U.S. patent application number 12/395299 was filed with the patent office on 2009-09-10 for constant cluster seeding.
This patent application is currently assigned to Illumina, Inc.. Invention is credited to Roberto Rigatti, Andrea Sabot, Min-Jui Richard Shen.
Application Number | 20090226975 12/395299 |
Document ID | / |
Family ID | 41054007 |
Filed Date | 2009-09-10 |
United States Patent
Application |
20090226975 |
Kind Code |
A1 |
Sabot; Andrea ; et
al. |
September 10, 2009 |
CONSTANT CLUSTER SEEDING
Abstract
The invention provides methods for controlling the density of
different molecular species on the surface of a solid support. A
first mixture of different molecular species is attached to a solid
support under conditions to attach each species at a desired
density, thereby producing a derivatized support having attached
capture molecules. The derivatized support is treated with a second
mixture of different molecular species, wherein different molecular
species in the second mixture bind specifically to the different
capture molecules attached to the solid support. One or more of the
capture molecules can be reversibly modified such that the capture
molecules have a different activity before and after the second
mixture of molecular species are attached. In particular
embodiments, the different molecular species are nucleic acids that
are reversibly modified to have different activity in an
amplification reaction.
Inventors: |
Sabot; Andrea; (Essex,
GB) ; Rigatti; Roberto; (Essex, GB) ; Shen;
Min-Jui Richard; (Poway, CA) |
Correspondence
Address: |
ILLUMINA, INC.;LEGAL DEPARTMENT
9885 TOWNE CENTRE DRIVE
SAN DIEGO
CA
92121-1975
US
|
Assignee: |
Illumina, Inc.
San Diego
CA
|
Family ID: |
41054007 |
Appl. No.: |
12/395299 |
Filed: |
February 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61035254 |
Mar 10, 2008 |
|
|
|
Current U.S.
Class: |
435/91.5 |
Current CPC
Class: |
C12Q 1/6837 20130101;
C12Q 1/6837 20130101; C12Q 2565/543 20130101 |
Class at
Publication: |
435/91.5 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A method of amplifying polynucleotides, comprising: (a)
providing a plurality of oligonucleotides immobilised to a solid
support, wherein the plurality of oligonucleotides comprises first
oligonucleotide species comprising a capture sequence and second
oligonucleotide species comprising an amplification sequence,
wherein the first oligonucleotide species are immobilised on the
solid support at a lower density than the second oligonucleotide
species; (b) applying a plurality of template polynucleotide
molecules to the solid support under conditions wherein the
template polynucleotide molecules hybridise to the capture sequence
of the first oligonucleotide species but do not hybridise to the
second oligonucleotide species; (c) extending the first
oligonucleotide species to generate polynucleotide complements of
the template polynucleotide molecules, wherein the polynucleotide
complements are immobilised on the solid support; and (d)
amplifying the polynucleotide complements under conditions wherein
the polynucleotide complements hybridize to the amplification
sequences of the second oligonucleotide species.
2. The method of claim 1, wherein the plurality of oligonucleotides
further comprises a third oligonucleotide species.
3. The method of claim 2, wherein the third oligonucleotide species
comprises a second amplification sequence.
4. The method of claim 3, wherein the amplifying occurs under
conditions wherein the template polynucleotide molecules hybridize
to the second amplification sequences of the third oligonucleotide
species.
5. The method of claim 1, wherein the first oligonucleotide species
is immobilised at a density of at least 1000 fold lower than the
amplification sequences.
6. The method of claim 1, wherein the oligonucleotide species is
immobilised at a density of 10.sup.6-10.sup.9 copies per
cm.sup.2.
7. The method of claim 1, wherein the capture sequence is longer
than the amplification sequences.
8. The method of claim 1, wherein the second oligonucleotide
species comprises a reversible blocking group.
9. The method of claim 8, wherein the reversible blocking group
comprises a chemical species attached to the 3' end of the second
oligonucleotide species.
10. The method of claim 9, wherein the chemical species is a
phosphate group.
11. The method of claim B, wherein the reversible blocking group
comprises a self-complementary hairpin structure.
12. The method of claim 11, wherein the second oligonucleotide
species is cleaved after extension of the capture sequence, thereby
removing the self-complementary hairpin structure from the
amplification sequence on the solid support.
13. The method of claim 1, wherein the amplification is
isothermal.
14. The method of claim 13, wherein the isothermal amplification
comprises cycles of polymerase extension and chemical
denaturation.
15. The method of claim 1, wherein the template polynucleotide
molecules that are applied to the solid support are single
stranded.
Description
[0001] This application is based on, and claims the benefit of,
U.S. Provisional Application Ser. No. 61/035,254, filed Mar. 10,
2008 and entitled "Constant Cluster Seeding," the entirety of which
is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The current invention relates to the field of nucleic acid
amplification. More specifically, the present invention provides
methods for optimising the density of nucleic acid clusters
produced on a solid support whilst eliminating the need for
multiple sample titration steps.
BACKGROUND TO THE INVENTION
[0003] Several publications and patent documents are referenced in
this application in order to more fully describe the state of the
art to which this invention pertains. The disclosure of each of
these publications and documents is incorporated by reference
herein.
[0004] A number of methods for high throughput nucleic acid
sequencing rely on a universal amplification reaction, whereby a
DNA sample is randomly fragmented, then treated such that the ends
of the different fragments all contain the same DNA sequence.
Fragments with universal ends can then be amplified in a single
reaction with a single pair of amplification primers. Separation of
the library of fragments to the single molecule level prior to
amplification ensures that the amplified molecules form discrete
populations that can then be further analysed. Such separations can
be performed either in emulsions, or on a surface.
[0005] Polynucleotide arrays have been formed based on
`solid-phase` nucleic acid amplification. For example, a bridging
amplification reaction can be used wherein a template immobilised
on a solid support is amplified and the amplification products are
formed on the solid support in order to form arrays comprised of
nucleic acid clusters or `colonies`. Each cluster or colony on such
an array is formed from a plurality of identical immobilised
polynucleotide strands and a plurality of identical immobilised
complementary polynucleotide strands. The arrays so formed are
generally referred to herein as `clustered arrays.`
[0006] In common with several other amplification techniques,
solid-phase bridging amplification uses forward and reverse
amplification primers which include `template specific` nucleotide
sequences which are capable of annealing to sequences in the
template to be amplified, or the complement thereof, under the
conditions of the annealing steps of the amplification reaction.
The sequences in the template to which the primers anneal under
conditions of the amplification reaction may be referred to herein
as `primer binding` sequences.
