of PCR
reagents
Sample
Preparation:
all samples and
controls processed
High Risk
Templates:
plasmid DNA or
synthetic controls
at high
concentrations
Nested PCR:
handling of first
round PCR
products
Dirty Room:
PCR amplification
and handling of
amplified products
•Work flow in unidirectional moving from clean to dirty
•No exchange of equipment,
materials or lab jackets
Quality Assurance / Quality Control for the Fish and Wildlife Fish Health Laboratories:
http://www.fws.gov/aah/PDF/QI-FWS%20AAHP%20QA%20Program.pdf
Analytical Validation
Validation encompasses assay development, assay
optimization, analytical performance at the bench-top
scale, and diagnostic performance to establish the
fitness of a new diagnostic assay for its intended
purpose

Important to evaluate properties of specificity,
sensitivity and repeatability for all diagnostic tests

http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/1.1.2_VALID.pdf
Analytical Validation

Definition of important terms
Term
Definition
Fitness of purpose
The intended purpose of the assay
Analytical sensitivity (ASe)
The minimum number of copies reliably detected by the assay
Analytical specificity (ASp)
The degree to which the assay does not detect (amplify) other
pathogens
Limit of detection (LOD)
Another term to describe analytical sensitivity
Repeatability
Agreement between sample replicates, both within an assay run and
between independent assay runs, when tested by the same laboratory
Reproducibility
Agreement among test results when the same samples is tested by
different laboratories
Ruggedness
Reproducibility of an assay using different reagent brands or batches
and different equipment
http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/1.1.2_VALID.pdf
Analytical Validation

Analytical sensitivity (ASe) / limit of detection (LOD)
 Theoretically one copy of the target must be
present in the reaction for PCR to occur but this
copy number will not be reliably detected
 Samples at or below the LOD typically have poor
repeatability
 Extending the assay cycle numbers well beyond
the LOD may produce spurious results
http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/1.1.2_VALID.pdf
Sampling and Template Preparation
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Sample acquisition represents the first source
of experimental variability

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Laboratories need clear acceptance / rejection
criteria for a sample
Sample integrity must be maintained between
collection, transport and receipt of sample
Nucleic acid degrading solution (e.g. sodium
hypochlorite or commercial product) should be used
to clean non-disposable sampling tools and work
spaces between samples

Alcohol and/or flaming tools is not sufficient to prevent
cross-contamination of samples
Quality Assurance / Quality Control for the Fish and Wildlife Fish Health Laboratories:
http://www.fws.gov/aah/PDF/QI-FWS%20AAHP%20QA%20Program.pdf
Sampling and Template Preparation

Stabilizing nucleic acids

RNA degrades rapidly and should be stabilized
immediately

Common stabilization methods for RNA



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Snap-freezing in liquid nitrogen
RNA stabilizing solution (e.g. RNAlater®)
Long-term storage at -80°C
DNA is more stable but can degrade if not properly
handled

Common stabilization methods for DNA



Freezing at -20°C or -80°C
95% ethanol
Drying on special filters (e.g. FTA® Cards)
Sampling and Template Preparation

Important to be familiar with general principles of
working with RNA:
 Avoid RNAses
 Always wear gloves when handling reagents or
equipment that will be used in the RNA extraction
and reverse transcription procedures
 RNAse-free water can be commercially purchased
or nanopure water can be treated with diethyl
pyrocarbonate (DEPC)
http://www.promega.com/~/media/files/resources/product%20guides/rna%20analysis%20notebook/workingwithrna
.ashx?la=en
Sampling and Template Preparation
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A variety of commercial kits exist to extract
nucleic acids
New extraction methodologies need to be
evaluated to assess impact on assay sensitivity
High throughput methods need careful
evaluation to ensure that no crosscontamination occurs among samples
Spectrophotometric analysis to obtain DNA
concentration is useful for monitoring
extraction efficiency
http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf
Primers



