U.S. patent application number 15/824885 was filed with the patent office on 2018-03-15 for nanopore-based nucleic acid analysis with mixed fret detection.
This patent application is currently assigned to Quantapore, Inc.. The applicant listed for this patent is Quantapore, Inc.. Invention is credited to Bason E. CLANCY, Paul HARDENBOL, Martin HUBER.
Application Number | 20180073072 15/824885 |
Document ID | / |
Family ID | 51934368 |
Filed Date | 2018-03-15 |
United States Patent
Application |
20180073072 |
Kind Code |
A1 |
HUBER; Martin ; et
al. |
March 15, 2018 |
NANOPORE-BASED NUCLEIC ACID ANALYSIS WITH MIXED FRET DETECTION
Abstract
Various methods, systems and devices for optical detection and
analysis of polymers, such as polynucleotides, using nanopores,
e.g., for determining sequences of nucleic acids, are provided
herein. In certain variations, methods and systems for determining
a nucleotide sequence of a polynucleotide, which include measuring
mixed FRET signals as a polynucleotide translocates through a
nanopore and determining a nucleotide sequence of the
polynucleotide from the mixed FRET signals, are provided.
Inventors: |
HUBER; Martin; (Menlo Park,
CA) ; CLANCY; Bason E.; (Redwood City, CA) ;
HARDENBOL; Paul; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Quantapore, Inc. |
Menlo Park |
CA |
US |
|
|
Assignee: |
Quantapore, Inc.
Menlo Park
CA
|
Family ID: |
51934368 |
Appl. No.: |
15/824885 |
Filed: |
November 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14786518 |
Oct 22, 2015 |
9862997 |
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PCT/US2014/039444 |
May 23, 2014 |
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15824885 |
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61827519 |
May 24, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6869 20130101; C12Q 2565/101 20130101; C12Q 2565/631
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-15. (canceled)
16. A system for determining a nucleotide sequence of a
polynucleotide, the system comprising: a nanopore providing fluid
communication between a first chamber and a second chamber and
through which a polynucleotide can be translocated, the nanopore
being dimensioned so that nucleotides of the polynucleotide pass
through an exit of the nanopore in sequence and whenever
nucleotides of the polynucleotide are labeled with FRET acceptors,
FRET is suppressed between such FRET acceptors inside the nanopore
and a FRET donor outside the nanopore; a polynucleotide with
acceptor-labeled nucleotides wherein different kinds of nucleotides
are labeled with acceptors that generate distinguishable signals; a
FRET donor disposed within a FRET distance of the exit of the
nanopore, such that a plurality of nucleotides pass within a FRET
distance of the FRET donor upon emerging from the exit of the
nanopore so that a mixed FRET signal is generated; a light source
for exciting the FRET donor; and a detector for collecting mixed
FRET signals and separating distinguishable signals from two or
more different acceptors.
17. The system of claim 16, wherein said nanopore is disposed in a
solid phase membrane and wherein said FRET donor is attached to the
solid phase membrane adjacent to said nanopore.
18. The system of claim 16, wherein said nanopore is a protein
nanopore and wherein said FRET donor is attached to the protein
nanopore.
19. The system of claim 16, wherein the polynucleotide is a single
stranded polynucleotide.
20. (canceled)
21. The system of claim 16, wherein at least one of said acceptors
is a fluorescent organic dye.
22. The system of claim 16, wherein said FRET donor is a quantum
dot.
23. The system of claim 16, wherein said step of exciting said FRET
donor includes exposing said FRET donor to an illumination
beam.
24-26. (canceled)
27. The system of claim 16, wherein said nucleotide sequence of
said polynucleotide is determined from said separated
distinguishable signals collected from every nucleotide of said
polynucleotide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/786,518, filed on Oct. 22, 2015, which is a U.S.
national phase under 35 U.S.C. 371 of PCT International Patent
Application No. PCT/US2014/039444, filed on May 23, 2014, which
claims benefit of priority to U.S. Provisional Patent Application
No. 61/827,519 filed on May 24, 2013, the contents of which are
incorporated herein by reference in their entireties.
BACKGROUND
[0002] DNA sequencing technologies developed over the last decade
have revolutionized the biological sciences, e.g. Lerner et al, The
Auk, 127: 4-15 (2010); Metzker, Nature Review Genetics, 11: 31-46
(2010); Holt et al, Genome Research, 18: 839-846 (2008); and have
the potential to revolutionize many aspects of medical practice,
e.g. Voelkerding et al, Clinical Chemistry, 55: 641-658 (2009);
Anderson et al, Genes, 1: 38-69 (2010); Freeman et al, Genome
Research, 19: 1817-1824 (2009); Tucker et al, Am. J. Human Genet.,
85: 142-154 (2009). However, to realize such potential there are
still a host of challenges that must be addressed, including
reduction of per-run sequencing cost, simplification of sample
preparation, reduction of run time, increasing read lengths,
improving data analysis, and the like, e.g. Baker, Nature Methods,
7: 495-498 (2010); Kircher et al, Bioessays, 32: 524-536 (2010);
Turner et al, Annual Review of Genomics and Human Genetics, 10:
263-284 (2009). Single molecule sequencing using nanopores may
address some of these challenges, e.g., Maitra et al,
Electrophoresis, 33: 3418-3428 (2012); Venkatesan et al, Nature
Nanotechnology, 6: 615-624 (2011); however, this approach has its
own set of technical difficulties, such as, reliable nanopore
fabrication, control of DNA translocation rates, nucleotide
discrimination, detection of electrical signals from large arrays
of nanopore sensors, and the like, e.g. Branton et al, Nature
Biotechnology, 26(10): 1146-1153 (2008); Venkatesan et al (cited
above).
[0003] Optical detection of nucleotides has been proposed as a
potential solution to some of the technical difficulties in the
field of nanopore sequencing, e.g. Huber, International patent
publication WO 2011/040996; Russell, U.S. Pat. No. 6,528,258;
Pittaro, U.S. patent publication 2005/0095599; Joyce, U.S. patent
publication 2006/0019259; Chan, U.S. Pat. No. 6,355,420; McNally et
al, Nano Lett., 10(6): 2237-2244 (2010); and the like. However,
optically-based nanopore sequencing has not been realized for a
variety of reasons, including the lack of suitable fabrication
techniques and understanding of how elements of such systems
interact.
[0004] In view of the above, it would be advantageous to nanopore
sensor technology in general and its particular applications, such
as optically based nanopore sequencing, if there were available
materials and configurations of optical elements that permitted
successful optical sensing and analysis of analytes, such as
sequences of nucleic acids.
SUMMARY
[0005] Various methods for optical detection and analysis of
polymers, such as polynucleotides, in microfluidic and/or
nanofluidic devices, such as those using nanopores for determining
sequences of nucleic acids are provided herein.
[0006] In certain variations, a method of determining a nucleotide
sequence of a polynucleotide comprises the following steps: (a)
translocating a polynucleotide, e.g., a single or double stranded
polynucleotide, through a nanopore so that nucleotides of the
polynucleotide pass in sequence by a first member of a FRET pair
positioned adjacent to the nanopore, a plurality of the nucleotides
being within a FRET distance of the first member of the FRET pair
as the nucleotides exit the nanopore and at least a portion of the
nucleotides being labeled with a second member of the FRET pair;
(b) exposing the FRET pairs adjacent to the nanopore to a light
beam so that FRET occurs between the first and a plurality of
second members of the FRET pair within the FRET distance to
generate a mixed FRET signal; (c) measuring mixed FRET signals as
the polynucleotide translocates through the nanopore; and (d)
determining a nucleotide sequence of the polynucleotide from the
mixed FRET signals. In some embodiments, the nanopore is disposed
in a solid phase membrane and the first member of a FRET pair is
attached to the solid phase membrane adjacent to said nanopore. In
other embodiments, the nanopore is a protein nanopore and the first
member of a FRET pair is attached to the protein nanopore.
[0007] In another variation, a method of determining a nucleotide
sequence of a polynucleotide comprises the following steps: (a)
translocating a polynucleotide, e.g., a single or double stranded
polynucleotide, through a nanopore having an exit so that
nucleotides of the polynucleotide pass in sequence through a FRET
zone upon exiting the nanopore, the FRET zone encompassing a
plurality of the nucleotides during such passage and at least a
portion of the nucleotides being labeled with at least one second
member of a FRET pair and at least one first member of the FRET
pair being in the FRET zone; (b) exposing the first and second
members of the FRET pair in the FRET zone to a light beam so that
FRET occurs between first and second members of the FRET pair to
generate a mixed FRET signal; (c) measuring mixed FRET signals as
the polynucleotide moves through the FRET zone; and (d) determining
a nucleotide sequence of the polynucleotide from the mixed FRET
signals.
[0008] In another variation, a method of determining a nucleotide
sequence of a polynucleotide comprises the following steps: (a)
translocating a polynucleotide, e.g., a single or double stranded
polynucleotide, with labeled nucleotides through a nanopore
dimensioned so that labels on the nucleotides are constrained to
suppress FRET reactions, the labels on the nucleotides being second
members of a FRET pair, and so that nucleotides of the
polynucleotide pass in sequence through a FRET zone upon exiting
the nanopore, the FRET zone encompassing a plurality of the
nucleotides during such passage and at least one first member of
the FRET pair being in the FRET zone; (b) exposing the first and
second members of the FRET pair in the FRET zone to a light beam so
that FRET occurs between the first and second members to generate a
mixed FRET signal; (c) measuring mixed FRET signals as the
polynucleotide moves through the FRET zone; and (d) determining a
nucleotide sequence of the polynucleotide from the mixed FRET
signals.
[0009] Various methods, systems and devices are exemplified in a
number of implementations and applications, some of which are
summarized below and throughout the specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A illustrates schematically one embodiment with mixed
FRET signal collection with a polynucleotide analyte labeled with a
single kind of acceptor molecule.
[0011] FIG. 1B shows data of mixed FRET signals of a test
polynucleotide labeled with a single kind acceptor molecule.