[0007] Certain embodiments of clustering methods make use of
`universal` primers to amplify a variable template portion that is
to be amplified and that is flanked 5' and 3' by common or
`universal` primer binding sequences. The `universal` forward and
reverse primers include sequences capable of annealing to the
`universal` primer binding sequences in the template construct. The
variable template portion, or `target` may itself be of known,
unknown or partially known sequence. This approach has the
advantage that it is not necessary to design a specific pair of
primers for each target sequence to be amplified; the same primers
can be used for amplification of different templates provided that
each template is modified by addition of the same universal
primer-binding sequences to its 5' and 3' ends. The variable target
sequence can therefore be any DNA fragment of interest. An
analogous approach can be used to amplify a mixture of templates
(targets with known ends), such as a plurality or library of target
nucleic acid molecules (e.g. genomic DNA fragments), using a single
pair of universal forward and reverse primers, provided that each
template molecule in the mixture is modified by the addition of the
same universal primer-binding sequences.
[0008] Such `universal primer` approaches to PCR amplification, and
in particular solid-phase bridging amplification, are advantageous
since they enable multiple template molecules of the same or
different, known or unknown sequence to be amplified in a single
amplification reaction, which may be carried out on a solid support
bearing a single pair of `universal` primers. Simultaneous
amplification of a mixture of templates of different sequences can
otherwise be carried out with a plurality of primer pairs, each
pair being complementary to each unique template in the mixture.
The generation of a plurality of primer pairs for each individual
template can be cumbersome and expensive for complex mixtures of
templates.
[0009] In preparing a clustered array, typically the higher the
concentration of template used, the higher the density of clusters
that will be produced on a clustered array. If the density of
clusters is too great, it may be difficult to individually resolve
each cluster and overlapping colonies may be formed. A titration
can be performed to determine the optimal template concentration to
achieve an optimal cluster density on the array wherein each
cluster can be separately resolved. However, such titrations can
lead to a loss of valuable flow cell channels due to a cluster
density that is too high or too low, a loss of template sample, an
increase in the level of reagents required or increase in sample
processing time.
[0010] Thus, there is a need for a method of controlling and
achieving desired cluster density that is independent of the
concentration of the original nucleic acid sample and avoids
nucleic acid titration steps. The present invention satisfies this
need and provides other advantages as well.
SUMMARY OF THE INVENTION
[0011] The invention provides a method of amplifying
polynucleotides. The method can include (a) providing a plurality
of oligonucleotides immobilised to a solid support, wherein the
plurality of oligonucleotides includes first species having a
capture sequence and second species having an amplification
sequence, wherein the first species are immobilised at a lower
density than the second species; (b) applying a plurality of single
stranded template polynucleotides to the solid support under
conditions wherein the single stranded template polynucleotide
molecules hybridise to the capture sequence but do not hybridise to
the amplification sequence; (c) extending the first species to
generate polynucleotide complements of the single stranded
polynucleotide template molecules, wherein the polynucleotide
complements are immobilised on the solid support; and (d)
amplifying the polynucleotide complements, wherein the amplifying
includes hybridizing the amplification sequences to the
complements.
[0012] In a particular aspect, the invention provides a method of
controlling the density of colonies of amplified single stranded
polynucleotides formed on a solid support. The method can include
the steps of (a) providing a plurality of single stranded template
polynucleotides; (b) providing a plurality of at least three
oligonucleotides immobilised to a solid support wherein at least
one of the oligonucleotides is a capture sequence capable of
hybridising to the single stranded template polynucleotides, and at
least two of the oligonucleotides are amplification sequences which
are incapable of hybridising to the single stranded template
polynucleotides, wherein the capture sequences are immobilised at a
lower density than the amplification sequences; (c) applying the
single stranded template polynucleotides to the solid support under
suitable conditions such that the single stranded template
polynucleotide molecules hybridise to the capture sequences; (d)
extending the capture sequences using a nucleic acid polymerase to
generate double stranded extension products complementary to the
single stranded template polynucleotides; (e) denaturing the double
stranded extension products to remove the hybridised single
stranded polynucleotide template molecules from the extension
products to produce single stranded template molecules immobilised
on the solid support; and (f) amplifying the single stranded
template molecules immobilised on the solid support using the two
or more amplification sequences immobilised on the solid support;
wherein the density of the immobilised colonies is controlled by
the density of the capture primers rather than the concentration of
the single stranded template polynucleotides.
[0013] The invention further provides a flow cell uniformly grafted
with a plurality of oligonucleotides, wherein the plurality
includes three species of oligonucleotides having different
sequences, wherein one of the three species is present at a lower
density than the other two species.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows a method of the invention wherein the capture
sequence is longer than the amplification sequences, and the
template selectively hybridises to the capture sequence that
extends beyond the amplification sequence. The capture sequence is
extended opposite the template strand, and the template strand is
denatured and removed. The immobilised template copy can hybridise
to one of the immobilised amplification sequences, and the
amplification sequence can be extended. The capture sequence also
comprises a sequence corresponding to one of the amplification
sequences, and hence upon synthesising a duplex from the
immobilised template copy, both ends of the immobilised duplex can
comprise sequences complementary to one of the amplification
sequences.
[0015] FIG. 2 shows an exemplary method of preparing a single
stranded template library suitable for amplification.
[0016] FIG. 3 shows an exemplary method of the invention wherein
one of the amplification primers is initially blocked from strand
elongation. After extending the immobilised template strand, the
block is removed and the sample can proceed through cycles of
bridge amplification.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The invention relates to methods for controlling the density
of different molecular species derivatized on a surface. In
particular embodiments, the molecular species are nucleic acids
having different sequences. The invention is particularly useful
for controlling the density of nucleic acid clusters produced on a
solid support. An advantage of the methods is the reduction or even
elimination of the need for multiple sample titration steps for
controlling density of molecules on surfaces.
[0018] In embodiments wherein surfaces are derivatized with nucleic
acids for subsequent formation of amplified clusters, the density
of the cluster on the support is controlled by the density of one
of the immobilised primers used for capturing the template samples.
The density of primers on every chip can be controlled during
manufacturing, simply by the ratio of the three or more immobilized
primers, and hence the density of clusters is independent of the
concentration or dilution of the template sample. This
concentration independence removes the need to accurately measure
the initial concentration of double stranded template, and is
independent of the accurate dilution of the sample. The density of
clusters on multiple chips can be made substantially uniform by
controlling the ratio and concentration of capture sequences to
amplification sequences attached to the chip surface. Because
primers can typically be synthesized and manipulated under more
controlled conditions than template samples that are derived from
different biological sources, the methods set forth herein provide
increased reproducibility in creating cluster arrays. Further
advantages are provided by creating pools of primers in a desired
ratio that can be reused for creating multiple cluster arrays
having reproducible density.
[0019] In accordance with the methods set forth herein a plurality
of oligonucleotides can be immobilised to a solid support. The
plurality can include different species of oligonucleotide molecule
each having a different sequence. For example, a plurality of
oligonucleotides can include at least two different species, at
least three different species or more, wherein a first species has
a different sequence than the other species in the plurality. It
will be understood that different species of oligonucleotide can
share a common sequence so long as there is a sequence difference
between at least a portion of the different species. For example,
as shown in FIG. 3, the two species identified as P5' and
P5'HybBlocked share a common sequence but the P5'HybBlocked species
has an additional hairpin forming sequence not found in the P5'
species.
