U.S. patent application number 12/400749 was filed with the patent office on 2009-12-17 for apparatus and method for specific release of captured extension products.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION, a Delaware Corporation. Invention is credited to Jer-Kang Chen, Janice G. Shigeura, John Shigeura.
Application Number | 20090308736 12/400749 |
Document ID | / |
Family ID | 22831921 |
Filed Date | 2009-12-17 |
United States Patent
Application |
20090308736 |
Kind Code |
A1 |
Shigeura; Janice G. ; et
al. |
December 17, 2009 |
APPARATUS AND METHOD FOR SPECIFIC RELEASE OF CAPTURED EXTENSION
PRODUCTS
Abstract
Apparatus and methods for separating different polynucleotide
populations in a mixture are provided, wherein different
polynucleotides or polynucleotide populations are captured on
different solid support. After hybridization, polynucleotides are
selectively released from a selected support by altering a physical
property of that support. The released polynucleotides can be
eluted from a common flow path and isolated.
Inventors: |
Shigeura; Janice G.;
(Portola Valley, CA) ; Chen; Jer-Kang; (Palo Alto,
CA) ; Shigeura; John; (Portola Valley, CA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES CORPORATION, a
Delaware Corporation
Carlsbad
CA
|
Family ID: |
22831921 |
Appl. No.: |
12/400749 |
Filed: |
March 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09908994 |
Jul 17, 2001 |
7501284 |
|
|
12400749 |
|
|
|
|
60222371 |
Jul 31, 2000 |
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Current U.S.
Class: |
204/228.6 ;
210/143; 210/149 |
Current CPC
Class: |
G01N 30/50 20130101;
G01N 30/6065 20130101; G01N 2030/528 20130101; G01N 30/466
20130101; G01N 30/6069 20130101; C40B 40/00 20130101; G01N 30/02
20130101; C12N 15/101 20130101; G01N 30/02 20130101; C12Q 1/6837
20130101; G01N 30/6043 20130101; C12Q 1/6874 20130101; G01N 1/405
20130101; G01N 30/6052 20130101; Y10T 436/143333 20150115; G01N
30/6091 20130101; C12Q 1/6834 20130101; G01N 2030/3061 20130101;
G01N 30/50 20130101; G01N 2030/521 20130101; G01N 30/6047 20130101;
G01N 30/463 20130101; C07B 2200/11 20130101; G01N 2030/562
20130101; G01N 2030/0035 20130101 |
Class at
Publication: |
204/228.6 ;
210/143; 210/149 |
International
Class: |
B01D 35/30 20060101
B01D035/30; C25B 15/02 20060101 C25B015/02 |
Claims
1. An apparatus for separating one or more different-sequence
polynucleotides from a polynucleotide mixture, comprising: (a) a
flow path, (b) a plurality of solid supports which are disposed in
series in the flow path, each support having bound thereto a
sequence-specific capture agent that is complementary to a
different-sequence target that may be present in the polynucleotide
mixture, and (c) a control mechanism in communication with the
supports for altering a physical property of each support,
separately from the other supports, between a target-binding state
and a target-nonbinding state, whereby (i) passage of such a
mixture through the plurality of solid supports is effective to
specifically bind different-sequence targets to a complementary
capture agent on each support when the supports are each in a
target-binding state, (ii) alteration of a physical property of a
first selected support to a target-non-binding state is effective
to release bound polynucleotides from that support, (iii) the
polynucleotides released thereby can be eluted from that support by
passage of a liquid medium through the flow path while
polynucleotides captured on the other supports remain bound to
those supports, and (iv) bound polynucleotides on the remaining
supports can be released and eluted separately by repetition of
steps (ii) and (iii) on the remaining supports.
2. The apparatus of claim 1, wherein the control mechanism is
capable of performing steps (ii) and (iii) simultaneously.
3. The apparatus of claim 1, wherein the physical property is
temperature, and the control mechanism comprises a temperature
control element for selectively heating a selected support to
release polynucleotides from that support.
4. The apparatus of claim 1, wherein the control mechanism
comprises a plurality of heating elements, one for each support,
and is operable to activate the heating elements to release
polynucleotides from one support at a time.
5. The apparatus of claim 1, wherein the physical property is
voltage potential, and the control mechanism comprises a voltage
control element for setting individual electrical potentials of the
solid supports to release polynucleotides from that support.
6. The apparatus of claim 1, wherein each support comprises a
frits, bead, or powder cluster.
7. The apparatus of claim 1, comprising a processor in
communication with the control mechanism for controlling the
functions of the control mechanism.
