U.S. patent application number 14/797895 was filed with the patent office on 2015-10-29 for non-mass determined base compositions for nucleic acid detection.
The applicant listed for this patent is IBIS BIOSCIENCES, INC.. Invention is credited to Christopher C. Sappenfield.
Application Number | 20150307935 14/797895 |
Document ID | / |
Family ID | 43544967 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150307935 |
Kind Code |
A1 |
Sappenfield; Christopher
C. |
October 29, 2015 |
NON-MASS DETERMINED BASE COMPOSITIONS FOR NUCLEIC ACID
DETECTION
Abstract
The present invention provides systems, methods, and
compositions for nucleic acid detection based on non-mass
determined base compositions. For example, in certain embodiments,
base count data for a template nucleic acid is generated using an
approach that does not measure molecular mass of the template
nucleic acid (e.g., by sequencing the template nucleic acid) and a
database comprising base count entries is queried to identify the
target nucleic acid. In particular embodiments, sequencing is
employed which is conducted in substantially real-time.
Inventors: |
Sappenfield; Christopher C.;
(Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IBIS BIOSCIENCES, INC. |
Carlsbad |
CA |
US |
|
|
Family ID: |
43544967 |
Appl. No.: |
14/797895 |
Filed: |
July 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13389139 |
Apr 4, 2012 |
9080209 |
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PCT/US2010/044774 |
Aug 6, 2010 |
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14797895 |
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61231907 |
Aug 6, 2009 |
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Current U.S.
Class: |
506/2 ;
435/6.12 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6869 20130101; C12Q 2535/101 20130101; C12Q 1/6869 20130101;
C12Q 2563/107 20130101; C12Q 2565/625 20130101; C12Q 2535/122
20130101; C12Q 2565/631 20130101; C12Q 2565/301 20130101; C12Q
2565/518 20130101; C12Q 2537/165 20130101; C12Q 2521/101 20130101;
C12Q 2563/125 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of identifying a template nucleic acid, the method
comprising: (a) determining at least a partial base count of at
least a subsequence of at least one template nucleic acid and/or a
complement thereof, using an approach that does not measure
molecular mass of the template nucleic acid, to produce base count
data; and (b) querying a database comprising at least one base
count entry corresponding to an identified nucleic acid to produce
a match of the base count data with the base count entry, thereby
identifying the template nucleic acid.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S. patent
application Ser. No. 13/389,139 filed Apr. 4, 2012, which is a
national phase application under 35 U.S.C. .sctn.371 of PCT
International Application No. PCT/US2010/044774, filed on 6 Aug.
2010, which claims priority to U.S. Provisional Patent Application
Ser. No. 61/231,907 filed Aug. 6, 2009, each of which are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to systems, methods, and
compositions for nucleic acid detection based on non-mass
determined base compositions. For example, in certain embodiments,
base count data for a template nucleic acid is generated using an
approach that does not measure molecular mass of the template
nucleic acid (e.g., by sequencing the template nucleic acid) and a
database comprising base count entries is queried to identify the
target nucleic acid. In particular embodiments, sequencing is
employed which is conducted in substantially real-time.
BACKGROUND OF THE INVENTION
[0003] In the United States, hospitals report well over 5 million
cases of recognized infectious disease-related illnesses annually.
Significantly greater numbers remain undetected, both in the
inpatient and community setting, resulting in substantial morbidity
and mortality. Critical intervention for infectious disease relies
on rapid, sensitive and specific detection of the offending
pathogen, and is central to the mission of microbiology
laboratories at medical centers. Unfortunately, despite the
recognition that outcomes from infectious illnesses are directly
associated with time to pathogen recognition, as well as accurate
identification of the class and species of microbe, and ability to
identify the presence of drug resistance isolates, conventional
hospital laboratories often remain encumbered by traditional slow
multi-step culture based assays. Other limitations of the
conventional laboratory which have become increasingly apparent
include: extremely prolonged wait-times for pathogens with long
generation time (up to several weeks); requirements for additional
testing and wait times for speciation and identification of
antimicrobial resistance; diminished test sensitivity for patients
who have received antibiotics; and absolute inability to culture
certain pathogens in disease states associated with microbial
infection.
[0004] For more than a decade, molecular testing has been heralded
as the diagnostic tool for the new millennium, whose ultimate
potential could include forced obsolescence of traditional hospital
laboratories. However, despite the fact that significant advances
in clinical application of nucleic acid amplification techniques
have occurred, the practicing physician still relies principally on
standard techniques, such as culturing. As such, what is needed are
rapid sensitive diagnostics systems and methods.
SUMMARY OF THE INVENTION
[0005] The present invention provides systems, methods, and
compositions for nucleic acid detection based on non-mass
determined base compositions. For example, in certain embodiments,
base count data for a template nucleic acid is generated using an
approach that does not measure molecular mass of the template
nucleic acid (e.g., by sequencing the template nucleic acid) and a
database comprising base count entries is queried to identify the
target nucleic acid. Essentially any sequencing approach is
optionally utilized in the present invention, including, for
example, pyrosequencing, zero-mode waveguide type sequencing,
nanopore sequencing, and the like. In particular embodiments,
sequencing is employed which is conducted in substantially
real-time.
[0006] In some embodiments, the present invention provides methods
of identifying a template nucleic acid comprising: (a) determining
at least a partial base count (e.g., a partial base count or a full
base count) of at least a subsequence (e.g., a sub-sequence or a
full sequence) of at least one template nucleic acid and/or a
complement thereof, using an approach that does not measure
molecular mass (e.g., without using mass spectrometry type methods)
of the template nucleic acid, to produce base count data; and (b)
querying a database comprising at least one base count entry (e.g.,
comprising 1 . . . 5 . . . 10 . . . 100 . . . 1000 . . . 10,000 . .
. 50,000 . . . or more base count entries) corresponding to an
identified nucleic acid to produce a match of the base count data
with the base count entry, thereby identifying the template nucleic
acid.
[0007] In certain embodiments, the base count or partial base count
is determined by sequencing. In other embodiments, the base count
or partial base count is determined by liquid chromatography, such
as High Performance Liquid Chromatography (HPLC). In further
embodiments, the base count or partial base count is determined by
paper chromatography (e.g., 2-D paper chromatography).
[0008] In particular embodiments, at least a partial base count
only includes the number of A's present in a template nucleic acid
(e.g., A.sub.14). In particular embodiments, at least a partial
base count only includes the number of C's present in a template
nucleic acid (e.g., C.sub.17). In particular embodiments, at least
a partial base count only includes the number of G's present in a
template nucleic acid (e.g., G.sub.10). In particular embodiments,
at least a partial base count only includes the number of T's
present in a template nucleic acid (e.g., T.sub.19). In other
embodiments, at least a partial base count only includes two of the
canonical bases (e.g., A.sub.14, C.sub.17). In further embodiments,
at least a partial base count only includes three of the canonical
bases (e.g., A.sub.14, C.sub.17, G.sub.10). In additional exemplary
embodiments, at least a partial base count includes all four
canonical bases (e.g., A.sub.14, C.sub.17, G.sub.10, T.sub.19).
[0009] In certain embodiments, the template nucleic acid comprises
DNA or RNA. In other embodiments, the determining is performed in
real-time or substantially real-time (e.g., using a device
comprising a zero-mode waveguide). In particular embodiments, the
template nucleic acid comprises a mammalian nucleic acid, a
bacterial nucleic acid, a viral nucleic acid, a fungal nucleic
acid, or a protozoal nucleic acid. In further embodiments, the
template nucleic acid is attached to a solid support. In additional
embodiments, the methods further comprise amplifying the template
nucleic acid prior to or during (a). In certain embodiments, the
method further comprise obtaining the template nucleic acid from
one or more sample sources selected from the group consisting of:
an environmental sample and a sample derived from a subject.
[0010] In particular embodiments, step (a) comprises contacting the
template nucleic acid with at least one nucleotide incorporating
biocatalyst, labeled nucleotides, and at least one primer nucleic
acid that is at least partially complementary to at least a
subsequence of the template nucleic acid, under conditions whereby
the nucleotide incorporating biocatalyst extends the primer nucleic
acid to produce an extended primer nucleic acid by incorporating
the labeled nucleotides at a terminal end of the extended primer
nucleic acid, wherein nucleotides that comprise different
nucleobases comprise different labels, wherein the different labels
produce detectable signals as or after the labeled nucleotides are
incorporated at the terminal end of the extended primer nucleic
acid, which detectable signals identify the labeled nucleotides
incorporated at the terminal end of the extended primer nucleic
acid and/or complementary nucleotides in the template nucleic acid,
and wherein the detectable signals are detected as or after the
labeled nucleotides are incorporated at the terminal end of the
extended primer nucleic acid to thereby determine the base count of
the subsequence of the template nucleic acid and/or the complement
thereof. In other embodiments, the labels comprise different
fluorescent labels and wherein the detectable signals are detected
using a fluorescence microscope. In some embodiments, the at least
one primer nucleic acid is a primer pair, wherein the primer pair
is configured to hybridize with conserved regions of the two or
more different bioagents and flank variable regions of the two or
more different bioagents.
[0011] In particular embodiments, the terminal end of the extended
primer nucleic acid is the 3' terminal end. In further embodiments,
the nucleotide incorporating biocatalyst comprises an enzyme
selected from the group consisting of: a polymerase, a terminal
transferase, a reverse transcriptase, a polynucleotide
phosphorylase, and a telomerase. In additional embodiments, the
nucleotide incorporating biocatalyst comprises one or more
modifications. In some embodiments, the nucleotide incorporating
biocatalyst is an enzyme derived from an organism that is selected
from the group consisting of: Thermus antranikianii, Thermus
aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus
filiformis, Thermus flavus, Thermus igniterrae, Thermus lacteus,
Thermus oshimai, Thermus ruber, Thermus rubens, Thermus
scotoductus, Thermus silvanus, Thermus species Z05, Thermus species
sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga
neapolitana, Thermosipho africanus, Anaerocellum thermophilum,
Bacillus caldotenax, and Bacillus stearothermophilus.
[0012] In certain embodiments, the nucleotide incorporating
biocatalyst comprises a .PHI.29 DNA polymerase. In other
embodiments, a label is attached to one of a heterocyclic base of a
labeled nucleotide, a sugar moiety of a labeled nucleotide, and a
phosphate group of a labeled nucleotide. In further embodiments, a
linker attaches a label to a labeled nucleotide. In particular
embodiments, the extended primer nucleic acid is complementary to a
subsequence of the template nucleic acid. In further embodiments,
the extended primer nucleic acid is complementary to a full-length
sequence of the template nucleic acid. In certain embodiments, the
primer nucleic acid comprises an intelligent primer. In other
embodiments, the label comprises a fluorescent dye, a
non-fluorescent label, a colorimetric label, a chemiluminescent
label, a bioluminescent label, a radioisotope, an antibody, an
antigen, biotin, a hapten, or an enzyme. In other embodiments, the
label is a fluorescent dye selected from the group consisting of: a
rhodamine dye, a fluorescein dye, a halofluorescein dye, a
dichlororhodamine dye, an energy transfer dye, a Lucifer dye,
Oregon Green, and a cyanine dye. In some embodiments, the label is
a fluorescent dye selected from the group consisting of: JOE, VIC,
TET, HEX, PAM, R6G, R110, TAMRA, and ROX. In particular
embodiments, the label is a radioisotope selected from the group
consisting of: .sup.3H, .sup.14C, .sup.22Na, .sup.32P, .sup.33P,
.sup.35S, .sup.42K, .sup.45Ca, .sup.59Fe, .sup.125I, and
.sup.203Hg.
