U.S. patent application number 17/677844 was filed with the patent office on 2022-06-09 for pyrophosphorolytic sequencing.
The applicant listed for this patent is Illumina Cambridge Limited. Invention is credited to Wouter Meuleman.
Application Number | 20220177960 17/677844 |
Document ID | / |
Family ID | 1000006153595 |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220177960 |
Kind Code |
A1 |
Meuleman; Wouter |
June 9, 2022 |
PYROPHOSPHOROLYTIC SEQUENCING
Abstract
A method for determining the sequence of a target nucleic acid,
including steps of contacting a target nucleic acid with a
polymerase to sequentially remove nucleotide triphosphates from the
target nucleic acid, wherein the nucleotide triphosphates that are
removed have a variety of different base moieties; and
distinguishing the different base moieties for the nucleotide
triphosphates that are removed. Also provided is a apparatus
including a nanopore positioned in a fluid impermeable barrier to
form a passage through which a nucleotide triphosphate can pass
from a first fluid reservoir to a second fluid reservoir, and a
reaction mix in the first fluid reservoir that includes a
polymerase, target nucleic acid having two strands, and
pyrophosphorolytic concentration of pyrophosphate.
Inventors: |
Meuleman; Wouter; (Nr
Saffron Walden, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Illumina Cambridge Limited |
Essex |
|
GB |
|
|
Family ID: |
1000006153595 |
Appl. No.: |
17/677844 |
Filed: |
February 22, 2022 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16657931 |
Oct 18, 2019 |
11286521 |
|
|
17677844 |
|
|
|
|
15847362 |
Dec 19, 2017 |
10450605 |
|
|
16657931 |
|
|
|
|
14286447 |
May 23, 2014 |
|
|
|
15847362 |
|
|
|
|
61827175 |
May 24, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869
20130101 |
International
Class: |
C12Q 1/6869 20060101
C12Q001/6869 |
Claims
1. A apparatus, comprising (a) a fluid impermeable barrier
separating a first fluid reservoir from a second fluid reservoir;
(b) a nanopore positioned in the fluid impermeable barrier to form
a passage through which a nucleotide triphosphate can pass from the
first fluid reservoir to the second fluid reservoir; (c) a reaction
mix in the first fluid reservoir, the reaction mix comprising a
polymerase, a target nucleic acid having two strands, and a
pyrophosphorolytic concentration of pyrophosphate; and (d) at least
one pump fluidly connected to the first fluid reservoir for adding
and/or removing a component of the reaction mix.
2. The apparatus of claim 1, further comprising electrodes
positioned to create difference in potential for the first fluid
reservoir compared to the second fluid reservoir.
3. The apparatus of claim 1, wherein the polymerase is attached to
the nanopore.
4. The apparatus of claim 1, wherein the fluid impermeable barrier
comprises a membrane.
5. The apparatus of claim 4, wherein the nanopore comprises a
protein nanopore that is embedded in the membrane.
6. The apparatus of claim 4, wherein a strand of the target nucleic
acid is attached to the membrane.
7. The apparatus of claim 6, wherein the target nucleic acid
includes at least one base moiety that is non-naturally occurring
in DNA or RNA.
8. The apparatus of claim 1, wherein the nanopore comprises a solid
state nanopore.
9. The apparatus of claim 1, wherein the pyrophosphorolytic
concentration comprises at least 100 .mu.M pyrophosphate.
10. The apparatus of claim 1, wherein the polymerase lacks 3' to 5'
exo nuclease activity.
11. The apparatus of claim 1, wherein the at least one pump
provides pyrophosphate to the first fluid reservoir.
12. The apparatus of claim 1, wherein the target nucleic acid is
DNA.
13. The apparatus of claim 12, wherein the target nucleic acid
comprises at least one base moiety that is non-naturally occurring
in DNA.
14. The apparatus of claim 12, wherein the at least one of the base
moieties comprises 5-methyl cytosine or
5-hydroxymethylcytosine.
15. The apparatus of claim 14, wherein the 5-methyl cytosine or
5-hydroxymethylcytosine is distinguished from cytosine, thereby
facilitating an epigenetic analysis.
16. The apparatus of claim 1, wherein the pump cycles pyrophosphate
into and out of the first fluid reservoir under conditions to pause
pyrophosphorolytic cleavage.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/657,931, filed Oct. 18, 2019, which
application is a continuation of U.S. patent application Ser. No.
15/847,362, filed Dec. 19, 2017, which application is a
continuation of U.S. patent application Ser. No. 14/286,447, filed
May 23, 2014, which application is based on, and claims the benefit
of, U.S. Provisional Application No. 61/827,175, filed May 24,
2013, the disclosure of which are incorporated herein by
reference.
BACKGROUND
[0002] This disclosure relates generally to nucleic acid analysis,
and more specifically to nucleic acid synthesis using
nanopores.
[0003] Currently available commercial platforms for sequencing DNA
are relatively costly. These platforms use a `sequencing by
synthesis` approach, so called because DNA polymers are synthesized
while detecting the addition of each monomer (i.e. nucleotide) to
the growing polymer structure. Because a template DNA strand
strictly directs synthesis of a new DNA polymer, one can infer the
sequence of the template DNA from the series of nucleotide monomers
that were added to the growing strand during the synthesis. The
ability to detect monomer additions is facilitated by specially
engineered variants of the biochemical components that normally
carry out DNA synthesis in biological systems. These engineered
components are expensive to make and are consumed in relatively
large amounts during sequencing by synthesis. Furthermore,
monitoring the reaction uses relatively expensive hardware such as
lasers, detection optics and complex fluid delivery systems. The
most successful commercial platforms to date also require expensive
reagents and hardware to amplify the DNA templates before
sequencing by synthesis can even begin.
[0004] Other sequencing methods have been considered in order to
reduce cost, increase throughput, and/or simplify the process. One
of these approaches is based on threading a single strand of DNA
through a nanopore and identifying its sequence from the variation
in the ionic current flowing through the pore as the strand is
threaded. An alternative to this `nanopore-strand` sequencing
approach is `nanopore-exonuclease` sequencing, which involves
exonuclease catalyzed removal of nucleotide monophosphates, one at
a time, from a DNA strand and sequentially passing the released
nucleotide monophosphates through a nanopore. However, the
resulting variations in the ionic current flowing through the
nanopores are quite small and it is difficult to distinguish one
nucleotide from another. Attempts have been made to modify the DNA
before digestion or to modify the nucleotide monophosphates once
they have been released. However despite these efforts,
nanopore-exonuclease sequencing has not yet been demonstrated at a
commercially viable level to date.
[0005] Thus, there exists a need for more cost effective, rapid and
convenient platforms that provide an alternative to those currently
available for sequencing nucleic acids. The present disclosure
addresses this need and provides other advantages as well.
BRIEF SUMMARY
[0006] The present disclosure provides a method for determining the
sequence of a target nucleic acid. The method can include the steps
of (a) providing a target nucleic acid; (b) contacting the target
nucleic acid with a polymerase to sequentially remove nucleotide
triphosphates from the target nucleic acid, wherein the nucleotide
triphosphates that are removed have a variety of different base
moieties; and (c) distinguishing the different base moieties for
the nucleotide triphosphates that are removed, thereby determining
the sequence of the target nucleic acid.
[0007] In some embodiments a method for determining the sequence of
a target nucleic acid can be carried out using the steps of (a)
providing a target nucleic acid having two strands; (b) contacting
the target nucleic acid with a polymerase under conditions to
sequentially remove nucleotides from the first of the two strands
by pyrophosphorolysis, thereby sequentially producing nucleotide
triphosphates having a variety of different base moieties; and (c)
distinguishing the different base moieties for the sequentially
produced nucleotide triphosphates, thereby determining the sequence
of the target nucleic acid.