8. The apparatus of claim 7, wherein the processor executes a
program of instructions to control the functions of the control
mechanism.
9. The apparatus of claim 8, wherein the program of instructions
are conveyed to the processor by a processor-readable medium.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of non-provisional
application Ser. No. 09/908,994, filed on Jul. 17, 2001, which
claims priority to provisional application No. 60/222,371 filed
Jul. 31, 2000, both of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to apparatus and methods for
separating different polynucleotide populations from each other,
wherein polynucleotide capture and elution are performed in a
common flow path. The invention also relates to a programmed device
and a program of instructions (e.g., software) that is executed by
the device for implementing methods of the invention.
Introduction
[0003] Polynucleotide sequencing is an important, well established
technique in molecular biology and has become integral to virtually
all aspects of molecular genetics. With the undertaking of massive
sequencing projects, such as the Human Genome Project, as well as
comparative sequencing of known genes for diagnostic purposes, the
demand to reduce the time and overhead associated with such
sequencing is greatly increasing.
[0004] To analyze multiple polynucleotide sequences in a sample, it
is desirable to use as few reaction vessels as possible in order to
use reagents efficiently and reduce liquid manipulations. For
example, DNA sequencing using the Sanger method has been
dramatically simplified using dye terminators labeled with
base-specific fluorescent dyes for each of the four standard bases
(A, C, G and T). This approach has made possible the performance of
template-dependent primer extension in a single reaction mixture in
the presence of four terminators, followed by sequence analysis of
the resulting fragment mixture in a single electrophoretic path.
However, this approach has usually required a different sequencing
reaction mixture for each different target. One attempt to address
this problem was proposed by Church et al. (Science 240:185, 1988;
and U.S. Pat. No. 5,149,625), wherein sequencing fragments are
produced by different, sequence-specific sequencing primers
containing distinct tag sequences to identify extension products
from each template-specific primer. After gel electrophoresis, the
separated fragments are transferred onto a membrane and iteratively
hybridized with different tag-specific probes to serially determine
the sequence of each different target, one at a time.
Unfortunately, this method is cumbersome to practice and also
requires four different extension reaction mixtures per target
template, since four different primer tags are required to identify
the four possible 3' terminators at the ends of the fragments.
[0005] DNA sequencing is but one example of methods that involve
mixtures of different polynucleotides. More generally, it is often
desirable to simultaneously generate a plurality of polynucleotide
populations in a single reaction mixture, followed by isolation of
the different populations from the mixture for further analysis or
manipulation. Ideally, such a method should be convenient to
perform and should allow the isolation and separation of the
different polynucleotide populations in analytical or preparative
amounts.
SUMMARY OF THE INVENTION
[0006] The present invention provides apparatus and methods whereby
the separation and isolation of different polynucleotide
populations can be achieved using a single flow path.
[0007] In one aspect, the invention includes an apparatus for
separating one or more different-sequence polynucleotides from a
polynucleotide mixture. The apparatus comprises (a) a flow path,
(b) a plurality of solid supports which are disposed in series in
the flow path, each support having bound thereto a
sequence-specific capture agent that is complementary to a
different-sequence target that may be present in the polynucleotide
mixture. In a preferred, optional embodiment, the apparatus further
comprises (c) a control mechanism in communication with the
supports for altering a physical property of each support,
separately from the other supports, between a target-binding state
and a target-nonbinding state. In operating the apparatus, passage
of the mixture through the plurality of solid supports is effective
to specifically bind different-sequence targets to a complementary
capture agent on each support when the supports are each in a
target-binding state. Thereafter, a physical property of a first
selected support can be altered to a target-non-binding state to
release bound polynucleotides from that support. The released
polynucleotides can be eluted from that support by passage of a
liquid medium (solvent) through the flow path while polynucleotides
captured on the other supports remain bound to those supports Bound
polynucleotides on the remaining supports can be released and
eluted separately by repetition of the foregoing steps.
[0008] In one embodiment, the control mechanism is capable of
performing steps (ii) and (iii) simultaneously.
[0009] In another embodiment, the physical property is temperature,
and the control mechanism comprises a temperature control element
for selectively heating a selected support to release
polynucleotides from that support. For example, the control
mechanism can comprise a plurality of heating elements, one for
each support, and may be operable to activate the heating elements
to release polynucleotides from one support at a time.
[0010] In another embodiment, the physical property is voltage
potential, and the control mechanism comprises a voltage control
element for setting individual electrical potentials of the solid
supports to release polynucleotides from that support.