[0012] FIG. 1C illustrates schematically another embodiment with
mixed FRET signal collection with a polynucleotide analyte labeled
with two kinds of acceptor molecules.
[0013] FIG. 1D shows data of mixed FRET signals of a test
polynucleotide labeled with two kinds of acceptor molecules.
[0014] FIGS. 2A-2C illustrate one embodiment of a hybrid
biosensor.
[0015] FIG. 2D illustrates an embodiment of a device with
positioning of a member of a FRET pair using oligonucleotide
hybridization.
[0016] FIG. 2E illustrates one embodiment of a hybrid nanopore
where the surface of the solid state membrane (201) is coated with
a hydrophobic layer (202) to which a lipid layer is adhered (203).
The lipids form a gigaohm seal with the inserted pore protein.
DETAILED DESCRIPTION
[0017] While the various methods, systems and devices described
herein are amenable to various modifications and alternative forms,
specifics thereof have been shown by way of example in the drawings
and will be described in detail. It should be understood, however,
that the intention is not to be limited to the particular
embodiments described. On the contrary, the intention is to cover
all modifications, equivalents, and alternatives falling within the
spirit and scope of the invention. For example, particular nanopore
types and numbers, particular labels, FRET pairs, detection
schemes, and fabrication approaches are shown for purposes of
illustration. It should be appreciated, however, that the
disclosure is not intended to be limiting in this respect, as other
types of nanopores, arrays of nanopores, and other fabrication
technologies may be utilized to implement various aspects of the
systems discussed herein. Guidance for certain aspects is found in
many available references and treatises well known to those with
ordinary skill in the art, including, for example, Cao,
Nanostructures & Nanomaterials (Imperial College Press, 2004);
Levinson, Principles of Lithography, Second Edition (SPIE Press,
2005); Doering and Nishi, Editors, Handbook of Semiconductor
Manufacturing Technology, Second Edition (CRC Press, 2007); Sawyer
et al, Electrochemistry for Chemists, 2.sup.nd edition (Wiley
Interscience, 1995); Bard and Faulkner, Electrochemical Methods:
Fundamentals and Applications, 2.sup.nd edition (Wiley, 2000);
Lakowicz, Principles of Fluorescence Spectroscopy, 3.sup.rd edition
(Springer, 2006); Hermanson, Bioconjugate Techniques, Second
Edition (Academic Press, 2008); and the like, which relevant parts
are hereby incorporated by reference.
[0018] Various methods and systems described herein relate to the
use of nanopores and FRET pairs to measure properties of analytes,
such as polymer analytes. A FRET pair generally is one or more FRET
donors and one or more FRET acceptors where each donor is capable
of a FRET reaction with each acceptor. In one aspect, this means
that the donors of the FRET pair have an emission spectrum that
substantially overlaps the absorption spectrum of the acceptors. In
another aspect, the transition dipole of the donor and the acceptor
have to be aligned in a way that allows efficient energy transfer.
Certain variations in part are based on the recognition and
appreciation of the use of FRET pairs under conditions where a
plurality of FRET acceptors generate FRET signals during a
detection event so that mixed FRET signals are collected. In some
aspects, certain variations in part are also based on the discovery
and appreciation of a FRET suppressing property of nanopores and
the application of this property to enable detection of labeled
analytes translocating through a nanopore. It is believed, although
the variations described herein are not intended to be limited
thereby, that a nanopore may be selected with a bore dimensioned so
that a FRET pair label cannot orient to engage in a FRET
interaction while translocating through the nanopore. The dipoles
of the labels of the polynucleotide in the bore of the nanopore are
constrained in their rotational freedom based on the limited
diameter of the nanopore. This reduction in dipole alignment with
the alignment of the corresponding FRET pair attached to the
nanopore limits the FRET efficiency dramatically. Labeled
polynucleotides can engage in a FRET interaction after exiting the
nanopore at which point the FRET acceptor or donor on the analyte
(e.g. polynucleotide) regains rotational freedom which allows for
mixed FRET events.
[0019] A wide range of embodiments are contemplated depending on
the type of analytes being detected, the types of donors and
acceptors employed, the physical arrangement of the nanopore, donor
and acceptors, whether analytes are labeled with donors or with
acceptors, and the like. In one embodiment, analytes measured are
acceptor-labeled polymers, especially acceptor-labeled
polynucleotides. In one species of the latter embodiment, different
nucleotides of a polynucleotide analyte are labeled with one or
more different kinds of acceptors, so that a nucleotide sequence of
the polynucleotide may be determined from measuring mixed FRET
signals generated as it translocates through a nanopore. In another
embodiment, analytes measured are donor-labeled polymers,
especially donor-labeled polynucleotides. The sequence of the
polynucleotide may be determined from measuring mixed FRET signals
as it translocates through a nanopore. In yet another embodiment,
at least one of the four nucleotides of a polynucleotide analyte is
labeled with a member of a FRET pair. The positions of the labeled
nucleotides in the polynucleotide are determined by translocating
the labeled polynucleotide through a labeled nanopore and measuring
FRET events. By labeling the remaining nucleotides of the same
polynucleotide sample and subsequently translocating said samples
through a labeled nanopore, sub-sequences of the polynucleotide are
generated. Such sub-sequences can be re-aligned resulting in a full
sequence of the polynucleotide.
[0020] Some of the above aspects and embodiments are illustrated
diagrammatically in FIG. 1A. Polymer analyte (1000), such as a
polynucleotide, is driven, e.g. electrophoretically, through
nanopore (1002), which constrains the conformation of polymer
(1000) so that its monomeric units translocate through the nanopore
in the same order as their primary sequence in the polymer.
Moreover, as mentioned above, whenever an acceptor-labeled
monomeric unit is within the bore of nanopore (1002), FRET
interactions between such acceptors and the donors of its FRET pair
(e.g. 1012) are suppressed. Such suppression typically means that
no detectable FRET signal is produced even if such acceptors are
within a FRET distance of a donor due to unfavorable orientation of
the acceptor and donor dipoles. On the other hand, as soon as an
acceptor-labeled monomeric unit emerges from the bore of the
nanopore into FRET zone (1008), a strong FRET signal is immediately
produced (due to the proximity of donor (1012)), after which the
signal decreases rapidly as the distance between the acceptor and
donor increases, because translocation of polymer (1000) carries
acceptors out of FRET zone (1008). FRET zone (1008), which is a
spatial region immediately adjacent to exit (1015) of nanopore
(1002), is defined by the FRET distances between donor (1012) and
the acceptor labels attached to polymer (1000) as it translocates
through and away from nanopore (1002). In FIG. 1A, only one type of
monomeric unit, illustrated as solid circles (1004) is labeled; the
rest of the monomeric units, illustrated as speckled circles
(1006), are unlabeled. As illustrated, three labeled monomeric
units (denoted "1", "2" and "3") are in FRET zone (1008). When
donor (1012) is excited by excitation beam (1014), FRET
interactions (1010) are generated and the three acceptors on the
monomeric units produce FRET emissions (1016) that are collected by
detector (1018) and recorded as mixed FRET signal intensity (1029).
Signal intensity contributions from acceptors on monomeric units 1,
2 and 3 are illustrated by curves (1031, 1032 and 1033,
respectively), which are combined by detector (1018) to give a
mixed FRET signal shown by dashed curve (1022). In the example
described below, an embodiment corresponding to that of FIG. 1A
produced data shown in FIG. 1B for sequence (1082)
3'-AACGGCCCTTCGATCTCATTGAGGATGAGAGGAGAGTCAAAGGAAGA-ACGAGGATGAGAGGAGAGTGAG-
AGCAAAGGAAGAACGAGGATGAGAGG-AGAGTGAGAGCAAAGGAAGAA-5'(SEQ ID NO: 1),
in which only cytosines are labeled. In FIG. 1B, only the relative
positions of the labeled C's are shown so that the correspondence
between such positions and peaks in the data can be appreciated.
Intensity peaks are indicated by asterisks, such as that of (1080).
The data is a plot of relative mixed FRET signal intensity versus
time for the translocation in a 3'-first orientation of sequence
(1082).
[0021] Embodiments are provided where different acceptor labels are
attached to different kinds of monomeric units, so that signals
having different characteristics, e.g. frequency, intensity,
wavelength, etc., are generated for different kinds of monomeric
units, thereby permitting the different kinds of monomeric units to
be distinguished. In one such embodiment, at least two different
acceptor labels are used to label different nucleotides of a target
polynucleotide. An apparatus for such an embodiment is illustrated
in FIG. 1C. Polynucleotide (1070) comprises cytosines (or cytidines
or deoxycytidines) labeled with a first acceptor (solid circles,
1064), Thymidines or thymines labeled with a second acceptor
(cross-hatched circles, 1066), and Guanines and Adenines unlabeled
(speckled circles, 1068). As above, as polynucleotide (1070)
translocates through nanopore (1002), nucleotides exit into FRET
zone (1008) where acceptors (if present) become capable of engaging
in a FRET reaction and generating FRET emissions (1062). Such
emissions are collected by detector (1060) which has conventional
optical components for separating FRET emissions (1062) in
accordance with the different signal characteristics of the
different acceptor labels being employed, such as wavelength which
can be separated, for example, by a dichroic mirror and/or filters.
As a result, an initially collected mixed FRET signal is split into
two or more signals representing mixed FRET signals from different
acceptors, which may be further processed by conventional
components (1072). Also described more fully in the example below,
an embodiment corresponding to that of FIG. 1C produced data shown
in FIG. 1D for sequence (1092) 5'
GCTATGTGGCGCGGTATTATTAAGAAGGAGACTGAGAGGAGAGAAGGAGCAAGA
AGGAAATGAGAGCGAGAGGAGAAGAAGGAGGAAGAAG 3'(SEQ ID NO: 2), in which
only cytosines (or cytidines or deoxycytidines) and thymidines or
thymines are labeled. Signals from first acceptors attached to T's
are indicated by dashed line (1095) and signals from first
acceptors attached to C's are indicated by solid line (1096) In
FIG. 1D, the positions of the labeled T's and C's are shown as
bolded letters (1051, 1052, 1053, 1054, 1055 and 1056,
respectively). Intensity peaks in the plots corresponding to the
labeled T's and C's are indicated by the same reference numbers.