A variety of commercial companies can
synthesize oligonucleotide primers
Primers typically arrive lyophilized, are
rehydrated with nuclease-free water, and stored
at -20°C
‘Dilution’ and ‘Resupension’ online calculators
to assist in primer dilution

http://www.idtdna.com/analyzer/Applications/DilutionCalc/

http://www.idtdna.com/analyzer/Applications/resuspensioncalc/
Standards, controls and normalization

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

Standard: a sample of a known concentration/copy
number used to construct the standard curve
Control: various samples that ensure the validity of
positive and negative results
Normalization: corrects for variation in template
quantity and/or template quality
Endogenous: target naturally present in sample
(e.g. host gene)
Exogenous: artificial target that is spiked into the
sample
*See reference below for in depth discussion
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Standard: a sample of a known concentration/copy
number used to construct the standard curve
Standards are typically used when quantitative results are desired
= quantitative PCR
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

A good standard:
 Stable
 Mimics the biological target
 Can be accurately quantified
 New batches can be reliably produced
 Not a high contamination risk for the
laboratory
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Standards for DNA targets



Plasmid DNA containing PCR target
Single-stranded oligodeoxynucleotides
Quantified pathogen culture




e.g. Bacterium quantified by FAT
e.g. CFU or PFU quantified pathogen
e.g. Purified parasite spores
Standards for RNA target


Same as above
In vitro transcript generated from plasmid
(synthesized using T3 or T7 RNA polymerase)
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Control: various samples that ensure the validity of
positive and negative results
Controls Distinguish:
true positives and true negatives
from
false positives and false negatives
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Optimal set of controls:
 Processing positive control
Control for false negatives and extraction efficiency

Processing negative control
Control for false positives (extraction contamination)

PCR no template control
Control for false positives (PCR contamination)

Standards diluted to the detection limit
Control for false negatives

Internal positive control (IPC)
Irrelevant template and primers that are added to the assay
Detects assay inhibitors (leading to false negatives)
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Normalization: corrects for variation in template
quantity and/or template quality
Normalization is typically only
performed when data are quantitative
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization


External (Exogenous) Normalizing Variables
 Tissue weight extracted
 Nucleic acid concentration
Internal (Endogenous) Normalizing Variables
 RNA: endogenous host gene (housekeeping
gene)
 DNA: can be done but not common
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Normalizing to tissue weight

Advantages


Typical ‘Fish Health Units’
 e.g. CFU/g tissue  gene copies/g tissue
Disadvantages

Extraction efficiency may vary
Does not detect degradation of sample or inhibitors
Normalizing to nucleic acid concentration
 Advantages
 Independent of extraction efficiency
 Done correctly, can be fairly reliable
 Disadvantages
 Time consuming to quantify samples
 Accuracy of spectrophotometer
 Impact of contaminating nucleic acids
 Does not detect degradation of sample or inhibitors


Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Normalizing to endogenous host gene
 Not recommended because expression of the typical
endogenous normalizing gene varies considerably
 Inappropriate in field samples to use as a measure of
‘RNA quantity’
 Results should not be used to ‘normalize’ pathogen copy
number
 Amplification of a housekeeping gene can be used to assess
RNA quality (i.e. as a ‘control’)
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Standards, controls and normalization

Recommendations for the use of controls, standards and normalization
Category
Standards
Type
Standard curve
Positive processing
sample
Negative processing
sample
No template control for
reaction
Internal positive
control (IPC)
Recommendation
Always recommended when quantitative results are
desired
Always recommended to include a minimum of one
positive reference sample per assay run
Always recommended to verify nucleic acid extraction
effectiveness
Always recommended to detect contamination during
extraction process
Always recommended on every assay run to detect
contamination in reagents
Good practice for detecting false negative results if IPC
does not interfere with assay sensitivity
Amplification of
endogenous gene
Good practice for ensuring nucleic acid integrity and
troubleshooting
Exogenous
normalization variables
Good practice to track tissue weight and nucleic acid
concentration; normalizing copy number to these variables
is dependent on goals
Not recommended to normalize copy number to
endogenous gene expression in field samples
Reference sample
Controls
Normalization
Normalization to
endogenous gene
Purcell, M.K. et al. (2011) Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic
laboratory setting. J. Aq. An. Health. 23:148-161. http://www.tandfonline.com/doi/abs/10.1080/08997659.2011.620217
Quantitative PCR – in depth