[0011] In another aspect, the invention includes a method for
isolating one or more different-sequence polynucleotides from a
mixture. In the method, the mixture is flowed through a flow path
containing a plurality of solid supports which are located in
series in the flow path, each support having bound thereto a
sequence-specific capture agent complementary to a
different-sequence polynucleotide, under conditions effective to
specifically bind different-sequence polynucleotides to
corresponding sequence-specific capture agents on one or more of
the supports. After binding is complete, bound polynucleotides can
be released from a selected support by altering a physical property
of that support while leaving unaltered the same physical property
of one or more of the other supports. The released polynucleotides
are eluted through the flow path such that the eluted
polynucleotides can be isolated in separated form.
[0012] In one embodiment, (i) the polynucleotide mixture comprises
a plurality of different polynucleotide populations, each different
polynucleotide population comprising a plurality of different
polynucleotides that contain a distinct sequence associated with
that population, and (ii) different sequence-specific capture
agents on the different solid supports are complementary to
different polynucleotide populations in the mixture. An example of
such populations is a mixture of sequencing ladders as discussed
further below. In another embodiment, the polynucleotide mixture
comprises a plurality of PCR products. In yet another embodiment,
the polynucleotide mixture comprises a plurality of ligation
products.
[0013] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 illustrates a sectional view of a column defining an
exemplary flow path in accordance with the invention.
[0015] FIG. 2 illustrates a perspective view of a plurality of flow
paths in accordance with the invention.
[0016] FIG. 3 illustrates an exploded view of another embodiment of
the invention comprising a plurality of flow paths that can be
formed by combination of two opposing pieces.
[0017] FIG. 4 illustrates another embodiment of a column, showing a
perspective view of a plurality of columns formed together, with
portions of one column broken away, showing an exploded view of a
support/insulator assembly.
[0018] FIG. 5 illustrates yet another embodiment of a column,
showing a perspective/exploded view of a plurality of columns.
[0019] FIGS. 6 and 7 show functional block diagrams of different
control mechanism in accordance with the invention.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0020] "Oligonucleotide" and "polynucleotides" are used
interchangeably herein and are intended to have the same meaning.
As used herein, these terms refer to naturally occurring
polynucleotides, e.g., DNA or RNA, and analogs thereof. Such
analogs include, but are not limited to, phosphoramidates,
peptide-nucleic acids, phosphorothioates, methylphosphonates, and
the like. In addition to having non-naturally occurring backbones,
analogs may also comprise base analogs such as 7-deazaguanosine,
5-methyl cytosine, inosine, and the like. Descriptions of how to
synthesize polynucleotides can be found, among other places, in
U.S. Pat. Nos. 4,373,071; 4,401,796; 4,415,732; 4,458,066;
4,500,707; 4,668,777; and 4,973,679.
[0021] "Recovery tag" as used herein refers to a compound (or
portion of a compound) that is a member of a specific binding pair
of molecules. A recovery tag may belong to any class of
macromolecule, e.g., polynucleotides, carbohydrates, polypeptides,
and the like. Alternatively, recovery tags may belong to a class of
non-naturally occurring molecules. Preferably, recovery tags are
oligonucleotides. When the recovery tag is an oligonucleotide, the
recovery tag may comprise none, part, or all of the
template-annealing sequence of a recoverable primer. Recovery tags
(and their respective recovery tag binding compounds) are selected
so as to avoid the binding of the recovery tags at improper
locations, e.g., different recovery tag oligonucleotides are
preferably non-cross hybridizing.
[0022] When the recovery tag is an oligonucleotide, the recovery
tag may optionally comprise a "balancing polynucleotide" sequence.
"Balancing polynucleotide" refers to polynucleotides that hybridize
to the recovery tag binding compound, but do not specifically
hybridize to the sequencing (or amplification) template. Balancing
polynucleotides are optionally present on recoverable primers. The
balancing polynucleotide may be used to equalize, i.e., balance,
the melting temperatures of the duplex (or triplex) formed between
the different recovery tag and the recovery tag binding compound
pairs used together in the same reaction vessel. Similarly, the
balancing polynucleotide may be used to equalize, i.e., balance,
the melting temperatures of the duplex (or triplex) formed between
the different recovery tag and the recovery tag binding compound
pairs that are to be denatured under similar conditions.