[0020] The term `immobilised` as used herein is intended to
encompass direct or indirect attachment to a solid support via
covalent or non-covalent bond(s). In certain embodiments of the
invention, covalent attachment may be used, but generally all that
is required is that the molecules (for example, nucleic acids)
remain immobilised or attached to a support under conditions in
which it is intended to use the support, for example in
applications requiring nucleic acid amplification and/or
sequencing. Typically oligonucleotides are immobilized such that a
3' end is available for enzymatic extension and at least a portion
of the sequence is capable of hybridizing to a complementary
sequence. Immobilization can occur via hybridization to a surface
attached oligonucleotide. Alternatively, immobilization can occur
by means other than base-pairing hybridization, such as the
covalent attachment set forth above.
[0021] The term `solid support` as used herein refers to any
insoluble substrate or matrix to which molecules can be attached,
such as for example latex beads, dextran beads, polystyrene
surfaces, polypropylene surfaces, polyacrylamide gel, gold
surfaces, glass surfaces and silicon wafers. The solid support may
be a planar glass surface. The solid support may be mounted on the
interior of a flow cell to allow the interaction with solutions of
various reagents.
[0022] In certain embodiments the solid support may comprise an
inert substrate or matrix which has been `functionalised`, for
example by the application of a layer or coating of an intermediate
material comprising reactive groups that permit covalent attachment
to molecules such as polynucleotides. By way of non-limiting
example such supports may include polyacrylamide hydrogel layers on
an inert substrate such as glass. In such embodiments the molecules
(for example, polynucleotides) may be directly covalently attached
to the intermediate layer (for example, a hydrogel) but the
intermediate layer may itself be non-covalently attached to other
layers of the substrate or matrix (for example, a glass substrate).
Covalent attachment to a solid support is to be interpreted
accordingly as encompassing this type of arrangement.
[0023] `Primer oligonucleotides` or `amplification sequences` are
polynucleotide sequences that are capable of annealing specifically
to a single stranded polynucleotide sequence to be amplified under
conditions encountered in a primer annealing step of an
amplification reaction. Generally, the terms "nucleic acid,"
"polynucleotide" and "oligonucleotide" are used interchangeably
herein. The different terms are not intended to denote any
particular difference in size, sequence, or other property unless
specifically indicated otherwise. For clarity of description the
terms may be used to distinguish one species of molecule from
another when describing a particular method or composition that
includes several molecular species.
[0024] A polynucleotide sequence that is to be amplified is
generally referred to herein as a "template." A template can
include primer binding sites that flank a template sequence that is
to be amplified. In particular embodiments, as set forth in further
detail below, a plurality of template polynucleotides includes
different species that differ in their template sequences but have
primer binding sites that are the same for two or more of the
different species. The two primer binding sites that flank a
particular template sequence can have the same sequence, such as a
palindromic sequence or homopolymeric sequence, or the two primer
binding sites can have different sequences. Accordingly, a
plurality of different template polynucleotides can have the same
primer binding sequence or two different primer binding sequences
at each end of the template sequence. Thus, species in a plurality
of template polynucleotides can include regions of known sequence
that flank regions of unknown sequence that are to be evaluated,
for example, by sequencing.
[0025] Generally amplification reactions use at least two
amplification primers, often denoted `forward` and `reverse`
primers. Generally amplification sequences are single stranded
polynucleotide structures. They may also contain a mixture of
natural or non-natural bases and also natural and non-natural
backbone linkages, provided, at least in some embodiments, that any
non-natural modifications do not permanently or irreversibly
preclude function as a primer--that being defined as the ability to
anneal to a template polynucleotide strand during conditions of an
extension or amplification reaction and to act as an initiation
point for the synthesis of a new polynucleotide strand
complementary to the annealed template strand. That being said, in
certain embodiments the present invention may involve the use of a
subset of primers, either forward or reverse, that have been
modified to preclude hybridisation to a template polynucleotide
strand, the modification being altered or reversed at some point
such that hybridisation is no longer precluded.
[0026] Primers may additionally comprise non-nucleotide chemical
modifications, for example to facilitate covalent attachment of the
primer to a solid support. Certain chemical modifications may
themselves improve the function of the molecule as a primer or may
provide some other useful functionality, such as providing a
cleavage site that enables the primer (or an extended
polynucleotide strand derived therefrom) to be cleaved from a solid
support. Useful chemical modifications can also provide reversible
modifications that prevent hybridisation or extension of the primer
until the modification is removed or reversed. Similarly, other
molecules attached to a surface in accordance with the invention
can include cleavable linker moieties and or reversible
modifications that alter a particular chemical activity of function
of the molecule.
[0027] A plurality of oligonucleotides used in the methods set
forth herein can include species that function as capture
oligonucleotides. The capture oligonucleotides may include a
`template specific portion`, namely a sequence of nucleotides
capable of annealing to a primer binding sequence in a single
stranded polynucleotide molecule of interest such as one that is to
be amplified. The primer binding sequences will generally be of
known sequence and will therefore be complementary to a region of
known sequence of the single stranded polynucleotide molecule. The
capture oligonucleotides may include a capture sequence and an
amplification sequence. For example, as shown in FIG. 1, a capture
oligonucleotide may be of greater length than amplification primers
that are attached to the same substrate, in which case the 5' end
of the capture sequences may comprise a region with the same
sequence as one of the amplification primers. A portion of a
template, such as the 3' end of the template, may be complementary
to the 3' of the capture sequences. The 5' end of the template may
contain a region that comprises a sequence identical to one of the
amplification primers such that upon copying the template, the copy
can hybridise to the immobilised amplification primer. Thus, an
oligonucleotide species that is useful in the methods set forth
herein can have a capture sequence, an amplification sequence or
both. Conversely, an oligonucleotide species can lack a capture
sequence, an amplification sequence or both. In this way the
hybridization specificity of an oligonucleotide species can be
tailored for a particular application of the methods.
[0028] The length of primer binding sequences need not be the same
as those of known sequences of polynucleotide template molecules
and may be shorter, being particularly 16-50 nucleotides, more
particularly 16-40 nucleotides and yet more particularly 20-30
nucleotides in length. The desired length of the primer
oligonucleotides will depend upon a number of factors. However, the
primers are typically long (complex) enough so that the likelihood
of annealing to sequences other than the primer binding sequence is
very low. Accordingly, known sequences that flank a template
sequence can include a primer binding portion and other portions
such as a capture sequence, tag sequence or combination
thereof.
[0029] `Solid phase amplification,` when used in reference to
nucleic acids, refers to any nucleic acid amplification reaction
carried out on or in association with a solid support. Typically,
all or a portion of the amplified products are synthesised by
extension of an immobilised primer. In particular the term
encompasses solid phase amplification reactions analogous to
standard solution phase amplifications except that at least one of
the amplification primers is immobilised on the solid support.
