U.S. patent application number 15/213109 was filed with the patent office on 2016-11-10 for bioagent detection systems, devices, and methods.
The applicant listed for this patent is IBIS BIOSCIENCES, INC.. Invention is credited to Lawrence B. Blyn, David J. Ecker, Mark W. Eshoo, Thomas A. Hall, Steven A. Hofstadler, Rangarajan Sampath.
Application Number | 20160325283 15/213109 |
Document ID | / |
Family ID | 42828667 |
Filed Date | 2016-11-10 |
United States Patent
Application |
20160325283 |
Kind Code |
A1 |
Ecker; David J. ; et
al. |
November 10, 2016 |
BIOAGENT DETECTION SYSTEMS, DEVICES, AND METHODS
Abstract
The present invention relates to portable systems and devices,
and corresponding methods, for detecting bioagents. In particular,
the present invention provides systems, devices, and methods that
utilize one or more of a sample preparation component, sample
analysis component employing broad range primers, and sample
detection component.
Inventors: |
Ecker; David J.; (Encinitas,
CA) ; Hofstadler; Steven A.; (Vista, CA) ;
Sampath; Rangarajan; (San Diego, CA) ; Blyn; Lawrence
B.; (Mission Viejo, CA) ; Hall; Thomas A.;
(Oceanside, CA) ; Eshoo; Mark W.; (Solana Beach,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IBIS BIOSCIENCES, INC. |
Carlsbad |
CA |
US |
|
|
Family ID: |
42828667 |
Appl. No.: |
15/213109 |
Filed: |
July 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13259350 |
Nov 28, 2011 |
9393564 |
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PCT/US10/29241 |
Mar 30, 2010 |
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15213109 |
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61164773 |
Mar 30, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/5027 20130101;
H01J 49/0022 20130101; B01L 3/52 20130101; B01L 3/502761 20130101;
B01L 2200/10 20130101; B01L 2400/0415 20130101; B01L 2300/027
20130101; B01L 2200/028 20130101; B01L 7/52 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; H01J 49/00 20060101 H01J049/00; B01L 7/00 20060101
B01L007/00 |
Claims
1. A portable system or device comprising: a) a first chamber
configured for isolation of nucleic acid molecule from a sample; b)
a second chamber configured for analysis of isolated nucleic acid,
the first and second chambers in liquid communication with one
another; c) a third chamber configured for receiving a removable
reagent cartridge, said third chamber in liquid communication with
each of said first and second chambers; d) a user interface for
receiving processing instruction from a user; e) a display; and f)
a processor configured to display an identity of a bioagent on said
display.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 13/259,350 filed Nov. 28, 2011, which claims
priority to PCT Patent Application No. PCT/US2010/029241 filed Mar.
30, 2010, which claims priority to U.S. Provisional Application
Ser. No. 61/164,773 filed Mar. 30, 2009, the entirety of each of
which is herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to portable systems and
devices, and corresponding methods, for detecting bioagents. In
particular, the present invention provides systems, devices, and
methods that utilize one or more of a sample preparation component,
sample analysis component employing broad range primers, and sample
detection component.
BACKGROUND OF THE INVENTION
[0003] Rapid and definitive microbial identification is desirable
for a variety of industrial, medical, environmental, quality, and
research reasons. Traditionally, the microbiology laboratory has
functioned to identify the etiologic agents of infectious diseases
through direct examination and culture of specimens. Since the
mid-1980s, researchers have repeatedly demonstrated the practical
utility of molecular biology techniques, many of which form the
basis of clinical diagnostic assays. Some of these techniques
include nucleic acid hybridization analysis, restriction enzyme
analysis, genetic sequence analysis, and separation and
purification of nucleic acids (See, e.g., J. Sambrook, E. F.
Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual,
2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989). These procedures, in general, are time-consuming and
tedious and require large and complex analytical equipment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] The foregoing summary and detailed description is better
understood when read in conjunction with the accompanying drawings
which are included by way of example and not by way of
limitation.
[0005] FIG. 1 shows a process diagram illustrating one embodiment
of the primer pair selection process.
[0006] FIG. 2 shows a process diagram illustrating one embodiment
of the primer pair validation process. Here, select primers are
shown meeting test criteria. Criteria include but are not limited
to, the ability to amplify targeted organism nucleic acid, the
ability to exclude non-target bioagents, the ability to not produce
unexpected amplicons, the ability to not dimerize, the ability to
have analytical limits of detection of .ltoreq.100 genomic
copies/reaction, and the ability to differentiate amongst different
target organisms.
[0007] FIG. 3 shows a process diagram illustrating an embodiment of
the calibration method.
[0008] FIG. 4 shows a block diagram showing a representative
system.
[0009] FIG. 5 shows an exemplary handheld device of the
invention.
[0010] FIG. 6 shows an exemplary handheld device of the invention
with consumables.
[0011] FIG. 7 shows an internal configuration of an exemplary
handheld device.
DESCRIPTION OF THE INVENTION
[0012] The present invention relates to portable systems and
devices, and corresponding methods, for detecting bioagents. In
particular, the present invention provides systems, devices, and
methods that utilize one or more of a sample preparation component,
sample analysis component employing broad range primers, and sample
detection component.
[0013] In some embodiments, the systems, devices, and methods are
embodied in a portable format. The portable systems and devices may
be hand-held sized or may be larger. Portability permits the use of
the systems and devices outside of traditional laboratory settings.
In some embodiments, devices are provided having a length, a width,
and depth. In some embodiments, the length, width, and depth are
each, independently, less than 0.5 meters (e.g., less than 0.3
meters, less than 0.2 meters, less than 0.1 meters, less than 0.05
meters, less than 0.03 meters, less than 0.02 meters, less than
0.01 meters, or less than 0.005 meters). In some embodiments, the
weight of the device is less than 10 kg (e.g., less than 5 kg, less
than 3 kg, less than 2 kg, less than 1 kg, less than 0.5 kg, less
than 0.3 kg, less than 0.2 kg, or less than 0.1 kg).
[0014] In some embodiments, the systems and device combine one or
more of sample preparation, sample analysis, and sample detection.
For example, in some embodiments, the systems and devices combine
sample preparation and single molecule-based analysis and detection
of nucleic acid molecules. In some embodiments, the small size of
the systems and devices is achieved by minimizing the need to
extensively move sample and fluid through large numbers of
different compartments. For example, in some embodiments, the
systems and devices use three or fewer chambers to process samples:
a sample preparation chamber, a sample analysis chamber, and a
sample detection chamber. One or more of these functionalities may
be combined (i.e., a single chamber provide two or all three of
these functions). Chambers are preferably fluidicly connected by
microchannels. Miniaturization is further enhanced by the use of
consumable kit cartridges that provide target-specific and general
reagents. An example comprises the uses of electrodynamic fields
(e.g., SCODA) for nucleic acid isolation, PCR with broad range
primers for nucleic acid amplification and next-generation
sequencing approaches for nucleic acid analysis, and detection via
electrostatic fields and nanopores.
[0015] An exemplary handheld device is shown in FIG. 5. This
embodiment provides a user interface that includes a keypad, which
can be a physical keypad or a touchscreen, and a display screen.
The keypad permits the user to input instructions or data into the
device. Such instructions and data include, but are not limited to,
sample identification, date, time, user name, selection of sample
type, selection of analysis type, selection of sample processing
conditions, selection of sample analysis conditions (e.g., number
of cycles of an amplification reaction), selection of detection
conditions, selection of data display formats, and the like. In
some embodiments, the device comprises computer memory that stores
data. In some embodiments, the device comprises a sample input
port. The sample input port may be configured in any desired manner
to accept desired sample types. Exemplary sample input ports permit
sample input from syringes, hoses, droppers, pipettes, and the
like. In some embodiments, the devices further comprise a kit
cartridge input port. Such ports permit addition of single-use or
multi-use reagents to the device for carrying out one or more
sample preparation, analysis, or detection steps. Cassettes may
provide target-specific reagents (e.g., primers for detection of
particular pathogens). Thus, in some embodiments, the device is
able to detect any desired target analyte through the addition of
interchangeable, consumable, target-specific cassettes containing
appropriate reagents (e.g., target-specific reagents, general
reagents, buffers, positive and negative control reagents, etc.)
for the target of interest. FIG. 6 provides an exemplary device
showing consumable sample input and reagent cartridges.
[0016] In some embodiments, the systems and devices are configured
to carry out sample preparation and processing, but not analysis.
In some such embodiments, the sample is prepared in a manner that
permits its transfer to different analytical equipment for
analysis. For example, in some embodiments, the device permits
nucleic acid isolation and amplification (e.g., using broad range
primers) and the amplified nucleic acid molecules are packaged for
transfer to a different analytical device (e.g., a mass
spectrometer).
[0017] In some embodiments, the systems and devices comprise
wireless communication components to permit wireless transfer of
data, instructions, or other information. For example, in some
embodiments, data collected by the system or device is transmitted
to a remote processing location. In some embodiments, the data is
compressed prior to transfer. In some embodiments, the transferred
data is processed (e.g., compared to a database to identify or
otherwise characterize an unknown target nucleic acid molecule) and
the processed data is presented to the user. In some embodiments,
the data is presented by transfer back to the device and the
analysis is displayed on the device. In other embodiments, the data
is made available over a public or private electronic communication
system (e.g., Internet, phone, etc.).
[0018] The internal layout of the device is configured with one or
more chambers for storing reagents and carrying out the processing
steps. An exemplary configuration is shown in FIG. 7. In this
embodiment, a first region comprises a power source. In some
embodiments, the power source comprises one or more batteries. In
some embodiments, the power source is configured for receipt of
power from an external power source. A second region provides a
computer and other necessary electronics. The computer comprises a
processor and computer memory. The device may contain a wired or
wireless data transfer component to permit transfer of data to
and/or from the computer. A third region provides a sample
preparation chamber in communication with the sample input port.
The sample preparation chamber is in liquid communication with a
sample preparation reagent housing of the kit cartridge that
contains reagents for sample preparation. In some embodiments, the
sample preparation chamber isolates and purifies nucleic acid
molecules from samples. A fourth region, a sample analysis chamber,
is in liquid communication with the sample preparation chamber and
receives purified nucleic acid molecules from the sample
preparation chamber. FIG. 7 exemplifies the analysis chamber as a
polymerase chain reaction (PCR) chamber for carrying out nucleic
acid amplification and post-amplification clean-up. The analysis
chamber is in liquid communication with reagent chambers in the kit
cartridge that provide PCR reagents and PCR clean-up reagents. A
fifth region, a sample detector region, is in liquid communication
with the sample analysis chamber and receives amplified nucleic
acid from the analysis chamber. The detector contains optical,
fluorescent, luminescent, or other signal detection components to
detect the presence of, or identity of, the target nucleic acid
molecule. The detection component is in liquid communication with a
waste container in the kit cartridge such that all reagents may be
removed and disposed with the consumable kit cartridge. In some
embodiments, the kit cartridge contains a wash reservoir that
provides a wash solution to clean all chambers of the device.
[0019] The systems and devices of the present invention may be
configured to work with a wide variety of sample types, analysis
methods, and detection systems. Non-limiting examples of each are
provided below.