[0023] "Recovery tag binding compound" refers to the member of a
specific binding pair that is not the recovery tag on a given
recoverable primer. In embodiments of the invention employing
polynucleotides as recovery tags, the recovery tag binding compound
comprises a polynucleotide sequence that is complementary or
partially complementary to the recovery tag polynucleotide of
interest. Individual recovery tag binding compound molecules may
comprise multiple copies of the complementary (or partially
complementary) polynucleotide sequence. Branched polynucleotides,
for example as described in published PCT patent application WO
96/016104 and published European patent application EP 646595, may
be used to increase the effective concentration of binding sites
for recovery tags.
[0024] "Recoverable primer" refers to an oligonucleotide primer
that comprises a recovery tag. Recoverable primers may be used to
specifically prime a polynucleotide sequencing reaction, a
polynucleotide amplification reaction, or other primer extension
reaction, i.e., recoverable primers comprise a polynucleotide
sequence that can specifically bind to a specific (usually
pre-determined) site on a template for sequencing (or
amplification). The portion of the recoverable primer that may
site-specifically hybridize to a template is referred to herein as
the "template-annealing sequence" of the recoverable primer. The
template-annealing sequence is of sufficient length to specifically
hybridize to a site or sites on the template of interest, typically
18-36 nucleotides in length. Template-annealing sequences for use
in polynucleotide sequencing must hybridize to unique sites on the
template of interest. The recovery tag is coupled to the primers in
such a way as to avoid having the recovery tag interfere with the
ability of the recoverable primer to site-specifically hybridize to
the priming site, e.g., the recovery tag may be joined at, or
proximal to, the 5' end of the recoverable primer. The particular
means of coupling a recovery tag to an oligonucleotide primer
depends upon the class of compound to which the recoverable tag
belongs. When the recovery tag is a polynucleotide, the recovery
tag is preferably coupled by polynucleotide linkage, e.g., a
phosphate linkage. When the recovery tag is a protein, the recovery
tag is preferably coupled by a bifunctional crosslinking agent such
as DSS (disuccinimidyl suberate), SPDP (N-succinimidyl 3-(2
pyridyldithio propionate)), SATA (N-succinimidyl
S-acetylthioacetate), and the like. Detailed protocols for methods
of attaching labels to polynucleotides can be found in, among other
places, G. T. Hermanson, Bioconjugate Techniques, Academic Press,
San Diego (1996).
[0025] When the recovery tag on a recoverable primer is a
polynucleotide, the recovery tag may comprise all, part, or none of
the template-annealing sequence of the recoverable primer. In some
embodiments of the invention, the recovery tag may consist of some
or all of the sequence of the recoverable primer. In other
embodiments of the invention, the recovery tag does not comprise
any portion of the template-annealing sequence of the recoverable
primer. In still other embodiments of the invention, the recovery
tag comprises a balancing polynucleotide. The entire recovery tag
may be a balancing polynucleotide. Alternatively, the recovery tag
may consist of a balancing polynucleotide and a portion of the
template-annealing sequence adjacent to the balancing
polynucleotide.
[0026] Recoverable primers are capable of specifically hybridizing
to target polynucleotide sequences under a given set of
hybridization conditions. Criteria for designing sequence specific
primers are well known to persons of ordinary skill in the art.
Detailed descriptions of primer design criteria that provide for
site-specific annealing can be found, among other places, in
Dieffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold
Spring Harbor, Cold Spring Harbor Press (1995), and Kwok et al,
Nuc. Ac. Res. 18:999-1005 (1990). The template-annealing sequence
portions of the primers are of sufficient length to permit
site-specific annealing to template sites of interest. Primers for
sequencing are designed to uniquely hybridize to a single template
site. The template-annealing sequence of the recoverable primers
may be either completely complementary or partially complementary
to the bases of the target sequence, i.e., the annealing site.
Preferably, the template-annealing sequence of a recoverable primer
is completely complementary to the bases of the corresponding
target sequence.
[0027] "Sequencing ladder" as used herein refers to a set of
polynucleotides that is produced from a sequencing reaction, either
a chain termination sequencing reaction, e.g., dideoxy sequencing,
or from chemical cleavage sequencing, e.g., Maxam and Gilbert
sequencing. The process of producing a sequencing ladder is
referred to herein as "sequencing ladder generation" or "generating
a sequence ladder." Methods for generating polynucleotide
sequencing ladders are well known to persons of ordinary skill in
the art. Examples of methods of generating a sequencing ladder can
be found among other places, in Sambrook et al, Molecular Cloning
Methods: A Laborato Manual Coldspring Harbor, Coldspring Harbor
Press (1989). The different pplynucleotides, i.e., members, of a
specific sequencing ladder, differ in length from one another, but
all members of the same ladder comprise the same oligonucleotide
primer from which that sequencing ladder is derived. Thus,
generating sequencing ladders from a first recoverable primer and a
second recoverable primer that anneal to the same template priming
site, but differ with respect to the identity of the recovery tags,
are said to result in the synthesis of two different sequencing
ladders. In addition to being derived from the same primer, the
members of a given polynucleotide sequencing ladder are also
derived from the same sequencing template. In labeled primer
sequencing, four different sequencing ladders, each using a
different dideoxy terminating base are generated separately (and
may subsequently be combined prior to analysis), even though only a
single completed sequence is obtained from combining the
information in the four constituent sequencing ladders. When the
same recoverable sequencing primer and template are used to
generate sequencing ladders in separate reaction vessels, the
sequencing ladders produced are said to be different sequencing
ladders.