[0008] The present disclosure also provides a apparatus that
includes (a) a fluid impermeable barrier separating a first fluid
reservoir from a second fluid reservoir; (b) a nanopore positioned
in the fluid impermeable barrier to form a passage through which a
nucleotide triphosphate can pass from the first fluid reservoir to
the second fluid reservoir; and (c) a reaction mix in the first
fluid reservoir, the reaction mix comprising a polymerase, target
nucleic acid having two strands, and pyrophosphorylitic
concentration of pyrophosphate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows a diagram of a pyrophosphorolytic sequencing
reaction using a polymerase attached to a nanopore.
[0010] FIG. 2 shows a diagram of a pyrophosphorolytic sequencing
reaction using a polymerase attached to a nanopore and a template
nucleic acid attached to a fluid impermeable barrier.
[0011] FIG. 3. shows pyrophosphorolytic sequencing with membrane
seeding of multiple nucleic acid templates.
DETAILED DESCRIPTION
[0012] The present disclosure provides a method of sequencing
nucleic acids in a reverse fashion compared to standard sequencing
by synthesis (SBS) techniques. In particular embodiments, the
method of the present disclosure exploits a catalytic function of
polymerase known as pyrophosphorolysis that is typically maligned
as the culprit for unwanted artifacts in SBS techniques.
Pyrophosphorolysis results in the removal of nucleotide
triphosphates from a nucleic acid strand by a polymerase, and as
such is the reverse of the polymerization reaction that drives
standard SBS techniques.
[0013] Pyrophosphorolysis can be distinguished from exonuclease
activity (which is present in some polymerases), for example, based
on the different catalytic mechanism for the two reactions,
different active sites in the polymerase structure where the two
reactions occur, and the different products for the reactions.
Regarding the catalytic mechanism and active site differences, it
is known that exonuclease activity can be removed from many
polymerase species by deletion of certain domains, whereas
pyrophosphorolysis activity is believed to be catalyzed by the same
domain that catalyzes polymerization. Furthermore, the reaction
cycle catalyzed by exonuclease is the conversion of DNA.sub.n (DNA
of length n) to DNA.sub.n-1 (DNA that is one nucleotide shorter
than DNA of length n) and a nucleotide monophosphate. In contrast,
a cycle of pyrophosphorolysis produces DNA.sub.n-1 and a nucleotide
triphosphate from DNA and pyrophosphate. Notably, pyrophosphate is
consumed in a pyrophosphorolysis reaction but is not consumed in an
exonuclease reaction.
[0014] Particular embodiments of the pyrophosphorolytic sequencing
methods utilize nanopore detection. Nanopores can be used to
sequentially detect the nucleotide triphosphates that are released
from a nucleic acid in order to determine the sequence of the
nucleic acid. Such embodiments provide a combination of advantages
that are typically only partially satisfied by nanopore-exonuclease
sequencing or nanopore-strand sequencing. Specifically, the
pyrophosphorolytic sequencing methods of the present disclosure
address some of the challenges incurred in nanopore-exonuclease
sequencing, namely low capture and detection probabilities, while
retaining its main advantage over strand sequencing, namely single
base resolution. This advantage derives from the fact that the
affinity of nanopores for nucleotide monophosphates is rather weak
(on the order of micromolar affinity), even in the presence of an
am6-cyclodextrin adaptor that has been used to improve signal in
some nanopore systems. See Clarke et al., Nat. Nanotechnol. April;
4(4):265-70 (2009), which is incorporated herein by reference. For
successful distinction of different nucleotide types in a
sequencing context, nanomolar range affinity is desired. ATP has an
affinity that is in a good range, even without the use of an
adaptor in the pore. See Cheley et al., Chem. Biol. 9, 829-838
(2002), which is incorporated herein by reference. Without wishing
to be bound by theory, it is postulated that the improved affinity
of ATP over nucleotide monophosphate is due to the triple negative
charge carried by ATP, which may cause it to bind more strongly
inside the nanopore. Furthermore, the triple negative charge may
aid capturing of the molecule in the presence of an electric field,
especially when the field is very weak, as is the case outside of
the nanopore where the nucleotides are actually released.
[0015] In addition to the enhanced capture and detection of
nucleotide triphosphates, there are a number of other advantages
that can be provided by embodiments set forth herein, such as the
use of a polymerase as opposed to an exonuclease. Polymerases have
been shown to form a good "fit" with nanopores for the purpose of
nanopore-strand sequencing (Cherf et al., Nat. Biotech. 30:344-348
(2012); Manrao et al., Nat. Biotech. 30:349-353 (2012), each of
which is incorporated herein by reference). A similarly good fit is
yet to be demonstrated with exonucleases. Furthermore, the
substrate for polymerases is double stranded DNA which generally
does not enter the nanopore. In contrast, single stranded DNA, the
substrate for most exonucleases, can pose the problem of entering
and blocking the nanopore. Finally, unlike in either
exonuclease-based sequencing or polymerase-based strand sequencing,
the rate of a pyrophosphorolytic sequencing reaction can be
controlled by tuning the pyrophosphate concentration.
[0016] Terms used herein will be understood to take on their
ordinary meaning unless specified otherwise. Examples of several
terms used herein and their definitions are set forth below.
[0017] As used herein, the term "attached" is intended to mean
connected by forces that prevent separation by diffusion. The term
can include connections that are covalent or non-covalent in
nature. For example two proteins can be covalently attached through
their primary sequence (e.g. a peptide linkage or protein fusion)
or between their primary sequences (e.g. a disulfide linkage or
chemical crosslink via amino acid R groups). Two proteins can be
non-covalently attached, for example, via one or more of hydrogen
bonds, ionic bonds, van der Waals forces, hydrophobic bonds or the
like.
[0018] As used herein, the term "each," when used in reference to a
collection of items, is intended to identify an individual item in
the collection but does not necessarily refer to every item in the
collection. Exceptions can occur if explicit disclosure or context
unambiguously dictates otherwise.
[0019] As used herein, the term "exonuclease activity" is intended
to mean hydrolysis of the phosphodiester bond that attaches a
nucleotide to the end of a nucleic acid of length n to produce a
nucleotide monophosphate and a nucleic acid of length n-1. The
hydrolysis can occur at the bond that attaches the 5' nucleotide to
the nucleic acid (i.e. 5' to 3' exonuclease activity) or at the
bond that attaches the 3' nucleotide to the nucleic acid (i.e. 3'
to 5' exonuclease activity).
[0020] As used herein, the term "fluid impermeable barrier" is
intended to mean anything that prevents passage of a fluid. For
example, a fluid impermeable barrier can be present between two
reservoirs such that a fluid in the first reservoir is separated
from the fluid in the second reservoir, and the fluids do not mix.
A fluid impermeable barrier can include a pore or opening that
allows passage of a fluid that is otherwise obstructed by the
remainder of the barrier. In particular embodiments, the fluid can
be an aqueous fluid and the barrier can be impermeable to aqueous
fluids.
[0021] As used herein, the term "lacks exonuclease activity" is
intended to mean having no measurable exonuclease activity. For
example, a polymerase or other agent that lacks 3' to 5'
exonuclease activity will have no measurable 3' to 5' exonuclease
activity. Similarly, a polymerase or other agent that lacks 5' to
3' exonuclease activity will have no measurable 5' to 3'
exonuclease activity. Polymerases known in the art as "exo minus"
(or "exo-") whether naturally occurring or engineered are
non-limiting examples of polymerases that lack exonuclease
activity. Known variants include those that are 5' to 3' exo minus
and/or 3' to 5' exo minus.