Major assay types

Fluorogenic 5’ Nuclease Assay
 Basis of TaqMan® chemistry
 Uses two primers and an internal hydrolysis probe
 Most commonly used for fish health diagnostics

SYBR ® green dye chemistry
 Increased fluorescence when bound to dsDNA
 Slightly lower specificity
 Costs less
 May not be as sensitive as the 5’ nuclease assays
http://www.clinical-virology.org/pdfs/PCR_experience.pdf
Quantitative PCR – in depth

Fluorogenic 5’ Nuclease Assay
Step 1:
Anneal and
Polymerization
Forward Primer
R
Taq
Polymerase
Step 2:
Strand Displacement
Probe
Q
Energy from fluorophore
transferred to quencher
Reverse Primer
R
T
Q
R
Step 3:
Cleavage
Polymerization
Complete
Q
Probe must hybridize specifically for cleavage
A probe is cleaved each time a target is copied
Quantitative PCR – in depth

Dual-labeled internal hydrolysis probes
 5’ reporter dye (typically Fam/Vic etc.)
 3’ quencher (typically non-fluorescent)
 Can order from a range of oligo companies
 Many companies have proprietary modifications for
internal hydrolysis probes
 Minor Grove Binding (MGB) – Applied Biosystems Inc.
 The MGB linker raises the melting temperature of
the internal hydrolysis probe and increases probe
specificity
http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_083
618.pdf
Quantitative PCR – in depth


Most common to use a commercial real-time PCR master mix

Variety of vendors

Variety of proprietary formulations

Empirically evaluate how different formulations impact assay sensitivity
Most master mixes contain:

Passive normalizing dye to correct for variation in master mix
concentration

Hot-start Taq polymerase activation so reactions can be set-up at room
temperature

System to degrade post-PCR products

Uracil-N-Glycosylase (UNG) degrades amplified products that have dUTP
Quantitative PCR – in depth


Analysis of real-time PCR results are specific to the
instrument
Most instrument vendors provide training and
technical support
Quantitative PCR – in depth

Standards are needed if quantitative results are
desired
Standard curve that plots log copy number against
cycle threshold (CT)
35
y = -3.3169x + 38.322
30
R2 = 0.9989
25
CT

20
15
10
5
0
0.0
2.0
4.0
6.0
8.0
10.0
Log Copy #
Quantity is determined by equation of the line
Antilog ((CT-y int)/m)
Quantitative PCR – in depth
Reliable endpoint of assay should be defined
empirically during assay validation
5 plasmid copies
40.0
y = -3.5689x + 38.561
R2 = 0.99
35.0
30.0
25.0
CT

20.0
15.0
10.0
5.0
0.0
0.0
1.0
2.0
3.0
4.0
Log (RS plasmid copies)
5.0
6.0
Analytical sensitivity: the smallest number of genome copies
that can be (reliably) detected and distinguished from zero
Quantitative PCR – in depth

Low initial starting copy
numbers impacts the
accuracy and precision of
quantitative PCR


Statistical errors impact
quantification when starting
copy number is < 1000
Results are not always
reproducible beyond the
reliable endpoint of the assay
Random effects
in PCR
Acknowledgements

Prepared by:
Maureen Purcell
Western Fisheries Research Center
U.S. Geological Survey
6505 NE 65th St, Seattle WA 98034
mpurcell@usgs.gov

The use of trade, firm, or corporation names in this publication is for
the information and convenience of the reader. Such use does not
constitute an official endorsement or approval by the U.S.
Department of Interior or U.S. Geological Survey of any product or
service to the exclusion of others that may be suitable.