[0013] In certain embodiments, the methods comprise determining at
least partial base counts of subsequences of two or more template
nucleic acids and/or complements thereof, using an approach that
does not measure molecular mass of the template nucleic acids to
produce the base count data. In other embodiments, the methods
comprise querying the database to produce two or more matches of
the base count data with two or more base count entries to thereby
identify the template nucleic acid. In other embodiments,
identifying the template nucleic acid comprises identifying an
organismal source of the template nucleic acid and/or one or more
properties of the template nucleic acid. In some embodiments, the
properties of the template nucleic acid comprise a genotype, a
virulence property, a pathogenic property, and/or a drug resistance
property. In particular embodiments, the determining is performed
in substantially real-time.
[0014] In other embodiments, the organismal source comprises a
mammal, a bacterium, a virus, a fungus, or a protozoan. In further
embodiments, the organismal source is identified at one or more
taxonomic rank levels selected from the group consisting of: a
Domain, a Superphylum, a Superdivision, a Superclass, a Superorder,
a Superfamily, a Superspecies, a Kingdom, a Phylum, a Division, a
Class, a Legion, an Order, a Family, a Tribe, a Genus, a Species, a
Subkingdom, a Subphylum, a Subclass, a Cohort, a Suborder, a
Subfamily, a Subtribe, a Subgenus, a Subspecies, an Infrakingdom, a
Branch, an Infraphylum, an Infraclass, an Infraorder, an Alliance,
an Infraspecies, a Microphylum, a Pan/class, and a Parvorder.
[0015] In some embodiments, the present invention provides systems
comprising: at least one sequencing device; at least one primer
pair configured to hybridize with conserved regions of two or more
different bioagents and flank variable regions of the two or more
different bioagents; and a computer readable medium having one or
more logic instructions for directing a system to: (a) determine at
least a partial base count of at least a subsequence of at least
one template nucleic acid and/or a complement thereof, using an
approach that does not measure molecular mass of the template
nucleic acid, to produce base count data; and (b) query a database
comprising at least one base count entry corresponding to an
identified nucleic acid to produce a match of the base count data
with the base count entry to thereby identifying the template
nucleic acid.
[0016] In certain embodiments, the system comprise instructions for
determining at least a partial base count of at least a subsequence
of at least one template nucleic acid and/or a complement thereof,
in substantially real-time without measuring the molecular mass of
the template nucleic acid to produce base count data using the
zero-mode waveguide and the primer nucleic acid and for identifying
the template nucleic acid using the base count data. In certain
embodiments, the sequencing device comprises a zero-mode waveguide.
In other embodiments, the systems comprise at least one nucleotide
incorporating biocatalyst and/or labeled nucleotides. In further
embodiments, the systems further comprise one or more containers
for packaging the zero-mode waveguide, or the primer pair.
[0017] In certain embodiments, the present invention provides a
computer program product, comprising a computer readable medium
having one or more logic instructions for directing a system to:
(a) determine at least a partial base count of at least a
subsequence of at least one template nucleic acid and/or a
complement thereof (e.g., in substantially real-time), using an
approach that does not measure molecular mass of the template
nucleic acid, to produce base count data; and (b) query a database
comprising at least one base count entry corresponding to an
identified nucleic acid to produce a match of the base count data
with the base count entry to thereby identifying the template
nucleic acid.
[0018] In some embodiments, the present invention provides system
comprising: a reaction vessel or substrate; a detector configured
to detect detectable signals produced in or on the reaction vessel
or substrate, which detectable signals correspond to at least some
nucleobases incorporated into a nucleic acid to generate nucleobase
incorporation data; a database of base count entries indexed to
identified nucleic acids; and a controller configured to correlate
the nucleobase incorporation data with detected base counts and to
query the database for a match between the detected base counts and
the base count entries to thereby identify or detect the nucleic
acids.
[0019] In particular embodiments, the reaction vessel or substrate
comprises at least one zero-mode waveguide. In further embodiments,
the detector comprises a fluorescence microscope. In other
embodiments, the systems comprise at least one material transfer
component that transfers material to and/or from the reaction
vessel or substrate. In other embodiments, the systems comprise at
least one thermal modulator configured to modulate temperature in
the reaction vessel or substrate. In other embodiments, the
reaction vessel or substrate comprises at least one primer nucleic
acid is selected from the primer nucleic acids disclosed in one or
more of the published documents incorporated by reference herein.
In other embodiments, the systems comprise at least one nucleic
acid sample preparation component and/or at least one nucleic acid
amplification component.
[0020] In further embodiments, the present invention provides
methods of identifying a target nucleic acid in a sample
comprising: (a) at least partially sequencing the target nucleic
acid, or amplified sequences thereof, to generate sequence data;
(b) determining at least a partial base count from the sequence
data to generate base count data; and (c) querying a database
comprising at least one base count entry corresponding to the
target nucleic acid to produce a match of the base count data with
the base count entry, thereby identifying the target nucleic
acid.
[0021] In some embodiments, the present invention provides methods
of identifying a target nucleic acid in a sample comprising: (a)
amplifying one or more segments of the nucleic acid in the sample
using at least one purified oligonucleotide primer pair to generate
amplified sequences, wherein the primer pair is configured to
hybridize with conserved regions of two or more different bioagents
and flank variable regions of the two or more different bioagents;
(b) at least partially sequencing the amplified sequences to
generate sequence data; (c) determining at least a partial base
count from the sequence data to generate base count data; and (d)
querying a database comprising at least one base count entry
corresponding to the target nucleic acid to produce a match of the
base count data with the base count entry, thereby identifying the
target nucleic acid.
[0022] In certain embodiments, the molecular mass of the target
sequence or the amplified sequences is not determined in the
identifying the target nucleic acid. In further embodiments, the
sequencing is performed in real-time or substantially real-time. In
other embodiments, the identifying the target nucleic acid is
performed in substantially real-time. In further embodiments, the
at least partially sequencing the target nucleic acid is performed
with a device comprising at least one zero-mode waveguide. In some
embodiments, the primer pair comprises forward and reverse primers
that are about 20 to 35 nucleobases in length.
[0023] In other embodiments, the present invention provides systems
comprising: (a) a sequencing device configured to generate nucleic
acid sequence data corresponding to the nucleic acid sequence of
one or more amplicons produced using at least one purified
oligonucleotide primer pair that comprises forward and reverse
primers, wherein the primer pair comprises nucleic acid sequences
that are substantially complementary to nucleic acid sequences of
two or more different bioagents; and (b) a controller operably
connected to the sequencing device, the controller configured to:
i) determine at least a partial base count from the nucleic acid
sequence data to generate base count data, and ii) query a database
with the base count data, wherein the database comprises at least
one base count entry corresponding to a target nucleic acid.
[0024] In certain embodiments, the database comprises base
composition information (entries) for at least three different
bioagents. In other embodiments, the database comprises base
composition information for at least 2 . . . 10 . . . 50 . . . 100
. . . 1000 . . . 10,000, or 100,000 different bioagents. In some
embodiments, the base composition data comprises at least 10 . . .
50 . . . 100 . . . 500 . . . 1000 . . . 1000 . . . 10,000 . . . or
100,000 unique base compositions. In further embodiments, the
database is stored on a local computer. In particular embodiments,
the database is accessed from a remote computer over a network. In
further embodiments, the base composition information in the
database is associated with bioagent identity. In certain
embodiments, the base composition data in the database is
associated with bioagent geographic origin.
[0025] In some embodiments, the present invention provides methods
of identifying a target nucleic acid in a sample comprising: (a)
amplifying one or more segments of the target nucleic acid in the
sample using at least one purified oligonucleotide primer pair to
generate a sample comprising amplified sequences, wherein said
primer pair comprises a forward primer and a reverse primer and is
configured to hybridize with conserved regions of two or more
different bioagents and flank variable regions of the two or more
different bioagents; (b) treating the sample with reagents such
that the amplified sequences in the sample are digested into
monodeoxynucleosides; (c) quantitating the monodeoxynucleosides
using HPLC, paper chromatography, or similar non-mass spectrometry
based technique such that base count data is generated; and (f)
querying a database comprising at least one base count entry
corresponding to the target nucleic acid to produce a match of the
base count data with said base count entry, thereby identifying the
target nucleic acid. In certain embodiments, the methods further
comprise a step after step (a) but before step (b) of treating the
sample comprising amplified sequences such that either the sense
strands corresponding to the forward primer or said anti-sense
strands corresponding to the reverse primer of the amplified
sequences are removed from the sample in order to generate a
purified sample. In certain embodiments, step (c) employs a
phosphodiesterase (e.g., such as snake venom phosphodiestease
I).
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] 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.
[0027] FIG. 1 shows one embodiment of a bioagent identification
process of the present invention.
[0028] FIG. 2 shows one embodiment of a bioagent identification
process of the present invention.
[0029] FIG. 3 shows one embodiment of a sequencing system composed
of zero-move waveguides.
[0030] FIG. 4 shows a process diagram illustrating one embodiment
of the primer pair selection process.
[0031] FIG. 5 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 bioagent 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.
[0032] FIG. 6 shows a block diagram showing a representative
system.
DETAILED DESCRIPTION OF EMBODIMENTS
[0033] The present invention provides systems, methods, and
compositions for nucleic acid detection based on non-mass
determined base compositions. For example, in certain embodiments,
base count data for a template nucleic acid is generated without
measuring the molecular mass of the template nucleic acid (e.g., by
sequencing the template nucleic acid) and a database comprising
base count entries is queried to identify the target nucleic acid.
In particular embodiments, sequencing is employed which is
conducted in substantially real-time.
[0034] The present invention provides methods that are useful in
detecting and characterizing nucleic acids. The methods typically
involve determining at least partial base counts (i.e., partial or
complete base counts) and/or the sequence of template or target
nucleic acids in real-time without measuring the molecular mass of
the template nucleic acid. Exemplary applications of the method
include diagnosing infectious or genetic diseases, forensics,
genotyping, and companion diagnostics, among many other
applications. In addition, the invention also provides related
zero-mode waveguide (e.g., arrays of zero-mode waveguides), kits,
computer program products and systems.