Sample Preparation
[0020] The present invention is not limited by the nature of the
sample that is analyzed. Samples include both biological samples
(e.g., blood, sputum, urine, tissue, nasopharyngeal or nasal swabs,
nasal wash or aspirate, etc.) and environmental samples (e.g., air,
water, etc.).
[0021] The sample preparation component of the systems and devices
may include microfluidic channels and chambers to permit proper
processing of the sample. Exemplary microfluidic systems are
described in Ohno et al., Electrophoresis, 29:4443 (2008), Franke
and Wixforth, Chemphyschem., 24:2140 (2008), Crevillen et al.,
Talanta, 74:342 (2007), Ong and Du, Front Biosci., 13:2757 (2008),
and Chen and Day, Lab Chip, 7:1413 (2007), herein incorporated by
reference in their entireties.
[0022] In some embodiments, sample is exposed to appropriate
reagents to release (e.g., lyse) nucleic acid from cells, tissues,
or other sample types. In some embodiments, capture components or
molecules (e.g., beads) are used to isolate the nucleic acid from
the non-nucleic acid components of the sample. Any of a wide
variety of nucleic acid isolation or capture technologies may be
used in the sample preparation component of the systems, devices,
and methods.
[0023] In some embodiments, cell capture technologies are use to
isolate cells or other materials containing a target nucleic acid
away from other cells and sample material. For example, in some
embodiments, ADEMTECH VIRO ADEMBEADS are used for magnetic
separation of viral particles. In other embodiments, Si-pillar
arrays are used to capture cells (see e.g., Hwang et al., Anal.
Chem., 80:7786 (2008), herein incorporated by reference in its
entirety).
[0024] Cell lysis can be conducted using chemical (e.g., chaotropic
salts, GITC, guanidinium-HCl, urea, phenol, NaOH/KOH, detergents,
etc.), temperature (boiling, freeze/thaw, microwave), physical
(e.g., pressure, bead beating, French Press, sonication, grinding,
mortar/pestle/SiO.sub.2), enzymatic (e.g., lysozymes, glycanases,
proteases, Proteinase K), or osmosis (e.g., osmotic shock, low salt
buffers) approaches, or combinations thereof. Lysis can be
organisms-specific or non-organisms-specific.
[0025] Nucleic acid isolation from lysed cellular material or other
materials can be conducted by Solid Phase Reversible Immobilization
using magnetic microparticles (see e.g., U.S. Pat. No. 5,234,809,
herein incorporated by reference in its entirety). In some
embodiments, capture oligonucleotides complementary to a target
nucleic acid of interest are employed.
[0026] In some embodiments, sample preparation employs a SCODA
method. In certain embodiments, broad range primers (e.g., as
disclosed herein) are immobilized in a SCODA gel (e.g., by
cross-linking the primers in the gel). In this regard, immobilized
primers serve as broad capture oligonucleotides. In general, a
sample is loaded into such a SCODA gel, which not only allows total
nucleic acid to be purified and concentrated from contaminants, but
also allows the target nucleic acid (e.g., a portion of a pathogen
genome) to be selectively concentrated from other non-target
nucleic acid. In certain embodiments, the selectively concentrated
target nucleic acid is eluted from the SCODA gel and subjected to
amplification methods in order to detect the target nucleic acid.
In particular embodiments, the concentrated nucleic acid is
subjected to broad range priming, using, for example, at least some
of the same primers immobilized in the SCODA gel. In some
embodiments, the same set of immobilized primers is used as primers
to amplify the target nucleic acid. In certain embodiments, the
SCODA gel immobilized primers are: complementary to the broad range
primers described further below that are complementary to variable
regions that flank a conserved regions in target pathogens; are
complementary to the broad range primers used in the mass
spectrometry methods described below (e.g., IBIS TIGER methods);
used to capture based on other broadly conserved domains that flank
the primers generally employed in the mass spectrometry methods
described below; contain "wild-card" inosine bases; or are composed
of mixtures of oligonucleotides which take into account known
mixtures/heteroplasmies/SNPs in the capture sequences.
[0027] In particular embodiments, prior to loading a sample (e.g.,
a crude sample, such a blood, serum, saliva, air sample, water
sample, etc.) onto a SCODA gel, it is subjected to processing with
restriction enzymes. In other embodiments, the concentrated nucleic
acid eluted from the SCODA gel is subjected to processing by
restriction enzymes. Preferably, the restriction enzymes are
selected to ensure digestion around the target areas of interest
(e.g., regions that have primer binding sites that are variable,
but surround a conserved region).
[0028] In certain embodiments, the gel immobilized SCODA primers
(capture olignucleotides) are used to perform in situ PCR methods
in the SCODA gel in order to amplify the target sequence prior to
detection or elution and detection. In certain embodiments, the
electrical or other fields used in the SCODA method are used to
promote hybridization and disassociation of the target nucleic acid
and immobilized primers in order to facilitate rounds of PCR.
[0029] In other embodiments, the concentrated target nucleic acid
(e.g., bound to the capture oligonucleotides in the gel) are
directly detected without eluting from the gel. For example, in
certain embodiments, the capture oligonucletodies are detectably
labeled such that hybridization with target nucleic acid (if
present) can be directly detected.
[0030] As indicated above, embodiments of the present invention
provide for the use of SCODA methods with broad range primers
immobilized in a SCODA gel as capture oligonucleotides. SCODA is a
method of particle separation and concentration that may be used to
purify highly negatively charged molecules such as nucleic acid
(e.g., DNA). SCODA methods, compositions, and devices are described
in: U.S. Provisional Application 60/540,352, filed 2 Feb. 2004,
U.S. Provisional Application 60/634,604, filed Dec. 10, 2004;
Marziali, A.; et al., Electrophoresis, 2005, 26, 82-89; Broemeling
et al., JALA Charlottesv Va., 2008 February; 13(1):40-48,
WO06/081691, filed Feb. 7, 2006; and WO05/072854, filed Feb. 2,
2005, all of which are herein incorporated by reference in their
entireties as if fully set forth herein. SCODA can be used to
concentrate the particles in the vicinity of a point in a region of
a suitable material in which the particles have mobilities that
vary in response to an applied field or combination of applied
fields. Where the particles are electrically-charged molecules,
such as DNA, the applied fields may comprise electric fields. The
material may comprise a suitable gel such as an agarose gel, for
example. SCODA does not require electrodes to be present at the
location where particles are concentrated. In one embodiment, SCODA
provides focusing and concentration of molecules based on the
non-linear dependence of the particles' velocity on the strength of
an applied electric field. This can also be stated as being based
on the field dependence of the particles' mobility.
[0031] Particles may be injected into a region of a medium within
which the particles can be concentrated by SCODA by providing the
particles in an adjacent region and applying a field that causes
the particles to move into the region of the SCODA medium. The
adjacent region may be called a first region and the region of the
SCODA medium may be called a second region. The field that causes
the particles to move from the first region into the second region
may be called a first field. The first field may comprise any field
to which particles of interest respond by moving. Where the
particles are electrically charged, the first field may comprise an
electric field. Depending upon the nature of the particles of
interest, the first field may comprise any of: a magnetic field; an
electric field; a flow field; or combination thereof.
Sample Analysis
[0032] Purified nucleic acid molecules may be analyzed by a wide
variety of methods. In some embodiments, analysis comprises nucleic
acid amplification. In some embodiments, no nucleic acid
amplification is employed. In some embodiments, nucleic acid
sequence is determined. In some embodiments, sequence is not
determined. In some embodiments, broad range priming is used in
conjunction with amplification, sequencing, or other analysis
techniques.
[0033] Broad Range Primers
[0034] Embodiments of the present employ broad range primers as
capture oligonucleotides and/or amplification primers. Broad range
primers refer to primers that hybridize to regions of a target
nucleic acid that are conserved between two or more organisms or
cells or loci and that, when two primers are used, flank a variable
region that differs between said two or more organisms or cells or
loci. In some embodiments, the two or more organisms differ in
their genotype, strain, sub-species, species, genus, family, order,
class, phylum, or kingdom. For example, in some embodiments, a
first organism is a particular genus of bacteria and the second
organism is a different genus of bacteria. In other embodiments,
the first and second organisms are the same genus, but different
species of bacteria. In other embodiments, the first organism is a
bacterium and the second organism is a virus or a mammal. In some
embodiments, the broad range primers are used to generate amplicons
from target nucleic acid molecules in a sample to facilitate
analysis of or determine the presence of the target nucleic acid
molecules.
[0035] One with ordinary skill in the art of design of primers will
recognize that a given primer need not hybridize with 100%
complementarity in order to effectively prime the synthesis of a
complementary nucleic acid strand. Primer pair sequences may be a
"best fit" amongst the aligned bioagent sequences, thus they need
not be fully complementary to the hybridization region of any one
of the bioagents in the alignment. Moreover, a primer may hybridize
over one or more segments such that intervening or adjacent
segments are not involved in the hybridization event (e.g., for
example, a loop structure or a hairpin structure). The primers may
comprise at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95% or at least 99% sequence identity with a
target nucleic acid of interest. Thus, in some embodiments, an
extent of variation of 70% to 100%, or any range falling within, of
the sequence identity is possible relative to the specific primer
sequences disclosed herein. To illustrate, determination of
sequence identity is described in the following example: a primer
20 nucleobases in length which is identical to another 20
nucleobase primer having two non-identical residues has 18 of 20
identical residues (18/20=0.9 or 90% sequence identity). In another
example, a primer 15 nucleobases in length having all residues
identical to a 15 nucleobase segment of primer 20 nucleobases in
length would have 15/20=0.75 or 75% sequence identity with the 20
nucleobase primer. Percent identity need not be a whole number, for
example when a 28 consecutive nucleobase primer is completely
identical to a 31 consecutive nucleobase primer (28/31=0.9032 or
90.3% identical).
[0036] Percent homology, sequence identity or complementarity, can
be determined by, for example, the Gap program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, Madison Wis.), using default settings,
which uses the algorithm of Smith and Waterman (Adv. Appl. Math.,
1981, 2, 482-489). In some embodiments, complementarity of primers
with respect to the conserved priming regions of viral nucleic
acid, is between about 70% and about 80%. In other embodiments,
homology, sequence identity or complementarity, is between about
80% and about 90%. In yet other embodiments, homology, sequence
identity or complementarity, is at least 90%, at least 92%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
at least 99% or is 100%.
[0037] In some embodiments, the primers described herein comprise
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 92%, at least 94%, at least 95%, at least 96%, at
least 98%, or at least 99%, or 100% (or any range falling within)
sequence identity with the primer sequences specifically disclosed
herein.
[0038] In some embodiments, the oligonucleotide primers are 13 to
35 nucleobases in length (13 to 35 linked nucleotide residues).
These embodiments comprise oligonucleotide primers 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34 or 35 nucleobases in length, or any range therewithin.
[0039] In some embodiments, any given primer comprises a
modification comprising the addition of a non-templated T residue
to the 5' end of the primer (i.e., the added T residue does not
necessarily hybridize to the nucleic acid being amplified). The
addition of a non-templated T residue has an effect of minimizing
the addition of non-templated A residues as a result of the
non-specific enzyme activity of, e.g., Taq DNA polymerase (Magnuson
et al., Biotechniques, 1996: 21, 700-709), an occurrence which may
lead to ambiguous results arising from molecular mass analysis.