The data is a plot of relative mixed FRET signal intensity versus
time for the translocation in a 3'-first orientation of sequence
(1070).
[0022] As mentioned above, in one aspect, a method may be carried
out by the following steps: (a) translocating a polynucleotide,
e.g., a single stranded or double stranded polynucleotide, through
a nanopore so that nucleotides of the polynucleotide pass in
sequence by a first member of a FRET pair positioned adjacent to
the nanopore, a plurality of the nucleotides being within a FRET
distance of the first member of the FRET pair as the nucleotides
exit the nanopore and a portion of the nucleotides being labeled
with a second member of the FRET pair; (b) exposing the FRET pairs
adjacent to the nanopore to a light beam so that FRET occurs
between the first and a plurality of second members of the FRET
pair within the FRET distance to generate a mixed FRET signal; (c)
measuring mixed FRET signals as the polynucleotide translocates
through the nanopore; and (d) determining a nucleotide sequence of
the polynucleotide from the mixed FRET signals. In some
embodiments, a nanopore is a hybrid nanopore comprising a protein
nanopore inserted into a pore of a solid phase membrane, as
described more fully below. In hybrid nanopores, a first member of
a FRET pair may be attached directly to the protein nanopore, or
alternatively, directly to the solid phase membrane using
conventional linking chemistries, such as "click" chemistries, e.g.
Kolb et al, Angew. Chem. Int. Ed., 4): 2004-2021 (2001), or the
like. In one embodiment, a first member of a FRET pair is attached
directly or indirectly to the protein nanopore, for example, as
discussed in reference to FIG. 2D. In another embodiment, the first
member of the FRET pair is a donor, such as a quantum dot. Quantum
dots are typically much larger than acceptors, especially acceptors
that are organic dyes, which typically have molecular weights in
the range of from 200 to 2000 daltons. Thus, for FRET to occur
between a quantum dot donor and a multiply-labeled polymer analyte,
multiple acceptors are brought within a FRET distance of the
quantum dot at the same time. Under such circumstances multiple
FRET signals are generated within the same time interval over which
such signals are collected, thereby giving rise to a mixed FRET
signal.
Nanopores and Nanopore Sequencing
[0023] Nanopores used with various methods, systems and devices
described herein may be solid-state nanopores, protein nanopores,
or hybrid nanopores comprising protein nanopores configured in a
solid-state membrane, or like framework. Important features of
nanopores include (i) constraining analytes, particularly polymer
analytes, to pass through a detection zone in sequence, (ii)
compatibility with a translocating means, that is, whatever method
is used to drive an analyte through a nanopore, and (iii) FRET
suppression for members of FRET pairs within the lumen, or bore, of
the nanopore.
[0024] Nanopores may be fabricated in a variety of materials
including but not limited to, silicon nitride (Si.sub.3N.sub.4),
silicon dioxide (SiO.sub.2), and the like. The fabrication and
operation of nanopores for analytical applications, such as DNA
sequencing, are disclosed in the following exemplary references
that are incorporated by reference: Russell, U.S. Pat. No.
6,528,258; Feier, U.S. Pat. No. 4,161,690; Ling, U.S. Pat. No.
7,678,562; Hu et al, U.S. Pat. No. 7,397,232; Golovchenko et al,
U.S. Pat. No. 6,464,842; Chu et al, U.S. Pat. No. 5,798,042; Sauer
et al, U.S. Pat. No. 7,001,792; Su et al, U.S. Pat. No. 7,744,816;
Church et al, U.S. Pat. No. 5,795,782; Bayley et al, U.S. Pat. No.
6,426,231; Akeson et al, U.S. Pat. No. 7,189,503; Bayley et al,
U.S. Pat. No. 6,916,665; Akeson et al, U.S. Pat. No. 6,267,872;
Meller et al, U.S. patent publication 2009/0029477; Howorka et al,
International patent publication WO2009/007743; Brown et al,
International patent publication WO2011/067559; Meller et al,
International patent publication WO2009/020682; Polonsky et al,
International patent publication WO2008/092760; Van der Zaag et al,
International patent publication WO2010/007537; Yan et al, Nano
Letters, 5(6): 1129-1134 (2005); Iqbal et al, Nature
Nanotechnology, 2: 243-248 (2007); Wanunu et al, Nano Letters,
7(6): 1580-1585 (2007); Dekker, Nature Nanotechnology, 2: 209-215
(2007); Storm et al, Nature Materials, 2: 537-540 (2003); Wu et al,
Electrophoresis, 29(13): 2754-2759 (2008); Nakane et al,
Electrophoresis, 23: 2592-2601 (2002); Zhe et al, J. Micromech.
Microeng., 17: 304-313 (2007); Henriquez et al, The Analyst, 129:
478-482 (2004); Jagtiani et al, J. Micromech. Microeng., 16:
1530-1539 (2006); Nakane et al, J. Phys. Condens. Matter, 15
R1365-R1393 (2003); DeBlois et al, Rev. Sci. Instruments, 41(7):
909-916 (1970); Clarke et al, Nature Nanotechnology, 4(4): 265-270
(2009); Bayley et al, U.S. patent publication 2003/0215881; and the
like. Briefly, in one aspect, a 1-50 nm channel is formed through a
substrate, usually a membrane, through which an analyte, such as
DNA, is induced to translocate. The solid-state approach of
generating nanopores offers robustness and durability as well as
the ability to tune the size and shape of the nanopore, the ability
to fabricate high-density arrays of nanopores on a wafer scale,
superior mechanical, chemical and thermal characteristics compared
with lipid-based systems, and the possibility of integrating with
electronic or optical readout techniques. Biological nanopores on
the other hand provide reproducible narrow bores, or lumens,
especially in the 1-10 nanometer range, as well as techniques for
tailoring the physical and/or chemical properties of the nanopore
and for directly or indirectly attaching groups or elements, such
as FRET donors or acceptors, by conventional protein engineering
methods. Protein nanopores typically rely on delicate lipid
bilayers for mechanical support, and the fabrication of solid-state
nanopores with precise dimensions remains challenging. Combining
solid-state nanopores with a biological nanopore overcomes some of
these shortcomings, especially the precision of a biological pore
protein with the stability of a solid state nanopore. For optical
read out techniques a hybrid nanopore provides a precise location
of the nanopore which simplifies the data acquisition greatly. The
lateral diffusion of nanopore proteins inserted in a lipid bilayer
makes an optical detection challenging. Since the biological part
of a hybrid nanopore does not rely on the insertion in a lipid
bilayer the degrees of freedom for modifications made to such a
protein are greatly increased, e.g. a genetically modified nanopore
protein that does not spontaneously insert in a lipid bilayer may
still be used as a protein component of a hybrid nanopore. Bilayer
destabilizing agents such as quantum dots may be used to label a
protein component of a hybrid nanopore.
[0025] In one embodiment, a device or system for detecting one or
more analytes, such as a polynucleotide analyte, comprises the
following elements; (a) a solid phase membrane separating a first
chamber and a second chamber, the solid phase membrane having at
least one aperture connecting the first chamber and the second
chamber through a bore; and (b) a first member of a fluorescent
resonance energy transfer (FRET) pair attached to the at least one
aperture, so that whenever one or more analytes having a plurality
of second members of the FRET pair attached thereto traverses the
bore, the plurality of second members are constrained to pass in
sequence within a FRET distance of the first member of the FRET
pair. In some embodiments, the solid phase membrane has been
treated with a low energy ion beam to bleach its
autofluorescence.
[0026] In another embodiment, a device or system for detecting a
plurality of analytes, or a polymer analyte having a plurality of
linked monomer units, such as nucleotides, is provided. Such an
embodiment for determining a sequence of a polynucleotide may
comprise one or more of the following elements: (a) a solid phase
membrane separating a first chamber and a second chamber, the solid
phase membrane having at least one aperture connecting the first
chamber and the second chamber, and having a hydrophobic coating on
at least one surface; (b) a lipid layer may be disposed on the
hydrophobic coating; (c) a protein nanopore immobilized in the
aperture, the protein nanopore having a bore with an exit, and the
protein nanopore interacting with the lipid layer to form a seal
with the solid phase membrane in the aperture so that fluid
communication between the first chamber and the second chamber
occurs solely through the bore of the protein nanopore, and the
protein nanopore being dimensioned so that nucleotides of the
polynucleotide pass through the exit of the bore in sequence and so
that whenever nucleotides of the polynucleotide are labeled with
second members of a FRET pair, FRET is suppressed between such
second members inside the bore and first members of the FRET pair
outside the bore; and/or (d) a first member of the FRET pair
attached to the solid phase membrane or the protein nanopore, so
that whenever nucleotides of the polynucleotide emerge from the
bore, a plurality of the nucleotides are within a FRET distance of
the first member of the FRET pair.
[0027] In some embodiments, the hydrophobic coating is optional in
that the surface of the solid phase membrane is sufficiently
hydrophobic itself so that a lipid layer adheres to it stably. The
at least one aperture will have an inner surface, or wall,
connected to, or contiguous with the surfaces of the solid phase
membrane. In some embodiments, the at least one aperture will be a
plurality of apertures, and the plurality of apertures may be
arranged as a regular array, such as a rectilinear array of
apertures, the spacing of which depending in part on the number and
kind of FRET pairs employed and the optical detection system used.
Each of the apertures has a diameter, which in some embodiments is
such that a protein nanopore is substantially immobilized therein.