[0030] As will be appreciated by the skilled reader, a given
nucleic acid amplification reaction can be carried out with at
least one type of forward primer and at least one type of reverse
primer specific for the template to be amplified. However, in
certain embodiments, the forward and reverse primers may include
template specific portions of identical sequence. In other words,
it is possible to carry out solid phase amplification using only
one type of primer and such single primer methods are encompassed
within the scope of the invention. The one type of primer may
include (a) subset(s) of modified primer(s) that have been modified
to preclude hybridisation to a template polynucleotide strand, the
modification being removed, altered or reversed at some point such
that hybridisation is no longer precluded. Other embodiments may
use forward and reverse primers which contain identical template
specific sequences but which differ in some structural features.
For example, one type of primer may contain a non-nucleotide
modification which is not present in the other. In still yet
another embodiment, the template specific sequences are different
and only one primer is used in a method of linear amplification. In
other embodiments of the invention the forward and reverse primers
may contain specific portions of different sequence.
[0031] In certain embodiments of the invention, amplification
primers for solid phase amplification are immobilised by covalent
attachment to the solid support at or near the 5' end of the
primer, such that a portion of the primer is free to anneal to its
cognate template and the 3' hydroxyl group is free to function in
primer extension. Again, in certain embodiments there is provided a
subset of modified primers that are prevented from hybridisation
and/or extension until the modification is removed, reversed or
altered. In particular embodiments, the amplification primers will
be incapable of hybridisation to the initial single stranded
template. In such embodiments, hybridisation of the single stranded
template will typically be specific for the capture sequences such
that the amount of capture sequences on the surface determines the
amount of template captured and thus the density of the resulting
amplified clusters.
[0032] The chosen attachment chemistry will typically depend on the
nature of the solid support and any functionalization or
derivatization applied to it. In the case of nucleic acid
embodiments, the primer itself may include a moiety which may be a
non-nucleotide chemical modification to facilitate attachment. For
example, the primer may include a sulphur containing nucleophile
such as a phosphorothioate or thiophosphate at the 5' end. In the
case of solid supported polyacrylamide hydrogels, this nucleophile
may bind to a bromoacetamide group present in the hydrogel. In one
embodiment, the means of attaching primers to the solid support is
via St phosphorothioate attachment to a hydrogel comprised of
polymerised acrylamide and N-(5-bromoacetamidylpentyl) acrylamide
(BRAPA).
[0033] A uniform, homogeneously distributed `lawn` of immobilised
oligonucleotides may be formed by coupling (grafting) a solution of
oligonucleotide species onto the solid support. The solution can
contain a homogenous population of oligonucleotides but will
typically contain a mixture of different oligonucleotide species.
The mixture can include, for example, at least two, three or more
different species of oligonucleotide. Each surface that is exposed
to the solution therefore reacts with the solution to create a
uniform density of immobilised sequences over the whole of the
exposed solid support. As such, a portion of the surface having a
mixture of different immobilized sequences can be surrounded by an
area of the surface having a mixture of the same immobilized
sequences. A suitable density of amplification oligonucleotides is
at least 1 fmol/mm.sup.2 (6.times.10.sup.10 per cm.sup.2), or more
optimally at least 10 fmol/mm.sup.2 (6.times.10.sup.11 per
cm.sup.2). The density of the capture oligonucleotides can be
controlled to give an optimum cluster density of 10.sup.6-10.sup.9
clusters per cm.sup.2. The ratio of capture oligonucleotide species
to the amplification oligonucleotide species can be any desired
value including, but not limited to at least 1:100, 1:1000 or
1:100000 depending on the desired cluster density and brightness.
Similar densities or ratios of other molecular species can be used
in embodiments where molecules other than nucleic acids are
attached to a surface.
[0034] Previously, the density of attached single stranded
polynucleotide molecules and hence the density of clusters has been
controlled by altering the concentration of template polynucleotide
molecules applied to a support. It has now been discovered that by
utilising a modified primer or capture sequence, the density of
clusters on the amplified array can be controlled without relying
on careful titration of the starting concentration of template
polynucleotide strand applied to the solid support. This has the
significant advantage that the methods need not rely on accurate
concentration measurements and dilutions of the template
polynucleotide molecules, thereby leading to increased reliability,
reduction in dilution errors and a reduction in time and quantity
of reagents required in downstream processes. For each solid
support that contains too many or too few clusters, there is a
reduction in the amount of data generated for an analysis of the
clusters. This can mean that to generate the required depth of
coverage of the sample may require additional analytical runs that
would not be required if the cluster density was optimal. Too many
clusters gives optical saturation and an increase in overlap
between two amplified molecules; too few clusters gives undesirably
high amounts of dark space that do not generate any data, thereby
wasting reagents that are more efficiently used with a densely
populated surface.
[0035] In a particular embodiment, for each cluster, an immobilised
complementary copy of a single stranded polynucleotide template
molecule is attached to the solid support by a method of
hybridisation and primer extension. Methods of hybridisation for
formation of stable duplexes between complementary sequences by way
of Watson-Crick base-pairing are known in the art. The immobilised
capture oligonucleotides can include a region of sequence that is
complementary to a region or template specific portion of the
single stranded template polynucleotide molecule. An extension
reaction may then be carried out wherein the capture sequence is
extended by sequential addition of nucleotides to generate a
complementary copy of the single stranded polynucleotide sequence
attached to the solid support via the capture oligonucleotide. The
single stranded polynucleotide sequence not immobilised to the
support may be separated from the complementary sequence under
denaturing conditions and removed, for example by washing.
[0036] The terms `separate` and `separating,` when used in
reference to strands of a nucleic acid, refer to the physical
dissociation of the DNA bases that interact within for example, a
Watson-Crick DNA-duplex of the single stranded polynucleotide
sequence and its complement. The terms also refer to the physical
separation of these strands. Thus, the term can refer to the
process of creating a situation wherein annealing of another primer
oligonucleotide or polynucleotide sequence to one of the strands of
a duplex becomes possible. After the first extension reaction, the
duplex is immobilised through a single 5' attachment, and hence
strand separation can result in loss of one of the strands from the
surface. In cases where both strands of the duplex are immobilised,
separation of the strands means that the duplex is converted into
two immobilised single strands.
[0037] In one aspect of the invention, one or more of the
amplification primers can be modified to prevent hybridisation of a
region or template specific portion of the single stranded
polynucleotide molecule. Alternatively or additionally, one or more
of the amplification primers may be modified to prevent extension
of the primer during one or more extension reactions, thus
preventing copying of the hybridised templates. These modifications
can be temporary or permanent.
[0038] Generally, the capture sequences will include a region of
the same sequence as the plurality of amplification
oligonucleotides. Once the 3' end of the extended immobilised
template copy has hybridised to one of the amplification primers
and been extended, the resulting duplex will be immobilised at both
ends and all of the bases in the capture oligonucleotide sequence
will have been copied. Thus the capture oligonucleotide may include
both the amplification primer sequence, plus a further sequence
that is complementary to the end of the template. Typically the
sequence complementary to the end of the template will not be
present in any of the amplification primers. Alternatively, the
amplification primers can contain the sequences complementary to
the ends of the single stranded templates, but the amplification
primers can be reversibly blocked to prevent hybridisation and/or
extension during one or more extension step, such as a first
extension step in a particular amplification process.