[0040] Primers may contain one or more universal bases. Because any
variation (due to codon wobble in the third position) in the
conserved regions among species is likely to occur in the third
position of a DNA (or RNA) triplet, oligonucleotide primers can be
designed such that the nucleotide corresponding to this position is
a base which can bind to more than one nucleotide, referred to
herein as a "universal nucleobase." For example, under this
"wobble" base pairing, inosine (I) binds to U, C or A; guanine (G)
binds to U or C, and uridine (U) binds to U or C. Other examples of
universal nucleobases include nitroindoles such as 5-nitroindole or
3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995,
14, 1001-1003), the degenerate nucleotides dP or dK, an acyclic
nucleoside analog containing 5-nitroindazole (Van Aerschot et al.,
Nucleosides and Nucleotides., 1995, 14, 1053-1056) or the purine
analog 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide
(Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).
[0041] In some embodiments, to compensate for weaker binding by the
wobble base, oligonucleotide primers are configured such that the
first and second positions of each triplet are occupied by
nucleotide analogs which bind with greater affinity than the
unmodified nucleotide. Examples of these analogs include, but are
not limited to, 2,6-diaminopurine which binds to thymine,
5-propynyluracil which binds to adenine and 5-propynylcytosine and
phenoxazines, including G-clamp, which binds to G. Propynylated
pyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653
and 5,484,908, each of which is commonly owned and incorporated
herein by reference in its entirety. Propynylated primers are
described in U.S Pre-Grant Publication No. 2003-0170682; also
commonly owned and incorporated herein by reference in its
entirety. Phenoxazines are described in U.S. Pat. Nos. 5,502,177,
5,763,588, and 6,005,096, each of which is incorporated herein by
reference in its entirety. G-clamps are described in U.S. Pat. Nos.
6,007,992 and 6,028,183, each of which is incorporated herein by
reference in its entirety.
[0042] In some embodiments, non-template primer tags are used to
increase the melting temperature (T.sub.m) of a primer-template
duplex in order to improve amplification efficiency. A non-template
tag is at least three consecutive A or T nucleotide residues on a
primer which are not complementary to the template. In any given
non-template tag, A can be replaced by C or G and T can also be
replaced by C or G. Although Watson-Crick hybridization is not
expected to occur for a non-template tag relative to the template,
the extra hydrogen bond in a G-C pair relative to an A-T pair
confers increased stability of the primer-template duplex and
improves amplification efficiency for subsequent cycles of
amplification when the primers hybridize to strands synthesized in
previous cycles.
[0043] In other embodiments, propynylated tags may be used in a
manner similar to that of the non-template tag, wherein two or more
5-propynylcytidine or 5-propynyluridine residues replace template
matching residues on a primer. In other embodiments, a primer
contains a modified internucleoside linkage such as a
phosphorothioate linkage, for example.
[0044] In some embodiments, the primers contain mass- or
mobility-modifying tags. Addition of mass- or mobility-modifying
tags to certain nucleobases of a given primer can result in
simplification of analysis of a given bioagent identifying
amplicon.
[0045] In some embodiments, the mass- or mobility-modified
nucleobase comprises one or more of the following: for example,
7-deaza-2'-deoxyadenosine-5-triphosphate,
5-iodo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxycytidine-5'-triphosphate,
5-iodo-2'-deoxycytidine-5'-triphosphate,
5-hydroxy-2'-deoxyuridine-5'-triphosphate,
4-thiothymidine-5'-triphosphate,
5-aza-2'-deoxyuridine-5'-triphosphate,
5-fluoro-2'-deoxyuridine-5'-triphosphate,
O6-methyl-2'-deoxyguanosine-5'-triphosphate,
N2-methyl-2'-deoxyguanosine-5'-triphosphate,
8-oxo-2'-deoxyguanosine-5'-triphosphate or
thiothymidine-5'-triphosphate. In some embodiments, the
mass-modified nucleobase comprises .sup.15N or .sup.13C or both
.sup.13N and .sup.13C.
[0046] One embodiment of a process flow diagram used for primer
selection and validation process is depicted in FIGS. 1 and 2. For
each group of organisms, candidate target sequences are identified
(200) from which nucleotide sequence alignments are created (210)
and analyzed (220). Primers are then configured by selecting
priming regions (230) to facilitate the selection of candidate
primer pairs (240). The primer pair sequence is typically a "best
fit" amongst the aligned sequences, such that the primer pair
sequence may or may not be fully complementary to the hybridization
region on any one of the bioagents in the alignment. Thus, best fit
primer pair sequences are those with sufficient complementarity
with two or more bioagents to hybridize with the two or more
bioagents and generate an amplicon or hybridization complex. Where
amplification is desired, the primer pairs are then subjected to in
silico analysis by electronic PCR (ePCR) (300) wherein bioagent
identifying amplicons are obtained from sequence databases such as
GenBank or other sequence collections (310) and tested for
specificity in silico (320). Bioagent identifying amplicons
obtained from ePCR of GenBank sequences (310) may also be analyzed
by a probability model which predicts the capability of a given
amplicon to identify unknown bioagents. Where base composition
analysis is used, the base compositions of amplicons with favorable
probability scores are then stored in a base composition database
(325). Alternatively, base compositions of the bioagent identifying
amplicons obtained from the primers and GenBank sequences are
directly entered into the base composition database (330).
Candidate primer pairs (240) are validated by in vitro
amplification by a method such as PCR analysis (400) of nucleic
acid from a collection of organisms (410). Amplicons thus obtained
are analyzed to confirm the sensitivity, specificity and
reproducibility of the primers used to obtain the amplicons
(420).
[0047] Synthesis of primers is well known and routine in the art.
The primers may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed.
[0048] In some embodiments, a bioagent identifying amplicon or
hybridization complex may be produced using only a single primer
(either the forward or reverse primer of any given primer pair),
provided an appropriate amplification method is chosen, such as,
for example, low stringency single primer PCR (LSSP-PCR).
[0049] Examples of broad range primers, and methods of generating
and selecting broad range primers are described in U.S. Pat. Nos.
7,108,974; 7,217,510; 7,226,739; 7,255,992; 7,312,036; 7,339,051;
patent publication numbers 2003/0027135; 2003/0167133;
2003/0167134; 2003/0175695; 2003/0175696; 2003/0175697;
2003/0187588; 2003/0187593; 2003/0190605; 2003/0225529;
2003/0228571; 2004/0110169; 2004/0117129; 2004/0121309;
2004/0121310; 2004/0121311; 2004/0121312; 2004/0121313;
2004/0121314; 2004/0121315; 2004/0121329; 2004/0121335;
2004/0121340; 2004/0122598; 2004/0122857; 2004/0161770;
2004/0185438; 2004/0202997; 2004/0209260; 2004/0219517;
2004/0253583; 2004/0253619; 2005/0027459; 2005/0123952;
2005/0130196 2005/0142581; 2005/0164215; 2005/0266397;
2005/0270191; 2006/0014154; 2006/0121520; 2006/0205040;
2006/0240412; 2006/0259249; 2006/0275749; 2006/0275788;
2007/0087336; 2007/0087337; 2007/0087338 2007/0087339;
2007/0087340; 2007/0087341; 2007/0184434; 2007/0218467;
2007/0218467; 2007/0218489; 2007/0224614; 2007/0238116;
2007/0243544; 2007/0248969; 2008/0138808; 20080145847; 20080146455;
20080160512; 20080233570; 20080311558; 20090004643; 20090047665;
WO2002/070664; WO2003/001976; WO2003/100035; WO2004/009849;
WO2004/052175; WO2004/053076; WO2004/053141; WO2004/053164;
WO2004/060278; WO2004/093644; WO 2004/101809; WO2004/111187;
WO2005/023083; WO2005/023986; WO2005/024046; WO2005/033271;
WO2005/036369; WO2005/086634; WO2005/089128; WO2005/091971;
WO2005/092059; WO2005/094421; WO2005/098047; WO2005/116263;
WO2005/117270; WO2006/019784; WO2006/034294; WO2006/071241;
WO2006/094238; WO2006/116127; WO2006/135400; WO2007/014045;
WO2007/047778; WO2007/086904; WO2007/100397; WO2007/118222; Ecker
et al., Ibis T5000: a universal biosensor approach for
microbiology. Nat Rev Microbiol. 2008 Jun. 3; Ecker et al.,
Identification of Acinetobacter species and genotyping of
Acinetobacter baumannii by multilocus PCR and mass spectrometry. J
Clin Microbiol. 2006 August; 44(8):2921-32.; Ecker et al., Rapid
identification and strain-typing of respiratory pathogens for
epidemic surveillance. Proc Natl Acad Sci USA. 2005 May 31;
102(22):8012-7. Epub 2005 May 23.; Wortmann et al., Genotypic
Evolution of Acinetobacter baumannii Strains in an Outbreak
Associated With War Trauma. Infect Control Hosp Epidemiol. 2008
June; 29(6):553-555.; Hannis et al., High-resolution genotyping of
Campylobacter species by use of PCR and high-throughput mass
spectrometry. J Clin Microbiol. 2008 April; 46(4):1220-5.; Blyn et
al., Rapid detection and molecular serotyping of adenovirus by use
of PCR followed by electrospray ionization mass spectrometry. J
Clin Microbiol. 2008 February; 46(2):644-51.; Eshoo et al., Direct
broad-range detection of alphaviruses in mosquito extracts.
Virology. 2007 Nov. 25; 368(2):286-95.; Sampath et al., Global
surveillance of emerging Influenza virus genotypes by mass
spectrometry. PLoS ONE. 2007 May 30; 2(5):e489.; Sampath et al.,
Rapid identification of emerging infectious agents using PCR and
electrospray ionization mass spectrometry. Ann N Y Acad Sci. 2007
April; 1102:109-20.; Hujer et al., Analysis of antibiotic
resistance genes in multidrug-resistant Acinetobacter sp. isolates
from military and civilian patients treated at the Walter Reed Army
Medical Center. Antimicrob Agents Chemother. 2006 December;
50(12):4114-23.; Hall et al., Base composition analysis of human
mitochondrial DNA using electrospray ionization mass spectrometry:
a novel tool for the identification and differentiation of humans.
Anal Biochem. 2005 Sep. 1; 344(1):53-69.; Sampath et al., Rapid
identification of emerging pathogens: coronavirus. Emerg Infect
Dis. 2005 March; 11(3):373-9; each of which is herein incorporated
by reference in its entirety.
[0050] In some embodiments, nucleic acid molecules are analyzed and
characterized by any of a wide variety of methods, including, but
not limited to, sequencing, hybridization analysis, amplification
(e.g., via polymerase chain reaction (PCR), reverse transcription
polymerase chain reaction (RT-PCR), transcription-mediated
amplification (TMA), ligase chain reaction (LCR), strand
displacement amplification (SDA), and nucleic acid sequence based
amplification (NASBA)).
[0051] Nucleic acid may be amplified prior to or simultaneous with
detection. Illustrative non-limiting examples of nucleic acid
amplification techniques include, but are not limited to,
polymerase chain reaction (PCR), reverse transcription polymerase
chain reaction (RT-PCR), transcription-mediated amplification
(TMA), ligase chain reaction (LCR), strand displacement
amplification (SDA), and nucleic acid sequence based amplification
(NASBA). Those of ordinary skill in the art will recognize that
certain amplification techniques (e.g., PCR) require that RNA be
reversed transcribed to DNA prior to amplification (e.g., RT-PCR),
whereas other amplification techniques directly amplify RNA (e.g.,
TMA and NASBA).