In some embodiments, substantially immobilized means that a protein
nanopore may move no more than 5 nm in the plane of the solid phase
membrane relative to the wall of the aperture. In another
embodiment, substantially immobilized means that a protein nanopore
may move no more than 5 nm in the plane of the solid phase membrane
relative to the wall of the aperture. The protein nanopores each
have a bore, or passage, or lumen, which permits fluid
communication between the first and second chambers when the
protein nanopore is immobilized in an aperture. Generally, the bore
is coaxially aligned with the aperture. One function of the
hydrophobic layer is to provide a surface to retain lipids in
and/or immediately adjacent to the at least one aperture. Such
lipids, in turn, permit disposition and immobilization of a protein
nanopore within an aperture in a functional conformation and in a
manner that forms a fluid seal with the wall of the aperture. In
some embodiments, such seal also prevents electrical current
passing between the first and second chambers around the protein
nanopore. In some embodiments, charged analytes are disposed in an
electrolyte solution in the first chamber and are translocated
through the bore(s) of the protein nanopore(s) into an electrolytic
solution in the second chamber by establishing an electrical field
across the solid phase membrane. For convenience of manufacture, in
some embodiments the hydrophobic coating will be on one surface of
the solid phase membrane and the wall(s) of the aperture(s).
[0028] In some embodiments, the solid phase membrane is treated
with a low energy ion beam to bleach its autofluorescence, as
described more fully below.
[0029] FIGS. 2A-2C are diagrams of hybrid biosensors. A nanometer
sized hole (102) is drilled into a solid-state substrate, or solid
phase membrane, (103) which separates two chambers, or compartments
cis (101) and trans (107). A protein biosensor (e.g a protein
nanopore) (104) attached to a charged polymer (105), such as a
single or double-stranded DNA, is embedded into the solid-state
nanohole by electrophoretic transport. In FIG. 1C the protein
biosensor is inserted. In a nanometer sized hole which surface has
a hydrophobic coating (106) and may have a lipid layer (109)
attached thereto. A nanopore may have two sides, or orifices. One
side is referred to as the "cis" side and faces the (-) negative
electrode or a negatively charged buffer/ion compartment or
solution. The other side is referred to as the "trans" side and
faces the (+) electrode or a positively charged buffer/ion
compartment or solution. A biological polymer, such as a labeled
nucleic acid molecule or polymer can be pulled or driven through
the pore by an electric field applied through the nanopore, e.g.,
entering on the cis side of the nanopore and exiting on the trans
side of the nanopore.
[0030] FIG. 2D shows protein nanopore (104) inserted into an
aperture drilled in a solid state membrane (103). Attached to the
protein nanopore (104) is an oligonucleotide (108) to which a
complementary secondary oligonucleotide (111) is hybridized. Said
secondary oligonucleotide (111) has one or more first or second
members of a FRET pair (110) attached to it. Alternatively, a
member of a FRET pair may be directly attached to an amino acid of
a protein nanopore. For example, a hemolysin subunit may be
modified by conventional genetic engineering techniques to
substitute a cysteine for a suitably located amino acid adjacent to
the exit of the nanopore, e.g. the threonine 129. An
oligonucleotide or members of a FRET pair may be attached via the
thio group of the cysteine using conventional linker chemistries,
e.g. Hermanson (cited above).
[0031] In some embodiments, a hybrid nanopore is utilized,
particularly for optical-based nanopore sequencing of
polynucleotides. Such embodiments comprise a solid-state orifice,
or aperture, into which a protein biosensor, such as a protein
nanopore, is stably inserted. A protein nanopore (e.g. alpha
hemolysin) may be attached to a charged polymer (e.g. double
stranded DNA) which serves as a drag force in an applied electric
field, and which may be used to guide a protein nanopore into an
aperture in a solid-state membrane. In some embodiments, the
aperture in the solid-state substrate is selected to be slightly
smaller than the protein, thereby preventing it from translocating
through the aperture. Instead, the protein will be embedded into
the solid-state orifice. The solid-state substrate can be modified
to generate active sites on the surface that allow the covalent
attachment of the plugged-in protein biosensor resulting in a
stable hybrid biosensor.
[0032] The polymer attachment site in the biosensor can be
generated by protein engineering e.g. a mutant protein can be
constructed that will allow the specific binding of the polymer. As
an example, a cysteine residue may be inserted at the desired
position of the protein. The cysteine can either replace a natural
occurring amino acid or can be incorporated as an addition amino
acid. Care must be taken not to disrupt the biological function of
the protein. The terminal primary amine group of a polymer (i.e.
DNA) is then activated using a hetero-bifunctional crosslinker
(e.g. SMCC). Subsequently, the activated polymer is covalently
attached to the cysteine residue of the protein biosensor. In some
embodiments, the attachment of the polymer to the biosensor is
reversible. By implementing a cleavable crosslinker, an easily
breakable chemical bond (e.g. an S--S bond) is introduced and the
charged polymer may be removed after insertion of the biosensor
into the solid-state aperture.
[0033] For someone skilled in the art it is obvious that a wide
variety of different approaches for covalent or non-covalent
attachment methods of a charged polymer to the protein biosensor
are possible and the above described approach merely serves as an
example. The skilled artisan will also realize that a variety of
different polymers may be used as a drag force, including, but not
limited to, single or double stranded DNA, polyethyleneglycol
(PEG), polyvinylpyrrolidone (PVP), poly-L-lysine, linear
polysaccharides etc. It is also obvious that these polymers may
exhibit either a negative (-) or positive (+) charge at a given pH
and that the polarity of the electric field may be adjusted
accordingly to pull the polymer-biosensor complex into a
solid-state aperture.
[0034] In some embodiments, a donor fluorophore is attached to the
protein nanopore. This complex is then inserted into a solid-state
aperture or nanohole (3-10 nm in diameter) by applying an electric
field across the solid state nanohole until the protein nanopore is
transported into the solid-state nanohole to form a hybrid
nanopore. The formation of the hybrid nanopore can be verified by
(a) the inserting protein nanopore causing a drop in current based
on a partial blockage of the solid-state nanohole and by (b) the
optical detection of the donor fluorophore.
[0035] Once stable hybrid nanopores have formed single stranded,
fluorescently labeled (or acceptor labeled) DNA may be added to the
cis chamber (the chamber with the (+) electrode). The applied
electric field forces the negatively charged ssDNA to translocate
through the hybrid nanopore during which the labeled nucleotides
get in close vicinity of the donor fluorophore. In certain
variations, double stranded DNA may be utilized.
[0036] Solid state, or synthetic, nanopores may be prepared in a
variety of ways, as exemplified in the references cited above. In
some embodiments a helium ion microscope may be used to drill the
synthetic nanopores in a variety of materials, e.g. as disclosed by
Yang et al, Nanotechnolgy, 22: 285310 (2011), which is incorporated
herein by reference. A chip that supports one or more regions of a
thin-film material, e.g. silicon nitride, that has been processed
to be a free-standing membrane is introduced to the helium ion
microscope (HIM) chamber. HIM motor controls are used to bring a
free-standing membrane into the path of the ion beam while the
microscope is set for low magnification. Beam parameters including
focus and stigmation are adjusted at a region adjacent to the
free-standing membrane, but on the solid substrate. Once the
parameters have been properly fixed, the chip position is moved
such that the free-standing membrane region is centered on the ion
beam scan region and the beam is blanked. The HIM field of view is
set to a dimension (in .mu.m) that is sufficient to contain the
entire anticipated nanopore pattern and sufficient to be useful in
future optical readout (i.e. dependent on optical magnification,
camera resolution, etc.). The ion beam is then rastered once
through the entire field of view at a pixel dwell time that results
in a total ion dose sufficient to remove all or most of the
membrane autofluorescence. The field of view is then set to the
proper value (smaller than that used above) to perform
lithographically-defined milling of either a single nanopore or an
array of nanopores. The pixel dwell time of the pattern is set to
result in nanopores of one or more predetermined diameters,
determined through the use of a calibration sample prior to sample
processing. This entire process is repeated for each desired region
on a single chip and/or for each chip introduced into the HIM
chamber.
[0037] In some embodiments, the solid-state substrate may be
modified to generate active sites on the surface that allow the
covalent attachment of the plugged in protein biosensor or to
modify the surface properties in a way to make it more suitable for
a given application. Such modifications may be of covalent or
non-covalent nature. A covalent surface modification includes a
silanization step where an organosilane compound binds to silanol
groups on the solid surface. For instance, the alkoxy groups of an
alkoxysilane are hydrolyzed to form silanol-containing species.
Reaction of these silanes involves four steps. Initially,
hydrolysis of the labile groups occurs. Condensation to oligomers
follows. The oligomers then hydrogen bond with hydroxyl groups of
the substrate. Finally, during drying or curing, a covalent linkage
is formed with the substrate with concomitant loss of water. For
covalent attachment organosilanes with active side groups may be
employed. Such side groups consist of, but are not limited to epoxy
side chain, aldehydes, isocyanates, isothiocyanates, azides or
alkynes (click chemistry) to name a few. For someone skilled in the
art it is obvious that multiple ways of covalently attaching a
protein to a surface are possible. For instance, certain side
groups on an organosilane may need to be activated before being
capable of binding a protein (e.g. primary amines or carboxyl side
groups activated with an N-hydroxysuccinimidester). Another way of
attaching a protein to the solid surface may be achieved through
affinity binding by having one affinity partner attached to the
protein and the second affinity partner being located on the solid
surface. Such affinity pairs consist of the group of, but are not
limited to biotin-strepavidin, antigen-antibody and aptamers and
the corresponding target molecules.
[0038] In one embodiment, the surface modification of the solid
state nanopore includes treatment with an organosilane that renders
the surface hydrophobic. Such organosilanes include but are not
limited to, alkanesilanes (e.g. octadecyldimethylchlorosilane) or
modified alkanesilanes such as fluorinated alkanesilanes with an
alkane chain length of 5 to 30 carbons. The hydrophobic surface may
then be treated with a dilute solution of a lipid in pentane. After
drying of the solvent and immersing the surface in an aqueous
solution the lipid will spontaneously form a layer on the surface.
A layer of lipid on the solid surface might prove beneficial for
the formation of a hybrid nanopore. The lipid layer on the solid
phase might reduce the leak current between protein and solid state
nanopore and it might increase the stability of the inserted
protein pore. Combining a low capacitance solid substrate as well
as a lipid coating of said substrate may render the hybrid nanopore
system amenable to an electrical readout based on current
fluctuations generated by translocation of DNA through the hybrid
nanopore. To achieve electrical read out with such a system a means
of decreasing the translocation speed of unmodified DNA must be
combined with a lipid coated hybrid nanopore. Molecular motors such
as polymerases or helicases may be combined with a hybrid nanopore
and effectively reduce the translocation speed of DNA through the
hybrid nanopore. The lipids used for coating the surface may be
from the group of sphingolipids, phospholipids or sterols.