[0039] According to one aspect of the invention, one or more of the
amplification primers may include a modification that acts as a
reversible block to either template hybridisation or extension or
both. By way of non-limiting example, such modifications can be
presence of an additional sequence of nucleotides that is
complementary to the amplification primer. This additional sequence
can be present in a portion of the amplification primer and thus
acts as an intramolecular hairpin duplex, or a 3' blocking group
that prevents extension of the primer. Alternatively, the
additional sequence can be found on a separate oligonucleotide that
hybridizes to the amplification primer. A particular feature of
such a modification is that it can be removed, altered or reversed
such that the functionality of the modified primer oligonucleotide
is restored and the primer is able to undergo hybridisation and
extension during later steps of the methods. Among other examples,
the blocking group may be a small chemical species such as a 3'
phosphate moiety that can be removed enzymatically, may be an
abasic nucleotide such that the 3' end of the primer is not capable
of hybridisation (and thereby extension), or may be a sequence of
nucleotides that can be selectively excised from the immobilised
strands, for example, using restriction endonucleases that
selectively cleave particular sequences or deglycosylases that
selectively cleave oligonucleotides having exogenous bases such as
uracil deoxyribonucleotides or 8-oxoguanine.
[0040] In one embodiment a plurality of three types of
oligonucleotides (for example comprising capture sequences, forward
and reverse primers) are immobilised to a solid support.
Alternatively the three oligonucleotides may be forward
amplification, blocked forward amplification and reverse
amplification, where the unblocked forward primer acts as the
capture sequence.
[0041] The single stranded polynucleotide molecules may have
originated in single-stranded form, as DNA or RNA or may have
originated in double-stranded DNA (dsDNA) form (e.g. genomic DNA
fragments, PCR and amplification products and the like). Thus a
single stranded polynucleotide may be the sense or antisense strand
of a polynucleotide duplex. Methods of preparation of single
stranded polynucleotide molecules suitable for use in the method of
the invention using standard techniques are well known in the art.
The precise sequence of the primary polynucleotide molecules may be
known or unknown during different steps of the methods set forth
herein. It will be understood that a double stranded polynucleotide
molecule can be hybridized to an immobilized capture
oligonucleotide as exemplified herein for single stranded
polynucleotide molecules, so long as a single stranded region of
the double stranded polynucleotide is available and complementary
to the capture oligonucleotide sequence.
[0042] An exemplary method for the isolation of one strand of a
double stranded molecular construct is shown in FIG. 2. A sample of
unknown sequence may be fragmented, and adapters attached to the
ends of each fragment. One strand of the adapters may contain a
moiety for surface immobilisation, for example a biotin that can be
captured onto a streptavidin surface. The adapters may be mismatch
adapters, for example as described in copending application US
2007/0128G24, the contents of which are incorporated herein by
reference in their entirety. Amplification of the mismatch or
forked adapters using a pair of amplification primers, one of which
carries a biotin modification means that one strand of each duplex
carries a biotin modification. Immobilisation of the strands onto a
streptavidin surface means that the non-biotinylated strand can be
eluted simply by denaturation/strand separation. The eluted
constructs will be in single stranded form and upon exposure to
hybridisation conditions can be used to hybridise against the
immobilised capture sequences which can be extended.
[0043] In a particular embodiment, the single stranded
polynucleotide molecules are DNA molecules. More particularly, the
single stranded polynucleotide molecules represent genomic DNA
molecules, or amplicons thereof, which include both intron and exon
sequence (coding sequence), as well as non-coding regulatory
sequences such as promoter and enhancer sequences. Still yet more
particularly, the single stranded polynucleotide molecules are
human genomic DNA molecules, or amplicons thereof.
[0044] In a particular embodiment, a single stranded target
polynucleotide molecule has two regions of known sequence. Yet more
particularly, the regions of known sequence will be at the 5' and
3' termini of the single stranded polynucleotide molecule such that
the single stranded polynucleotide molecule will be of the
structure:
5'[known sequence I]-[target polynucleotide sequence]-[known
sequence II]-3'
[0045] Typically "known sequence I" and "known sequence II" will
consist of more than 20, or more than 40, or more than 50, or more
than 100, or more than 300 consecutive nucleotides. The precise
length of the two sequences may or may not be identical. The primer
binding sequences generally will be of known sequence and will
therefore particularly be complementary to a sequence within known
sequence I and known sequence II of the single stranded
polynucleotide molecule. The length of the primer binding sequences
need not be the same as those of known sequence I or II, and may be
shorter, being particularly 16-50 nucleotides, more particularly
16-40 nucleotides and yet more particularly 20-30 nucleotides in
length. Known sequence I can be the same as known sequence II or
the two can be different.
[0046] Methods of hybridisation for formation of stable duplexes
between complementary sequences by way of Watson-Crick base pairing
are known in the art. A region or part of the single stranded
polynucleotide template molecules can be complementary to at least
a part of the immobilised capture sequence oligonucleotides. Since
the amplification oligonucleotides are either modified to prevent
hybridisation and/or extension, or are non-complementary to the
known ends of the template strands, only the capture sequences will
be capable of hybridisation and extension. An extension reaction
may then be carried out wherein the capture sequence primer is
extended by sequential addition of nucleotides to generate a
complementary copy of the single stranded template polynucleotide
attached to the solid support via the capture sequence
oligonucleotide. The single stranded template polynucleotide
sequence not immobilised to the support may be separated from the
complementary sequence under denaturing conditions and removed, for
example by washing. The distance between the individual capture
sequence oligonucleotides on the surface therefore controls the
density of the single stranded template polynucleotides and hence
the density of clusters formed later on the surface is also
controlled.
[0047] In embodiments such as that shown in FIG. 3 wherein the
modified forward primer oligonucleotides are blocked and are unable
to be extended, generally all of the amplification primer
oligonucleotides will hybridise to the single stranded template
polynucleotides. When the extension reaction is carried out only
the unmodified forward capture primer oligonucleotides are extended
by sequential addition of nucleotides to generate a complementary
copy of the single stranded template polynucleotide attached to the
solid support via the unmodified forward primer oligonucleotide.
The single stranded template polynucleotide sequences not
hybridised to the support may be separated from the un-extended
blocked forward primer oligonucleotides under denaturing conditions
and removed, for example by washing with a chemical denaturant such
as formamide. The distance between the individual unmodified
forward primer oligonucleotides on the surface therefore controls
the density of the single stranded template polynucleotides and
hence the density of clusters formed later on the surface is also
controlled.