[0035] The term "partial base composition" or "partial base count"
refers to the number of each residue of at least one nucleobase
type (e.g., a given purine nucleobase type, a given pyrimidine
nucleobase type, a given nucleobase analog type, and/or the like),
but not each residue comprised in an amplicon or other nucleic acid
(e.g., for single or multiple strands of those nucleic acids),
without consideration for the linear arrangement of these residues
in the strand(s) of the amplicon. For example, if a given amplicon
or other nucleic acid includes four nucleobase types (e.g.,
adenosine (A), guanosine (G), cytidine, (C), and (deoxy)thymidine
(T)), a partial base count for that amplicon or other nucleic acid
would include the number of any one of those four nucleobase types
(e.g., [A.sub.w], [G.sub.x], [C.sub.y], or [T.sub.x]), any two of
those four nucleobase types (e.g., [A.sub.wG.sub.x],
[A.sub.wC.sub.y], [A.sub.wT.sub.z], [G.sub.xC.sub.y],
[G.sub.xT.sub.z], or [C.sub.yT.sub.z]), or at most any three of
those four nucleobase types (e.g., [A.sub.wG.sub.xC.sub.y],
[A.sub.wC.sub.yT.sub.z], [A.sub.wG.sub.xT.sub.z], or
[G.sub.xC.sub.yT.sub.z]), in which w, x, y and z are each
independently a whole number representing the number of said
nucleoside residues in that amplicon or other nucleic acid. To
further illustrate, if a nucleic acid has the following
composition: ATTGCCTAAGGTTAACG, then partial base counts for that
nucleic acid include [A.sub.5], [G.sub.4], [C.sub.3], [T.sub.5],
[A.sub.5G.sub.4], [A.sub.5C.sub.3], [A.sub.5T.sub.5],
[G.sub.4C.sub.3], [G.sub.4T.sub.5], [C.sub.3T.sub.5],
[A.sub.5G.sub.4C.sub.3], [A.sub.5C.sub.3T.sub.5],
[A.sub.5G.sub.4T.sub.5], or [G.sub.4C.sub.3T.sub.5].
[0036] FIG. 1 schematically illustrates an exemplary process
according to one embodiment of the invention. As shown, the process
includes obtaining a sample with template nucleic acids. The sample
is prepared, for example, by purifying or at partially isolating
the template nucleic acids. As shown, the template nucleic acids
are amplified, for example, in polymerase chain reactions or other
amplification approaches to produce amplicons. In some embodiments,
template nucleic acids are analyzed directly, for example, without
being amplified. Amplicon base counts and/or sequences are
determined, for example, using an array of zero-mode waveguides and
a fluorescent microscope (and, e.g., primer nucleic acids (e.g.,
intelligent primers), polymerases (e.g., .PHI.29 DNA polymerases,
etc.), phospholinked nucleotides, etc, as also described in, for
example, Korlach et al. (2008) "Selective aluminum passivation for
targeted immobilization of single DNA polymerase molecules in
zero-mode waveguide nanostructures" Proc. Nat'l. Acad. Sci. U.S.A.
105(4):1176-1181, herein incorporated by reference. The determined
amplicon base counts and/or amplicon sequence information is used
to query a base counts and/or sequence database to detect,
identify, characterize, etc. the template nucleic acids. See also,
FIG. 2 for additional exemplary variations.
[0037] Sequencing Technologies
[0038] As described above, embodiments of the present invention
involve determining the base composition, or partial base
composition, or a target sequence without determining the mass of
the target sequence (e.g., without using mass spectrometry related
methods). In certain embodiments, such methods of determining base
compositions employ sequencing methods. The present invention is
not limited by the type of sequencing method employed. Exemplary
sequencing methods are described below.
[0039] Illustrative non-limiting examples of nucleic acid
sequencing techniques include, but are not limited to, chain
terminator (Sanger) sequencing and dye terminator sequencing. Those
of ordinary skill in the art will recognize that because RNA is
less stable in the cell and more prone to nuclease attack
experimentally RNA is usually reverse transcribed to DNA before
sequencing.
[0040] Chain terminator sequencing uses sequence-specific
termination of a DNA synthesis reaction using modified nucleotide
substrates. Extension is initiated at a specific site on the
template DNA by using a short radioactive, or other labeled,
oligonucleotide primer complementary to the template at that
region. The oligonucleotide primer is extended using a DNA
polymerase, standard four deoxynucleotide bases, and a low
concentration of one chain terminating nucleotide, most commonly a
di-deoxynucleotide. This reaction is repeated in four separate
tubes with each of the bases taking turns as the
di-deoxynucleotide. Limited incorporation of the chain terminating
nucleotide by the DNA polymerase results in a series of related DNA
fragments that are terminated only at positions where that
particular di-deoxynucleotide is used. For each reaction tube, the
fragments are size-separated by electrophoresis in a slab
polyacrylamide gel or a capillary tube filled with a viscous
polymer. The sequence is determined by reading which lane produces
a visualized mark from the labeled primer as you scan from the top
of the gel to the bottom.
[0041] Dye terminator sequencing alternatively labels the
terminators. Complete sequencing can be performed in a single
reaction by labeling each of the di-deoxynucleotide
chain-terminators with a separate fluorescent dye, which fluoresces
at a different wavelength.
[0042] A set of methods referred to as "next-generation sequencing"
techniques have emerged as alternatives to Sanger and
dye-terminator sequencing methods (Voelkerding et al., Clinical
Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol.,
7: 287-296; each herein incorporated by reference in their
entirety). Next-generation sequencing (NGS) methods share the
common feature of massively parallel, high-throughput strategies,
with the goal of lower costs in comparison to older sequencing
methods. NGS methods can be broadly divided into those that require
template amplification and those that do not.
Amplification-requiring methods include pyrosequencing
commercialized by Roche as the 454 technology platforms (e.g., GS
20 and GS FLX), the Solexa platform commercialized by Illumina, and
the Supported Oligonucleotide Ligation and Detection (SOLiD)
platform commercialized by Applied Biosystems. Non-amplification
approaches, also known as single-molecule sequencing, are
exemplified by the HeliScope platform commercialized by Helicos
BioSciences, and emerging platforms commercialized by VisiGen,
Oxford Nanopore Technologies Ltd., and Pacific Biosciences,
respectively.
[0043] In pyrosequencing (Voelkerding et al., Clinical Chem., 55:
641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296;
U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; each herein
incorporated by reference in its entirety), template DNA is
fragmented, end-repaired, ligated to adaptors, and clonally
amplified in-situ by capturing single template molecules with beads
bearing oligonucleotides complementary to the adaptors. Each bead
bearing a single template type is compartmentalized into a
water-in-oil microvesicle, and the template is clonally amplified
using a technique referred to as emulsion PCR. The emulsion is
disrupted after amplification and beads are deposited into
individual wells of a picotitre plate functioning as a flow cell
during the sequencing reactions. Ordered, iterative introduction of
each of the four dNTP reagents occurs in the flow cell in the
presence of sequencing enzymes and luminescent reporter such as
luciferase. In the event that an appropriate dNTP is added to the
3' end of the sequencing primer, the resulting production of ATP
causes a burst of luminescence within the well, which is recorded
using a CCD camera. It is possible to achieve read lengths greater
than or equal to 400 bases, and 1.times.10.sup.6 sequence reads can
be achieved, resulting in up to 500 million base pairs (Mb) of
sequence.
[0044] In the Solexa/Illumina platform (Voelkerding et al.,
Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.
Microbiol., 7: 287-296; U.S. Pat. No. 6,833,246; U.S. Pat. No.
7,115,400; U.S. Pat. No. 6,969,488; each herein incorporated by
reference in its entirety), sequencing data are produced in the
form of shorter-length reads. In this method, single-stranded
fragmented DNA is end-repaired to generate 5'-phosphorylated blunt
ends, followed by Klenow-mediated addition of a single A base to
the 3' end of the fragments. A-addition facilitates addition of
T-overhang adaptor oligonucleotides, which are subsequently used to
capture the template-adaptor molecules on the surface of a flow
cell that is studded with oligonucleotide anchors. The anchor is
used as a PCR primer, but because of the length of the template and
its proximity to other nearby anchor oligonucleotides, extension by
PCR results in the "arching over" of the molecule to hybridize with
an adjacent anchor oligonucleotide to form a bridge structure on
the surface of the flow cell. These loops of DNA are denatured and
cleaved. Forward strands are then sequenced with reversible dye
terminators. The sequence of incorporated nucleotides is determined
by detection of post-incorporation fluorescence, with each fluor
and block removed prior to the next cycle of dNTP addition.
Sequence read length ranges from 36 nucleotides to over 50
nucleotides, with overall output exceeding 1 billion nucleotide
pairs per analytical run.
[0045] Sequencing nucleic acid molecules using SOLiD technology
(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et
al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148;
U.S. Pat. No. 6,130,073; each herein incorporated by reference in
their entirety) also involves fragmentation of the template,
ligation to oligonucleotide adaptors, attachment to beads, and
clonal amplification by emulsion PCR. Following this, beads bearing
template are immobilized on a derivatized surface of a glass
flow-cell, and a primer complementary to the adaptor
oligonucleotide is annealed. However, rather than utilizing this
primer for 3' extension, it is instead used to provide a 5'
phosphate group for ligation to interrogation probes containing two
probe-specific bases followed by 6 degenerate bases and one of four
fluorescent labels. In the SOLiD system, interrogation probes have
16 possible combinations of the two bases at the 3' end of each
probe, and one of four fluors at the 5' end. Fluor color and thus
identity of each probe corresponds to specified color-space coding
schemes. Multiple rounds (usually 7) of probe annealing, ligation,
and fluor detection are followed by denaturation, and then a second
round of sequencing using a primer that is offset by one base
relative to the initial primer. In this manner, the template
sequence can be computationally re-constructed, and template bases
are interrogated twice, resulting in increased accuracy. Sequence
read length averages 35 nucleotides, and overall output exceeds 4
billion bases per sequencing run.
[0046] In certain embodiments, nanopore sequencing in employed
(see, e.g., Astier et al., J Am Chem Soc. 2006 Feb. 8;
128(5):1705-10, herein incorporated by reference). The theory
behind nanopore sequencing has to do with what occurs when the
nanopore is immersed in a conducting fluid and a potential
(voltage) is applied across it: under these conditions a slight
electric current due to conduction of ions through the nanopore can
be observed, and the amount of current is exceedingly sensitive to
the size of the nanopore. If DNA molecules pass (or part of the DNA
molecule passes) through the nanopore, this can create a change in
the magnitude of the current through the nanopore, thereby allowing
the sequences of the DNA molecule to be determined.
[0047] HeliScope by Helicos BioSciences (Voelkerding et al.,
Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.
Microbiol., 7: 287-296; U.S. Pat. No. 7,169,560; U.S. Pat. No.
7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat. No. 7,501,245; U.S.
Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Pat. No.
7,501,245; each herein incorporated by reference in their entirety)
is the first commercialized single-molecule sequencing platform.
This method does not require clonal amplification. Template DNA is
fragmented and polyadenylated at the 3' end, with the final
adenosine bearing a fluorescent label. Denatured polyadenylated
template fragments are ligated to poly(dT) oligonucleotides on the
surface of a flow cell. Initial physical locations of captured
template molecules are recorded by a CCD camera, and then label is
cleaved and washed away. Sequencing is achieved by addition of
polymerase and serial addition of fluorescently-labeled dNTP
reagents. Incorporation events result in fluor signal corresponding
to the dNTP, and signal is captured by a CCD camera before each
round of dNTP addition. Sequence read length ranges from 25-50
nucleotides, with overall output exceeding 1 billion nucleotide
pairs per analytical run.
[0048] Another exemplary nucleic acid sequencing approach developed
by Stratos Genomics, Inc. that is also optionally adapted for use
with the present invention involves the use of Xpandomers. This
sequencing process typically includes providing a daughter strand
produced by a template-directed synthesis. The daughter strand
generally includes a plurality of subunits coupled in a sequence
corresponding to a contiguous nucleotide sequence of all or a
portion of a target nucleic acid in which 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 typically includes 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.
Additional details relating to Xpandomer-based approaches are
described in, for example, U.S. Patent Publication No. 20090035777,
entitled "HIGH THROUGHPUT NUCLEIC ACID SEQUENCING BY EXPANSION,"
that was filed Jun. 19, 2008, which is incorporated herein in its
entirety.