[0022] As used herein, the term "nanopore" is intended to mean a
small hole that allows passage of nucleotide triphosphates across
an otherwise impermeable barrier. The barrier is typically an
electrically insulating layer and the nanopore typically permits
ions to flow from one side of the barrier to the other, driven by
an applied potential. The nanopore preferably permits nucleotides
to flow through the nanopore lumen along the applied potential. The
nanopore may also allow a nucleic acid, such as DNA or RNA, to be
pushed or pulled through the lumen of the nanopore. However, in
particular embodiments the nanopore need not allow passage of a
double stranded or single stranded nucleic acid. A nanopore used in
a particular embodiment can have a minimum lumen diameter of no
more than 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm or less. One
type of nanopore is a "protein nanopore" which is a polypeptide or
a collection of polypeptides that forms the small hole to allow
passage of nucleotide triphosphates across a barrier such as a
lipid bilayer. Examples of protein nanopores include alpha
hemolysin nanopore, mycobacterium smegmatis porin A (MspA) and
variants thereof. Another type of nanopore is a "solid state
nanopore" which is a small hole fabricated through a solid
material. The solid material can be, for example, graphene or
silicon.
[0023] As used herein the term "nucleotide" is intended to include
ribonucleotides, deoxynucleotides or analogs thereof. For example
the term is used herein to generally refer to a nucleoside moiety
(whether ribose, deoxyribose, or analog thereof) including a base
moiety and optionally attached to one or more phosphate moieties.
Exemplary nucleotides include, but are not limited to,
ribonucleotide monophosphate (sometimes referred to as a
ribonucleoside monophosphate), ribonucleotide diphosphate
(sometimes referred to as a ribonucleoside diphosphate),
ribonucleotide triphosphate (sometimes referred to as a
ribonucleoside triphosphate), deoxynucleotide monophosphate
(sometimes referred to as a deoxynucleoside monophosphate),
deoxynucleotide diphosphate (sometimes referred to as a
deoxynucleoside diphosphate) and deoxynucleotide triphosphate
(sometimes referred to as a deoxynucleoside triphosphate). For
clarity when wishing to distinguish RNA components from DNA
components, the term "ribonucleotide" can be used to specify RNA
nucleotides, such as ribouridine triphosphate, riboguanidine
triphosphate, ribocytidine triphosphate or riboadenosine
triphosphate; and the term "deoxynucleotide" can be used to specify
DNA nucleotides, such as deoxythymidine triphosphate,
deoxyguanidine triphosphate, deoxycytidine triphosphate and
deoxyadenosine triphosphate. In particular embodiments, the
nucleotides are `extendable`, for example, lacking an extension
blocking moiety at the 3' hydroxyl or at any other position on the
nucleotide.
[0024] As used herein, the term "pyrophosphorolysis" is intended to
mean reaction between pyrophosphate and the 3'-nucleotide unit of a
nucleic acid to release the nucleotide in the form of the
corresponding nucleotide triphosphate. A further product of the
reaction is the nucleic acid lacking the corresponding nucleotide
(i.e. the reaction converts a nucleic acid of length n to a nucleic
acid of length n-1). The reaction is typically catalyzed by a
polymerase and is the reverse of the polymerization reaction
carried out by the polymerase under standard biological
conditions.
[0025] As used herein, the term "pyrophosphorolytic concentration,"
when used in reference to pyrophosphate, is intended to mean a
concentration of pyrophosphate that causes a pyrophosphorolysis
reaction to occur at a substantial level. For example, a
pyrophosphorylitic concentration of pyrophosphate can result in a
polymerase displaying a higher rate of pyrophosphorolysis than
polymerization. Thus, a pyrophosphorylitic concentration of
pyrophosphate can result in a substantial reversal of
polymerization activity that would otherwise be catalyzed by a
polymerase.
[0026] As used herein, the term "reservoir" is intended to mean a
receptacle or chamber for holding or restricting the flow of fluid.
A reservoir can be fully enclosed, at least temporarily.
Alternatively, a reservoir can be partially enclosed, for example,
having one or more opening (e.g. one or more inlets or outlets). A
fluid may flow through a reservoir, for example, in embodiments
where the reservoir is in a flow cell.
[0027] As used herein, the term "species" is used to identify
molecules that share the same chemical structure. For example, a
mixture of nucleotides can include several dCTP molecules. The dCTP
molecules will be understood to be the same species as each other.
Similarly, individual DNA molecules that have the same sequence of
nucleotides are the same species.
[0028] The embodiments set forth below and recited in the claims
can be understood in view of the above definitions.
[0029] The present disclosure provides a method for determining the
sequence of a target nucleic acid. The method can include the steps
of (a) providing a target nucleic acid; (b) contacting the target
nucleic acid with a polymerase to sequentially remove nucleotide
triphosphates from the target nucleic acid, wherein the nucleotide
triphosphates that are removed have a variety of different base
moieties; and (c) distinguishing the different base moieties for
the nucleotide triphosphates that are removed, thereby determining
the sequence of the target nucleic acid.
[0030] In some embodiments a method for determining the sequence of
a target nucleic acid can be carried out using the steps of (a)
providing a target nucleic acid having two strands; (b) contacting
the target nucleic acid with a polymerase under conditions to
sequentially remove nucleotides from the first of the two strands
by pyrophosphorolysis, thereby sequentially producing nucleotide
triphosphates having a variety of different base moieties; and (c)
distinguishing the different base moieties for the sequentially
produced nucleotide triphosphates, thereby determining the sequence
of the target nucleic acid.
[0031] An exemplary embodiment is shown in FIG. 1. As shown, a
polymerase binds to a double stranded DNA molecule and, in the
presence of excess pyrophosphate, catalyzes a pyrophosphorolysis
reaction to release nucleotide triphosphates from the 3' end of one
of the strands. In this example, a deoxyguanidine triphosphate has
been produced followed by a deoxythymidine triphosphate. The
polymerase is coupled to a nanopore and the deoxyguanidine
triphosphate is in the lumen of the nanopore, whereas the
deoxythymidine triphosphate is in the process of being released
into the nanopore lumen. As such the deoxynucleotide triphosphates
enter the nanopore sequentially, in the same order that they were
released from the DNA strand by the pyrophosphorolytic action of
the polymerase. The deoxynucleotide triphosphates have a net
negative charge due to the triphosphates and are driven through the
nanopore by a potential across the membrane (as indicated by the
positive sign on the side of the membrane where pyrophosphorolysis
occurs and a negative sign on the opposite side of the membrane).
Typically, reagent nucleotide triphosphates are not present in a
pyrophosphorolysis reaction. In some cases, trace amounts of
nucleotide triphosphates can be present, but at amounts that do not
substantially catalyze a forward primer extension reaction by
polymerase. Thus, in most embodiments the only nucleotide
triphosphates that are substantially present in a
pyrophosphorolysis reaction are those produced by the catalytic
action of the polymerase on the nucleic acid.
[0032] A similar reaction is exemplified in FIG. 2 except that the
template strand (i.e. the strand that is not being cleaved by
pyrophosphorolysis) is bound to the membrane. In this case, the
template strand has a covalently attached sterol moiety (e.g.
cholesterol) that binds to the hydrophobic interior of the
membrane's lipid bilayer.
[0033] A method of the present disclosure can be used with any of a
variety of target nucleic acids. The target nucleic acid can have a
naturally occurring structure as found for example in DNA or RNA.