[0052] The polymerase chain reaction (U.S. Pat. Nos. 4,683,195,
4,683,202, 4,800,159 and 4,965,188, each of which is herein
incorporated by reference in its entirety), commonly referred to as
PCR, uses multiple cycles of denaturation, annealing of primer
pairs to opposite strands, and primer extension to exponentially
increase copy numbers of a target nucleic acid sequence. In a
variation called RT-PCR, reverse transcriptase (RT) is used to make
a complementary DNA (cDNA) from mRNA, and the cDNA is then
amplified by PCR to produce multiple copies of DNA. For other
various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683,195,
4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155: 335
(1987); and, Murakawa et al., DNA 7: 287 (1988), each of which is
herein incorporated by reference in its entirety.
[0053] Transcription mediated amplification (U.S. Pat. Nos.
5,480,784 and 5,399,491, each of which is herein incorporated by
reference in its entirety), commonly referred to as TMA,
synthesizes multiple copies of a target nucleic acid sequence
autocatalytically under conditions of substantially constant
temperature, ionic strength, and pH in which multiple RNA copies of
the target sequence autocatalytically generate additional copies.
See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518, each of which is
herein incorporated by reference in its entirety. In a variation
described in U.S. Publ. No. 20060046265, herein incorporated by
reference in its entirety, TMA optionally incorporates the use of
blocking moieties, terminating moieties, and other modifying
moieties to improve TMA process sensitivity and accuracy.
[0054] The ligase chain reaction (Weiss, R., Science 254: 1292
(1991), herein incorporated by reference in its entirety), commonly
referred to as LCR, uses two sets of complementary DNA
oligonucleotides that hybridize to adjacent regions of the target
nucleic acid. The DNA oligonucleotides are covalently linked by a
DNA ligase in repeated cycles of thermal denaturation,
hybridization and ligation to produce a detectable double-stranded
ligated oligonucleotide product.
[0055] Strand displacement amplification (Walker, G. et al., Proc.
Natl. Acad. Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184
and 5,455,166, each of which is herein incorporated by reference in
its entirety), commonly referred to as SDA, uses cycles of
annealing pairs of primer sequences to opposite strands of a target
sequence, primer extension in the presence of a dNTPaS to produce a
duplex hemiphosphorothioated primer extension product,
endonuclease-mediated nicking of a hemimodified restriction
endonuclease recognition site, and polymerase-mediated primer
extension from the 3' end of the nick to displace an existing
strand and produce a strand for the next round of primer annealing,
nicking and strand displacement, resulting in geometric
amplification of product. Thermophilic SDA (tSDA) uses thermophilic
endonucleases and polymerases at higher temperatures in essentially
the same method (EP Pat. No. 0684315, herein incorporated by
reference in its entirety).
[0056] Other amplification methods include, for example: nucleic
acid sequence based amplification (U.S. Pat. No. 5,130,238, herein
incorporated by reference in its entirety), commonly referred to as
NASBA; one that uses an RNA replicase to amplify the probe molecule
itself (Lizardi et al., BioTechnol. 6: 1197 (1988), herein
incorporated by reference in its entirety), commonly referred to as
Q.beta.-replicase; a transcription based amplification method (Kwoh
et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989)); and,
self-sustained sequence replication (Guatelli et al., Proc. Natl.
Acad. Sci. USA 87: 1874 (1990), each of which is herein
incorporated by reference in its entirety). For further discussion
of known amplification methods see Persing, David H., "In Vitro
Nucleic Acid Amplification Techniques" in Diagnostic Medical
Microbiology: Principles and Applications (Persing et al., Eds.),
pp. 51-87 (American Society for Microbiology, Washington, D.C.
(1993)).
[0057] In some embodiments, the molecular mass of a given bioagent
identifying amplicon is determined by mass spectrometry. Mass
spectrometry is intrinsically a parallel detection scheme without
the need for radioactive or fluorescent labels, because an amplicon
is identified by its molecular mass. The current state of the art
in mass spectrometry is such that less than femtomole quantities of
material can be analyzed to provide information about the molecular
contents of the sample. An accurate assessment of the molecular
mass of the material can be quickly obtained, irrespective of
whether the molecular weight of the sample is several hundred, or
in excess of one hundred thousand atomic mass units (amu) or
Daltons.
[0058] In some embodiments, the present invention provides DNA or
gene sequencing methodologies and/or technologies. In some
embodiments, sequencing methodologies and technologies provided by
the present invention comprise traditional or first generation
sequencing technologies (Maxam & Gilbert, 1977, Proc Natl Acad
Sci USA 74: 560-564; Sanger et al., 1977, Proc Natl Acad Sci USA
74: 5463-5467; herein incorporated by reference in their
entireties) which utilize electrophoretic detection on a gel or
through capillary electrophoresis ((Smith et al., 1986, Nature 321:
674-679; herein incorporated by reference in its entirety). In some
embodiments, DNA sequencing methodologies provided by the present
invention comprise Second Generation (a.k.a. Next Generation or
Next-Gen), Third Generation (a.k.a. Next-Next-Gen), or Fourth
Generation (a.k.a. N.sub.3-Gen) sequencing technologies including
but not limited to pyrosequencing, sequencing-by-ligation, single
molecule sequencing, sequence-by-synthesis (SBS), massive parallel
clonal, massive parallel single molecule SBS, massive parallel
single molecule real-time, massive parallel single molecule
real-time nanopore technology, etc. Morozova and Marra provide a
review of some such technologies, Genomics, 92:255 (2008), herein
incorporated by reference in its entirety.
[0059] In some embodiments, the present invention provides DNA
sequencing by pyrosequencing (Ronaghi et al. 1998, Science 281:363,
365; Ronaghi et al. 1996, Analytical Biochemistry 242: 84; Nyren
2007, Methods Mol Biology 373: 1-14; herein incorporated by
reference in their entireties). Pyrosequencing is a method of DNA
sequencing based on the "sequencing by synthesis" principle, which
relies on detection of pyrophosphate release. "Sequencing by
synthesis" involves imobilizing a single strand of the DNA, and
synthesizing its complementary strand enzymatically. The
pyrosequencing method is based on detecting the activity of DNA
polymerase with a chemiluminescent enzyme. Pyrosequencing allows
sequencing of a single strand of DNA by synthesizing the
complementary strand along it, one base pair at a time, and
detecting which base added at each step. The template DNA is
immobilized, and solutions of A, C, G, and T nucleotides are added
and removed after the reaction, sequentially. Chemiluminescence is
produced when the nucleotide solution complements the next unpaired
base of the template. The sequence of solutions which produce
chemiluminescent signals providse sequence of the template.
[0060] In some embodiments, the present invention provides DNA
sequencing by 454 sequencing by ROCHE LIFE SCIENCES. 454 sequencing
by ROCHE LIFE SCIENCES provides SBS pyrosequencing which can be
performed in Polony beads deposited in 44 .mu.m picoliter wells,
provides very long read lengths (400-500 bases), and can yield
approximately 400-600 Mbases/run or 1 billion bases/day. 454
sequencing finds utility in de novo sequencing, resequencing,
expression tags, transcriptome sequencing, ChIP, methylation
analysis, etc. 454 sequencing involves annealing of ssDNA to an
excess of DNA capture beads, emulsification of beads and PCR
reagents in water-in-oil microreactors, clonal amplification,
breaking of microreactors, and enrichment for DNA positive beads.
454 sequencing is performed on a GENOME FLX SEQUENCER.
[0061] In some embodiments, the present invention provides DNA
sequencing by SOLID sequencing by APPLIED BIOSYSTEMS. SOLID
sequencing by APPLIED BIOSYSTEMS utilizes Polony-based sequencing
methodologies (Mitra & Church 1999 Nucleic Acids Res, 27:e34;
herein incorporated by reference in its entirety). Polony
sequencing provides a nonelectrophoretic sequencing method without
in vivo cloning artifacts at a low cost per base. In some
embodiments, an in vitro paired-tag library is constructed from
genomic DNA. Library molecules are clonally amplified on microbeads
by emulsion PCR, the clonal amplification yields polymerase
colonies, or polonies, that can be sequenced. Short reads are
generated in parallel from the microbeads via a cyclic DNA
sequencing strategy that utilizes T4 DNA ligase to selectively tag
each microbead with fluorescent labels that correlate with the
unique nucleotide sequence present on any given bead. SOLID
sequencing provides sequencing by ligation using T4 DNA ligase,
fluorescent-labeled degenerate nonamers, "Two Base Encoding" which
provides increased accuracy (>99.94%), read length up to 35
bases, and high throughput of 20 Gb/run. SOILD sequencing finds
utility in de novo sequencing, targeted and whole genome
resequencing, gene expression, transcriptome and methylation
analysis. SOLID sequencing is performed on a SOLID 3 platform.
[0062] In some embodiments, the present invention provides DNA
sequencing by ILLUMINA sequencing technology. ILLUMINA sequencing
technology utilizes massively parallel SBS using reverse terminator
chemistry. SBS is performed at 4 bases/cycle versus 1 base/cycle
for pyrosequencing. ILLUMINA sequencing relies on the attachment of
randomly fragmented genomic DNA to a planar, optically transparent
surface. Attached DNA fragments are extended and bridge amplified
to create an ultra-high density sequencing flow cell with 80-100
million clusters, each containing .about.1,000 copies of the same
template. These templates are sequenced using a four-color DNA SBS
technology that employs reversible terminators with removable
fluorescent dyes. In some embodiments, high-sensitivity
fluorescence detection is achieved using laser excitation and total
internal reflection optics. ILLUMINA sequencing provides read
lengths of up to 75 bases, throughput of approximately 10-15
Gb/run, and a paired end strategy allows sequencing from both ends.
ILLUMINA sequencing finds utility in de novo sequencing,
resequencing, transcriptome analysis, epigenomic/methylation
status. ILLUMIN sequencing is performed on a GENOME ANALYZER
platform.
[0063] In some embodiments, the present invention provides DNA
sequencing by TRUE SINGLE MOLECULE SEQUENCING (TSMS) by HELICOS
BIOSCIENCES. TSMS provides massive parallel single molecule SBS
using 1 base per cycle of pyrosequencing. TSMS does not require any
up-front library synthesis steps or PCR amplification, therefore
eliminating PCR errors. TSMS relies on attachment of billions of
single molecules of sample DNA on an application-specific
proprietary surface. The captured strands serve as templates for
the sequencing-by-synthesis process in which polymerase and one
fluorescently labeled nucleotide (C, G, A or T) are added,
polymerase catalyzes the sequence-specific incorporation of
fluorescent nucleotides into nascent complementary strands on all
the templates, free nucleotides are removed by washing,
incorporated nucleotides are imaged and positions recorded, the
fluorescent group is removed in a highly efficient cleavage process
leaving behind the incorporated nucleotide, and the process
continues through each of the other three bases. Multiple four-base
cycles result in complementary strands greater than 25 bases in
length synthesized on billions of templates, providing a greater
than 25-base read from each individual template. TSMS provides very
high density arrays (1 million/mm.sup.2), low cost/base, two laser
system (Cy3 and Cy5-labeled dNTP), and read lengths of read
length--20-55 bases. TSMS find utility in human genome
resequencing, de novo sequencing. TSMS is performed on the
HELISCOPE platform.