[0049] Other emerging single molecule sequencing methods include
real-time sequencing by synthesis using a VisiGen platform
(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; U.S. Pat.
No. 7,329,492; U.S. patent application Ser. No. 11/671,956; U.S.
patent application Ser. No. 11/781,166; each herein incorporated by
reference in their entirety) in which immobilized, primed DNA
template is subjected to strand extension using a
fluorescently-modified polymerase and florescent acceptor
molecules, resulting in detectible fluorescence resonance energy
transfer (FRET) upon nucleotide addition.
[0050] Another real-time single molecule sequencing system
developed by Pacific Biosciences (Voelkerding et al., Clinical
Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol.,
7: 287-296; U.S. Pat. No. 7,170,050; U.S. Pat. No. 7,302,146; U.S.
Pat. No. 7,313,308; U.S. Pat. No. 7,476,503; all of which are
herein incorporated by reference) utilizes reaction wells 50-100 nm
in diameter and encompassing a reaction volume of approximately 20
zeptoliters (10.times.10.sup.-21 L). Sequencing reactions are
performed using immobilized template, modified phi29 DNA
polymerase, and high local concentrations of fluorescently labeled
dNTPs. High local concentrations and continuous reaction conditions
allow incorporation events to be captured in real time by fluor
signal detection using laser excitation, an optical waveguide, and
a CCD camera.
[0051] In certain embodiments, the single molecule real time (SMRT)
DNA sequencing methods using zero-mode waveguides (ZMWs) developed
by Pacific Biosciences, or similar methods, are employed. With this
technology, DNA sequencing is performed on SMRT chips, each
containing thousands of zero-mode waveguides (ZMWs). A ZMW is a
hole, tens of nanometers in diameter, fabricated in a 100 nm metal
film deposited on a silicon dioxide substrate. Each ZMW becomes a
nanophotonic visualization chamber providing a detection volume of
just 20 zeptoliters (10-21 liters). At this volume, the activity of
a single molecule can be detected amongst a background of thousands
of labeled nucleotides.
[0052] The ZMW provides a window for watching DNA polymerase as it
performs sequencing by synthesis. Within each chamber, a single DNA
polymerase molecule is attached to the bottom surface such that it
permanently resides within the detection volume. Phospholinked
nucleotides, each type labeled with a different colored
fluorophore, are then introduced into the reaction solution at high
concentrations which promote enzyme speed, accuracy, and
processivity. Due to the small size of the ZMW, even at these high,
biologically relevant concentrations, the detection volume is
occupied by nucleotides only a small fraction of the time. In
addition, visits to the detection volume are fast, lasting only a
few microseconds, due to the very small distance that diffusion has
to carry the nucleotides. The result is a very low background.
[0053] As the DNA polymerase incorporates complementary
nucleotides, each base is held within the detection volume for tens
of milliseconds, which is orders of magnitude longer than the
amount of time it takes a nucleotide to diffuse in and out of the
detection volume. During this time, the engaged fluorophore emits
fluorescent light whose color corresponds to the base identity.
Then, as part of the natural incorporation cycle, the polymerase
cleaves the bond holding the fluorophore in place and the dye
diffuses out of the detection volume. Following incorporation, the
signal immediately returns to baseline and the process repeats.
[0054] Unhampered and uninterrupted, the DNA polymerase continues
incorporating bases at a speed of tens per second. In this way, a
completely natural long chain of DNA is produced in minutes.
Simultaneous and continuous detection occurs across all of the
thousands of ZMWs on the SMRT chip in real time. This approach has
the capability to produce reads thousands of nucleotides in
length.
[0055] Some aspects the invention are generally directed to optical
detection or monitoring systems (e.g., ZMVs discussed above),
methods enabled by such systems, and components of such systems for
monitoring, in real-time, optical signals that emanate from
multiple discrete sources of those optical signals. In particular,
the optical detection and monitoring systems are generally capable
of monitoring discrete signals from potentially very large numbers
of different signal sources, optionally separating and/or
deconvolving such signals into constituent signal events, and doing
so in real-time, despite that such signals may be changing rapidly,
over time.
[0056] The systems typically include all or a portion of a
collection of different functional elements. These elements include
the multiple discrete sources that include the capability of
generating optical signals. In some aspects, such sources include
chemical, biochemical and/or biological reactants, or mimics of
such reactants that are capable of generating optical signals that
are indicative of their presence, reaction or conversion. While the
sources may be capable of generating optical signals on their own,
in certain cases, a source of excitation radiation is also provided
to excite optical signals, e.g., fluorescence, within the
sources.
[0057] The systems also typically include optical elements that
direct, separate, and/or otherwise alter optical signals from these
sources (as well as excitation radiation directed at such sources),
in order to ultimately derive optimal amounts of information from
such signals when they are ultimately detected. Consequently, the
systems typically include an optical detection system for detecting
the potentially large numbers of signals that were directed from
the sources, and optionally separated and/or otherwise altered by
the optical elements. Signals detected by the optical detection
system are then recorded and processed by appropriate processing
systems and data management processes to provide output of the
system in user ready formats.
[0058] The systems are typically applied in the monitoring of
arrays or collections of spatially discrete chemical, biochemical
and/or biological reactions that generate optically detectable
signals, such as chromogenic reactions, luminescent or luminogenic
reactions, or fluorescent or fluorogenic reactions. A few examples
of reactions include those that are regularly performed in the
pharmaceutical, biotechnology and healthcare diagnostic fields,
i.e., base count analyses, immunoassays, enzymatic assays, receptor
assays, nucleic acid hybridization assays, nucleic acid synthesis
reactions, cellular assays, and many others.
[0059] Typically, the progress of the reactions used in application
of the systems described herein result in one or more of the
consumption, production and/or conversion of a material that is
capable of generating an optically detectable signal, either alone,
or in response to an external stimulus, e.g., excitation radiation.
By way of example, certain reactants may become fluorescent upon
reaction with another reactant, or may have their fluorescence
altered or reduced upon such reaction. As such, the fluorescence
emitted from the reaction in response to an excitation radiation
will change as the reaction progresses. The systems generally
provide for the source of such signals, e.g., the area in which the
reaction occurs, including optionally, the reactants and/or
products, the optical elements for collecting, directing and
optionally separating and/or altering such signals from such
sources, and the ultimate detection of such signals, as well as the
manipulation of the resulting data to yield optimal value and
information for the user.
[0060] The systems typically include all or a subset of a substrate
that includes all or a subset of the sources of optical signals, an
optional excitation light source, an optical train that includes
the various optical elements for collection, direction and/or
manipulation of the optical signals and optional excitation light,
optical detectors for receiving, detecting and recording (or
putting into a form for recordation) the optical signals, as well
as processors for processing data derived from the optical
detectors.
[0061] A general schematic representation of the system as set
forth above, is illustrated in FIG. 3. As shown, the system 100
includes a substrate 102 that includes a plurality of discrete
sources of optical signals, e.g., reaction wells or optical
confinements 104. An excitation light source, e.g., laser 106, is
optionally provided in the system and is positioned to direct
excitation radiation at the various signal sources. This is
typically done by directing excitation radiation at or through
appropriate optical components, e.g., dichroic 108 and objective
lens 110, that direct the excitation radiation at the substrate
102, and particularly the signal sources 104. Emitted signals from
source 104 are then collected by the optical components, e.g.,
objective 110, and passed through additional optical elements,
e.g., dichroic 108, prism 12 and lens 114, until they are directed
to and impinge upon an optical detection system, e.g., detector
array 116. The signals are then detected by detector array 116, and
the data from that detection is transmitted to an appropriate data
processing unit, e.g., computer 118, where the data is subjected to
interpretation, analysis, and ultimately presented in a user ready
format, e.g., on display 120, or printout 122, from printer
124.
[0062] Processes and systems for such real time sequencing that may
be adapted for use with the invention are described in, for
example, U.S. Pat. No. 7,405,281, entitled "Fluorescent nucleotide
analogs and uses therefor", issued Jul. 29, 2008 to Xu et al., U.S.
Pat. No. 7,315,019, entitled "Arrays of" optical confinements and
uses thereof, issued Jan. 1, 2008 to Turner et al., U.S. Pat. No.
7,313,308, entitled "Optical analysis of molecules", issued Dec.
25, 2007 to Turner et al., U.S. Pat. No. 7,302,146, entitled
"Apparatus and method for analysis of molecules", issued Nov. 27,
2007 to Turner et al., and U.S. Pat. No. 7,170,050, entitled
"Apparatus and methods for optical analysis of molecules", issued
Jan. 30, 2007 to Turner et al., U.S. Patent Publications Nos.
20080212960, entitled "Methods and systems for simultaneous
real-time monitoring of optical signals from multiple sources",
filed Oct. 26, 2007 by Lundquist et al., 20080206764, entitled
"Flowcell system for single molecule detection", filed Oct. 26,
2007 by Williams et al., 20080199932, entitled "Active surface
coupled polymerases", filed Oct. 26, 2007 by Hanzel et al.,
20080199874, entitled "CONTROLLABLE STRAND SCISSION OF MINI CIRCLE
DNA", filed Feb. 11, 2008 by Otto et al., 20080176769, entitled
"Articles having localized molecules disposed thereon and methods
of producing same", filed Oct. 26, 2007 by Rank et al.,
20080176316, entitled "Mitigation of photodamage in analytical
reactions", filed Oct. 31, 2007 by Eid et al., 20080176241,
entitled "Mitigation of photodamage in analytical reactions", filed
Oct. 31, 2007 by Eid et al., 20080165346, entitled "Methods and
systems for simultaneous real-time monitoring of optical signals
from multiple sources", filed Oct. 26, 2007 by Lundquist et al.,
20080160531, entitled "Uniform surfaces for hybrid material
substrates and methods for making and using same", filed Oct. 31,
2007 by Korlach, 20080157005, entitled "Methods and systems for
simultaneous real-time monitoring of optical signals from multiple
sources", filed Oct. 26, 2007 by Lundquist et al., 20080153100,
entitled "Articles having localized molecules disposed thereon and
methods of producing same", filed Oct. 31, 2007 by Rank et al.,
20080153095, entitled "CHARGE SWITCH NUCLEOTIDES", filed Oct. 26,
2007 by Williams et al., 20080152281, entitled "Substrates, systems
and methods for analyzing materials", filed Oct. 31, 2007 by
Lundquist et al., 20080152280, entitled "Substrates, systems and
methods for analyzing materials", filed Oct. 31, 2007 by Lundquist
et al., 20080145278, entitled "Uniform surfaces for hybrid material
substrates and methods for making and using same", filed Oct. 31,
2007 by Korlach, 20080128627, entitled "SUBSTRATES, SYSTEMS AND
METHODS FOR ANALYZING MATERIALS", filed Aug. 31, 2007 by Lundquist
et al., 20080108082, entitled "Polymerase enzymes and reagents for
enhanced nucleic acid sequencing", filed Oct. 22, 2007 by Rank et
al., 20080095488, entitled "SUBSTRATES FOR PERFORMING ANALYTICAL
REACTIONS", filed Jun. 11, 2007 by Foquet et al., 20080080059,
entitled "MODULAR OPTICAL COMPONENTS AND SYSTEMS INCORPORATING
SAME", filed Sep. 27, 2007 by Dixon et al., 20080050747, entitled
"Articles having localized molecules disposed thereon and methods
of producing and using same", filed Aug. 14, 2007 by Korlach et
al., 20080032301, entitled "Articles having localized molecules
disposed thereon and methods of producing same", filed Mar. 29,
2007 by Rank et al., 20080030628, entitled "Methods and systems for
simultaneous real-time monitoring of optical signals from multiple
sources", filed Feb. 9, 2007 by Lundquist et al., 20080009007,
entitled "CONTROLLED INITIATION OF PRIMER EXTENSION", filed Jun.