DNA naturally contains monomers having thymine, guanine, cytosine,
or adenine bases. A DNA strand that is subjected to
pyrophosphorolysis can produce deoxythymidine triphosphate,
deoxyguanidine triphosphate, deoxycytidine triphosphate and
deoxyadenosine triphosphate, respectively. DNA can also contain
some variants of the four nucleotide bases such as 5-methyl
cytosine, 5-hydroxymethylcytosine and other methylated bases.
Deoxynucleotide triphosphates having these variant bases can be
produced and/or detected using a method or apparatus set forth
herein. For example, the presence or absence of methylation on
cytosine can be distinguished to facilitate epigenetic analyses.
RNA naturally contains monomers having uracil, guanine, cytosine,
or adenine bases. An RNA strand that is subjected to
pyrophosphorolysis can produce ribouridine triphosphate,
riboguanidine triphosphate, ribocytidine triphosphate or
riboadenosine triphosphate, respectively.
[0034] A nucleic acid can include non-naturally occurring
modifications such as non-native bases, modifications to the
phosphate moieties or modifications to the sugar moieties.
Exemplary non-native bases that can be included in a nucleic acid,
whether having a native backbone or analog structure, include,
without limitation, inosine, xathanine, hypoxathanine, isocytosine,
isoguanine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine,
2-propyl guanine, 2-propyl adenine, 2-thioLiracil, 2-thiothymine,
2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil,
5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine,
5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine
or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or
guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil
or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine,
8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,
3-deazaadenine or the like.
[0035] Non-native bases can be selected, for example, to impart
larger or smaller size, or to impart increased or decreased charge,
so as to influence the ability of the resulting nucleotide
triphosphate analogs to be distinguished by a nanopore or other
detection component. Similarly, such bases can be beneficial if
selected to impart a desired rate of pyrophosphorolysis. Non-native
bases can be incorporated into a nucleic acid using known methods
such as amplification or replication of a template nucleic acid in
the presence of the nucleotide analogs. One or more of the
resulting nucleic acid copies can then be used as target nucleic
acid(s) in apparatus or sequencing method set forth herein.
[0036] In particular embodiments, a nucleic acid that is used in a
method or apparatus herein will lack one or more of the non-native
bases or other non-native moieties set forth herein. For example,
in particular embodiments the methods are not used to remove a
terminator nucleotide from the 3' end of a nucleic acid strand.
Accordingly, in some embodiments, an apparatus or method may not
include any nucleic acid(s) having a terminator nucleotide at its
3' end. Alternatively or additionally, in some embodiments, an
apparatus or method may not include any terminator
nucleotide(s).
[0037] As exemplified in FIG. 1 and elsewhere herein, a target
nucleic acid can be double stranded DNA, for example, when using a
DNA polymerase for pyrophosphorolysis. A heteroduplex, formed
between a DNA strand and RNA strand, can also be used. For example,
an RNA polymerase can be used to catalyze pyrophosphorolysis at the
3' end of an RNA strand that is hybridized to a DNA template
strand, thereby producing ribonucleotide triphosphates.
[0038] A nucleic acid that is used in a method or apparatus herein
can be isolated from a biological source and used directly or
processed to produce an amplified or modified product.
Alternatively a synthetic nucleic acid can be used, again, directly
or after processing. Processing can include, for example, one or
more of isolation from native components, cleavage to form
fragments, amplification (e.g. via PCR, Rolling Circle or other
known techniques), ligation of adapter sequences or tag sequences,
tagmentation using a transposon, or "sample preparation" methods
used prior to nucleic acid sequencing techniques. Useful processing
techniques are known in the art as set forth, for example, in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd
edition, Cold Spring Harbor Laboratory, New York (2001) or in
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1998), each of which is incorporated
herein by reference.
[0039] Examples of sample preparation methods that can be used to
process nucleic acids prior to pyrophosphorolysis-based sequencing
include methods that have been developed for whole genome
amplification or whole exome amplification in combination with
massively parallel sequencing techniques. For example, commercially
available sample preparation techniques from Illumina, Inc. (San
Diego, Calif.), Life Technologies (Carlsbad, Calif.), 454 Life
Sciences (a subsidiary of Roche, Basel, Switzerland) or New England
Biolabs (Ipswich, Mass.) can be used. Further useful sample
preparation methods that can be used are described, for example, in
U.S. Pat. Nos. 6,107,023 or 7,741,463; or US Pat. App. Pub. No.
2010/0120098 A1, each of which is incorporated herein by reference.
Targeted sample preparation methods can be used as well in order to
isolate a subset of the sequence content of a complex sample for
subsequent sequencing. Exemplary commercial methods that can be
used for targeted capture of a subset of nucleic acid fragments
include, but are not limited to SureSelect.TM.kits (Agilent, Santa
Clara, Calif.), TruSeq Enrichment Kits (Illumina, Inc., San Diego,
Calif.) or Nextera.RTM. Enrichment Kits (Illumina, Inc., San Diego,
Calif.).
[0040] Nucleic acids can be isolated from any of a variety of
sources including, without limitation, a mammal such as a rodent,
mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat,
cow, cat, dog, primate, human or non-human primate; a plant such as
Arabidopsis thaliana, corn (Zea mays), sorghum, oat (oryza sativa),
wheat, rice, canola, or soybean; an algae such as Chlamydomonas
reinhardtii; a nematode such as Caenorhabditis elegans; an insect
such as Drosophila melanogaster, mosquito, fruit fly, honey bee or
spider; a fish such as zebrafish (Danio rerio); a reptile; an
amphibian such as a frog or Xenopus laevis; a dictyostelium
discoideum; a fungi such as pneumocystis carinii, Takifugu
rubripes, yeast, Saccharamoyces cerevisiae or Schizosaccharomyces
pombe; or a plasmodium falciparum. Nucleic acids can also be
derived from smaller genomes such as those from a prokaryote such
as a bacterium, Escherichia coli, staphylococci or mycoplasma
pneumoniae; an archae; a virus such as Hepatitis C virus or human
immunodeficiency virus; or a viroid.
[0041] Any of a variety of polymerases can be used in a method or
apparatus set forth herein including, for example, protein-based
enzymes isolated from biological systems and functional variants
thereof. Reference to a particular polymerase, such as those
exemplified below, will be understood to include functional
variants thereof unless indicated otherwise. Examples of useful
polymerases include DNA polymerases and RNA polymerases. Exemplary
DNA polymerases include those that have been classified by
structural homology into families identified as A, B, C, D, X, Y,
and RT. DNA Polymerases in Family A include, for example, T7 DNA
polymerase, eukaryotic mitochondrial DNA Polymerase .gamma., E.
coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus
stearothermophilus Pol I. DNA Polymerases in Family B include, for
example, eukaryotic DNA polymerases .alpha., .delta., and
.epsilon.; DNA polymerase .zeta.; T4 DNA polymerase, Phi29 DNA
polymerase, and RB69 bacteriophage DNA polymerase. Family C
includes, for example, the E. coli DNA Polymerase III alpha
subunit. Family D includes, for example, polymerases derived from
the Euryarchaeota subdomain of Archaea. DNA Polymerases in Family X
include, for example, eukaryotic polymerases Pol .beta., pol
.sigma., Pol .lamda., and Pol .mu., and S. cerevisiae Pol4. DNA
Polymerases in Family Y include, for example, Pol .eta., Pol iota,
Pol kappa, E. coli Pol IV (DINB) and E. coli Pol V (UmuD'2C). The
RT (reverse transcriptase) family of DNA polymerases includes, for
example, retrovirus reverse transcriptases and eukaryotic
telomerases. Exemplary RNA polymerases include, but are not limited
to, viral RNA polymerases such as T7 RNA polymerase; Eukaryotic RNA
polymerases such as RNA polymerase I, RNA polymerase II, RNA
polymerase III, RNA polymerase IV, and RNA polymerase V; and
Archaea RNA polymerase.