[0064] In some embodiments, the present invention provides DNA
sequencing by VISIGEN BIOTECHNOLOGIES. VISIGEN BIOTECHNOLOGIES
sequencing provides massive parallel single molecule sequencing in
real-time through engineered DNA polymerases and nucleoside
triphosphates which function as direct molecular sensors of DNA
base identity. Genetically engineered polymerase is fixed on the
surface during synthesis. Fluorescence resonance energy transfer
(FRET) is detected between the immobilized polymerase and labeled
dNTP as they are incorporated. VISIGEN sequencing provides no
up-front amplification or cloning steps, read lengths of 1,000
bases, massive parallel arrays (1 Mb/sec/instrument), and no
sequential reagent addition during synthesis. VISIGEN sequencing
finds utility in de novo sequencing, resequencing, personalized
medicine, clinical diagnostics, forensics, basic research, etc.
[0065] In some embodiments, the present invention provides single
molecule real time (SMRT) sequencing by PACIFIC BIOSCIENCES. SMRT
provides massive parallel single molecule sequencing in real-time.
Thousands of zero-mode waveguides (ZMWs) in zeptoliter wells are
contained on an array. A single DNA polymerase molecule is attached
to the bottom of each waveguide. DNA is synthesized using
.gamma.-phosphate group labeled with base-specific fluorophores.
Upon incorporation of a phospholinked nucleotide, the DNA
polymerase cleaves the dye molecule from the nucleotide when it
cleaves the phosphate chain. Fluorophores are detected upon
incorporation of the corresponding base by the immobilized
polymerase. SMRT provides low reaction volumes, very low
fluorescence background, fast cycle times, with long read lengths
(approx. 1,000 bases), and no sequential reagent addition during
synthesis. SMRT find utility in de novo sequencing, resequencing,
etc.
[0066] In some embodiments, the Xpandomer technology of STRATOS is
used (see e.g., U.S. Pat. Publn. No. 20090035777, herein
incorporated by reference in its entirety). In this approach,
methods for sequencing a target nucleic acid comprise providing a
daughter strand produced by a template-directed synthesis, the
daughter strand comprising a plurality of subunits coupled in a
sequence corresponding to a contiguous nucleotide sequence of all
or a portion of the target nucleic acid, wherein the individual
subunits comprise a tether, at least one probe or nucleobase
residue, and at least one selectively cleavable bond. The
selectively cleavable bond(s) is/are cleaved to yield an Xpandomer
of a length longer than the plurality of the subunits of the
daughter strand, the Xpandomer comprising the tethers and reporter
elements for parsing genetic information in a sequence
corresponding to the contiguous nucleotide sequence of all or a
portion of the target nucleic acid. Reporter elements of the
Xpandomer are then detected.
Sample Detection
[0067] Detectors are typically structured to detect detectable
signals produced, e.g., in or proximal to another component of the
given assay system (e.g., in a container and/or on a solid
support). Suitable signal detectors that are optionally utilized,
or adapted for use, herein detect, e.g., fluorescence,
phosphorescence, radioactivity, absorbance, refractive index,
luminescence, or mass. Detectors optionally monitor one or a
plurality of signals from upstream and/or downstream of the
performance of, e.g., a given assay step. For example, detectors
optionally monitor a plurality of optical signals, which correspond
in position to "real-time" results. Example detectors or sensors
include photomultiplier tubes, CCD arrays, optical sensors,
temperature sensors, pressure sensors, pH sensors, conductivity
sensors, or scanning detectors. Detectors are also described in,
e.g., Skoog et al., Principles of Instrumental Analysis, 5.sup.th
Ed., Harcourt Brace College Publishers (1998), Currell, Analytical
Instrumentation: Performance Characteristics and Quality, John
Wiley & Sons, Inc. (2000), Sharma et al., Introduction to
Fluorescence Spectroscopy, John Wiley & Sons, Inc. (1999),
Valeur, Molecular Fluorescence: Principles and Applications, John
Wiley & Sons, Inc. (2002), and Gore, Spectrophotometry and
Spectrofluorimetry: A Practical Approach, 2.sup.nd Ed., Oxford
University Press (2000), which are each incorporated by
reference.
[0068] Non-amplified or amplified nucleic acids can be detected by
any conventional means. For example, in some embodiments, nucleic
acids are detected by hybridization with a detectably labeled probe
and measurement of the resulting hybrids. Illustrative non-limiting
examples of detection methods are described below.
[0069] One illustrative detection method, the Hybridization
Protection Assay (HPA) involves hybridizing a chemiluminescent
oligonucleotide probe (e.g., an acridinium ester-labeled (AE)
probe) to the target sequence, selectively hydrolyzing the
chemiluminescent label present on unhybridized probe, and measuring
the chemiluminescence produced from the remaining probe in a
luminometer. See, e.g., U.S. Pat. No. 5,283,174 and Norman C.
Nelson et al., Nonisotopic Probing, Blotting, and Sequencing, ch.
17 (Larry J. Kricka ed., 2d ed. 1995, each of which is herein
incorporated by reference in its entirety).
[0070] Another illustrative detection method provides for
quantitative evaluation of the amplification process in real-time.
Evaluation of an amplification process in "real-time" involves
determining the amount of amplicon in the reaction mixture either
continuously or periodically during the amplification reaction, and
using the determined values to calculate the amount of target
sequence initially present in the sample. A variety of methods for
determining the amount of initial target sequence present in a
sample based on real-time amplification are well known in the art.
These include methods disclosed in U.S. Pat. Nos. 6,303,305 and
6,541,205, each of which is herein incorporated by reference in its
entirety. Another method for determining the quantity of target
sequence initially present in a sample, but which is not based on a
real-time amplification, is disclosed in U.S. Pat. No. 5,710,029,
herein incorporated by reference in its entirety.
[0071] Amplification products may be detected in real-time through
the use of various self-hybridizing probes, most of which have a
stem-loop structure. Such self-hybridizing probes are labeled so
that they emit differently detectable signals, depending on whether
the probes are in a self-hybridized state or an altered state
through hybridization to a target sequence. By way of non-limiting
example, "molecular torches" are a type of self-hybridizing probe
that includes distinct regions of self-complementarity (referred to
as "the target binding domain" and "the target closing domain")
which are connected by a joining region (e.g., non-nucleotide
linker) and which hybridize to each other under predetermined
hybridization assay conditions. In a preferred embodiment,
molecular torches contain single-stranded base regions in the
target binding domain that are from 1 to about 20 bases in length
and are accessible for hybridization to a target sequence present
in an amplification reaction under strand displacement conditions.
Under strand displacement conditions, hybridization of the two
complementary regions, which may be fully or partially
complementary, of the molecular torch is favored, except in the
presence of the target sequence, which will bind to the
single-stranded region present in the target binding domain and
displace all or a portion of the target closing domain. The target
binding domain and the target closing domain of a molecular torch
include a detectable label or a pair of interacting labels (e.g.,
luminescent/quencher) positioned so that a different signal is
produced when the molecular torch is self-hybridized than when the
molecular torch is hybridized to the target sequence, thereby
permitting detection of probe:target duplexes in a test sample in
the presence of unhybridized molecular torches. Molecular torches
and a variety of types of interacting label pairs are disclosed in
U.S. Pat. No. 6,534,274, herein incorporated by reference in its
entirety.
[0072] Another example of a detection probe having
self-complementarity is a "molecular beacon." Molecular beacons
include nucleic acid molecules having a target complementary
sequence, an affinity pair (or nucleic acid arms) holding the probe
in a closed conformation in the absence of a target sequence
present in an amplification reaction, and a label pair that
interacts when the probe is in a closed conformation. Hybridization
of the target sequence and the target complementary sequence
separates the members of the affinity pair, thereby shifting the
probe to an open conformation. The shift to the open conformation
is detectable due to reduced interaction of the label pair, which
may be, for example, a fluorophore and a quencher (e.g., DABCYL and
EDANS). Molecular beacons are disclosed in U.S. Pat. Nos. 5,925,517
and 6,150,097, herein incorporated by reference in its
entirety.
[0073] Other self-hybridizing probes are well known to those of
ordinary skill in the art. By way of non-limiting example, probe
binding pairs having interacting labels, such as those disclosed in
U.S. Pat. No. 5,928,862 (herein incorporated by reference in its
entirety) might be adapted for use in the present invention.
[0074] In some embodiments, intact molecular ions are generated
from amplicons using one of a variety of ionization techniques to
convert the sample to the gas phase. These ionization methods
include, but are not limited to, electrospray ionization (ESI),
matrix-assisted laser desorption ionization (MALDI) and fast atom
bombardment (FAB). Upon ionization, several peaks are observed from
one sample due to the formation of ions with different charges.
Averaging the multiple readings of molecular mass obtained from a
single mass spectrum affords an estimate of molecular mass of the
bioagent identifying amplicon. Electrospray ionization mass
spectrometry (ESI-MS) is particularly useful for very high
molecular weight polymers such as proteins and nucleic acids having
molecular weights greater than 10 kDa, since it yields a
distribution of multiply-charged molecules of the sample without
causing a significant amount of fragmentation.
[0075] The mass detectors used include, but are not limited to,
Fourier transform ion cyclotron resonance mass spectrometry
(FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic
sector, Q-TOF, and triple quadrupole.
[0076] In some embodiments, assignment of previously unobserved
base compositions (also known as "true unknown base compositions")
to a given phylogeny can be accomplished via the use of pattern
classifier model algorithms. Base compositions, like sequences, may
vary slightly from strain to strain within species, for example. In
some embodiments, the pattern classifier model is the mutational
probability model. In other embodiments, the pattern classifier is
the polytope model. A polytope model is the mutational probability
model that incorporates both the restrictions among strains and
position dependence of a given nucleobase within a triplet. In
certain embodiments, a polytope pattern classifier is used to
classify a test or unknown organism according to its amplicon base
composition.
[0077] In some embodiments, it is possible to manage this diversity
by building "base composition probability clouds" around the
composition constraints for each species. A "pseudo
four-dimensional plot" may be used to visualize the concept of base
composition probability clouds. Optimal primer design typically
involves an optimal choice of bioagent identifying amplicons and
maximizes the separation between the base composition signatures of
individual bioagents. Areas where clouds overlap generally indicate
regions that may result in a misclassification, a problem which is
overcome by a triangulation identification process using bioagent
identifying amplicons not affected by overlap of base composition
probability clouds.
[0078] In some embodiments, base composition probability clouds
provide the means for screening potential primer pairs in order to
avoid potential misclassifications of base compositions. In other
embodiments, base composition probability clouds provide the means
for predicting the identity of an unknown bioagent whose assigned
base composition has not been previously observed and/or indexed in
a bioagent identifying amplicon base composition database due to
evolutionary transitions in its nucleic acid sequence. Thus, in
contrast to probe-based techniques, mass spectrometry determination
of base composition does not require prior knowledge of the
composition or sequence in order to make the measurement.