[0048] Following the attachment of the complementary single
stranded template polynucleotides, the modified/blocked primers can
be treated to reverse, remove or alter the modification such that
they become functionally equivalent to the unmodified forward
primer oligonucleotides. For example, the double stranded structure
may be removed either by denaturation, for example by heating or
treatment with an alkaline solution when it is formed by a separate
hybridised polynucleotide. Alternatively, where the hybridised
polynucleotide is covalently linked, enzymatic digestion could be
used to sequence-selectively cleave the strand, followed by
denaturation. Such methods for removing the double stranded
structure are known in the art and would be apparent to the skilled
person (Sambrook and Russell, Molecular Cloning, A Laboratory
Manual, third edition, Cold Spring Harbor Laboratory Press
(2001)).
[0049] In one embodiment of the invention, the single stranded
template polynucleotide molecule can be attached to the solid
support by ligation to double stranded primers immobilised to the
solid support using ligation methods known in the art (Sambrook and
Russell, supra). Such methods utilise ligase enzymes such as DNA
ligase to effect or catalyse the joining of the ends of the two
polynucleotide strands, in this case, the single stranded template
polynucleotide molecule and the primer oligonucleotide ligate such
that covalent linkages are formed. In this context "joining" means
covalent linkage of two polynucleotide strands that were not
previously covalently linked. Thus, an aim of the invention can
also be achieved by modifying the 3' end of a subset of primer
oligonucleotides such that they are unable to ligate to the single
stranded template polynucleotides. By way of non-limiting example,
the addition of 2'3'dideoxy AMP (dideoxyAMP) by the enzyme terminal
deoxynucleotidyl transferase (TdT) effectively prevents T4 DNA
ligase from ligating treated molecules together.
[0050] An alternative method would be to have the capture sequences
as duplex strands and the amplification sequences as single
strands. Upon ligation of the single strands to the capture
duplexes (which would be the only immobilised species carrying a
free 5' phosphate) the 3' end of the immobilised strand can be
extended as described above. Upon denaturation of the hybridised
template sequence, amplification of the immobilised strand can
proceed as described. Other such methods for attaching single
strands will be apparent to others skilled in the art.
[0051] In a next step according to particular embodiments of the
present invention, suitable conditions are applied to the
immobilised single stranded polynucleotide molecule and the
plurality of amplification primer oligonucleotides such that the
single stranded polynucleotide molecule hybridises to an
amplification primer oligonucleotide to form a complex in the form
of a bridge structure. Suitable conditions such as neutralising
and/or hybridising buffers are well known in the art (See Sambrook
et al., supra; Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (1998)). The
neutralising and/or hybridising buffer may then be removed.
[0052] Next by applying suitable conditions for extension an
extension reaction is performed. The primer oligonucleotide of the
complex is extended by sequential addition of nucleotides to
generate an extension product complimentary to the single stranded
polynucleotide molecule. The resulting duplex is immobilised at
both 5' ends such that each strand is immobilised.
[0053] Suitable conditions such as extension buffers/solutions
comprising an enzyme with polymerase activity are well known in the
art (See Sambrook et al., supra; Ausubel et al. supra). In a
particular embodiment dNTP's may be included in the extension
buffer. In a further embodiment dNTP's could be added prior to the
extension buffer. This bridge amplification technique can be
carried out as described, for example, in U.S. Pat. No. 7,115,400
and US 2005/0100900 A1, the contents of which are incorporated
herein by reference.
[0054] Examples of enzymes with polymerase activity which can be
used in the present invention are DNA polymerase (Klenow fragment,
T4 DNA polymerase), heat-stable DNA polymerases from a variety of
thermostable bacteria (such as Taq, VENT, Pfu, or Tfl DNA
polymerases) as well as their genetically modified derivatives
(TaqGold, VENTexo, or Pfu exo). A combination of RNA polymerase and
reverse transcriptase can also be used to generate the extension
products. Particularly the enzyme has strand displacement activity,
more particularly the enzyme will be active at a pH of about 7 to
about 9, particularly pH 7.9 to pH 8+, yet more particularly the
enzymes are Est or Klenow.
[0055] The nucleoside triphosphate molecules used are typically
deoxyribonucleotide triphosphates, for example dATP, dTTP, dCTP,
dGTP, or are ribonucleoside triphosphates for example ATP, UTP,
CTP, GTP, The nucleoside triphosphate molecules may be naturally or
non-naturally occurring.
[0056] After the hybridisation and extension steps, the support and
attached nucleic acids can be subjected to denaturation conditions.
A flow cell can be used such that, the extension buffer is
generally removed by the influx of the denaturing buffer. Suitable
denaturing buffers are well known in the art (See Sambrook et al.,
supra; Ausubel et al. supra). By way of example it is known that
alterations in pH and low ionic strength solutions can denature
nucleic acids at substantially isothermal temperatures. Formamide
and urea form new hydrogen bonds with the bases of nucleic acids
disrupting hydrogen bonds that lead to Watson-Crick base pairing.
In a particular embodiment the concentration of formamide is 50% or
more. These result in single stranded nucleic acid molecules. If
desired, the strands may be separated by treatment with a solution
of very low salt (for example less than 0.01 M cationic conditions)
and high pH (>12) or by using a chaotropic salt (e.g.
guanidinium hydrochloride). In a particular embodiment a strong
base is used. A strong base is a basic chemical compound that is
able to deprotonate very weak acids in an acid base reaction. The
strength of a base is indicated by its pK.sub.b value, compounds
with a pK.sub.b value of less than about 1 are called strong bases
and are well known to one skilled in the art. In a particular
embodiment the strong base is Sodium Hydroxide (NaOH) solution used
at a concentration of from 0.05 M to 0.25 M, particularly 0.1
M.
[0057] Following the hybridization, extension and denaturation
steps exemplified above, two immobilised nucleic acids will be
present, the first being the first template single stranded
polynucleotide molecule (that was initially immobilised) and the
second being a nucleic acid complementary thereto, extending from
one of the immobilised primer oligonucleotides. Both the original
immobilised single stranded polynucleotide molecule and the
immobilised extended primer oligonucleotide formed are then able to
initiate further rounds of amplification by subjecting the support
to further cycles of hybridisation, extension and denaturation.
[0058] It may be advantageous to perform optional washing steps in
between each step of the amplification method. For example an
extension buffer without polymerase enzyme with or without dNTP's
could be applied to the solid support before being removed and
replaced with the full extension buffer.
[0059] Such further rounds of amplification can be used to produce
a nucleic acid colony or "cluster" comprising multiple immobilised
copies of the single stranded polynucleotide sequence and its
complementary sequence.
[0060] The initial immobilisation of the single stranded
polynucleotide molecule means that the single stranded
polynucleotide molecule can hybridise with primer oligonucleotides
located at a distance within the total length of the single
stranded polynucleotide molecule. Other surface bound primers that
are out of reach will not hybridize to the polynucleotide. Thus the
boundary of the nucleic acid colony or cluster formed is limited to
a relatively local area surrounding the location in which the
initial single stranded polynucleotide molecule was
immobilised.