[0042] The above classifications are provided for illustrative
purposes. It will be understood that variations in the
classification system are possible. For example, in at least one
classification system Family C polymerases have been categorized as
a subcategory of Family X. Furthermore, polymerases can be
classified according to other characteristics, whether functional
or structural, that may or may not overlap with the structural
characteristics exemplified above. Some exemplary characteristics
are set forth in further detail below.
[0043] Many polymerases have an intrinsic 3' to 5' proofreading
exonuclease activity which can be useful for some embodiments.
Polymerases that substantially lack 3' to 5' proofreading
exonuclease activity are preferred in some embodiments, for
example, in most sequencing embodiments. Absence of exonuclease
activity can be a wild type characteristic or a characteristic
imparted by a variant or engineered polymerase. For example, exo
minus Klenow fragment is a mutated version of Klenow fragment that
lacks 3' to 5' proofreading exonuclease activity. Klenow fragment
and its exo minus variant can be useful in a method or apparatus
set forth herein. Polymerases that catalyze pyrophosphorolysis, the
direct reversal of polymerization in the same active site, are
particularly useful.
[0044] Depending on the embodiment that is to be used, a polymerase
can be either thermophilic or heat inactivatable. Thermophilic
polymerases are typically useful for high temperature conditions or
in thermocycling conditions such as those employed for polymerase
chain reaction (PCR) techniques. Examples of thermophilic
polymerases include, but are not limited to 9.degree. N DNA
Polymerase, Taq DNA polymerase, Phusion.RTM. DNA polymerase, Pfu
DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase, and
VentR.RTM. DNA polymerase. Most polymerases isolated from
non-thermophilic organisms are heat inactivatable. Heat
inactivation can be useful to stop a sequencing reaction set forth
herein. The reaction can optionally be re-started by adding a new
supply of polymerase to the reaction at the appropriately
permissive temperature. Examples of heat inactivatable polymerases
are those from phage. It will be understood that polymerases from
any of a variety of sources can be modified to increase or decrease
their tolerance to high temperature conditions.
[0045] An engineered variant of a polymerase having increased
pyrophosphorolysis activity can be used. Exemplary variants are
those that have been created for use in PCR techniques including,
but not limited to the variants described in U.S. Pat. No.
8,071,536, which is incorporated herein by reference.
[0046] A polymerase can be induced to sequentially remove
nucleotides from one of two nucleic acid strands by
pyrophosphorolysis in a method set forth herein. The polymerase can
be placed under conditions for pyrophosphorolysis to occur. For
example, the polymerase can be contacted with a double stranded
nucleic acid and a pyrophosphorolytic concentration of
pyrophosphate. Any concentration of pyrophosphate that causes a
pyrophosphorolysis reaction to occur at a substantial level can be
used including, but not limited to, at least about 100 .mu.M
pyrophosphate. Higher concentrations of pyrophosphate can be
employed, for example, to drive pyrophosphorolysis at a faster
rate. Accordingly, a concentration of at least about 250 .mu.M
pyrophosphate, at least about 500 .mu.M pyrophosphate, at least
about 750 .mu.M pyrophosphate, at least about 1 mM pyrophosphate,
at least about 2 mM pyrophosphate, at least about 5 mM
pyrophosphate, at least about 10 mM pyrophosphate, at least about
20 mM pyrophosphate or higher can be used.
[0047] The ability to alter the rate of pyrophosphorolysis is an
advantage for tuning the rate of the sequencing reaction, for
example, to accommodate an optimal or desired rate of nucleotide
triphosphate detection for a given detection device. For example,
the rate of pyrophosphorolysis can be decreased by using a lower
concentration of pyrophosphate than those set forth above. Thus, as
an alternative or addition to the threshold concentrations set
forth above, a maximum concentration of pyrophosphate present in a
apparatus or method set forth herein can be at most about 20 mM
pyrophosphate, at most about 10 mM pyrophosphate, at most about 5
mM pyrophosphate, at most about 2 mM pyrophosphate, at most about 1
mM pyrophosphate, at most about 750 .mu.M pyrophosphate, at most
about 500 .mu.M pyrophosphate, at most about 250 .mu.M
pyrophosphate, at most about 100 .mu.M pyrophosphate, or less.
[0048] Reagent nucleotide triphosphates are typically absent under
pyrophosphorolysis conditions. Thus, in most embodiments the only
nucleotide triphosphates that are substantially present in a
pyrophosphorolysis reaction are those produced by the catalytic
action of the polymerase on the nucleic acid. If nucleotide
triphosphates are present under pyrophosphorolysis conditions, the
nucleotide triphosphates will be present at what can be considered
a non-catalytic concentration. A non-catalytic concentration of
nucleotide triphosphate is a concentration that is insufficient to
allow substantial or detectable polymerase extension activity to
occur. For example, a non-catalytic concentration of nucleotide
triphosphate is a concentration that is substantially below the
association binding constant for binding of the nucleotide
triphosphates to polymerase. Accordingly, pyrophosphorolysis can be
carried out by contacting a polymerase with a double stranded
nucleic acid in the presence of a pyrophosphorolytic concentration
of pyrophosphate and no more than a non-catalytic concentration of
nucleotide triphosphate.
[0049] Exemplary conditions for inducing pyrophosphorolysis that
can be used herein are set forth in Patel et al. Biochem.
30:511-525 (1991), or U.S. Pat. Nos. 7,090,975; 7,914,995 or
8,071,536, each of which is incorporated herein by reference. In
several cases these references describe reactions that also include
components used for extension of a nucleic acid. It will be
understood that extension is not utilized in particular embodiments
of the present disclosure. One or more of the components that are
described in Patel et al. Biochem. 30:511-525 (1991) or U.S. Pat.
Nos. 7,090,975; 7,914,995 or 8,071,536, including, for example,
components used for polymerase extension reaction, can be excluded
from a method or apparatus set forth herein.
[0050] Buffers, salts, metal ions, glycerol, DMSO and other
components typically included in polymerase storage or reaction
mixtures can be included in a apparatus or method of the present
disclosure, as desired. The quantities and amounts of components to
be included will be apparent to those skilled in the art or
determinable, for example, via routine titration techniques.
[0051] A beneficial aspect of some embodiments is the ability to
stop or pause the sequencing method by altering the reaction
conditions to inhibit pyrophosphorolysis. The sequencing method can
then optionally be restarted by altering the reaction conditions to
allow pyrophosphorolysis to resume. Any of a variety of the
reaction conditions set forth herein, or in the references cited
herein, can be altered between paused pyrophosphorolysis and
resumed pyrophosphorolysis, thereby allowing an effective toggle
between paused and active sequencing, respectively.
[0052] In particular embodiments, a method of the present
disclosure can include a step of pausing the sequential production
of nucleotide triphosphates by removing pyrophosphate from contact
with a polymerase that is catalyzing pyrophosphorolysis, followed
by a step of resuming the sequential production of the nucleotide
triphosphates by contacting the polymerase with pyrophosphate.