15, 2007 by Lyle et al., 20070238679, entitled "Articles having
localized molecules disposed thereon and methods of producing
same", filed Mar. 30, 2006 by Rank et al., 20070231804, entitled
"Methods, systems and compositions for monitoring enzyme activity
and applications thereof", filed Mar. 31, 2006 by Korlach et al.,
20070206187, entitled "Methods and systems for simultaneous
real-time monitoring of optical signals from multiple sources",
filed Feb. 9, 2007 by Lundquist et al., 20070196846, entitled
"Polymerases for nucleotide analogue incorporation", filed Dec. 21,
2006 by Hanzel et al., 20070188750, entitled "Methods and systems
for simultaneous real-time monitoring of optical signals from
multiple sources", filed Jul. 7, 2006 by Lundquist et al.,
20070161017, entitled "MITIGATION OF PHOTODAMAGE IN ANALYTICAL
REACTIONS", filed Dec. 1, 2006 by Eid et al., 20070141598, entitled
"Nucleotide Compositions and Uses Thereof", filed Nov. 3, 2006 by
Turner et al., 20070134128, entitled "Uniform surfaces for hybrid
material substrate and methods for making and using same", filed
Nov. 27, 2006 by Korlach, 20070128133, entitled "Mitigation of
photodamage in analytical reactions", filed Dec. 2, 2005 by Eid et
al., 20070077564, entitled "Reactive surfaces, substrates and
methods of producing same", filed Sep. 30, 2005 by Roitman et al.,
20070072196, entitled "Fluorescent nucleotide analogs and uses
therefore", filed Sep. 29, 2005 by Xu et al., and 20070036511,
entitled "Methods and systems for monitoring multiple optical
signals from a single source", filed Aug. 11, 2005 by Lundquist et
al., and Korlach et al. (2008) "Selective aluminum passivation for
targeted immobilization of single DNA polymerase molecules in
zero-mode waveguide nanostructures" Proc. Nat'l. Acad. Sci. U.S.A.
105(4): 11761181--all of which are herein incorporated by reference
in their entireties.
DEFINITIONS AND FURTHER DESCRIPTION
[0063] 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.
[0064] 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.
[0065] 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 HPV 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
bioagent nucleic acid. As such, the 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 the base composition data. Bioagent identifying
amplicons generate base compositions that are preferably unique to
the identity of a bioagent.
[0066] 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.
[0067] The term "attached" refers to interactions including, but
not limited to, covalent bonding, ionic bonding, chemisorption,
physisorption, and combinations thereof.
[0068] As used herein, "viral nucleic acid" includes, but is not
limited to, DNA, RNA, or DNA that has been obtained from viral RNA,
such as, for example, by performing a reverse transcription
reaction. Viral RNA can either be single-stranded (of positive or
negative polarity) or double-stranded.
[0069] As used herein, the term "base composition" or "base count"
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.
[0070] 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.
[0071] As used herein, the term "base composition signature" refers
to the base composition generated by any one particular
amplicon.
[0072] 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).
[0073] 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.
[0074] 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,
Glade, 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. Broad range survey primers may be used in the non-mass
determined base compositions methods and systems of the present
invention.
[0075] 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 binding between nucleic acids.
[0076] 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.
[0077] As used herein, in some embodiments the term "database" is
used to refer to a collection of base composition and/or partial
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 at least one type
of nucleoside in an amplicon (e.g., A.sub.17) 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. Note that base composition entries in the
database may be derived from sequencing data (i.e., known sequence
information). 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.
[0078] 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.
[0079] As used herein, the term "etiology" refers to the causes or
origins, of diseases or abnormal physiological conditions.
[0080] Nucleic acids are "extended" or "elongated" when additional
nucleotides (or other analogous molecules) are incorporated into
the nucleic acids. For example, a nucleic acid is optionally
extended by a nucleotide incorporating biocatalyst, such as a
polymerase that typically adds nucleotides at the 3' terminal end
of a nucleic acid.
[0081] An "extended primer nucleic acid" refers to a primer nucleic
acid to which one or more additional nucleotides have been added or
otherwise incorporated (e.g., covalently bonded to).
[0082] 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.
[0083] A "genotype" refers to all or part of the genetic
constitution of a nucleic acid molecule, cell or subject, or group
of nucleic acid molecules, cells or subjects. For example, a
genotype includes the particular mutations and/or alleles (e.g.,
polymorphisms, such as single nucleotide polymorphisms (SNPs) or
the like) present at a given locus or distributed in a genome.
[0084] A "heterocyclic ring" refers to a monocyclic or bicyclic
ring that is either saturated, unsaturated, or aromatic, and which
comprises one or more heteroatoms independently selected from
nitrogen, oxygen and sulfur. A heterocyclic ring may be attached to
the sugar moiety, or analog thereof, of a nucleotide of the
invention via any heteroatom or carbon atom. Exemplary heterocyclic
rings include morpholinyl, pyrrolidinonyl, pyrrolidinyl,
piperidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl,
indolyl, isoindolyl, azaindolyl, pyridyl, quinolinyl,
isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl, pyrazolyl,
imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl,
isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,
cinnolinyl, phthalazinyl, quinazolinyl, and the like.
[0085] A "homocyclic ring" refers to a saturated or unsaturated
(but not aromatic) carbocyclic ring, such as cyclopropane,
cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene,
and the like.
[0086] 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).
[0087] 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.
[0088] 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.
[0089] 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.
[0090] A "label" refers to a moiety attached (covalently or
non-covalently), or capable of being attached, to a molecule, which
moiety provides or is capable of providing information about the
molecule (e.g., descriptive, identifying, etc. information about
the molecule). Exemplary labels include fluorescent labels,
non-fluorescent labels, colorimetric labels, chemiluminescent
labels, bioluminescent labels, radioactive labels, mass-modifying
groups, antibodies, antigens, biotin, haptens, and enzymes
(including, e.g., peroxidase, phosphatase, etc.).
[0091] The term "kit" is used in reference to a combination of
articles that facilitate a process, method, assay, analysis or
manipulation of a sample. Kits can contain instructions describing
how to use the kit (e.g., instructions describing the methods of
the invention), zero-mode waveguides, primer nucleic acids,
nucleotide incorporating biocatalysts, labeled nucleotides,
chemical reagents, as well as any other components.
[0092] A "linker" or "spacer" refers to a chemical moiety that
covalently or non-covalently (e.g., ionically, etc.) attaches a
compound or substituent group to, e.g., a solid support, another
compound or group, or the like. For example, a linker optionally
attaches a label (e.g., a fluorescent dye, a radioisotope, etc.) to
a nucleotide or the like. Linkers are typically bifunctional
chemical moieties and in certain embodiments, they comprise
cleavable attachments, which can be cleaved by, e.g., heat, an
enzyme, a chemical agent, electromagnetic radiation, etc. to
release materials or compounds from, e.g., a solid support, another
compound, etc. A careful choice of linker allows cleavage to be
performed under appropriate conditions compatible with the
stability of the compound and assay method. Generally a linker has
no specific biological activity other than to, e.g., join chemical
species together or to preserve some minimum distance or other
spatial relationship between such species. However, the
constituents of a linker may be selected to influence some property
of the linked chemical species such as three-dimensional
conformation, net charge, hydrophobicity, etc. Additional
description of linker molecules is provided in, e.g., Lyttle et al.
(1996) Nucleic Acids Res. 24(14):2793, Shchepino et al. (2001)
Nucleosides, Nucleotides, & Nucleic Acids 20:369, Doronina et
al (2001) Nucleosides, Nucleotides, & Nucleic Acids 20:1007,
Trawick et al. (2001) Bioconjugate Chem. 12:900, Olejnik et al.
(1998) Methods in Enzymology 291:135, Pljevaljcic et al. (2003) J.
Am. Chem. Soc. 125(12):3486, Ward, et. al., U.S. Pat. No.
4,711,955, Stavrianopoulos, U.S. Pat. No. 4,707,352, and
Stavrianopoulos, U.S. Pat. No. 4,707,440, which are each
incorporated by reference.
[0093] A "mass modifying" group modifies the mass, typically
measured in terms of molecular weight as daltons, of a molecule
that comprises the group. For example, mass modifying groups that
increase the discrimination between at least two nucleic acids with
single base differences in size or sequence can be used to
facilitate sequencing using, e.g., molecular weight
determinations.
[0094] A "mixture" refers to a combination of two or more different
components. A "reaction mixture" refers a mixture that comprises
molecules that can participate in and/or facilitate a given
reaction.
[0095] The term "molecular mass" refers to the mass of a compound
(e.g., a nucleic acid, etc.) as determined, for example, using mass
spectrometry.
[0096] A "modified" enzyme refers to an enzyme comprising a monomer
sequence in which at least one monomer of the sequence differs from
a monomer in a reference sequence, such as a native or wild-type
form of the enzyme or another modified form of the enzyme, e.g.,
when the two sequences are aligned for maximum identity. Exemplary
modifications include monomer insertions, deletions, and
substitutions. The modified enzymes (i.e., protein- or nucleic
acid-based catalysts) of the invention have been or are optionally
created by various diversity generating methods. Although
essentially any method can be used to produce a modified enzyme,
certain exemplary techniques include recombining (e.g., via
recursive recombination, synthetic recombination, or the like) two
or more nucleic acids encoding one or more parental enzymes, or by
mutating one or more nucleic acids that encode enzymes, e.g., using
recursive ensemble mutagenesis, cassette mutagenesis, random
mutagenesis, in vivo mutagenesis, site directed mutagenesis, or the
like. A nucleic acid encoding a parental enzyme typically includes
a gene that, through the mechanisms of transcription and
translation, produces an amino acid sequence corresponding to a
parental enzyme, e.g., a native form of the enzyme. Modified
enzymes also include chimeric enzymes that have identifiable
component sequences (e.g., structural and/or functional domains,
etc.) derived from two or more parents. Also included within the
definition of modified enzymes are those comprising chemical
modifications (e.g., attached substituent groups, altered
substituent groups, etc.) relative to a reference sequence.
[0097] A "moiety" or "group" refers to one of the portions into
which something, such as a molecule, is divided (e.g., a functional
group, substituent group, or the like). For example, a nucleotide
typically comprises a basic group (e.g., adenine, thymine,
cytosine, guanine, uracil, or an analog basic group), a sugar
moiety (e.g., a moiety comprising a sugar ring or an analog
thereof), and one or more phosphate groups.
[0098] As used herein, the term "primer" or "primer nucleic acid"
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.
[0099] 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.
[0100] 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.
[0101] The term "nucleic acid" or "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-N.sup.6-methyladenosine,
aziridinylcytosine, pseudoisocytosine,
5-(carboxyhydroxyl-methyl)-uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,
N.sup.6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methyl-cytosine,
5-methylcytosine, N.sup.6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N-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.