[0061] Once more copies of the single stranded polynucleotide
molecule and its complement have been synthesised by carrying out
further rounds of amplification, i.e. further rounds of
hybridisation, extension and denaturation, then the boundary of the
nucleic acid colony or cluster being generated will be able to be
extended further, although the boundary of the colony formed is
still limited to a relatively local area around the location in
which the initial single stranded polynucleotide molecule was
immobilised. For example the size of each amplified cluster may be
0.5-5 microns.
[0062] It can thus be seen that the method of the present invention
allows the generation of a plurality of nucleic acid colonies from
multiple single immobilised single stranded polynucleotide
molecules and that the density of these colonies can be controlled
by altering the proportions of modified capture/amplification
oligonucleotides used to graft the surface of the solid
support.
[0063] In one embodiment, the hybridisation, extension and
denaturation steps are all carried out at the same, substantially
isothermal temperature. For example the temperature is from
37.degree. C. to about 75.degree. C., particularly from 50.degree.
C. to 70.degree. C., yet more particularly from 60.degree. C. to
65.degree. C. In a particular embodiment the substantially
isothermal temperature may be the optimal temperature for the
desired polymerase.
[0064] In a particular aspect, the method according to the first
aspect of the invention is used to prepare clustered arrays of
nucleic acid colonies, analogous to those described in U.S. Pat.
No. 7,115,400, US 2005/0100900 A1, WO 00/18957 and WO 98/44151 (the
contents of which are herein incorporated by reference), by
solid-phase amplification.
[0065] In yet another aspect more than one capture sequences and
more than two amplification sequences, for example, at least three
or four or more, different amplification primer sequences may be
grafted to the solid support. In this manner more than one library,
with common sequences which differ between the libraries, could be
utilised to prepare clusters, such as, for example libraries
prepared from two different patients. Whilst the cluster may
overlap in space, they would be able to be sequenced one after the
other due to the differences between the ends of the templates. For
example, two different samples can be captured using two different
capture sequences. These can be amplified from the same two
amplification primers. The samples can be differentiated due to the
two different capture sequences, which can be used as the sites for
hybridisation of two different sequencing primers. The use of
different capture sequences thereby gives rise to a method of
sample indexing using different sequencing primers.
[0066] Clustered arrays formed by the methods of the invention are
suitable for use in applications usually carried out on ordered
arrays such as micro-arrays. Such applications by way of
non-limiting example include hybridisation analysis, gene
expression analysis, protein binding analysis, sequencing,
genotyping, nucleic acid methylation analysis and the like. The
clustered array may be sequenced before being used for downstream
applications such as, for example, hybridisation with fluorescent
RNA or binding studies using fluorescent labelled proteins.
Sequencing Methods
[0067] The invention also encompasses methods of sequencing
amplified nucleic acids generated by solid-phase amplification.
Thus, the invention provides a method of nucleic acid sequencing
comprising amplifying a pool of nucleic acid templates using
solid-phase amplification as described above and carrying out a
nucleic acid sequencing reaction to determine the sequence of the
whole or a part of at least one amplified nucleic acid strand
produced in the solid-phase amplification reaction.
[0068] Sequencing can be carried out using any suitable sequencing
technique. A particularly useful method is one wherein nucleotides
are added successively to a free 31 hydroxyl group, resulting in
synthesis of a polynucleotide chain in the 5' to 3' direction. The
nature of the nucleotide added may be determined after each
nucleotide addition or at the end of the sequencing process.
Sequencing techniques using sequencing by ligation, wherein not
every contiguous base is sequenced, and techniques such as
massively parallel signature sequencing (MPSS) where bases are
removed from, rather than added to the strands on the surface are
also within the scope of the invention.
[0069] The initiation point for the sequencing reaction may be
provided by annealing of a sequencing primer to a product of the
solid-phase amplification reaction. In this connection, one or both
of the adaptors added during formation of the template library may
include a nucleotide sequence which permits annealing of a
sequencing primer to amplified products derived by whole genome or
solid-phase amplification of the template library.
[0070] The products of solid-phase amplification reactions wherein
both forward and reverse amplification primers are covalently
immobilised on the solid surface are so-called `bridged` structures
formed by annealing of pairs of immobilised polynucleotide strands
and immobilised complementary strands, both strands being attached
to the solid support at the 5' end. Arrays comprised of such
bridged structures provide inefficient templates for typical
nucleic acid sequencing techniques, since hybridisation of a
conventional sequencing primer to one of the immobilised strands is
not favoured compared to annealing of this strand to its
immobilised complementary strand under standard conditions for
hybridisation.
[0071] In order to provide more suitable templates for nucleic acid
sequencing, it may be advantageous to remove or displace
substantially all or at least a portion of one of the immobilised
strands in the `bridged` structure in order to generate a template
which is at least partially single-stranded. The portion of the
template which is single-stranded will thus be available for
hybridisation to a sequencing primer. The process of removing all
or a portion of one immobilised strand in a `bridged`
double-stranded nucleic acid structure may be referred to herein as
`linearization`, and is described in further detail in WO07010251,
the contents of which are incorporated herein by reference in their
entirety.
[0072] Bridged template structures may be linearized by cleavage of
one or both strands with a restriction endonuclease or by cleavage
of one strand with a nicking endonuclease. Other methods of
cleavage can be used as an alternative to restriction enzymes or
nicking enzymes, including inter alia chemical cleavage (e.g.
cleavage of a diol linkage with periodate), cleavage of abasic
sites by cleavage with endonuclease (for example `USER`, as
supplied by NEB, part number M5505S), or by exposure to heat or
alkali, cleavage of ribonucleotides incorporated into amplification
products otherwise comprised of deoxyribonucleotides, photochemical
cleavage or cleavage of a peptide linker.
[0073] Following the cleavage step, regardless of the method used
for cleavage, the product of the cleavage reaction may be subjected
to denaturing conditions in order to remove the portion(s) of the
cleaved strand(s) that are not attached to the solid support.
Suitable denaturing conditions, for example sodium hydroxide
solution, formamide solution or heat, will be apparent to the
skilled reader with reference to standard molecular biology
protocols (Sambrook et al., supra; Ausubel et al. supra).
Denaturation results in the production of a sequencing template
which is partially or substantially single-stranded. A sequencing
reaction may then be initiated by hybridisation of a sequencing
primer to the single-stranded portion of the template.
[0074] Thus, the invention encompasses methods wherein the nucleic
acid sequencing reaction comprises hybridising a sequencing primer
to a single-stranded region of a linearized amplification product,
sequentially incorporating one or more nucleotides into a
polynucleotide strand complementary to the region of amplified
template strand to be sequenced, identifying the base present in
one or more of the incorporated nucleotide(s) and thereby
determining the sequence of a region of the template strand.