[0039] A method and/or system for sequencing a biological polymer
or molecule (e.g., a nucleic acid) may include exciting one or more
donor labels attached to a pore or nanopore. A biological polymer
may be translocated through the pore or nanopore, where a monomer
of the biological polymer is labeled with one or more acceptor
labels. Energy may be transferred from the excited donor label to
the acceptor label of the monomer as, after the labeled monomer
passes through, exits or enters the pore or nanopore. Energy
emitted by the acceptor label as a result of the energy transfer
may be detected, where the energy emitted by the acceptor label may
correspond to or be associated with a single or particular monomer
(e.g., a nucleotide) of a biological polymer. The sequence of the
biological polymer may then be deduced or sequenced based on the
detection of the emitted energy from the monomer acceptor label
which allows for the identification of the labeled monomer. A pore,
nanopore, channel or passage, e.g., an ion permeable pore,
nanopore, channel or passage may be utilized in the systems and
methods described herein.
[0040] The nanopore may have one or more labels attached. In some
embodiments, the label is a member of a Forster Resonance Energy
Transfer (FRET) pair. Such labels may comprise organic
fluorophores, chemiluminescent labels, quantum dots, metallic
nanoparticles and fluorescent proteins. The nucleic acid may have
one distinct label per nucleotide. The labels attached to the
nucleotides consist of the group of organic fluorophores,
chemiluminescent labels, quantum dots, metallic nanoparticles and
fluorescent proteins. The label attachment site in the pore protein
can be generated by protein engineering e.g. a mutant protein can
be constructed that will allow the specific binding of the label.
As an example, a cysteine residue may be inserted at the desired
position of the protein which inserts a thiol (SH) group that can
be used to attach a label. The cysteine can either replace a
natural occurring amino acid or can be incorporated as an addition
amino acid. Care must be taken not to disrupt the biological
function of the protein. A malemeide-activated label is then
covalently attached to the thiol residue of the protein nanopore.
In one embodiment, the attachment of the label to the protein
nanopore or the label on the nucleic acid is reversible. By
implementing a cleavable crosslinker, an easily breakable chemical
bond (e.g. an S--S bond or a pH labile bond) is introduced and the
label may be removed when the corresponding conditions are met.
[0041] A nanopore, or pore, may be labeled with one or more donor
labels. For example, the cis side or surface and/or trans side or
surface of the nanopore may be labeled with one or more donor
labels. The label may be attached to the base of a pore or nanopore
or to another portion or monomer making up the nanopore or pore A
label may be attached to a portion of the membrane or substrate
through which a nanopore spans or to a linker or other molecule
attached to the membrane, substrate or nanopore. The nanopore or
pore label may be positioned or attached on the nanopore, substrate
or membrane such that the pore label can come into proximity with
an acceptor label of a biological polymer, e.g., a nucleic acid,
which is translocated through the pore. The donor labels may have
the same or different emission or absorption spectra. The labeling
of a pore structure may be achieved via covalent or non-covalent
interactions.
[0042] A donor label may be placed as close as possible to the
aperture of a nanopore without causing an occlusion that impairs
translocation of a nucleic acid through the nanopore. A pore label
may have a variety of suitable properties and/or characteristics.
For example, a pore label may have energy absorption properties
meeting particular requirements. A pore label may have a large
radiation energy absorption cross-section, ranging, for example,
from about 0 to 1000 nm or from about 200 to 500 nm. A pore label
may absorb radiation within a specific energy range that is higher
than the energy absorption of the nucleic acid label. The
absorption energy of the pore label may be tuned with respect to
the absorption energy of a nucleic acid label in order to control
the distance at which energy transfer may occur between the two
labels. A pore label may be stable and functional for at least 10 6
or 10 9 excitation and energy transfer cycles.
Treating Solid Phase Membranes to Reduce Autofluorescence
[0043] In some embodiments, a solid phase membrane of a
microelectromechanical system (MEMS) material is treated with a low
energy ion beam to bleach its autofluorescence. Typically such
treatment is carried out by directing an ion beam to a surface
region of the MEMS material, at a sufficiently high energy to cause
a physical change in the MEMS material at its surface or near its
surface to disrupt or inactivate structures contributing to
autofluorescence, but not with such high energy that melting,
vaporization, significant deformations or sputtering occur. The
minimal energy required may be readily determined on a
material-by-material basis by gradually increasing beam energy
starting from zero and measuring reduction in autofluorescence with
increasing beam energy. As used herein, the term "autofluorescence"
is used synonymously with "background fluorescence" to mean
fluorescence emanating from a source at or near a surface of a MEMS
material upon excitation with a light source selected to excite a
fluorescent label that is not a part of the MEMS material. Thus,
autofluorescence in a MEMS material depends on the frequency of the
light source. In one aspect, the frequency of the light source is
selected to excite organic fluorescent dyes, so that the method
reduces autofluorescence of frequencies in the visible range of
light as well as frequencies from the near infrared to the near
ultraviolet. MEMS materials include a wide variety of solids
capable of microfabrication and use in analytical techniques using
optical detection. Exemplary MEMS materials are silicon-based
substrates, such as silicon nitride and silicon dioxide or metal
based substrates, such as aluminum oxide. In one aspect, MEMS
materials are processed and used in the form of a membrane. In one
embodiment, the MEMS material is silicon nitride. A wide variety of
focused ion beams may be employed for such bleaching and guidance
for the production and application of such beams at various
energies may be found in such references as, Natasi et al, Ion
Solid Interactions: Fundamentals and Applications (Cambridge
University Press, 1996), and like references. Exemplary focused ion
beams include helium ion beams, neon ion beams and gallium ion
beams. In one embodiment, a helium ion beam is used in the method.
Helium ion beams may be produced with a commercially available ion
beam microscope (HIM) (e.g. Zeiss Orion Nanofab). The amount of
energy or dosage delivered to a surface of a MEMS material, such as
silicon nitride, to reduce autofluorescence may be in the range of
from 2e-10 to 8e-10 nC/nm 2.
Labels for Nanopores and Analytes
[0044] In some embodiments, a nanopore may be labeled with one or
more quantum dots. In particular, in some embodiments, one or more
quantum dots may be attached to a nanopore, or attached to a solid
phase support adjacent to (and within a FRET distance of an
entrance or exit of a nanopore), and employed as donors in FRET
reactions with acceptors on analytes. Such uses of quantum dots are
well known and are described widely in the scientific and patent
literature, such as, in U.S. Pat. Nos. 6,252,303; 6,855,551;
7,235,361; and the like, which are incorporated herein by
reference.
[0045] One example of a Quantum dot which may be utilized as a pore
label is a CdTe quantum dot which can be synthesized in an aqueous
solution. A CdTe quantum dot may be functionalized with a
nucleophilic group such as primary amines, thiols or functional
groups such as carboxylic acids. A CdTe quantum dot may include a
mercaptopropionic acid capping ligand, which has a carboxylic acid
functional group that may be utilized to covalently link a quantum
dot to a primary amine on the exterior of a protein pore. The
cross-linking reaction may be accomplished using standard
cross-linking reagents (homo-bifunctional as well as
hetero-bifunctional) which are known to those having ordinary skill
in the art of bioconjugation. Care may be taken to ensure that the
modifications do not impair or substantially impair the
translocation of a nucleic acid through the nanopore. This may be
achieved by varying the length of the employed crosslinker molecule
used to attach the donor label to the nanopore.
[0046] The primary amine of the Lysin residue 131 of the natural
alpha hemolysin protein (Song, L. et al., Science 274, (1996):
1859-1866) may be used to covalently bind carboxy modified CdTe
Quantum dots via 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride/N-hydroxysulfosuccinimide (EDC/NHS) coupling
chemistry. Alternatively, amino acid 129 (threonine) may be
exchanged into cysteine. Since there is no other cysteine residue
in the natural alpha hemolysin protein the thiol side group of the
newly inserted cysteine may be used to covalently attach other
chemical moieties.
[0047] A variety of methods, mechanisms and/or routes for attaching
one or more pore labels to a pore protein may be utilized. A pore
protein may be genetically engineered in a manner that introduces
amino acids with known properties or various functional groups to
the natural protein sequence. Such a modification of a naturally
occurring protein sequence may be advantageous for the
bioconjugation of Quantum dots to the pore protein. For example,
the introduction of a cysteine residue would introduce a thiol
group that would allow for the direct binding of a Quantum dot,
such as a CdTe quantum dot, to a pore protein. Also, the
introduction of a Lysin residue would introduce a primary amine for
binding a Quantum dot. The introduction of glutamic acid or
aspartic acid would introduce a carboxylic acid moiety for binding
a Quantum dot. These groups are amenable for bioconjugation with a
Quantum dot using either homo- or hetero-bifunctional crosslinker
molecules. The insertions of poly-histidines allow the direct
binding of Quantum dots to a protein pore via metal-histidine
coordination. Such modifications to pore proteins aimed at the
introduction of functional groups for bioconjugation are known to
those having ordinary skill in the art. Care should be taken to
ensure that the modifications do not impair or substantially impair
the translocation of a nucleic acid through the nanopore.
[0048] The nanopore label can be attached to a protein nanopore
before or after insertion of said nanopore into a lipid bilayer.
Where a label is attached before insertion into a lipid bilayer,
care may be taken to label the base of the nanopore and avoid
random labeling of the pore protein. This can be achieved by
genetic engineering of the pore protein to allow site specific
attachment of the pore label (see section 0047). An advantage of
this approach is the bulk production of labeled nanopores.
Alternatively, a labeling reaction of a pre-inserted nanopore may
ensure site-specific attachment of the label to the base
(trans-side) of the nanopore without genetically engineering the
pore protein.