[0102] 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.
[0103] A "nucleoside" refers to a nucleic acid component that
comprises a base or basic group (e.g., comprising at least one
homocyclic ring, at least one heterocyclic ring, at least one aryl
group, and/or the like) covalently linked to a sugar moiety (e.g.,
a ribose sugar, etc.), a derivative of a sugar moiety, or a
functional equivalent of a sugar moiety (e.g., an analog, such as
carbocyclic ring). For example, when a nucleoside includes a sugar
moiety, the base is typically linked to a 1'-position of that sugar
moiety. As described above, a base can be naturally occurring
(e.g., a purine base, such as adenine (A) or guanine (G), a
pyrimidine base, such as thymine (T), cytosine (C), or uracil (U)),
or non-naturally occurring (e.g., a 7-deazapurine base, a
pyrazolo[3,4-d]pyrimidine base, a propynyl-dN base, etc.).
Exemplary nucleosides include ribonucleosides,
deoxyribonucleosides, dideoxyribonucleosides, carbocyclic
nucleosides, etc.).
[0104] A "nucleotide" refers to an ester of a nucleoside, e.g., a
phosphate ester of a nucleoside. For example, a nucleotide can
include 1, 2, 3, or more phosphate groups covalently linked to a 5'
position of a sugar moiety of the nucleoside.
[0105] A "nucleotide incorporating biocatalyst" refers to a
catalyst that catalyzes the incorporation of nucleotides into a
nucleic acid. Nucleotide incorporating biocatalysts are typically
enzymes. An "enzyme" is a protein- and/or nucleic acid-based
catalyst that acts to reduce the activation energy of a chemical
reaction involving other compounds or "substrates." A "nucleotide
incorporating enzyme" refers to an enzyme that catalyzes the
incorporation of nucleotides into a nucleic acid. Exemplary
nucleotide incorporating enzymes include, e.g., DNA polymerases,
RNA polymerases, terminal transferases, reverse transcriptases,
telomerases, polynucleotide phosphorylases, and the like.
[0106] 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',
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.
[0107] 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. Preferably, the
samples comprise nucleic acids (e.g., DNA, RNA, cDNAs, etc.) from
one or more bioagents. Samples can include, for example, blood,
saliva, urine, feces, anorectal swabs, vaginal swabs, cervical
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.
[0108] 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.
[0109] 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.
[0110] A "solid support" refers to a solid material which can be
derivatized with, or otherwise attached to, a chemical moiety, such
as a primer nucleic acid, a template nucleic acid, or the like.
Exemplary solid supports include a zero-mode waveguide array, a
plate, a bead, a microbead, a fiber, a whisker, a comb, a
hybridization chip, a membrane, a single crystal, a ceramic layer,
a self-assembling monolayer, and the like.
[0111] 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 viral strain may be
distinguished from another viral 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.
[0112] A "subsequence" or "fragment" refers to any portion of an
entire nucleic acid sequence.
[0113] 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.
[0114] 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.
[0115] A "template nucleic acid" refers to a nucleic acid to which
a primer nucleic acid can hybridize and be extended. Accordingly,
template nucleic acids include subsequences that are at least
partially complementary to the primer nucleic acids. Template
nucleic acids can be derived from essentially any source. To
illustrate, template nucleic acids are optionally derived or
isolated from, e.g., cultured microorganisms, uncultured
microorganisms, complex biological mixtures, tissues, sera, pooled
sera or tissues, multispecies consortia, ancient, fossilized or
other nonliving biological remains, environmental isolates, soils,
groundwaters, waste facilities, deep-sea environments, or the like.
Further, template nucleic acids optionally include or are derived
from, e.g., individual cDNA molecules, cloned sets of cDNAs, cDNA
libraries, extracted RNAs, natural RNAs, in vitro transcribed RNAs,
characterized or uncharacterized genomic DNAs, cloned genomic DNAs,
genomic DNA libraries, enzymatically fragmented DNAs or RNAs,
chemically fragmented DNAs or RNAs, physically fragmented DNAs or
RNAs, or the like. Template nucleic acids can also be chemically
synthesized using techniques known in the art. In addition,
template nucleic acids optionally correspond to at least a portion
of a gene or are complementary thereto.
[0116] 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 base composition analysis
(e.g., which does not involve molecular mass analysis). The
combination of pooled amplicons can be chosen such that the
expected ranges of base compositions 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] In certain embodiments, provided herein are methods,
compositions, kits, and related systems for the detection and
identification of bioagents (e.g., species of HPV) using bioagent
identifying amplicons. In some embodiments, primers are selected to
hybridize to conserved sequence regions of nucleic acids derived
from a bioagent and which flank variable sequence regions to yield
a bioagent identifying amplicon which can be amplified and which is
amenable to base composition analysis. In some embodiments, the
corresponding base composition of one or more different amplicons
is queried against a database of base compositions indexed to
bioagents and to the primer pair used to generate the amplicon. A
match of the measured base composition to a database entry base
composition associates the sample bioagent to an indexed bioagent
in the database. Thus, the identity of the unknown bioagent is
determined. No prior knowledge of the unknown bioagent is necessary
to make an identification. In some instances, the measured base
composition associates with more than one database entry base
composition. Thus, a second/subsequent primer pair is generally
used to generate an amplicon, and its measured base composition is
similarly compared to the database to determine its identity in
triangulation identification. Furthermore, the methods and other
aspects of the invention can be applied to rapid parallel multiplex
analyses, the results of which can be employed in a triangulation
identification strategy. Thus, in some embodiments, the present
invention provides rapid throughput and does not require nucleic
acid sequencing or knowledge of the linear sequences of nucleobases
of the amplified target sequence for bioagent detection and
identification.
[0121] Methods of employing base compositions, databases containing
base composition entries, and triangulation using primers, are
described in the following patents, patent applications and
scientific publications, all of which are herein incorporated by
reference as if fully set forth herein: U.S. Pat. Nos. 7,108,974;
7,217,510; 7,226,739; 7,255,992; 7,312,036; 7,339,051; US 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; 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; and
WO2007/118222, all of which are herein incorporated by
reference.
[0122] Exemplary base-count related methods and other aspects of
use in the methods, systems, and other aspects of the invention are
also described in, for example, Ecker et al., Ibis T5000: a
universal biosensor approach for microbiology. Nat Rev Microbiol.
2008 Jun. 3.; Ecker et al., The Microbial Rosetta Stone Database: A
compilation of global and emerging infectious microorganisms and
bioterrorist threat agents.; Ecker et al., The Ibis T5000 Universal
Biosensor: An Automated Platform for Pathogen Identification and
Strain Typing.; Ecker et al., The Microbial Rosetta Stone Database:
A common structure for microbial biosecurity threat agents.; 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 NY 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.; Jiang Y, Hofstadler S A. A highly
efficient and automated method of purifying and desalting PCR
products for analysis by electrospray ionization mass spectrometry;
Jiang et al., Mitochondrial DNA mutation detection by electrospray
mass spectrometry; Russell et al., Transmission dynamics and
prospective environmental sampling of adenovirus in a military
recruit setting; Hofstadler et al., Detection of microbial agents
using broad-range PCR with detection by mass spectrometry: The
TIGER concept. Chapter in; Hofstadler et al., Selective ion
filtering by digital thresholding: A method to unwind complex
ESI-mass spectra and eliminate signals from low molecular weight
chemical noise.; Hofstadler et al., TIGER: The Universal
Biosensor.; Van Ert et al., Mass spectrometry provides accurate
characterization of two genetic marker types in Bacillus
anthracis.; Sampath et al., Forum on Microbial Threats: Learning
from SARS: Preparing for the Next Disease Outbreak--Workshop
Summary (ed. Knobler S E, Mahmoud A, Lemon S.) The National
Academies Press, Washington, D.C. 2004. 181-185.
[0123] In some embodiments, amplicons corresponding to bioagent
identifying amplicons are obtained using the polymerase chain
reaction (PCR). Other amplification methods may be used such as
ligase chain reaction (LCR), low-stringency single primer PCR, and
multiple strand displacement amplification (MDA). (Michael, S F.,
Biotechniques (1994), 16:411-412 and Dean et al., Proc. Natl. Acad.
Sci. U.S.A. (2002), 99, 5261-5266).
[0124] One embodiment of a process flow diagram used for primer
selection and validation process is depicted in FIGS. 4 and 5. 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. 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. Preferably, 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).
[0125] 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.
[0126] The primers, in some embodiments, are employed as
compositions for use in methods for identification of bioagents as
follows: a primer pair composition is contacted with nucleic acid
of an unknown isolate suspected of comprising a target bioagent.
The nucleic acid is then amplified by a nucleic acid amplification
technique, such as PCR for example, to obtain an amplicon that
represents a bioagent identifying amplicon. The base composition of
the double-stranded amplicon, or single strand corresponding to
only the forward or reverse primer, is determined by techniques
such as sequencing, HPLC, and paper chromatography (see, e.g.,
Voelkerding et al., Clinical Chem., "Next-generation sequencing:
from basic research to diagnostics," 55: 641-658, 2009; MacLean et
al., Nature Rev. Microbiol., 7: 287-296, and Manderville and
Kropinski, "Approaches to the Compositional Analysis of DNA,"
Methods Mol Biol. 2009; 502:11-7, all of which are herein
incorporated by reference). A measured base composition calculated
therefrom is then compared with a database of base compositions
indexed to primer pairs and to known bioagents. A match between the
base composition of the amplicon and the database base composition
for that indexed primer pair correlates the measured base
composition with an indexed bioagent, thus identifying the unknown
bioagent (e.g. the species of virus). In some embodiments, the
method is repeated using a different primer pair to resolve
possible ambiguities in the identification process or to improve
the confidence level for the identification assignment
(triangulation identification). In some embodiments, for example,
where the unknown is a novel, previously uncharacterized organism,
the base composition from an amplicon generated from the unknown is
matched with one or more best match base compositions from a
database to predict a family, genus, species, sub-type, etc. of the
unknown. Such information may assist further characterization of
the unknown or provide a physician treating a patient infected by
the unknown with a therapeutic agent best calculated to treat the
patient.
[0127] In certain embodiments, the bioagent is detected with the
systems and methods of the present invention in combination with
other bioagents, including viruses, bacteria, fungi, or other
bioagents. In particular embodiments, a panel is employed that
includes a first bioagent and other related or un-related
bioagents. Such panels may be specific for a particular type of
bioagent, or specific for a specific type of test (e.g., for
testing the safety of blood, one may include commonly present viral
pathogens such as HCV, HIV, and bacteria that can be contracted via
a blood transfusion).
[0128] In some embodiments, a bioagent identifying amplicon 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).
[0129] In some embodiments, the oligonucleotide primers are broad
range survey primers which hybridize to conserved regions of
nucleic acid. The broad range primer may identify the unknown
bioagent depending on which bioagent is in the sample. In other
cases, the base composition of an amplicon does not provide
sufficient resolution to identify the unknown bioagent as any one
bioagent at or below the species level. These cases generally
benefit from further analysis of one or more amplicons generated
from at least one additional broad range survey primer pair, or
from at least one additional division-wide primer pair, or from at
least one additional drill-down primer pair. Identification of
sub-species characteristics may be required, for example, to
determine a clinical treatment of patient, or in rapidly responding
to an outbreak of a new species, sub-type, etc. of pathogen to
prevent an epidemic or pandemic.