[0079] Provided herein is bioagent classifying information at a
level sufficient to identify a given bioagent. Furthermore, the
process of determining a previously unknown base composition for a
given bioagent (for example, in a case where sequence information
is unavailable) has utility by providing additional bioagent
indexing information with which to populate base composition
databases. The process of future bioagent identification is thus
improved as additional base composition signature indexes become
available in base composition databases.
[0080] In some embodiments, the identity and quantity of an unknown
bioagent may be determined using the process illustrated in FIG. 3.
Primers (500) and a known quantity of a calibration polynucleotide
(505) are added to a sample containing nucleic acid of an unknown
bioagent. The total nucleic acid in the sample is then subjected to
an amplification reaction (510) to obtain amplicons. The molecular
masses of amplicons are determined (515) from which are obtained
molecular mass and abundance data. The molecular mass of the
bioagent identifying amplicon (520) provides for its identification
(525) and the molecular mass of the calibration amplicon obtained
from the calibration polynucleotide (530) provides for its
quantification (535). The abundance data of the bioagent
identifying amplicon is recorded (540) and the abundance data for
the calibration data is recorded (545), both of which are used in a
calculation (550) which determines the quantity of unknown bioagent
in the sample.
[0081] In certain embodiments, a sample comprising an unknown
bioagent is contacted with a primer pair which amplifies the
nucleic acid from the bioagent, and a known quantity of a
polynucleotide that comprises a calibration sequence. The
amplification reaction then produces two amplicons: a bioagent
identifying amplicon and a calibration amplicon. The bioagent
identifying amplicon and the calibration amplicon are
distinguishable by molecular mass while being amplified at
essentially the same rate. Effecting differential molecular masses
can be accomplished by choosing as a calibration sequence, a
representative bioagent identifying amplicon (from a specific
species of bioagent) and performing, for example, a 2-8 nucleobase
deletion or insertion within the variable region between the two
priming sites. The amplified sample containing the bioagent
identifying amplicon and the calibration amplicon is then subjected
to molecular mass analysis by mass spectrometry, for example. The
resulting molecular mass analysis of the nucleic acid of the
bioagent and of the calibration sequence provides molecular mass
data and abundance data for the nucleic acid of the bioagent and of
the calibration sequence. The molecular mass data obtained for the
nucleic acid of the bioagent enables identification of the unknown
bioagent by base composition analysis. The abundance data enables
calculation of the quantity of the bioagent, based on the knowledge
of the quantity of calibration polynucleotide contacted with the
sample.
[0082] In some embodiments, construction of a standard curve in
which the amount of calibration or calibrant polynucleotide spiked
into the sample is varied provides additional resolution and
improved confidence for the determination of the quantity of
bioagent in the sample. Alternatively, the calibration
polynucleotide can be amplified in its own reaction vessel or
vessels under the same conditions as the bioagent. A standard curve
may be prepared there from, and the relative abundance of the
bioagent determined by methods such as linear regression. In some
embodiments, multiplex amplification is performed where multiple
bioagent identifying amplicons are amplified with multiple primer
pairs which also amplify the corresponding standard calibration
sequences. In this or other embodiments, the standard calibration
sequences are optionally included within a single construct
(preferably a vector) which functions as the calibration
polynucleotide.
[0083] In some embodiments, the calibrant polynucleotide is used as
an internal positive control to confirm that amplification
conditions and subsequent analysis steps are successful in
producing a measurable amplicon. Even in the absence of copies of
the genome of a bioagent, the calibration polynucleotide gives rise
to a calibration amplicon. Failure to produce a measurable
calibration amplicon indicates a failure of amplification or
subsequent analysis step such as amplicon purification or molecular
mass determination. Reaching a conclusion that such failures have
occurred is, in itself, a useful event. In some embodiments, the
calibration sequence is comprised of DNA. In some embodiments, the
calibration sequence is comprised of RNA.
[0084] In some embodiments, a calibration sequence is inserted into
a vector which then functions as the calibration polynucleotide. In
some embodiments, more than one calibration sequence is inserted
into the vector that functions as the calibration polynucleotide.
Such a calibration polynucleotide is herein termed a "combination
calibration polynucleotide." It should be recognized that the
calibration method should not be limited to the embodiments
described herein. The calibration method can be applied for
determination of the quantity of any bioagent identifying amplicon
when an appropriate standard calibrant polynucleotide sequence is
designed and used.
[0085] As mentioned above, the systems of the invention also
typically include controllers that are operably connected to one or
more components (e.g., detectors, databases, thermal modulators,
fluid transfer components, robotic material handling devices, and
the like) of the given system to control operation of the
components. More specifically, controllers are generally included
either as separate or integral system components that are utilized,
e.g., to receive data from detectors (e.g., molecular masses,
etc.), to effect and/or regulate temperature in the containers, or
to effect and/or regulate fluid flow to or from selected
containers. Controllers and/or other system components are
optionally coupled to an appropriately programmed processor,
computer, digital device, information appliance, or other logic
device (e.g., including an analog to digital or digital to analog
converter as needed), which functions to instruct the operation of
these instruments in accordance with preprogrammed or user input
instructions, receive data and information from these instruments,
and interpret, manipulate and report this information to the user.
Suitable controllers are generally known in the art and are
available from various commercial sources.
[0086] Any controller or computer optionally includes a monitor,
which is often a cathode ray tube ("CRT") display, a flat panel
display (e.g., active matrix liquid crystal display or liquid
crystal display), or others. Computer circuitry is often placed in
a box, which includes numerous integrated circuit chips, such as a
microprocessor, memory, interface circuits, and others. The box
also optionally includes a hard disk drive, a floppy disk drive, a
high capacity removable drive such as a writeable CD-ROM, and other
common peripheral elements. Inputting devices such as a keyboard or
mouse optionally provide for input from a user. These components
are illustrated further below.
[0087] The computer typically includes appropriate software for
receiving user instructions, either in the form of user input into
a set of parameter fields, e.g., in a graphic user interface (GUI),
or in the form of preprogrammed instructions, e.g., preprogrammed
for a variety of different specific operations. The software then
converts these instructions to appropriate language for instructing
the operation of one or more controllers to carry out the desired
operation. The computer then receives the data from, e.g.,
sensors/detectors included within the system, and interprets the
data, either provides it in a user understood format, or uses that
data to initiate further controller instructions, in accordance
with the programming.
[0088] FIG. 4 is a schematic showing a representative system that
includes a logic device in which various aspects of the present
invention may be embodied. As will be understood by practitioners
in the art from the teachings provided herein, aspects of the
invention are optionally implemented in hardware and/or software.
In some embodiments, different aspects of the invention are
implemented in either client-side logic or server-side logic. As
will be understood in the art, the invention or components thereof
may be embodied in a media program component (e.g., a fixed media
component) containing logic instructions and/or data that, when
loaded into an appropriately configured computing device, cause
that device to perform as desired. As will also be understood in
the art, a fixed media containing logic instructions may be
delivered to a viewer on a fixed media for physically loading into
a viewer's computer or a fixed media containing logic instructions
may reside on a remote server that a viewer accesses through a
communication medium in order to download a program component.
[0089] More specifically, FIG. 4 schematically illustrates computer
1000 to which mass spectrometer 1002 (e.g., an ESI-TOF mass
spectrometer, etc.), fluid transfer component 1004 (e.g., an
automated mass spectrometer sample injection needle or the like),
and database 1008 are operably connected. Optionally, one or more
of these components are operably connected to computer 1000 via a
server (not shown in FIG. 4). During operation, fluid transfer
component 1004 typically transfers reaction mixtures or components
thereof (e.g., aliquots comprising amplicons) from multi-well
container 1006 to mass spectrometer 1002. Mass spectrometer 1002
then detects molecular masses of the amplicons. Computer 1000 then
typically receives this molecular mass data, calculates base
compositions from this data, and compares it with entries in
database 1008 to identify the nucleic acid in a given sample. It
will be apparent to one of skill in the art that one or more
components of the system schematically depicted in FIG. 4 are
optionally fabricated integral with one another (e.g., in the same
housing).
DEFINITIONS
[0090] It is to be understood that the terminology used herein is
for the purpose of describing particular embodiments only, and is
not intended to be limiting. Further, unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention pertains. In describing and claiming the present
invention, the following terminology and grammatical variants will
be used in accordance with the definitions set forth below.
[0091] As used herein, the term "about" means encompassing plus or
minus 10%. For example, about 200 nucleotides refers to a range
encompassing between 180 and 220 nucleotides.
[0092] As used herein, the term "amplicon" or "bioagent identifying
amplicon" refers to a nucleic acid generated using the primer pairs
described herein. The amplicon is typically double stranded DNA;
however, it may be RNA and/or DNA:RNA. In some embodiments, the
amplicon comprises DNA complementary to target RNA, DNA, or cDNA.
In some embodiments, the amplicon comprises sequences of conserved
regions/primer pairs and intervening variable region. As discussed
herein, primer pairs are configured to generate amplicons from
target nucleic acid. As such, the identity or base composition of
any given amplicon may include the primer pair, the complement of
the primer pair, the conserved regions and the variable region from
the bioagent that was amplified to generate the amplicon. One
skilled in the art understands that the incorporation of the
designed primer pair sequences into an amplicon may replace the
native sequences at the primer binding site, and complement
thereof. In certain embodiments, after amplification of the target
region using the primers the resultant amplicons having the primer
sequences are used to generate signal that detects, identifies, or
otherwise analyzes the nucleic acid from the tested sample.
[0093] Amplicons typically comprise from about 45 to about 200
consecutive nucleobases (i.e., from about 45 to about 200 linked
nucleosides), although a wide variety of lengths may be used
depending on the detection and analysis methods desired. One of
ordinary skill in the art will appreciate that this range expressly
embodies compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,
118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,
131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,
144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,
170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,
183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,
196, 197, 198, 199, and 200 nucleobases in length. One of ordinary
skill in the art will further appreciate that the above range is
not an absolute limit to the length of an amplicon, but instead
represents a preferred length range.
[0094] The term "amplifying" or "amplification" in the context of
nucleic acids refers to the production of multiple copies of a
polynucleotide, or a portion of the polynucleotide, typically
starting from a small amount of the polynucleotide (e.g., a single
polynucleotide molecule), where the amplification products or
amplicons are generally detectable. Amplification of
polynucleotides encompasses a variety of chemical and enzymatic
processes. The generation of multiple DNA copies from one or a few
copies of a target or template DNA molecule during a polymerase
chain reaction (PCR) or a ligase chain reaction (LCR) are forms of
amplification. Amplification is not limited to the strict
duplication of the starting molecule. For example, the generation
of multiple cDNA molecules from a limited amount of RNA in a sample
using reverse transcription (RT)-PCR is a form of amplification.
Furthermore, the generation of multiple RNA molecules from a single
DNA molecule during the process of transcription is also a form of
amplification.
[0095] As used herein, "bacterial nucleic acid" includes, but is
not limited to, DNA, RNA, or DNA that has been obtained from
bacterial RNA, such as, for example, by performing a reverse
transcription reaction. Bacterial RNA can either be single-stranded
(of positive or negative polarity) or double-stranded.