[0049] A biological polymer, e.g., a nucleic acid molecule or
polymer, may be labeled with one or more acceptor labels. For a
nucleic acid molecule, each of the four nucleotides or building
blocks of a nucleic acid molecule may be labeled with an acceptor
label thereby creating a labeled (e.g., fluorescent) counterpart to
each naturally occurring nucleotide. The acceptor label may be in
the form of an energy accepting molecule which can be attached to
one or more nucleotides on a portion or on the entire strand of a
converted nucleic acid.
[0050] A variety of methods may be utilized to label the monomers
or nucleotides of a nucleic acid molecule or polymer. A labeled
nucleotide may be incorporated into a nucleic acid during synthesis
of a new nucleic acid using the original sample as a template
("labeling by synthesis"). For example, the labeling of nucleic
acid may be achieved via PCR, whole genome amplification, rolling
circle amplification, primer extension or the like or via various
combinations and extensions of the above methods known to persons
having ordinary skill in the art.
[0051] Labeling of a nucleic acid may be achieved by replicating
the nucleic acid in the presence of a modified nucleotide analog
having a label, which leads to the incorporation of that label into
the newly generated nucleic acid. The labeling process can also be
achieved by incorporating a nucleotide analog with a functional
group that can be used to covalently attach an energy accepting
moiety in a secondary labeling step. Such replication can be
accomplished by whole genome amplification (Zhang, L. et al., Proc.
Natl. Acad. Sci. USA 89 (1992): 5847) or strand displacement
amplification such as rolling circle amplification, nick
translation, transcription, reverse transcription, primer extension
and polymerase chain reaction (PCR), degenerate oligonucleotide
primer PCR (DOP-PCR) (Telenius, H. et al., Genomics 13 (1992):
718-725) or combinations of the above methods.
[0052] A label may comprise a reactive group such as a nucleophile
(amines, thiols etc.). Such nucleophiles, which are not present in
natural nucleic acids, can then be used to attach fluorescent
labels via amine or thiol reactive chemistry such as NHS esters,
maleimides, epoxy rings, isocyanates etc. Such nucleophile reactive
fluorescent dyes (i.e. NHS-dyes) are readily commercially available
from different sources. An advantage of labeling a nucleic acid
with small nucleophiles lies in the high efficiency of
incorporation of such labeled nucleotides when a "labeling by
synthesis" approach is used. Bulky fluorescently labeled nucleic
acid building blocks may be poorly incorporated by polymerases due
to steric hindrance of the labels during the polymerization process
into newly synthesized DNA.
[0053] DNA can be directly chemically modified without polymerase
mediated incorporation of labeled nucleotides. One example of a
modification includes cis-platinum containing dyes that modify
Guanine bases at their N7 position (Hoevel, T. et al., Bio
Techniques 27 (1999): 1064-1067). Another example includes the
modifying of pyrimidines with hydroxylamine at the C6 position
which leads to 6-hydroxylamino derivatives. The resulting amine
groups can be further modified with amine reactive dyes (e.g.
NHS-Cy5). Yet another example are azide or alkyne modified
nucleotides which are readily incorporated by polymerases (Gierlich
et al., Chem. Eur. J., 2007, 13, 9486-0404). The alkyne or azide
modified polynucleotide is subsequently labeled with an azide or
alkyne modified fluorophore following well established click
chemistry protocols.
[0054] A nucleic acid molecule may be directly modified with
N-Bromosuccinimide which upon reacting with the nucleic acid will
result in 5-Bromocystein, 8-Bromoadenine and 8-Bromoguanine. The
modified nucleotides can be further reacted with di-amine
nucleophiles. The remaining nucleophile can then be reacted with an
amine reactive dye (e.g. NHS-dye) (Hermanson G. in Bioconjugate
Techniques, Academic Press 1996, ISBN 978-0-12-342336-8).
[0055] A combination of 1, 2, 3 or 4 nucleotides in a nucleic acid
strand may be exchanged with their labeled counterpart. The various
combinations of labeled nucleotides can be sequenced in parallel,
e.g., labeling a source nucleic acid or DNA with combinations of 2
labeled nucleotides in addition to the four single labeled samples,
which will result in a total of 10 differently labeled sample
nucleic acid molecules or DNAs (G, A, T, C, GA, GT, GC, AT, AC,
TC). The resulting sequence pattern may allow for a more accurate
sequence alignment due to overlapping nucleotide positions in the
redundant sequence read-out.
[0056] In certain variations, a method for sequencing a polymer,
such as a nucleic acid molecule, may include providing a nanopore
or pore protein (or a synthetic pore) inserted in a membrane or
membrane like structure or other substrate. The base or other
portion of the pore may be modified with one or more pore labels.
The base may refer to the Trans side of the pore. Optionally, the
Cis and/or Trans side of the pore may be modified with one or more
pore labels. Nucleic acid polymers to be analyzed or sequenced may
be used as a template for producing a labeled version of the
nucleic acid polymer, in which one of the four nucleotides or up to
all four nucleotides in the resulting polymer is/are replaced with
the nucleotide's labeled analogue(s). An electric field is applied
to the nanopore which forces the labeled nucleic acid polymer
through the nanopore, while an external monochromatic or other
light source may be used to illuminate the nanopore, thereby
exciting the pore label. As, after or before labeled nucleotides of
the nucleic acid pass through, exit or enter the nanopore, energy
is transferred from the pore label to a nucleotide label, which
results in emission of lower energy radiation. The nucleotide label
radiation is then detected by a confocal microscope setup or other
optical detection system or light microscopy system capable of
single molecule detection known to people having ordinary skill in
the art. Examples of such detection systems include but are not
limited to confocal microscopy, epifluorescent microscopy and total
internal reflection fluorescent (TIRF) microscopy. Other polymers
(e.g., proteins and polymers other than nucleic acids) having
labeled monomers may also be sequenced according to the methods
described herein.
[0057] Energy may be transferred from a pore or nanopore donor
label (e.g., a Quantum Dot) to an acceptor label on a polymer
(e.g., a nucleic acid) when an acceptor label of an acceptor
labeled monomer (e.g., nucleotide) of the polymer interacts with
the donor label as, after or before the labeled monomer exits,
enters or passes through a nanopore. For example, the donor label
may be positioned on or attached to the nanopore on the cis or
trans side or surface of the nanopore such that the interaction or
energy transfer between the donor label and acceptor label does not
take place until the labeled monomer exits the nanopore and comes
into the vicinity or proximity of the donor label outside of the
nanopore channel or opening. As a result, interaction between the
labels, energy transfer from the donor label to the acceptor label,
emission of energy from the acceptor label and/or measurement or
detection of an emission of energy from the acceptor label may take
place outside of the passage, channel or opening running through
the nanopore, e.g., within a cis or trans chamber on the cis or
trans sides of a nanopore. The measurement or detection of the
energy emitted from the acceptor label of a monomer may be utilized
to identify the monomer.
[0058] The nanopore label may be positioned outside of the passage,
channel or opening of the nanopore such that the label may be
visible or exposed to facilitate excitation or illumination of the
label. The interaction and energy transfer between a donor label
and accepter label and the emission of energy from the acceptor
label as a result of the energy transfer may take place outside of
the passage, channel or opening of the nanopore. This may
facilitate ease and accuracy of the detection or measurement of
energy or light emission from the acceptor label, e.g., via an
optical detection or measurement device. The donor and acceptor
label interaction may take place within a channel of a nanopore and
a donor label could be positioned within the channel of a
nanopore.
[0059] A donor label may be attached in various manners and/or at
various sites on a nanopore. For example, a donor label may be
directly or indirectly attached or connected to a portion or unit
of the nanopore. Alternatively, a donor label may be positioned
adjacent to a nanopore.
[0060] Each acceptor labeled monomer (e.g., nucleotide) of a
polymer (e.g., nucleic acid) can interact sequentially with a donor
label positioned on or next to or attached directly or indirectly
to a nanopore or channel through which the polymer is translocated.
The interaction between the donor and acceptor labels may take
place outside of the nanopore channel or opening, e.g., after the
acceptor labeled monomer exits the nanopore or before the monomer
enters the nanopore. The interaction may take place within or
partially within the nanopore channel or opening, e.g., while the
acceptor labeled monomer passes through, enters or exits the
nanopore.
[0061] When one of the four nucleotides of a nucleic acid is
labeled, the time dependent signal arising from the single
nucleotide label emission is converted into a sequence
corresponding to the positions of the labeled nucleotide in the
nucleic acid sequence. The process is then repeated for each of the
four nucleotides in separate samples and the four partial sequences
are then aligned to assemble an entire nucleic acid sequence.
[0062] When multi-color labeled nucleic acid (DNA) sequences are
analyzed, the energy transfer from one or more donor labels to each
of the four distinct acceptor labels that may exist on a nucleic
acid molecule may result in light emission at four distinct
wavelengths or colors (each associated with one of the four
nucleotides) which allows for a direct sequence read-out.
Translocation Speed
[0063] A major obstacle associated with Nanopore based sequencing
approaches is the high translocation velocity of nucleic acid
through a nanopore (.about.500.000-1.000.000 nucleotides/sec) which
doesn't allow for direct sequence readout due to the limited
bandwidth of the recording equipment. A way of slowing down the
nucleic acid translocation with two different nanopore proteins was
recently shown by Cherf et al. (Nat Biotechnol. 2012 Feb. 14;
30(4):344-8) and Manrao et al. (Nat Biotechnol. 2012 Mar. 25;
30(4):349-53) and are incorporated herein by reference. Both groups
used a DNA polymerase to synthesize a complementary strand from a
target template which resulted in the step-wise translocation of
the template DNA through the nanopore. Hence, the synthesis speed
of the nucleic acid polymerase (10-500 nucleotides/sec) determined
the translocation speed of the DNA and since it's roughly 3-4
orders of magnitude slower than direct nucleic acid translocation
the analysis of single nucleotides became feasible. However, the
polymerase-aided translocation requires significant sample
preparation to generate a binding site for the polymerase and the
nucleic acid synthesis has to be blocked in bulk and can only start
once the nucleic acid-polymerase complex is captured by the
nanopore protein. This results in a rather complex set-up which
might prevent the implementation in a commercial setting.