[0130] One with ordinary skill in the art of design of
amplification 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 in an
amplification reaction. 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). 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).
[0131] 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%.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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).
[0136] 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.
[0137] 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.
[0138] 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.
[0139] In some embodiments, the primers contain mass-modifying
tags. Reducing the total number of possible base compositions of a
nucleic acid of specific molecular weight provides a means of
avoiding a possible source of ambiguity in the determination of
base composition of amplicons.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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 base composition while being amplified at
essentially the same rate. Effecting differential base compositions
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, a calibration sequence with a different base
composition due to base substitutions. The amplified sample
containing the bioagent identifying amplicon and the calibration
amplicon is then subjected to base composition analysis (e.g.,
sequencing, HPLC, paper chromatography, etc.) without determining
the molecular mass of the sequences. The resulting base composition
analysis of the nucleic acid of the bioagent and of the calibration
sequence provides base composition data and abundance data for the
nucleic acid of the bioagent and of the calibration sequence. The
base composition 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.
[0145] 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.
[0146] 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 base
composition 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.
[0147] 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.
[0148] In certain embodiments, primer pairs are configured to
produce bioagent identifying amplicons within more conserved
regions of a bioagent, while others produce bioagent identifying
amplicons within regions that are may evolve more quickly. Primer
pairs that characterize amplicons in a conserved region with low
probability that the region will evolve past the point of primer
recognition are useful, e.g., as a broad range survey-type primer.
Primer pairs that characterize an amplicon corresponding to an
evolving genomic region are useful, e.g., for distinguishing
emerging bioagent strain variants.
[0149] The primer pairs described herein provide reagents, e.g.,
for identifying diseases caused by emerging types of bioagents.
Base composition analysis eliminates the need for prior knowledge
of bioagent sequence to generate hybridization probes. Thus, in
another embodiment, there is provided a method for determining the
etiology of a particular stain when the process of identification
of is carried out in a clinical setting, and even when a new strain
is involved. This is possible because the methods may not be
confounded by naturally occurring evolutionary variations.
[0150] Another embodiment provides a means of tracking the spread
of any species or strain of particular bioagents when a plurality
of samples obtained from different geographical locations are
analyzed by methods described above in an epidemiological setting.
For example, a plurality of samples from a plurality of different
locations may be analyzed with primers which produce bioagent
identifying amplicons, a subset of which identifies a specific
strain. The corresponding locations of the members of the
strain-containing subset indicate the spread of the specific strain
to the corresponding locations.
[0151] Also provided are kits for carrying out the methods
described herein. In some embodiments, the kit may comprise a
sufficient quantity of one or more primer pairs to perform an
amplification reaction on a target polynucleotide from a bioagent
to form a bioagent identifying amplicon. In some embodiments, the
kit may comprise from one to twenty primer pairs, from one to ten
primer pairs, from one to eight pairs, from one to five primer
pairs, from one to three primer pairs, or from one to two primer
pairs.
[0152] In some embodiments, the kit may also comprise a sufficient
quantity of reverse transcriptase, a DNA polymerase, suitable
nucleoside triphosphates (including any of those described above),
a DNA ligase, and/or reaction buffer, or any combination thereof,
for the amplification processes described above. The kit may also
comprise reagents necessary for performing sequencing methods, or
HPLC or paper chromatography (see, e.g., Voelkerding et al.,
Clinical Chem., "Next-generation sequencing: from basic research to
diagnostics," 55: 641-658, 2009; MacLean et al., Nature Rev.
Microbiol., 7: 287-296, and Manderville and Kropinski, "Approaches
to the Compositional Analysis of DNA," Methods Mol Biol. 2009;
502:11-7, all of which are herein incorporated by reference).
[0153] A kit may further include instructions pertinent for the
particular embodiment of the kit, such instructions describing the
primer pairs and amplification conditions for operation of the
method. In some embodiments, the kit further comprises instructions
for analysis, interpretation and dissemination of data acquired by
the kit. In other embodiments, instructions for the operation,
analysis, interpretation and dissemination of the data of the kit
are provided on computer readable media. A kit may also comprise
amplification reaction containers such as microcentrifuge tubes,
microtiter plates, and the like. A kit may also comprise reagents
or other materials for isolating bioagent nucleic acid or bioagent
identifying amplicons from amplification reactions, including, for
example, detergents, solvents, or ion exchange resins which may be
linked to magnetic beads. A kit may also comprise a table of
measured or calculated base compositions of bioagents using the
primer pairs of the kit.
[0154] The invention also provides systems that can be used to
perform various assays relating to bioagent detection or
identification. In certain embodiments, systems include sequencing
devices (or HPLC equipment or paper chromatography equipment)
configured to detect base compositions of amplicons produced using
purified oligonucleotide primer pairs described herein. Other
devices/equipment that are optionally adapted for use in the
systems of the invention are described further below. In some
embodiments, systems also include controllers operably connected to
sequencing devices and/or other system components. In some of these
embodiments, controllers are configured to correlate the base
compositions of the amplicons with bioagents to effect detection or
identification. As described herein, the base compositions
generally correspond to the bioagent species identities. In certain
embodiments, controllers include, or are operably connected to,
databases of known base compositions of amplicons of known species
of bioagents produced with the primer pairs described herein.
Controllers are described further below.
[0155] In certain embodiments, the oligonucleotides are arrayed on
solid supports, whereas in others, they are provided in one or more
containers, e.g., for assays performed in solution. In certain
embodiments, the systems also include at least one detector or
detection component (e.g., a spectrometer) that is configured to
detect detectable signals produced in the container or on the
support. In addition, the systems also optionally include at least
one thermal modulator (e.g., a thermal cycling device) operably
connected to the containers or solid supports to modulate
temperature in the containers or on the solid supports, and/or at
least one fluid transfer component (e.g., an automated pipettor)
that transfers fluid to and/or from the containers or solid
supports, e.g., for performing one or more assays (e.g., nucleic
acid amplification, real-time amplicon detection, etc.) in the
containers or on the solid supports.
[0156] Examples of suitable thermocycling devices that are
optionally utilized are available from many different commercial
suppliers, including Mastercycler.RTM. devices (Eppendorf North
America, Westbury, N.Y., U.S.A.), the COBAS.RTM. AMPLICOR Analyzer
(Roche Molecular Systems, Inc., Pleasanton, Calif., U.S.A.),
MyCycler and iCycler Thermal Cyclers (Bio-Rad Laboratories, Inc.,
Hercules, Calif., U.S.A.), and the SmartCycler System (Cepheid,
Sunnyvale, Calif. U.S.A.), among many others. In other exemplary
embodiments, sample preparation components, nucleic acid
amplification components, and related fluid handling or material
transfer components are integrated with the systems described
herein, e.g., to fully automate a given nucleic acid amplification
and analysis process. Instruments that can be adapted for this
purpose include, for example, the m2000.TM. automated instrument
system (Abbott Laboratories, Abbott Park, Ill., U.S.A.), the
GeneXpert System (Cepheid, Sunnyvale, Calif. U.S.A.), and the
COBAS.RTM. AmpliPrep.RTM. System (Roche Molecular Systems, Inc.,
Pleasanton, Calif., U.S.A.), and the like.
[0157] 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, 6.sup.th
Ed., Brooks Cole (2006), Currell, Analytical
[0158] 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.
[0159] 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 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.
[0160] 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.
[0161] 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.
[0162] FIG. 6 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.
[0163] More specifically, FIG. 6 schematically illustrates computer
1000 to which sequencing device or system 1002 (e.g., SMRT
detection array from Pacific Biosciences), fluid transfer component
1004 (e.g., a 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. 6). During operation, fluid transfer component
1004 typically transfers reaction mixtures or components thereof
(e.g., aliquots comprising amplicons) from multi-well container
1006 to sequencing device. Sequencing device 1002 then detects the
nucleic acid sequence of the amplicons. Computer 1000 then
typically receives this sequence data, calculates base compositions
from this data, and compares it with entries in database 1008 to
identify species or strains of bioagents 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. 6 are optionally
fabricated integral with one another (e.g., in the same
housing).
[0164] While the present invention has been described with
specificity in accordance with certain of its embodiments, the
following examples serve only to illustrate the invention and are
not intended to limit the same. In order that the invention
disclosed herein may be more efficiently understood, examples are
provided below. It should be understood that these examples are for
illustrative purposes only and are not to be construed as limiting
the invention in any manner.
Example 1
Non-Mass Determined Base Composition Analysis for Target Sequence
Detection
[0165] This example illustrates that pathogens can be identified
and distinguished from one another using complete or partial base
composition or count information without the need for mass
determination of bases. For example, Table I shows base composition
(single strand) results for 16S.sub.--1100-1188 (16S rRNA) primer
amplification reactions for different species of bacteria. Such
base composition data can be generated by any type of sequencing
methodology or system (see, e.g., Voelkerding et al., Clinical
Chem., "Next-generation sequencing: from basic research to
diagnostics," 55: 641-658, 2009; and MacLean et al., Nature Rev.
Microbiol., 7: 287-296). Species which are repeated in the table
(e.g., Clostridium botulinum) are different strains which have
different base compositions in the 16S.sub.--1100-1188 region. As
shown in Table I, for example, a complete base composition using
this primer amplification reaction will uniquely identify and
distinguish Mycobacterium avium (i.e.,
A.sub.16G.sub.32C.sub.18T.sub.16) from the other organisms listed
in Table I, as will various partial base compositions using the
same primer amplification reaction (e.g., A.sub.16G.sub.32,
A.sub.16C.sub.18T.sub.16, G.sub.32C.sub.18T.sub.16,
C.sub.18T.sub.16, etc.).
TABLE-US-00001 TABLE I Organism name Base comp. Organism name Base
comp. Mycobacterium A.sub.16G.sub.32C.sub.18T.sub.16 Vibrio
cholerae A.sub.23G.sub.30C.sub.21T.sub.16 avium Streptomyces sp.