[0075] One sequencing method which can be used in accordance with
the invention relies on the use of modified nucleotides having
removable 3' blocks, for example as described in WO04018497, US
2007/0166705A1 and U.S. Pat. No. 7,057,026, the contents of which
are incorporated herein by reference in their entirety. Once the
modified nucleotide has been incorporated into the growing
polynucleotide chain complementary to the region of the template
being sequenced there is no free 3'-OH group available to direct
further sequence extension and therefore the polymerase can not add
further nucleotides. Once the nature of the base incorporated into
the growing chain has been determined, the 3' block may be removed
to allow addition of the next successive nucleotide. By ordering
the products derived using these modified nucleotides, it is
possible to deduce the DNA sequence of the DNA template. Such
reactions can be done in a single experiment if each of the
modified nucleotides has a different label attached thereto, known
to correspond to the particular base, to facilitate discrimination
between the bases added during each incorporation step.
Alternatively, a separate reaction may be carried out containing
each of the modified nucleotides separately.
[0076] The modified nucleotides may carry a label to facilitate
their detection. A fluorescent label, for example, may be used for
detection of modified nucleotides. Each nucleotide type may thus
carry a different fluorescent label, for example, as described in
U.S. Provisional Application No. 60/801,270 (Novel dyes and the use
of their labelled conjugates), published as WO07135368, the
contents of which are incorporated herein by reference in their
entirety. The detectable label need not, however, be a fluorescent
label. Any label can be used which allows the detection of an
incorporated nucleotide.
[0077] One method for detecting fluorescently labelled nucleotides
comprises using laser light of a wavelength specific for the
labelled nucleotides, or the use of other suitable sources of
illumination. The fluorescence from the label on the nucleotide may
be detected by a CCD camera or other suitable detection means.
Suitable instrumentation for recording images of clustered arrays
is described in U.S. Provisional Application No. 60/788,248
(Systems and devices for sequence by synthesis analysis), published
as WO07123744, the contents of which are incorporated herein by
reference in their entirety.
[0078] The invention is not intended to be limited to use of the
sequencing method outlined above, as essentially any sequencing
methodology which relies on successive incorporation of nucleotides
into a polynucleotide chain can be used. Suitable alternative
techniques include, for example, Pyrosequencing.TM., FISSEQ
(fluorescent in situ sequencing), MPSS and sequencing by
ligation-based methods, for example as described in U.S. Pat. No.
6,306,597 which is incorporated herein by reference.
[0079] The nucleic acid sample may be further analysed to obtain a
second read from the opposite end of the fragment. Methodology for
sequencing both ends of a cluster are described in co-pending
applications WO07010252 and PCTGB2007/003798, the contents of which
are incorporated by reference herein in their entirety. In one
example, the series of steps may be performed as follows; generate
clusters, linearize, hybridise first sequencing primer and obtain
first sequencing read. The first sequencing primer can be removed,
a second primer hybridised and the tag sequenced. The nucleic acid
strand may then be `inverted` on the surface by synthesising a
complementary copy from the remaining immobilised primers used in
cluster amplification. This process of strand resynthesis
regenerates the double stranded cluster. The original template
strand can be removed, to linearize the resynthesized strand that
can then be annealed to a sequencing primer and sequenced in a
third sequencing run.
[0080] In the cases where strand resynthesis is employed, both
strands can be immobilised to the surface in a way that allows
subsequent release of a portion of the immobilised strand. This can
be achieved through a number of mechanisms as described in
WO07010251, the contents of which are incorporated herein by
reference in their entirety. For example, one primer can contain a
uracil nucleotide, which means that the strand can be cleaved at
the uracil base using the enzymes uracil glycosylase (UDG) which
removes the nucleoside base, and endonuclease VIII that excises the
abasic nucleotide. This enzyme combination is available as USER.TM.
from New England Biolabs (NEB part number M5505). The second primer
may comprise an 8-oxoguanine nucleotide, which is then cleavable by
the enzyme FPG (NEB part number M0240). This design of primers
gives control of which primer is cleaved at which point in the
process, and also where in the cluster the cleavage occurs. The
primers may also be chemically modified, for example with a
disulfide or diol modification that allows chemical cleave at
specific locations.
Flow Cells
[0081] The invention also relates to flow cells for the preparation
of amplified arrays of nucleic acids wherein the flow cells contain
a uniform coating of three or more immobilised primers. Thus a
substrate described herein can occur within or as a part of a flow
cell and the methods set forth herein can be carried out in a flow
cell. In contrast to spotted arrays of multiple sequences, the
three or more oligonucleotides can be coated over the whole of the
array surface rather than in discreet locations that comprise
different sequences in each small location. The arrays may be of a
size of 1 cm.sup.2 or greater whereby the whole 1 cm.sup.2 or
greater comprises a homogeneous coating of multiple copies of the
same three or more sequences. A flow cell can be distinguished from
a `spotted array` or photolithographically synthesised array due to
the fact that the oligonucleotides are attached to each and every
surface; top, bottom, walls and ends of the flow cell chamber,
rather than being an array that is mounted in a housing. However,
if desired a flow cell that is used in a method set forth herein
can have surfaces with different reactivity for oligonucleotides
such that the oligonucleotides are only attached to one or a subset
of the aforementioned surfaces or even to only a subset of regions
within these surfaces.
[0082] The flow cell may be coated with exactly three
oligonucleotide species of different sequence composition, namely
two amplification primers and a capture primer. The capture primer
may be present at a lower concentration than the amplification
primer, for example at least 100, 1000 or 100,000 fold lower
relative concentration. The two amplification primers may be
present at similar ratios to each other, for example varying by
less than a factor of two. The capture primers may be longer than
the amplification primers, and may comprise the amplification
primer sequence region plus a capture sequence region, as shown for
example in FIG. 1. Alternatively or additionally, the amplification
primers may be blocked to prevent hybridisation and/or
extension.
[0083] Although the invention has been exemplified herein for
embodiments using nucleic acid species, it will be understood that
the same principles can be applied to other molecular species. For
example, surfaces of substrates can be derivatized with other
synthetic molecules such as peptides, small molecule ligands,
saccharides or the like. By controlling the amount of different
species of such molecules in the derivatization step, a desired
density of each species can result. Samples of molecules that bind
to one or more of these solid phase molecules can be used without
the need for titrating the samples because the density of molecules
from the sample that bind to the surfaces will be controlled by the
density of their binding partners on the surface. Accordingly,
attachment of molecules from the sample can be controlled
thermodynamically in a process that is allowed to proceed to
equilibrium as opposed to a kinetic process that requires more
precise control of reaction conditions and incubation times. Once
bound to the surface the molecules from the sample can be
subsequently modified or detected. In such embodiments, the surface
can include reversibly modified synthetic molecules such that
altering or removing the modification can allow the molecules from
the sample to be modified or detected for a particular analytical
assay or step.
[0084] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above may be used in various
combinations. All publications, patents, patent applications, or
other documents cited in this application are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication, patent, patent application, or
other document were individually indicated to be incorporated by
reference for all purposes.
* * * * *