Furthermore, fluctuation in polymerase synthesis reactions such as
a stalled polymerization as well as the dissociation of the
polymerase from the nucleic acid may hamper the sequence read-out
resulting in a high error rate and reduced read-length,
respectively. Optical Nanopore sequence as described in this
application uses a different way of slowing down the DNA
translocation. A target nucleic acid is enzymatically copied by
incorporating fluorescent modified nucleotides. The resulting
labeled nucleic acid has an increased nominal diameter which
results in a decreased translocation velocity when pulled through a
nanopore. The preferred translocation rate for optical sequencing
lies in the range of 1-1000 nucleotides per second with a more
preferred range of 200-800 nucleotides per second and a most
preferred translocation rate of 200-600 nucleotides per second.
[0064] Alternatively, translocation speed of a polynucleotide,
especially a single stranded polynucleotide, may be controlled by
employing a nanopore dimensioned so that adducts and/or labels,
e.g. organic dyes attached to bases, inhibit but do not prevent
polynucleotide translocation. A translocation speed may be selected
by attaching labels and/or adducts at a predetermined density. Such
labels and/or adducts may have regular spaced attachments, e.g.
every third nucleotide or the like, or they may have random, or
pseudorandom attachments, e.g. every C may be labeled. In some
embodiments, a selected number of different nucleotides may be
labeled, e.g. every A and C, or every A and G, or every A and T, or
every C, or the like, that results in an average translocation
speed. Such average speed may be decreased by attaching adducts to
unlabeled nucleotides. Adducts include any molecule, usually and
organic molecule, that may be attached to a nucleotide using
conventional chemistries. Typically adducts have a molecular weight
in the same range as common organic dyes, e.g. fluorescein, Cy3, or
the like. Adducts may or may not be capable of generating signals,
that is, serving as a label. In some embodiments, adducts and/or
labels are attached to bases of nucleotides. In other embodiments,
labels and/or adducts may be attached to linkages between
nucleosides in a polynucleotide. In one aspect, a method of
controlling translocation velocity of a single stranded
polynucleotide through a nanopore comprises the step of attaching
adducts to the polynucleotide at a density, wherein translocation
velocity of the single stranded polynucleotide monotonically
decreases with a larger number of adducts attached, or with the
density of adducts attached. In some embodiments, not every kind of
nucleotide of a polynucleotide is labeled. For example, four
different sets of a polynucleotide may be produced where
nucleotides of each set are labeled with the same molecule, e.g. a
fluorescent organic dye acceptor, but in each set a different kind
of nucleotide will be labeled. Thus, in set 1 only A's may be
labeled; in set 2 only C's may be labeled; in set 3 only G's may be
labeled; and so on. After such labeling, the four sets of
polynucleotides may then be analyzed separately in accordance with
the methods and systems described herein and a nucleotide sequence
of the polynucleotide determined from the data generated in the
four analysis. In such embodiments, and similar embodiments, e.g.
two labels are used, where some of the nucleotides of a
polynucleotide are not labeled, translocation speed through a
nanopore will be affected by the distribution of label along the
polynucleotide. To prevent such variability in translocation speed,
in some embodiments, nucleotides that are not labeled with an
acceptor or donor for generating signals to determine nucleotide
sequence, may be modified by attaching a non-signal-producing
adduct that has substantially the same effect on translocation
speed as the signal-producing labels.
Example
[0065] In this example, a nanopore apparatus is described for
determining a sequence of acceptor-labeled nucleotides of a
polynucleotide, after which it is used to detect a sequence of
acceptor-labeled cytosines in a first polynucleotide and to detect
a sequence of first acceptor-labeled thymines or thymidines and
second acceptor-labeled cytosines in a second polynucleotide.
[0066] HIM drilling to form nanopore(s) in a silicon nitride
membrane: A 3 mm Si chip (Protochips, NC) with a 50.times.50 um
etched window spanned by a 30 nm Si.sub.3N.sub.4 membrane is
cleaned with oxygen plasma prior to the drilling process. The
cleaned chip is inserted into the vacuum chamber of a Helium ion
Microscope (Orion, Zeiss). After insertion, the nanoholes are
drilled with a focused ion stream with a beam current of .about.5
pA through a 20 um aperture and with an exposure time calibrated to
result in 4+/-2 nm holes
[0067] Protein nanopores: A cloned variant of the wt alpha
hemolysin protein is used as a template for an in vitro
transcription/translation reaction. The resulting monomers are
heptamerized by a stepwise addition of sodium deoxycholate to a
final concentration of 6.25 mM and a subsequent incubation for 12
hours at 4 C. The resulting heptamer is attached to an
amine-modified oligonucleotide using a heterobifunctional
crosslinker (SMCC, Sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate). This oligonucleotide serves as a
hybridization site for a 3 kb ds-DNA fragment which is used as a
drag force to pull the protein nanopore into the drilled holes in
the Si.sub.3N.sub.4 membrane. In addition to the oligonucleotide
modification, the base of the protein nanopore is also modified
with one or more maleimide activated fluorescent dyes. The attached
fluorophores can either serve as donor or acceptor in a FRET
reaction.
[0068] Hybrid Nanopore: TEM grids with drilled holes are cleaned by
submersing in 90 C hot Piranha solution (3:1 Sulfuric
acid:H.sub.2O.sub.2, v/v) for 15 min or by an air plasma for 5 min.
After extensive rinsing with water the TEM grid is installed in a
Delrin holder separating a cis and trans chamber. The trans chamber
is sealed with a cover slip that allows the optical interrogation
of the nanopores. Each chamber is filled with Ethanol to promote
wetting of the nanopores. The Ethanol is subsequently exchanged
with water and then a buffered 1 M KCl solution. In the trans
chamber the buffered KCl contains 50% glycerol to facilitate TIR
(total internal reflection) imaging. Both the cis and trans chamber
harbor a Ag/AgCl electrode in contact with the buffer solution with
the cathode (+) in the cis and the anode (-) in the trans chamber.
The nanopore protein is added to the cis side of the SiN membrane
and by applying an electric field (200-600 mV) the protein nanopore
is plugged into the drilled nanoholes forming a hybrid
nanopore.
[0069] After 5-25 min at 600 mV usually 50-75% of the drilled holes
have a protein nanopore inserted. The formation of the hybrid
nanopores is checked on an inverted microscope (Olympus IX71)
equipped with an APON 60.times.TIRF oil-immersion objective and a
532 nm diode laser which is used to excite the fluorophore attached
to the protein nanopore. The buffer in the cis chamber is exchanged
to remove excess nanopore protein. Labeled single stranded (ss) DNA
at a final concentration of 10 nM is added to the cis chamber. The
electric field is reduced to 200-400 mV which promotes the
translocation of ssDNA through the hybrid nanopore. The labeled
DNA, when exiting the nanopore, comes in close proximity to the
excited donor fluorophore. A FRET reaction occurs which results in
the photon emission from the labeled DNA. Emitted photons are
collected, filtered and imaged using an Orca Flash 4.0 cMOS camera
(Hamamatsu) at a frame rate of 500-5000 Hz. Data is extracted from
a 5.times.5 pixel area covering the entire hybrid nanopore from the
raw tiff images using ImageJ. Raw traces are normalized and a peak
find algorithm is used to identify FRET signal. Base calling is
performed on the identified peaks.
[0070] Each cytosine of the following 107-mer single stranded
polynucleotide (SEQ ID NO: 1) was labeled with a Cy5
fluorophore.
TABLE-US-00001 3'-AACGGCCCTTCGATCTCATTGAGGATGAGAGGAGAGTCAAAGGAAGA
ACGAGGATGAGAGGAGAGTGAGAGCAAAGGAAGAACGAGGATGAGAGGAG
AGTGAGAGCAAAGGAAGAA-5'
[0071] The labeled polynucleotide was translocated in a 3'-first
orientation through the hybrid nanopore and mixed FRET signals from
the labeled cytosines was collected. Raw data is shown in FIG. 1B.
When translocated through the hybrid nanopore multiple peaks are
observed which correspond to the number of cytosines in the DNA.
Remarkably, the homopolymer at the 3' end is perfectly
resolved.
[0072] After the 20.sup.th nucleotide position from the 5' end,
every cytosine and every thymidine of the following single stranded
polynucleotide (SEQ ID NO: 2) were labeled with Cy5 and Atto700,
respectively.
TABLE-US-00002 5'-GCTATGTGGCGCGGTATTATTAAGAAGGAGACTGAGAGGAGAGAA
GGAGCAAGAAGGAAATGAGAGCGAGAGGAGAGAAGGAGGAAGAAG-3'
The labeled polynucleotide was translocated in a 3'-first
orientation through the hybrid nanopore and mixed FRET signals from
the labeled cytosines and thymidines were collected. Raw data is
shown in FIG. 1D. The peaks in this raw data trace show a pattern
that resembles the position of the labeled nucleotides in the
template strand.
[0073] This disclosure is not intended to be limited to the scope
of the particular forms set forth, but is intended to cover
alternatives, modifications, and equivalents of the variations
described herein. Further, the scope of the disclosure fully
encompasses other variations that may become obvious to those
skilled in the art in view of this disclosure. The scope of the
present invention is limited only by the appended claims.