[0096] As used herein, the term "base composition" refers to the
number of each residue comprised in an amplicon or other nucleic
acid, without consideration for the linear arrangement of these
residues in the strand(s) of the amplicon. The amplicon residues
comprise, adenosine (A), guanosine (G), cytidine, (C),
(deoxy)thymidine (T), uracil (U), inosine (I), nitroindoles such as
5-nitroindole or 3-nitropyrrole, dP or dK (Hill F et al.,
Polymerase recognition of synthetic oligodeoxyribonucleotides
incorporating degenerate pyrimidine and purine bases. Proc Natl
Acad Sci USA. 1998 Apr. 14; 95(8):4258-63), an acyclic nucleoside
analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides
and Nucleotides, 1995, 14, 1053-1056), the purine analog
1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide,
2,6-diaminopurine, 5-propynyluracil, 5-propynylcytosine,
phenoxazines, including G-clamp, 5-propynyl deoxy-cytidine,
deoxy-thymidine nucleotides, 5-propynylcytidine, 5-propynyluridine
and mass tag modified versions thereof, including
7-deaza-2'-deoxyadenosine-5-triphosphate,
5-iodo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxyuridine-5'-triphosphate,
5-bromo-2'-deoxycytidine-5'-triphosphate,
5-iodo-2'-deoxycytidine-5'-triphosphate,
5-hydroxy-2'-deoxyuridine-5'-triphosphate,
4-thiothymidine-5'-triphosphate,
5-aza-2'-deoxyuridine-5'-triphosphate,
5-fluoro-2'-deoxyuridine-5'-triphosphate,
O6-methyl-2'-deoxyguanosine-5'-triphosphate,
N2-methyl-2'-deoxyguanosine-5'-triphosphate,
8-oxo-2'-deoxyguanosine-5'-triphosphate or
thiothymidine-5'-triphosphate. In some embodiments, the
mass-modified nucleobase comprises .sup.15N or .sup.13C or both
.sup.15N and .sup.13C. In some embodiments, the non-natural
nucleosides used herein include 5-propynyluracil,
5-propynylcytosine and inosine. Herein the base composition for an
unmodified DNA amplicon is notated as A.sub.wG.sub.xC.sub.yT.sub.z,
wherein w, x, y and z are each independently a whole number
representing the number of said nucleoside residues in an amplicon.
Base compositions for amplicons comprising modified nucleosides are
similarly notated to indicate the number of said natural and
modified nucleosides in an amplicon. Base compositions are
calculated from a molecular mass measurement of an amplicon, as
described below. The calculated base composition for any given
amplicon is then compared to a database of base compositions. A
match between the calculated base composition and a single database
entry reveals the identity of the bioagent.
[0097] As used herein, a "base composition probability cloud" is a
representation of the diversity in base composition resulting from
a variation in sequence that occurs among different isolates of a
given species, family or genus. Base composition calculations for a
plurality of amplicons are mapped on a pseudo four-dimensional
plot. Related members in a family, genus or species typically
cluster within this plot, forming a base composition probability
cloud.
[0098] As used herein, the term "base composition signature" refers
to the base composition generated by any one particular
amplicon.
[0099] As used herein, a "bioagent" means any biological organism
or component thereof or a sample containing a biological organism
or component thereof, including microorganisms or infectious
substances, or any naturally occurring, bioengineered or
synthesized component of any such microorganism or infectious
substance or any nucleic acid derived from any such microorganism
or infectious substance. Those of ordinary skill in the art will
understand fully what is meant by the term bioagent given the
instant disclosure. Still, a non-exhaustive list of bioagents
includes: cells, cell lines, human clinical samples, mammalian
blood samples, cell cultures, bacterial cells, viruses, viroids,
fungi, protists, parasites, rickettsiae, protozoa, animals, mammals
or humans. Samples may be alive, non-replicating or dead or in a
vegetative state (for example, vegetative bacteria or spores).
[0100] As used herein, a "bioagent division" is defined as group of
bioagents above the species level and includes but is not limited
to, orders, families, genus, classes, clades, genera or other such
groupings of bioagents above the species level.
[0101] As used herein, "broad range survey primers" are primers
designed to identify an unknown bioagent as a member of a
particular biological division (e.g., an order, family, class,
clade, or genus). However, in some cases the broad range survey
primers are also able to identify unknown bioagents at the species
or sub-species level. As used herein, "division-wide primers" are
primers designed to identify a bioagent at the species level and
"drill-down" primers are primers designed to identify a bioagent at
the sub-species level. As used herein, the "sub-species" level of
identification includes, but is not limited to, strains, subtypes,
variants, and isolates. Drill-down primers are not always required
for identification at the sub-species level because broad range
survey intelligent primers may, in some cases provide sufficient
identification resolution to accomplishing this identification
objective.
[0102] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, the sequence "5'-A-G-T-3'," is complementary to the
sequence "3'-T-C-A-5'." Complementarity may be "partial," in which
only some of the nucleic acids' bases are matched according to the
base pairing rules. Or, there may be "complete" or "total"
complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend
upon
[0103] The term "conserved region" in the context of nucleic acids
refers to a nucleobase sequence (e.g., a subsequence of a nucleic
acid, etc.) that is the same or similar in two or more different
regions or segments of a given nucleic acid molecule (e.g., an
intramolecular conserved region), or that is the same or similar in
two or more different nucleic acid molecules (e.g., an
intermolecular conserved region). To illustrate, a conserved region
may be present in two or more different taxonomic ranks (e.g., two
or more different genera, two or more different species, two or
more different subspecies, and the like) or in two or more
different nucleic acid molecules from the same organism. To further
illustrate, in certain embodiments, nucleic acids comprising at
least one conserved region typically have between about 70%-100%,
between about 80-100%, between about 90-100%, between about
95-100%, or between about 99-100% sequence identity in that
conserved region. A conserved region may also be selected or
identified functionally as a region that permits generation of
amplicons via primer extension through hybridization of a
completely or partially complementary primer to the conserved
region for each of the target sequences to which conserved region
is conserved.
[0104] The term "correlates" refers to establishing a relationship
between two or more things. In certain embodiments, for example,
detected molecular masses of one or more amplicons indicate the
presence or identity of a given bioagent in a sample. In some
embodiments, base compositions are calculated or otherwise
determined from the detected molecular masses of amplicons, which
base compositions indicate the presence or identity of a given
bioagent in a sample.
[0105] As used herein, in some embodiments the term "database" is
used to refer to a collection of base composition molecular mass
data. In other embodiments the term "database" is used to refer to
a collection of base composition data. The base composition data in
the database is indexed to bioagents and to primer pairs. The base
composition data reported in the database comprises the number of
each nucleoside in an amplicon that would be generated for each
bioagent using each primer. The database can be populated by
empirical data. In this aspect of populating the database, a
bioagent is selected and a primer pair is used to generate an
amplicon. The amplicon's molecular mass is determined using a mass
spectrometer and the base composition calculated therefrom without
sequencing i.e., without determining the linear sequence of
nucleobases comprising the amplicon. Note that base composition
entries in the database may be derived from sequencing data (i.e.,
known sequence information), but the base composition of the
amplicon to be identified is determined without sequencing the
amplicon. An entry in the database is made to associate correlate
the base composition with the bioagent and the primer pair used.
The database may also be populated using other databases comprising
bioagent information. For example, using the GenBank database it is
possible to perform electronic PCR using an electronic
representation of a primer pair. This in silico method may provide
the base composition for any or all selected bioagent(s) stored in
the GenBank database. The information may then be used to populate
the base composition database as described above. A base
composition database can be in silico, a written table, a reference
book, a spreadsheet or any form generally amenable to databases.
Preferably, it is in silico on computer readable media.
[0106] The term "detect", "detecting" or "detection" refers to an
act of determining the existence or presence of one or more targets
(e.g., bioagent nucleic acids, amplicons, etc.) in a sample.
[0107] As used herein, the term "etiology" refers to the causes or
origins, of diseases or abnormal physiological conditions.
[0108] As used herein, the term "gene" refers to a nucleic acid
(e.g., DNA) sequence that comprises coding sequences necessary for
the production of a polypeptide, precursor, or RNA (e.g., rRNA,
tRNA). The polypeptide can be encoded by a full length coding
sequence or by any portion of the coding sequence so long as the
desired activity or functional properties (e.g., enzymatic
activity, ligand binding, signal transduction, immunogenicity,
etc.) of the full-length sequence or fragment thereof are retained.
As used herein, the term "heterologous gene" refers to a gene that
is not in its natural environment. For example, a heterologous gene
includes a gene from one species introduced into another species. A
heterologous gene also includes a gene native to an organism that
has been altered in some way (e.g., mutated, added in multiple
copies, linked to non-native regulatory sequences, etc).
Heterologous genes are distinguished from endogenous genes in that
the heterologous gene sequences are typically joined to nucleic
acid sequences that are not found naturally associated with the
gene sequences in the chromosome or are associated with portions of
the chromosome not found in nature (e.g., genes expressed in loci
where the gene is not normally expressed).
[0109] The terms "homology," "homologous" and "sequence identity"
refer to a degree of identity. There may be partial homology or
complete homology. A partially homologous sequence is one that is
less than 100% identical to another sequence. Determination of
sequence identity is described in the following example: a primer
20 nucleobases in length which is otherwise identical to another 20
nucleobase primer but having two non-identical residues has 18 of
20 identical residues (18/20=0.9 or 90% sequence identity). In
another example, a primer 15 nucleobases in length having all
residues identical to a 15 nucleobase segment of a primer 20
nucleobases in length would have 15/20=0.75 or 75% sequence
identity with the 20 nucleobase primer. In context of the present
invention, sequence identity is meant to be properly determined
when the query sequence and the subject sequence are both described
and aligned in the 5' to 3' direction. Sequence alignment
algorithms such as BLAST, will return results in two different
alignment orientations. In the Plus/Plus orientation, both the
query sequence and the subject sequence are aligned in the 5' to 3'
direction. On the other hand, in the Plus/Minus orientation, the
query sequence is in the 5' to 3' direction while the subject
sequence is in the 3' to 5' direction. It should be understood that
with respect to the primers of the present invention, sequence
identity is properly determined when the alignment is designated as
Plus/Plus. Sequence identity may also encompass alternate or
"modified" nucleobases that perform in a functionally similar
manner to the regular nucleobases adenine, thymine, guanine and
cytosine with respect to hybridization and primer extension in
amplification reactions. In a non-limiting example, if the
5-propynyl pyrimidines propyne C and/or propyne T replace one or
more C or T residues in one primer which is otherwise identical to
another primer in sequence and length, the two primers will have
100% sequence identity with each other. In another non-limiting
example, Inosine (I) may be used as a replacement for G or T and
effectively hybridize to C, A or U (uracil). Thus, if inosine
replaces one or more C, A or U residues in one primer which is
otherwise identical to another primer in sequence and length, the
two primers will have 100% sequence identity with each other. Other
such modified or universal bases may exist which would perform in a
functionally similar manner for hybridization and amplification
reactions and will be understood to fall within this definition of
sequence identity.
[0110] As used herein, "housekeeping gene" or "core viral gene"
refers to a gene encoding a protein or RNA involved in basic
functions required for survival and reproduction of a bioagent.