Pyrophosphate can be delivered to a reaction using techniques
appropriate for the fluid system being used including, for example,
pipetting fluid aliquots, movement of fluid boluses under positive
or negative pressure (e.g. via pumps or gravity), electrophoresis,
isotachophoresis, droplet manipulation (e.g. electrowetting) or the
like. Similar fluidic techniques can be used to remove
pyrophosphate, for example, by displacement and/or replacement with
a wash solution. Of course, such fluidic techniques can be used to
add or remove other components used in the methods and apparatus
set forth herein.
[0053] Alternatively or additionally to the fluidic methods set
forth above, pyrophosphate can be removed from the reaction by
sequestration, degradation or inactivation. For example, physical
manipulations can be used such as adsorption to a sequestering
agent, or degradation by heat, light or electricity. Chemical
methods can be used to modify the structure or activity of
pyrophosphate or to degrade the molecule. Enzymatic methods can
also be used such as degradation by pyrophosphatase, as shown for
PCR reactions in U.S. Pat. Nos. 4,800,159 and 5,498,523 and for gel
based sequencing reactions in U.S. Pat. No. 4,962,020, each of
which is incorporated herein by reference.
[0054] Alternatively or additionally to the techniques set forth
above, pyrophosphorolysis can be stopped or paused by removing
other reaction components. For example, polymerase can be removed
from a reaction and optionally replaced or returned to an active
state. For example, polymerase can be removed by fluidic,
sequestration, degradation or inactivation methods such as those
exemplified above for pyrophosphate. In particular embodiments, a
heat sensitive (non-thermophilic) polymerase can be used in a
pyrophosphorolysis reaction and then heat inactivated. Similarly, a
polymerase can be degraded by chemical modification or enzymatic
degradation (e.g. via a protease). Whether degraded by physical,
chemical or enzymatic techniques, the spent polymerase can be
washed away and then pyrophosphorolysis can be resumed by addition
of new polymerase to the nucleic acid being sequenced. Polymerase
activity can also be toggled by addition and removal of inhibitors,
toggling between permissive and non-permissive temperatures for
heat stable polymerases, or presence and absence of a sequestering
agent or competitive substrate. Pyrophosphorolysis can also be
stopped and started by denaturation and renaturation, respectively,
of the nucleic acid that is being sequenced.
[0055] Although methods and apparatus have been exemplified herein
for embodiments that use pyrophosphate to drive pyrophosphorolysis,
it will be understood that analogs of pyrophosphate can be used
instead. An exemplary analog is pyrovanadate, which can be used,
for example, as described in Akabayov et al. J. Biol. Chem.
286:29146-29157 (2011), which is incorporated herein by reference.
As further examples, analogs of pyrophosphate having additional
moieties can be used. Generally pyrophosphate analogs are selected
that do not entirely inhibit pyrophosphorolysis or passage of the
resulting nucleotide triphosphates, or analogs thereof, through a
nanopore. However, pyrophosphate analogs can alter characteristics
of pyrophosphorolysis and/or nanopore detection. For example, it
may be beneficial to use a pyrophosphate analog to slow down or
speed up pyrophosphorolysis to provide a desired or optimal
detection rate. Similarly, analogs of nucleotide triphosphates that
result when a pyrophosphate analog is used in a pyrophosphorolysis
reaction can also impart desired characteristics for nanopore
detection. For example, moieties that alter charge or size,
compared to diphosphate alone, can increase or decrease the rate of
passage of nucleotide triphosphate analogs through a nanopore, or
otherwise alter interactions of the nucleotide triphosphate analogs
with the nanopore, to provide improved sequencing results.
[0056] However, in some embodiments a method or apparatus of the
present disclosure will exclude pyrophosphate having any added
moieties. For example, pyrophosphate that lacks an optically
detectable moiety, such as a fluorescent moiety, can be used.
[0057] In particular embodiments, nucleotide triphosphates are
detected using nanopores. For example, nucleotide triphosphates
that are sequentially removed from a nucleic acid via
pyrophosphorolysis can be passed through a nanopore for detection.
By use of an appropriate nanopore, different base moieties of the
nucleotide triphosphates can be distinguished to allow sequence
detection. Generally an apparatus can be used that includes a first
and a second compartment separated by a physical barrier, wherein
the barrier has one or more nanopores. The first compartment can
include components used for a pyrophosphorolysis reaction. The
apparatus can be configured to apply an electric field across the
barrier so that nucleotide triphosphates are driven from the first
compartment through the pore to the second compartment. The
apparatus can be configured for measuring the electronic signature
of a nucleotide triphosphate passing through the nanopore.
Accordingly, a useful apparatus can include an electrical circuit
capable of applying a potential and measuring an electrical signal
across a barrier and nanopore. The methods may be carried out using
a patch clamp or a voltage clamp.
[0058] A method of the present disclosure can be carried out using
any suitable system in which a pore penetrates through a barrier.
The barrier in many embodiments is preferably a lipid bilayer.
Lipid bilayers can be made using methods known in the art, for
example, as described in Montal and Mueller Proc. Natl. Acad. Sci.
USA 69:3561-3566 (1972) or WO 2008/102120, each of which is
incorporated herein by reference. Lipid bilayers can be formed from
any of a variety of lipids including, but not limited to,
phospholipids, glycolipids, cholesterol and mixtures thereof.
[0059] Exemplary nanopores that can be used include, for example,
protein based nanopores such as alpha hemolysin nanopore,
mycobacterium smegmatis porin A (MspA) and variants thereof. Alpha
hemolysin nanopore and variants of the native nanopore that are
particularly useful are described, for example, in US Pat. App.
Pub. No. 2011/0229877 A1, or U.S. Pat. Nos. 6,916,665; 7,867,716;
7,947,454; or 8,105,846, each of which is incorporated herein by
reference. MspA and variants of the native nanopore that are
particularly useful are described, for example, in US Pat. App.
Pub. No. 2012/0055792 A1, which is incorporated herein by
reference. Solid state nanopores can also be useful including, for
example, those described in U.S. Pat. Nos. 6,413,792; 7,444,053; or
7,582,490, each of which is incorporated herein by reference.
[0060] Detection of nucleotide triphosphates can exploit
interaction with a nanopore that results in changes to the current
flowing through the nanopore in a manner that is specific to each
species of nucleotide triphosphate. For example, a first nucleotide
triphosphate species may reduce the current flowing through the
nanopore for a particular mean time period or to a particular
extent. A second species of nucleotide triphosphate can be
distinguished by virtue of a different mean time period or a
different extent of current alteration when passing through the
nanopore. Thus, different nucleotide triphosphate species can be
distinguished based on distinctive alterations of the current
flowing through a nanopore.
[0061] Nanopore detection can be carried out using any of a variety
of apparatus known in the art including for example, those
described in US Pat. App. Pub. Nos. 2011/0229877 A1; or
2012/0055792 A1; or U.S. Pat. No. 6,413,792; 6,916,665; 7,867,716;
7,444,053; 7,582,490; 7,947,454; or 8,105,846, each of which is
incorporated herein by reference.
[0062] A polymerase that is used in an apparatus or method set
forth herein can be present in solution such that it is relatively
free to diffuse, at least within a reaction chamber or it can be
relatively limited in its ability to diffuse by being attached to a
solid phase support, nanopore, barrier or other component of a
method or apparatus set forth herein. Limiting diffusion by
attachment can provide an advantage of closely coupling the point
of nucleotide triphosphate production (e.g. a polymerase catalyzing
pyrophosphorolysis) with the point of nucleotide triphosphate
detection (e.g. a nanopore through which the nucleotide
triphosphates pass). A polymerase can be attached to a nanopore for
example via a recombinant protein fusion to a subunit of a
nanopore, chemical crosslinkage or adapter moiety. Useful methods
for attaching polymerases to nanopores and polymerase-nanopore
components are set forth, for example, in US Pat. App. Pub. Nos.