Definitions
[0074] "Nanopore" means any opening positioned in a substrate that
allows the passage of analytes through the substrate in a
predetermined or discernable order, or in the case of polymer
analytes, passage of their monomeric units through the substrate in
a predetermined or discernible order. In the latter case, a
predetermined or discernible order may be the primary sequence of
monomeric units in the polymer. Examples of nanopores include
proteinaceous or protein based nanopores, synthetic or solid state
nanopores, and hybrid nanopores comprising a solid state nanopore
having a protein nanopore embedded therein. A nanopore may have an
inner diameter of, e.g., 1-10 nm or 1-5 nm or 1-3 nm, or other
various sizes. Examples of protein nanopores include but are not
limited to, alpha-hemolysin, voltage-dependent mitochondrial porin
(VDAC), OmpF, OmpC, MspA and LamB (maltoporin), e.g. disclosed in
Rhee, M. et al., Trends in Biotechnology, 25(4) (2007): 174-181;
Bayley et al (cited above); Gundlach et al, U.S. patent publication
2012/0055792; and the like, which are incorporated herein by
reference. Any protein pore that allows the translocation of single
nucleic acid molecules may be employed. A nanopore protein may be
labeled at a specific site on the exterior of the pore, or at a
specific site on the exterior of one or more monomer units making
up the pore forming protein. Pore proteins are chosen from a group
of proteins such as, but not limited to, alpha-hemolysin, MspA,
voltage-dependent mitochondrial porin (VDAC), Anthrax porin, OmpF,
OmpC and LamB (maltoporin). Integration of the pore protein into a
solid state hole is accomplished by attaching a charged polymer to
the pore protein. After applying an electric field the charged
complex is electrophoretically pulled into the solid state hole. A
synthetic nanopore, or solid-state nanopore, may be created in
various forms of solid substrates, examples of which include but
are not limited to silicones (e.g. Si3N4, SiO2), metals, metal
oxides (e.g. Al2O3) plastics, glass, semiconductor material, and
combinations thereof. A synthetic nanopore may be more stable than
a biological protein pore positioned in a lipid bilayer membrane. A
synthetic nanopore may also be created by using a carbon nanotube
embedded in a suitable substrate such as but not limited to
polymerized epoxy. Carbon nanotubes can have uniform and
well-defined chemical and structural properties. Various sized
carbon nanotubes can be obtained, ranging from one to hundreds of
nanometers. The surface charge of a carbon nanotube is known to be
about zero, and as a result, electrophoretic transport of a nucleic
acid through the nanopore becomes simple and predictable (Ito, T.
et al., Chem. Commun. 12 (2003): 1482-83). The substrate surface of
a synthetic nanopore may be chemically modified to allow for
covalent attachment of the protein pore or to render the surface
properties suitable for optical nanopore sequencing. Such surface
modifications can be covalent or non-covalent. Most covalent
modification include an organosilane deposition for which the most
common protocols are described: 1) Deposition from aqueous alcohol.
This is the most facile method for preparing silylated surfaces. A
95% ethanol-5% water solution is adjusted to pH 4.5-5.5 with acetic
acid. Silane is added with stirring to yield a 2% final
concentration. After hydrolysis and silanol group formation the
substrate is added for 2-5 min. After rinsed free of excess
materials by dipping briefly in ethanol. Cure of the silane layer
is for 5-10 min at 110 degrees Celsius. 2) Vapor Phase Deposition.
Silanes can be applied to substrates under dry aprotic conditions
by chemical vapor deposition methods. These methods favor monolayer
deposition. In closed chamber designs, substrates are heated to
sufficient temperature to achieve 5 mm vapor pressure.
Alternatively, vacuum can be applied until silane evaporation is
observed. 3) Spin-on deposition. Spin-on applications can be made
under hydrolytic conditions which favor maximum functionalization
and polylayer deposition or dry conditions which favor monolayer
deposition.
[0075] "FRET" or "Forrester, or fluorescence, resonant energy
transfer" means a non-radiative dipole-dipole energy transfer
mechanism from a donor to acceptor fluorophore. The efficiency of
FRET may be dependent upon the distance between donor and acceptor
as well as the properties of the fluorophores (Stryer, L., Annu Rev
Biochem. 47 (1978): 819-846). "FRET distance" means a distance
between a FRET donor and a FRET acceptor over which a FRET
interaction can take place and a detectable FRET signal produced by
the FRET acceptor.
[0076] "Polynucleotide" or "oligonucleotide" are used
interchangeably and each mean a linear polymer of nucleotide
monomers. Monomers making up polynucleotides and oligonucleotides
are capable of specifically binding to a natural polynucleotide by
way of a regular pattern of monomer-to-monomer interactions, such
as Watson-Crick type of base pairing, base stacking, Hoogsteen or
reverse Hoogsteen types of base pairing, or the like. Such monomers
and their internucleosidic linkages may be naturally occurring or
may be analogs thereof, e.g. naturally occurring or non-naturally
occurring analogs. Non-naturally occurring analogs may include
PNAs, phosphorothioate internucleosidic linkages, bases containing
linking groups permitting the attachment of labels, such as
fluorophores, or haptens, and the like. Whenever the use of an
oligonucleotide or polynucleotide requires enzymatic processing,
such as extension by a polymerase, ligation by a ligase, or the
like, one of ordinary skill would understand that oligonucleotides
or polynucleotides in those instances would not contain certain
analogs of internucleosidic linkages, sugar moieties, or bases at
any or some positions. Polynucleotides typically range in size from
a couple or a few monomeric units, e.g. 5-40, when they are usually
referred to as "oligonucleotides," to several thousand monomeric
units. Whenever a polynucleotide or oligonucleotide is represented
by a sequence of letters (upper or lower case), such as "ATGCCTG,"
it will be understood that the nucleotides are in 5'.fwdarw.3'
order from left to right and that "A" denotes deoxyadenosine, "C"
denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes
thymidine, "I" denotes deoxyinosine, "U" denotes uridine, unless
otherwise indicated or obvious from context. Unless otherwise noted
the terminology and atom numbering conventions will follow those
disclosed in Strachan and Read, Human Molecular Genetics 2
(Wiley-Liss, New York, 1999). Usually polynucleotides comprise the
four natural nucleosides (e.g. deoxyadenosine, deoxycytidine,
deoxyguanosine, deoxythymidine for DNA or their ribose counterparts
for RNA) linked by phosphodiester linkages; however, they may also
comprise non-natural nucleotide analogs, e.g. including modified
bases, sugars, or internucleosidic linkages. It is clear to those
skilled in the art that where an enzyme has specific
oligonucleotide or polynucleotide substrate requirements for
activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,
then selection of appropriate composition for the oligonucleotide
or polynucleotide substrates is well within the knowledge of one of
ordinary skill, especially with guidance from treatises, such as
Sambrook et al, Molecular Cloning, Second Edition (Cold Spring
Harbor Laboratory, New York, 1989), and like references. Likewise,
the oligonucleotide and polynucleotide may refer to either a single
stranded form or a double stranded form (i.e. duplexes of an
oligonucleotide or polynucleotide and its respective complement).
It will be clear to one of ordinary skill which form or whether
both forms are intended from the context of the terms usage.
[0077] "Sequence determination", "sequencing" or "determining a
nucleotide sequence" or like terms in reference to polynucleotides
includes determination of partial as well as full sequence
information of the polynucleotide. That is, the terms include
sequences of subsets of the full set of four natural nucleotides,
A, C, G and T, such as, for example, a sequence of just A's and C's
of a target polynucleotide. That is, the terms include the
determination of the identities, ordering, and locations of one,
two, three or all of the four types of nucleotides within a target
polynucleotide. In some embodiments, the terms include the
determination of the identities, ordering, and locations of two,
three or all of the four types of nucleotides within a target
polynucleotide. In some embodiments sequence determination may be
accomplished by identifying the ordering and locations of a single
type of nucleotide, e.g. cytosines, within the target
polynucleotide "catcgc . . . " so that its sequence is represented
as a binary code, e.g. "100101 . . . " representing "c-(not c)(not
c)c-(not c)-c . . . " and the like. In some embodiments, the terms
may also include subsequences of a target polynucleotide that serve
as a fingerprint for the target polynucleotide; that is,
subsequences that uniquely identify a target polynucleotide within
a set of polynucleotides, e.g. all different RNA sequences
expressed by a cell.
[0078] Each of the individual variations and embodiments described
and illustrated herein has discrete components and features which
may be readily separated from or combined with the features of any
of the other variations. Modifications may be made to adapt a
particular situation, material, composition of matter, process,
process act(s) or step(s) to the objective(s), spirit or scope of
the present invention.
[0079] Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as the
recited order of events. Furthermore, where a range of values is
provided, every intervening value between the upper and lower limit
of that range and any other stated or intervening value in that
stated range is encompassed within the invention. Also, any
optional feature of the inventive variations described may be set
forth and claimed independently, or in combination with any one or
more of the features described herein.
[0080] All existing subject matter mentioned herein (e.g.,
publications, patents, patent applications and hardware) is
incorporated by reference herein in its entirety except insofar as
the subject matter may conflict with that of the present invention
(in which case what is present herein shall prevail). The
referenced items are provided solely for their disclosure prior to
the filing date of the present application. Nothing herein is to be
construed as an admission that the present invention is not
entitled to antedate such material by virtue of prior
invention.
[0081] Reference to a singular item, includes the possibility that
there are plural of the same items present. More specifically, as
used herein and in the appended claims, the singular forms "a,"
"an," "said" and "the" include plural referents unless the context
clearly dictates otherwise. It is further noted that the claims may
be drafted to exclude any optional element. As such, this statement
is intended to serve as antecedent basis for use of such exclusive
terminology as "solely," "only" and the like in connection with the
recitation of claim elements, or use of a "negative" limitation.
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 belongs.
[0082] This disclosure is not intended to be limited to the scope
of the particular forms set forth, but is intended to cover
alternatives, modifications, combinations of elements disclosed in
different variations, and equivalents of the variations described
herein. Further, the scope of the disclosure fully encompasses
other variations that may become obvious to those skilled in the
art in view of this disclosure. The scope of the present invention
is limited only by the appended claims.
Sequence CWU 1
1
21116DNAArtificial Sequencepolynucleotide 1aacggccctt cgatctcatt
gaggatgaga ggagagtcaa aggaagaacg aggatgagag 60gagagtgaga gcaaaggaag
aacgaggatg agaggagagt gagagcaaag gaagaa 116291DNAArtificial
Sequencepolynucleotide 2gctatgtggc gcggtattat taagaaggag actgagagga
gagaaggagc aagaaggaaa 60tgagagcgag aggagaagaa ggaggaagaa g 91
* * * * *