Housekeeping genes include, but are not limited to, genes encoding
RNA or proteins involved in translation, replication, recombination
and repair, transcription, nucleotide metabolism, amino acid
metabolism, lipid metabolism, energy generation, uptake, secretion
and the like.
[0111] As used herein, the term "hybridization" or "hybridize" is
used in reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is influenced by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the melting temperature
(T.sub.m) of the formed hybrid, and the G:C ratio within the
nucleic acids. A single molecule that contains pairing of
complementary nucleic acids within its structure is said to be
"self-hybridized." An extensive guide to nucleic hybridization may
be found in Tijssen, Laboratory Techniques in Biochemistry and
Molecular Biology-Hybridization with Nucleic Acid Probes, part I,
chapter 2, "Overview of principles of hybridization and the
strategy of nucleic acid probe assays," Elsevier (1993), which is
incorporated by reference.
[0112] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, that is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product that is
complementary to a nucleic acid strand is induced (e.g., in the
presence of nucleotides and an inducing agent such as a biocatalyst
(e.g., a DNA polymerase or the like) and at a suitable temperature
and pH). The primer is typically single stranded for maximum
efficiency in amplification, but may alternatively be double
stranded. If double stranded, the primer is generally first treated
to separate its strands before being used to prepare extension
products. In some embodiments, the primer is an
oligodeoxyribonucleotide. The primer is sufficiently long to prime
the synthesis of extension products in the presence of the inducing
agent. The exact lengths of the primers will depend on many
factors, including temperature, source of primer and the use of the
method.
[0113] As used herein, "intelligent primers" or "primers" or
"primer pairs," in some embodiments, are oligonucleotides that are
designed to bind to conserved sequence regions of one or more
bioagent nucleic acids to generate bioagent identifying amplicons.
In some embodiments, the bound primers flank an intervening
variable region between the conserved binding sequences. Upon
amplification, the primer pairs yield amplicons e.g., amplification
products that provide base composition variability between the two
or more bioagents. The variability of the base compositions allows
for the identification of one or more individual bioagents from,
e.g., two or more bioagents based on the base composition
distinctions. In some embodiments, the primer pairs are also
configured to generate amplicons amenable to molecular mass
analysis. Further, the sequences of the primer members of the
primer pairs are not necessarily fully complementary to the
conserved region of the reference bioagent. For example, in some
embodiments, the sequences are designed to be "best fit" amongst a
plurality of bioagents at these conserved binding sequences.
Therefore, the primer members of the primer pairs have substantial
complementarity with the conserved regions of the bioagents,
including the reference bioagent.
[0114] In some embodiments of the invention, the oligonucleotide
primer pairs described herein can be purified. As used herein,
"purified oligonucleotide primer pair," "purified primer pair," or
"purified" means an oligonucleotide primer pair that is
chemically-synthesized to have a specific sequence and a specific
number of linked nucleosides. This term is meant to explicitly
exclude nucleotides that are generated at random to yield a mixture
of several compounds of the same length each with randomly
generated sequence. As used herein, the term "purified" or "to
purify" refers to the removal of one or more components (e.g.,
contaminants) from a sample.
[0115] As used herein, the term "molecular mass" refers to the mass
of a compound as determined using mass spectrometry, for example,
ESI-MS. Herein, the compound is preferably a nucleic acid. In some
embodiments, the nucleic acid is a double stranded nucleic acid
(e.g., a double stranded DNA nucleic acid). In some embodiments,
the nucleic acid is an amplicon. When the nucleic acid is double
stranded the molecular mass is determined for both strands. In one
embodiment, the strands may be separated before introduction into
the mass spectrometer, or the strands may be separated by the mass
spectrometer (for example, electro-spray ionization will separate
the hybridized strands). The molecular mass of each strand is
measured by the mass spectrometer.
[0116] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule, including but not limited to,
DNA or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxyl-methyl) uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methyl-cytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil,
beta-D mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0117] As used herein, the term "nucleobase" is synonymous with
other terms in use in the art including "nucleotide,"
"deoxynucleotide," "nucleotide residue," "deoxynucleotide residue,"
"nucleotide triphosphate (NTP)," or deoxynucleotide triphosphate
(dNTP). As is used herein, a nucleobase includes natural and
modified residues, as described herein.
[0118] An "oligonucleotide" refers to a nucleic acid that includes
at least two nucleic acid monomer units (e.g., nucleotides),
typically more than three monomer units, and more typically greater
than ten monomer units. The exact size of an oligonucleotide
generally depends on various factors, including the ultimate
function or use of the oligonucleotide. To further illustrate,
oligonucleotides are typically less than 200 residues long (e.g.,
between 15 and 100), however, as used herein, the term is also
intended to encompass longer polynucleotide chains.
Oligonucleotides are often referred to by their length. For example
a 24 residue oligonucleotide is referred to as a "24-mer".
Typically, the nucleoside monomers are linked by phosphodiester
bonds or analogs thereof, including phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like, including associated counterions, e.g., H.sup.+,
NH.sub.4.sup.+, Na.sup.+, and the like, if such counterions are
present. Further, oligonucleotides are typically single-stranded.
Oligonucleotides are optionally prepared by any suitable method,
including, but not limited to, isolation of an existing or natural
sequence, DNA replication or amplification, reverse transcription,
cloning and restriction digestion of appropriate sequences, or
direct chemical synthesis by a method such as the phosphotriester
method of Narang et al. (1979) Meth Enzymol. 68:90-99; the
phosphodiester method of Brown et al. (1979) Meth Enzymol.
68:109-151; the diethylphosphoramidite method of Beaucage et al.
(1981) Tetrahedron Lett. 22:1859-1862; the triester method of
Matteucci et al. (1981) J Am Chem Soc 103:3185-3191; automated
synthesis methods; or the solid support method of U.S. Pat. No.
4,458,066, entitled "PROCESS FOR PREPARING POLYNUCLEOTIDES," issued
Jul. 3, 1984 to Caruthers et al., or other methods known to those
skilled in the art. All of these references are incorporated by
reference.
[0119] As used herein a "sample" refers to anything capable of
being analyzed by the methods provided herein. In some embodiments,
the sample comprises or is suspected to comprise one or more
nucleic acids capable of analysis by the methods. In certain
embodiments, for example, the samples comprise nucleic acids (e.g.,
DNA, RNA, cDNAs, etc.) from one or more organisms, tissues, or
cells. Samples can include, for example, blood, semen, saliva,
urine, feces, rectal swabs, and the like. In some embodiments, the
samples are "mixture" samples, which comprise nucleic acids from
more than one subject or individual. In some embodiments, the
methods provided herein comprise purifying the sample or purifying
the nucleic acid(s) from the sample. In some embodiments, the
sample is purified nucleic acid.
[0120] A "sequence" of a biopolymer refers to the order and
identity of monomer units (e.g., nucleotides, etc.) in the
biopolymer. The sequence (e.g., base sequence) of a nucleic acid is
typically read in the 5' to 3' direction.
[0121] As is used herein, the term "single primer pair
identification" means that one or more bioagents can be identified
using a single primer pair. A base composition signature for an
amplicon may singly identify one or more bioagents.
[0122] As used herein, a "sub-species characteristic" is a genetic
characteristic that provides the means to distinguish two members
of the same bioagent species. For example, one bacterial strain may
be distinguished from another bacterial strain of the same species
by possessing a genetic change (e.g., for example, a nucleotide
deletion, addition or substitution) in one of the viral genes, such
as the RNA-dependent RNA polymerase.
[0123] As used herein, in some embodiments the term "substantial
complementarity" means that a primer member of a primer pair
comprises between about 70%-100%, or between about 80-100%, or
between about 90-100%, or between about 95-100%, or between about
99-100% complementarity with the conserved binding sequence of a
nucleic acid from a given bioagent. These ranges of complementarity
and identity are inclusive of all whole or partial numbers embraced
within the recited range numbers. For example, and not limitation,
75.667%, 82%, 91.2435% and 97% complementarity or sequence identity
are all numbers that fall within the above recited range of 70% to
100%, therefore forming a part of this description.
[0124] A "system" in the context of analytical instrumentation
refers a group of objects and/or devices that form a network for
performing a desired objective.
[0125] As used herein, "triangulation identification" means the use
of more than one primer pair to generate a corresponding amplicon
for identification of a bioagent. The more than one primer pair can
be used in individual wells or vessels or in a multiplex PCR assay.
Alternatively, PCR reactions may be carried out in single wells or
vessels comprising a different primer pair in each well or vessel.
Following amplification the amplicons are pooled into a single well
or container which is then subjected to molecular mass analysis.
The combination of pooled amplicons can be chosen such that the
expected ranges of molecular masses of individual amplicons are not
overlapping and thus will not complicate identification of signals.
Triangulation is a process of elimination, wherein a first primer
pair identifies that an unknown bioagent may be one of a group of
bioagents. Subsequent primer pairs are used in triangulation
identification to further refine the identity of the bioagent
amongst the subset of possibilities generated with the earlier
primer pair. Triangulation identification is complete when the
identity of the bioagent is determined. The triangulation
identification process may also be used to reduce false negative
and false positive signals, and enable reconstruction of the origin
of hybrid or otherwise engineered bioagents. For example,
identification of the three part toxin genes typical of B.
anthracis (Bowen et al., J Appl Microbiol, 1999, 87, 270-278) in
the absence of the expected compositions from the B. anthracis
genome would suggest a genetic engineering event.
[0126] As used herein, the term "unknown bioagent" can mean, for
example: (i) a bioagent whose existence is not known (for example,
the SARS coronavirus was unknown prior to April 2003) and/or (ii) a
bioagent whose existence is known (such as the well known bacterial
species Staphylococcus aureus for example) but which is not known
to be in a sample to be analyzed. For example, if the method for
identification of coronaviruses disclosed in commonly owned U.S.
patent Ser. No. 10/829,826 (incorporated herein by reference in its
entirety) was to be employed prior to April 2003 to identify the
SARS coronavirus in a clinical sample, both meanings of "unknown"
bioagent are applicable since the SARS coronavirus was unknown to
science prior to April, 2003 and since it was not known what
bioagent (in this case a coronavirus) was present in the sample. On
the other hand, if the method of U.S. patent Ser. No. 10/829,826
was to be employed subsequent to April 2003 to identify the SARS
coronavirus in a clinical sample, the second meaning (ii) of
"unknown" bioagent would apply because the SARS coronavirus became
known to science subsequent to April 2003 because it was not known
what bioagent was present in the sample.
[0127] As used herein, the term "variable region" is used to
describe a region that falls between any one primer pair described
herein. The region possesses distinct base compositions between at
least two bioagents, such that at least one bioagent can be
identified at, for example, the family, genus, species or
sub-species level. The degree of variability between the at least
two bioagents need only be sufficient to allow for identification
using mass spectrometry analysis, as described herein.
[0128] As used herein, a "wobble base" is a variation in a codon
found at the third nucleotide position of a DNA triplet. Variations
in conserved regions of sequence are often found at the third
nucleotide position due to redundancy in the amino acid code.
[0129] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference
(including, but not limited to, journal articles, U.S. and non-U.S.
patents, patent application publications, international patent
application publications, gene bank accession numbers, internet web
sites, and the like) cited in the present application is
incorporated herein by reference in its entirety.
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