2011/0229877 A1; or 2012/0055792 A1; or U.S. Pat. No. 7,947,454,
each of which is incorporated herein by reference.
[0063] A polymerase can be attached to a bead or other solid
support that resides in a chamber where pyrophosphorolysis occurs.
Chemical linkers that are useful for attaching polymerases to beads
or solid supports include those that are commercially available,
for example, from Thermo Fisher (Rockford, Ill.) or Sigma Aldrich
(St. Louis, Mo.) or otherwise known in the art.
[0064] A polymerase can be attached to a barrier used in a nanopore
sequencing apparatus. For example, in embodiments that use a lipid
bilayer as the barrier, a lipophilic moiety can be attached to the
polymerase to localize the polymerase in proximity to the bilayer
due to interactions between the bilayer and lipophilic moiety.
Exemplary lipophilic moieties include, but are not limited to,
sterols or lipids. A further example of a lipophilic moiety is a
membrane protein (or portion thereof) that can be attached to a
polymerase, for example, via recombinant protein fusion. Linkages
such as those set forth above for beads and other solid supports
can be used to attach a polymerase to a barrier used in solid state
nanopore systems.
[0065] A nucleic acid that is sequenced in a method set forth
herein or present in a apparatus of the present disclosure can be
in solution such that it is relatively free to diffuse or it can be
relatively limited in its ability to diffuse by being attached to a
solid phase support, nanopore, barrier or other component of a
method or apparatus set forth herein. Attachments similar to those
set forth above for polymerases can be used for nucleic acids. For
example, a sterol, lipid or other lipophilic moiety can be attached
to a nucleic acid to localize it to a lipid bilayer. An example is
shown in FIG. 2, where the nucleic acid is localized to the
membrane via a sterol moiety attached to the template strand. As
exemplified by the figure, the nucleic acid can be attached via the
template strand, for example, at the 5' end of the template strand.
Attachment can also be made at a point on the template that is
between the location where the polymerase is bound to the template
and the 5' end of the template.
[0066] A lipophilic moiety can be attached to a nucleic acid using
methods known in the art for attaching other moieties such as
biotin or fluorophores. For example, a primer having the lipophilic
moiety can be used in an amplification, primer extension, or
ligation reaction. Alternatively or additionally, nucleotides
having the moiety can be used in an extension or amplification
reaction. If desired, a lipophilic moiety can be introduced prior
to sequencing and during a sample preparation step, such as those
set forth previously herein. For example, a targeted sequencing
technique can be employed wherein a subset of target nucleic acid
having desired sequences are to be selected from a more complex
sequence background. In this example, a lipophilic moiety can be
selectively introduced into the subset of target nucleic acids
using the targeting technique and this can allow the targets to be
selectively captured by a lipid bilayer, while other non-targeted
sequences are washed away due to not having been modified to
include the lipophilic moiety.
[0067] It can be beneficial in some embodiments to limit diffusion
of both the polymerase and the nucleic acid with respect to a
nanopore, for example, using one or more of the attachment means
set forth above.
[0068] As set forth previously herein, a method of the present
disclosure can include a step of contacting a target nucleic acid
with a polymerase under conditions to sequentially remove
nucleotides, thereby sequentially producing nucleotide
triphosphates having a variety of different base moieties. The
variety of different base moieties produced will depend on the
content of the target nucleic acid that is contacted with the
polymerase. For example, DNA typically includes the four common
bases guanine, cytosine, adenine and thymine such that
pyrophosphorolysis will produce deoxyguanidine triphosphate,
deoxycytidine triphosphate, deoxyadenosine triphosphate and
deoxythymidine triphosphate. In some cases the target nucleic acid
may not include all four of these base types such that no more than
3, 2 or even 1 type of deoxynucleotide triphosphate will be
produced by pyrophosphorolysis. In some cases, variants of one or
more of these four base types can be present in the target DNA and
accordingly pyrophosphorolysis can produce variant deoxynucleotide
triphosphates having, for example, methyl, hydroxymethyl or other
added moieties. Other variant bases known in the art such as those
set forth herein can also be present in the deoxynucleotide
triphosphates produced by pyrophosphorolysis.
[0069] Another example is RNA, which typically includes the four
common bases guanine, cytosine, adenine and uracil such that
pyrophosphorolysis will produce riboguanidine triphosphate,
ribocytidine triphosphate, riboadenosine triphosphate and
ribothymidine triphosphate. In some cases the target nucleic acid
may not include all four of these base types such that no more than
3, 2 or even 1 type of ribonucleotide triphosphate will be produced
by pyrophosphorolysis. Variant bases, such as those exemplified
herein, for example, with respect to deoxynucleotide triphosphates,
or otherwise known in the art, can be present in ribonucleotide
triphosphates. Generally, the nucleotide triphosphates produced by
pyrophosphorolysis (whether deoxynucleotide triphosphates or
ribonucleotide triphosphates) will include one, at least two, at
least three, at least four or more different base types.
[0070] A method of the present disclosure can be carried out under
conditions that sequentially remove a number of nucleotides from a
target nucleic acid, thereby sequentially producing that same
number of nucleotide triphosphates. Furthermore, at least that same
number of nucleotide triphosphates can be distinguished, for
example, via passage through a nanopore, to allow determination of
a sequence having a length that is at least equivalent to the
number nucleotides removed from the target nucleic acid. In
particular embodiments the number is at least 1, 2, 3, 4, 5, 10,
25, 50, 100, 200, 500, 1000, 10,000 or more up to and including the
length of the target nucleic acid. Alternatively or additionally,
the number may be no more than 1, 2, 3, 4, 5, 10, 25, 50, 100, 200,
500, 1000, or 10,000. The number may be, but need not be, between
any two of these values. As set forth previously herein, a variety
of techniques can be used to pause pyrophosphorolysis. This can
provide for control of the length of sequence determined using
embodiments of the present methods.
[0071] The number of nucleotide triphosphates released by
pyrophosphorolysis and/or detected in a method set forth herein may
be larger than the number of different types of nucleotide
triphosphates detected. However, the order and number of the
different nucleotide triphosphates detected can be correlated with
the sequence of the nucleic acid.
[0072] In some embodiments it may be beneficial to repeatedly
sequence a particular target nucleic acid. The repetition can be
achieved, for example, by repeatedly processing a target nucleic
acid molecule in a method set forth herein. For example, a method
can include the steps of (a) contacting a target nucleic acid with
a polymerase to sequentially remove nucleotide triphosphates from
the target nucleic acid, wherein the nucleotide triphosphates that
are removed have a variety of different base moieties; (b)
distinguishing the different base moieties for the nucleotide
triphosphates that are removed, thereby determining the sequence of
the target nucleic acid; (c) regenerating at least a portion of the
target nucleic acid; and repeating steps (a) and (b) using the
regenerated target nucleic acid. The target nucleic acid can be
regenerated for example by adding nucleotide triphosphates under
conditions for the polymerase (or a newly added polymerase) to
carry out a polymerization reaction to regenerate at least a
portion of a strand of the target nucleic acid that was previously
removed by pyrophosphorolysis. Typically pyrophosphate will be
substantially absent during the polymerization reaction.
[0073] Alternatively or additionally to the repeated processing
embodiment above, a target nucleic acid can be amplified or copied
to create multiple copies that are processed using a method of the
present disclosure. A diagrammatic example is shown in FIG. 3.