A.sub.17G.sub.38C.sub.27T.sub.14 Aeromonas hydrophila
A.sub.23G.sub.31C.sub.21T.sub.15 Ureaplasma
A.sub.18G.sub.30C.sub.17T.sub.17 Aeromonas
A.sub.23G.sub.31C.sub.21T.sub.15 urealyticum salmonicida
Streptomyces sp. A.sub.19G.sub.36C.sub.24T.sub.18 Mycoplasma
genitalium A.sub.24G.sub.19C.sub.12T.sub.18 Mycobacterium
A.sub.20G.sub.32C.sub.22T.sub.16 Clostridium botulinum
A.sub.24G.sub.25C.sub.18T.sub.20 leprae M. tuberculosis
A.sub.20G.sub.33C.sub.21T.sub.16 Bordetella
A.sub.24G.sub.26C.sub.19T.sub.14 bronchiseptica Nocardia
A.sub.20G.sub.33C.sub.21T.sub.16 Francisella tularensis
A.sub.24G.sub.26C.sub.19T.sub.19 asteroides Fusobacterium
A.sub.21G.sub.26C.sub.23T.sub.16 Bacillus anthracis
A.sub.24G.sub.26C.sub.20T.sub.18 necroforum Listeria
A.sub.21G.sub.27C.sub.19T.sub.19 Campylobacter jejuni
A.sub.24G.sub.26C.sub.20T.sub.18 monocytogenes Clostridium
A.sub.21G.sub.27C.sub.19T.sub.21 Staphylococcus aureus
A.sub.24G.sub.26C.sub.20T.sub.18 botulinum Neisseria
A.sub.21G.sub.28C.sub.21T.sub.18 Helicobacter pylori
A.sub.24G.sub.26C.sub.20T.sub.19 gonorrhoeae Bartonella
A.sub.21G.sub.30C.sub.22T.sub.16 Helicobacter pylori
A.sub.24G.sub.26C.sub.21T.sub.18 quintana Enterococcus
A.sub.22G.sub.27C.sub.20T.sub.19 Moraxella catarrhalis
A.sub.24G.sub.26C.sub.23T.sub.16 faecalis Bacillus
A.sub.22G.sub.28C.sub.20T.sub.18 Haemophilus
A.sub.24G.sub.28C.sub.20T.sub.17 megaterium influenzae Rd Bacillus
subtilis A.sub.22G.sub.28C.sub.21T.sub.17 Chlamydia trachomatis
A.sub.24G.sub.28C.sub.21T.sub.16 Pseudomonas
A.sub.22G.sub.29C.sub.23T.sub.15 Chlamydophila
A.sub.24G.sub.28C.sub.21T.sub.16 aeruginosa pneumoniae Legionella
A.sub.22G.sub.32C.sub.20T.sub.16 C. pneumonia Ar39
A.sub.24G.sub.28C.sub.21T.sub.16 pneumophila Mycoplasma
A.sub.23G.sub.20C.sub.14T.sub.16 Pseudomonas putida
A.sub.24G.sub.29C.sub.21T.sub.16 pneumoniae Clostridium
A.sub.23G.sub.26C.sub.20T.sub.19 Proteus vulgaris
A.sub.24G.sub.30C.sub.21T.sub.15 botulinum Enterococcus
A.sub.23G.sub.26C.sub.21T.sub.18 Yersinia pestis
A.sub.24G.sub.30C.sub.21T.sub.15 faecium Acinetobacter
A.sub.23G.sub.26C.sub.21T.sub.19 Yersinia
A.sub.24G.sub.30C.sub.21T.sub.15 calcoaceti pseudotuberculos
Leptospira A.sub.23G.sub.26C.sub.24T.sub.15 Clostridium botulinum
A.sub.25G.sub.24C.sub.18T.sub.21 borgpeterseni Leptospira
A.sub.23G.sub.26C.sub.24T.sub.15 Clostridium tetani
A.sub.25G.sub.25C.sub.16T.sub.20 interrogans Clostridium
A.sub.23G.sub.27C.sub.19T.sub.19 Francisella tularensis
A.sub.25G.sub.25C.sub.19T.sub.19 perfringens Bacillus anthracis
A.sub.23G.sub.27C.sub.20T.sub.18 Acinetobacter
A.sub.25G.sub.26C.sub.20T.sub.19 calcoacetic Bacillus cereus
A.sub.23G.sub.27C.sub.20T.sub.18 Bacteriodes fragilis
A.sub.25G.sub.27C.sub.16T.sub.22 Bacillus
A.sub.23G.sub.27C.sub.20T.sub.18 Chlamydophila psittaci
A.sub.25G.sub.27C.sub.21T.sub.16 thuringensis Acromonas
A.sub.23G.sub.29C.sub.21T.sub.16 Borrelia burgdorferi
A.sub.25G.sub.29C.sub.17T.sub.19 hydrophila Escherichia coli
A.sub.23G.sub.29C.sub.21T.sub.16 Streptbacillus monilifor
A.sub.26G.sub.26C.sub.20T.sub.16 Pseudomonas
A.sub.23G.sub.29C.sub.21T.sub.17 Rickettsia prowazekii
A.sub.26G.sub.28C.sub.18T.sub.18 putida Escherichia coli
A.sub.23G.sub.29C.sub.22T.sub.15 Rickettsia rickettsii
A.sub.26G.sub.28C.sub.20T.sub.16 Shigella
A.sub.23G.sub.29C.sub.22T.sub.15 Mycoplasma mycoides
A.sub.28G.sub.23C.sub.16T.sub.20 dysenteriae
Some of these organisms can be distinguished using multiple
primers. For example, Chlamydia trachomatis can be distinguished
from Chlamydia pneumoniae AR39 and other organisms listed in Table
II using the primer 16S.sub.--971-1062 and the primer 16S 1228-1310
in addition to 16S 1100-1188 based on a complete or partial base
composition for the amplicons.
TABLE-US-00002 TABLE II Organism 16S_971-1062 16S_1228-1310
16S_1100-1188 Acromonas hydrophila A.sub.21G.sub.29C.sub.22T.sub.20
A.sub.22G.sub.27C.sub.21T.sub.13 A.sub.23G.sub.31C.sub.21T.sub.15
Aeromonas salmonicida A.sub.21G.sub.29C.sub.22T.sub.20
A.sub.22G.sub.27C.sub.21T.sub.13 A.sub.23G.sub.31C.sub.21T.sub.15
Bacillus anthracis A.sub.21G.sub.27C.sub.22T.sub.22
A.sub.24G.sub.22C.sub.19T.sub.18 A.sub.23G.sub.27C.sub.20T.sub.18
Bacillus cereus A.sub.22G.sub.27C.sub.21T.sub.22
A.sub.24G.sub.22C.sub.19T.sub.18 A.sub.23G.sub.27C.sub.20T.sub.18
Bacillus thuringensis A.sub.22G.sub.27C.sub.21T.sub.22
A.sub.24G.sub.22C.sub.19T.sub.18 A.sub.23G.sub.27C.sub.20T.sub.18
Chlamydia trachomatis A.sub.22G.sub.26C.sub.20T.sub.23
A.sub.24G.sub.23C.sub.19T.sub.16 A.sub.24G.sub.28C.sub.21T.sub.16
Chlamydia pneumonia A.sub.26G.sub.23C.sub.20T.sub.22
A.sub.26G.sub.22C.sub.16T.sub.18 A.sub.24G.sub.28C.sub.21T.sub.16
Ar39 Leptospira A.sub.22G.sub.26C.sub.20T.sub.21
A.sub.22G.sub.25C.sub.21T.sub.15 A.sub.23G.sub.26C.sub.24T.sub.15
borgpetersenii Leptospira interrogans
A.sub.22G.sub.26C.sub.20T.sub.21 A.sub.22G.sub.25C.sub.21T.sub.15
A.sub.23G.sub.26C.sub.24T.sub.15 Mycoplasma genitalium
A.sub.28G.sub.23C.sub.15T.sub.22 A.sub.30G.sub.18C.sub.15T.sub.19
A.sub.24G.sub.19C.sub.12T.sub.18 Mycoplasma
A.sub.28G.sub.23C.sub.15T.sub.22 A.sub.27G.sub.19C.sub.16T.sub.20
A.sub.23G.sub.20C.sub.14T.sub.16 pneumoniae Escherichia coli
A.sub.22G.sub.28C.sub.20T.sub.22 A.sub.24G.sub.25C.sub.21T.sub.13
A.sub.23G.sub.29C.sub.22T.sub.15 Shigella dysenteriae
A.sub.22G.sub.28C.sub.21T.sub.21 A.sub.24G.sub.25C.sub.21T.sub.13
A.sub.23G.sub.29C.sub.22T.sub.15 Proteus vulgaris
A.sub.23G.sub.26C.sub.22T.sub.21 A.sub.26G.sub.24C.sub.19T.sub.14
A.sub.24G.sub.30C.sub.21T.sub.15 Yersinia pestis
A.sub.24G.sub.25C.sub.21T.sub.22 A.sub.25G.sub.24C.sub.20T.sub.14
A.sub.24G.sub.30C.sub.21T.sub.15 Yersinia
A.sub.24G.sub.25C.sub.21T.sub.22 A.sub.25G.sub.24C.sub.20T.sub.14
A.sub.24G.sub.30C.sub.21T.sub.15 pseudotuberculosis Francisella
tularensis A.sub.20G.sub.25C.sub.21T.sub.23
A.sub.23G.sub.26C.sub.17T.sub.17 A.sub.24G.sub.26C.sub.19T.sub.19
Rickettsia prowazekii A.sub.21G.sub.26C.sub.24T.sub.25
A.sub.24G.sub.23C.sub.16T.sub.19 A.sub.26G.sub.28C.sub.18T.sub.18
Rickettsia rickettsii A.sub.21G.sub.26C.sub.25T.sub.24
A.sub.24G.sub.24C.sub.17T.sub.17
A.sub.26G.sub.28C.sub.20T.sub.16
Example 2
HPLC Base Composition Determination
[0166] Example 1 can be repeated and instead of sequencing
methodologies, HPLC type methodologies can be used to determine
base compositions of the various amplicons. Methods for determining
base compositions with HPLC and UV-vis detection are described in
Manderville and Kropinski, "Approaches to the Compositional
Analysis of DNA," Methods Mol Biol. 2009; 502:11-7, which is herein
incorporated by reference. Briefly, a given PCR primer-pair
generated amplicon can be rendered single-stranded (e.g., by heat)
and then the forward primer strand or reverse primer strand can be
removed from the sample so only the sense strand or anti-sense
strand is present. This can be done, for example, by subjecting the
amplicon containing sample to a solid support that has the forward
primer linked to the solid support. Sense strands can then
hybridize to the solid support, while reverse strands are washed
away. This purified sample can then digested (e.g., to completion)
to generate monodeoxynucleosides (e.g., the sample can be expose to
alkaline phosphatase and a phosphodiesterase, see Huang et al.,
Free Radical Biology & Medicine, 31:1341-1351, 2001, herein
incorporated by reference). This sample may then be filtered to
remove enzymes. This filtered sample is then injected onto a HPLC
column (e.g., C18 column, such as 5 um Agilent ZORBAX Exlipse XDB
C18 column, 4.6 mm.times.150 mm) with 0.1M triethylammonium acetate
(pH 6.5) containing 5% CH3CN (buffer A) and 0.1M triethylammonium
acetate (pH 6.5) containing 65% CH3CN (buffer B) operated at a flow
rate of 1 ml/min and ambient temperature. This system may employ
isocratic elution with 95% buffer A and 5% buffer B. The order of
elution for these conditions is dC, dG, dT, dA. Standards for the
four deoxynucleosides should be used for comparison and to generate
standard curves that can be used for quantification. The amount of
each deoxynucleoside in the single stranded amplicon is determined
by integration to give the area under each peak in the HPLC trace.
These areas must be divided by the following extinction
coefficients at 254 nm to take into account the different
absorptions of the deoxynucleosides at the detection wavelength
(Connolly, 1991, "Oligonucleotides Containing Modified Bases, In F.
Eckstein (Ed.), Oligonucleotides and Analogues A Practical
Approach, Oxford University Press, New York, herein incorporated by
reference). Levels of" the normal nucleosides are quantified using
the standard curves derived from standards. The extinction
coefficients at 254 nm: dC (6.times.10.sup.3), dG
(13.5.times.10.sup.3), T (7.times.10.sup.3), and dA
(14.3.times.10.sup.3). Once the base compositions are determined in
this manner, they can be compared to database entries and unknown
bioagent amplicons can be identified.
[0167] 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.
Sequence CWU 1
1
1117DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1attgcctaag gttaacg 17
* * * * *