Multiple copies of a double stranded template nucleic acid are
localized to a barrier in a chamber having a nanopore-polymerase
fusion (step 1), one of the strands is captured by the polymerase
(step 2), pyrophosphorolysis-based sequencing occurs (step 3), the
template strand, being single stranded, can then be pulled through
the nanopore via electric force (step 4) until it is cleared from
the chamber where the other copies of the nucleic acid remain (n.b.
the other copies remain due to being double stranded and thus
resistant to passage through the nanopore) (step 5), and then
another copy of the double stranded template nucleic acid is
captured to initiate repetition of steps 2 et seq. Any of a variety
of methods known in the art for amplifying nucleic acids, such as
those set forth previously herein, can be used to create the
multiple copies of the target nucleic acid.
[0074] Although the system of FIG. 3 is exemplified for copies of a
single template, it will be understood that nucleic acid species
having different sequences can be used similarly. Thus, a variety
of different double stranded nucleic acid species can be localized
to a barrier in a chamber having a nanopore-polymerase fusion (step
1), a strand from a first species can be captured by the polymerase
(step 2), pyrophosphorolysis-based sequencing can occur (step 3),
the template strand of the first strand can then be pulled through
the nanopore via electric force (step 4) until it is cleared from
the chamber where the other nucleic acid species remain (step 5),
and then another double stranded nucleic acid species can be
captured to initiate repetition of steps 2 et seq.
[0075] The present disclosure also provides an apparatus that
includes (a) a fluid impermeable barrier separating a first fluid
reservoir from a second fluid reservoir; (b) a nanopore positioned
in the fluid impermeable barrier to form a passage through which a
nucleotide triphosphate can pass from the first fluid reservoir to
the second fluid reservoir; and (c) a reaction mix in the first
fluid reservoir, the reaction mix including a polymerase, target
nucleic acid having two strands, and pyrophosphorolytic
concentration of pyrophosphate. The components used in the
apparatus can be one or more of those exemplified above in the
context of various methods. Further components and configurations
are exemplified below for purposes of illustration.
[0076] A fluid impermeable barrier can be configured to separate
two reservoirs and to have a nanopore placed in the barrier to
provide a fluid connection between the reservoirs. Exemplary
nanopores and barriers are set forth above and in various
references set forth above. Generally, the two reservoirs will be
in fluid communication via a single nanopore. Thus, nucleotide
triphosphates produced in one of the reservoirs will have one and
only one fluid path to the second reservoir. The use of a single
nanopore in this way allows for convenient measurement of each
nucleotide triphosphate that passes from one reservoir to the other
due to changes in electrical properties at the nanopore, barrier
and/or reservoirs. However, it is also possible in some embodiments
to include more than one nanopore in the barrier that separates two
reservoirs. When multiple nanopores fluidly connect two reservoirs,
the passage of nucleotide triphosphates can be measured at the
individual nanopore using, for example, optical or electrical
measurements that resolve each nanopore.
[0077] A reservoir can create a chamber where fluid remains
contained for at least some of the time. For example, a chamber can
be configured to form a well, cavity, compartment etc. that
restricts the flow of fluid. Alternatively, a reservoir can be
configured for fluid flow. For example, the reservoir can be
configured as a tube, channel, or flow cell, thereby allowing flow
of fluids for convenient delivery and removal of components used in
a sequencing method. In particular embodiments, a first reservoir
that contains template nucleic acid, polymerase and pyrophosphate
will be configured for fluid flow, whereas the second reservoir,
which is connected to the first chamber via a nanopore, can be
configured as a chamber. The second reservoir need not be
configured for fluid flow, but optionally can be.
[0078] The present disclosure provides multiplex embodiments. For
example, the sequences for a plurality of target nucleic acids can
be determined in parallel. A multiplex method can include the steps
of (a) providing a plurality of target nucleic acids; (b)
contacting each of the target nucleic acids with a polymerase to
sequentially remove nucleotide triphosphates from each target
nucleic acid, wherein the nucleotide triphosphates that are removed
have a variety of different base moieties; and (c) distinguishing
the different base moieties for the nucleotide triphosphates that
are removed from each nucleic acid, thereby determining the
sequences of the target nucleic acids.
[0079] A further example of a multiplex method is one that includes
the steps of (a) providing a plurality of target nucleic acids each
having two strands; (b) contacting each of the target nucleic acids
with a polymerase under conditions to sequentially remove
nucleotides from the first of each of the two strands by
pyrophosphorolysis, thereby sequentially producing nucleotide
triphosphates having a variety of different base moieties; and (c)
distinguishing the different base moieties for the sequentially
produced nucleotide triphosphates, thereby determining the sequence
of the target nucleic acids.
[0080] A multiplex apparatus can include (a) a plurality of fluid
impermeable barriers that each separate a first fluid reservoir
from a second fluid reservoir; (b) a nanopore positioned in each of
the fluid impermeable barriers to form a passage through which a
nucleotide triphosphate can pass from the first fluid reservoir to
the second fluid reservoir; and (c) a reaction mix in each of the
first fluid reservoirs, each of the reaction mixes including a
polymerase, target nucleic acid having two strands, and
pyrophosphorolytic concentration of pyrophosphate.
[0081] The plexity (i.e. multiplex level) of a method or apparatus
can be selected to satisfy a particular use. For example, the
number of target nucleic acids that are processed or present
together can be determined from the complexity of the sample to be
evaluated. Exemplary complexity estimates for some of the genomes
that can be evaluated using methods or apparatus of the present
disclosure are about 3.1 Gbp (human), 2.7 Gbp (mouse), 2.8 Gbp
(rat), 1.7 Gbp (zebrafish), 165 Mbp (fruit fly), 13.5 Mbp (S.
cerevisiae), 390 Mbp (fugu), 278 Mbp (mosquito) or 103 Mbp (C.
elegans). Those skilled in the art will recognize that genomes
having sizes other than those exemplified above including, for
example, smaller or larger genomes, can be used in a method of the
invention. Typically a nucleic acid sample is fragmented prior to
use. The number of fragments to be handled in parallel will depend
on the complexity of the genome, the average fragment size and the
desired coverage. For example, 1x coverage of a human genome (3.1
Gbp) that is fragmented to an average size of 1000 nucleotides can
be achieved using plexity of 3 million fragments (i.e. ((3.1
billion/1000).times.1)=1 million). Using similar calculations one
can determine that a plexity of 30 million fragments (of 1000
nucleotides each) is sufficient to provide 30.times. coverage of a
human genome.
[0082] The methods and apparatus set forth herein can be configured
at a plexity level sufficient to provide at least 1x, 2x, 5x, 10x,
20x, 30x, 50x or more coverage of any of a variety of genomes
including, but not limited to, those exemplified herein. The
plexity can be a function of the number of various components set
forth herein such as the number of target nucleic acid fragments as
exemplified above. Other components that can be multiplexed
include, for example, the number of nanopores used, the number of
polymerases, the number of chambers having a barrier and nanopore
etc. The multiplex level of these or other components can be, for
example, at least 2, 5, 10, 100, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.9, or higher. Alternatively or additionally, the
multiplex level can be selected to be no more than 2, 5, 10, 100,
1.times.10.sup.3, 1.times.10.sup.4, 1.times.10.sup.5,
1.times.10.sup.6, or 1.times.10.sup.9.
[0083] Throughout this application various publications, patents
and patent applications have been referenced. The disclosures of
these publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0084] The term "comprising" is intended herein to be open-ended,
including not only the recited elements, but further encompassing
any additional elements.
[0085] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the invention.
Accordingly, the invention is limited only by the claims.
* * * * *