[0028] A sequencing ladder generated from a recoverable primer may
be referred to as a "recoverable sequencing ladder." "Recoverable
sequencing ladder" also includes sequencing ladders that comprise
the functional equivalent of recovery tags, such as sequencing
ladders produced during non-recovery tag multiplex methods.
[0029] A polynucleotide amplification product generated from a
recoverable primer may be referred to as a "recoverable
amplification product." "Recoverable amplification product"
includes amplification products that comprise the functional
equivalent of recovery tags, such as sequencing ladders produced
during non-recovery tag multiplex methods.
[0030] "Specific binding pair" refers to a pair of molecules that
specifically bind to one another. Binding between members of a
specific binding pair is usually non-covalent. Examples of specific
binding pairs include, but are not limited to antibody-antigen (or
hapten) pairs, ligand-receptor pairs, biotin-avidin pairs,
polynucleotides with complementary base pairs, and the like. Each
specific binding pair comprises two members, however, it may be
possible to find additional compounds that may specifically bind to
either member of a given specific binding pair.
[0031] "Polynucleotide amplification reaction" as used herein
refers to a broad range of techniques for the amplification of
specific polynucleotide sequences. Examples of such amplification
techniques include the polymerase chain reaction (PCR), ligase
chain reaction (e.g., EP 336731 (Wallace), EP 320308 (Backman), and
EP 439182 (Backman)), 3SR (Guatelli et al, Proc. Natl. Aca. Sci.
USA 87:1874-1878 (1990), and nucleic acid sequence-based
amplification (NASB) (van Gemen et al., J. Virol. Methods
45:177-188 (1993), for example.
[0032] "In separated form" refers to polynucleotides that are
eluted from a particular support without significant contamination
by polynucleotides that were captured on other supports in the flow
path.
[0033] "Target polynucleotide" means a polynucleotide that is to be
specifically bound to a complementary entity, such as a recovery
tag binding compound.
[0034] "Polynucleotide population" reference to a collection of
polynucleotides that are the same as or different from each other,
but which contain a common polynucleotide sequence or a recovery
tag. Different polynucleotide populations differ because each
different population has a different, distinct common sequence or
common recovery tag.
[0035] "Sequence-specific capture agent" has substantially the same
meaning as "recovery tag binding compound".
II. Apparatus
[0036] According to one aspect, the invention provides apparatus
which are useful for separating and isolating different-sequence
polynucleotides from each other, in separated form.
[0037] A first exemplary apparatus 12 is shown in FIGS. 1 and 2.
Apparatus 12 comprises a column 13 which defines a common flow path
14 extending therethrough. Column 13 includes an increased diameter
portion 15 which forms an inlet port 16 into which a polynucleotide
mixture can be introduced. In the embodiment shown, inlet port 16
includes a funnel-shaped bottom that is integrally formed with a
main body 17 of the column. The main body terminates with an outlet
port 18 from which different polynucleotides or different
polynucleotides populations can be eluted. A plurality of chambers
20, each of which contains a solid support 22, are provided in
series along flow path 14. Although four chambers and corresponding
supports are shown in FIGS. 1 and 2, it will be appreciated that
fewer or more chambers and supports can be employed, depending on
the number of different polynucleotides or populations to be
separated. Optionally, the chambers are separated from each other
by intervening segments 24 to help hold the supports in place, and
also to provide insulation regions between the chambers to improve
the specificity of control over the physical properties of the
different changes.
[0038] For example, main body 17 can be formed from an initially
cylindrical tube using a heat-shrinkable plastic. An end of the
tube is heated to form outlet region 18 having an inner diameter
that is smaller than the initial inner diameter of the tube. Region
18 thus includes a lower, outlet end for fluid egress, and an
upper, inner end which defines a lower end of a first chamber 20. A
first support 22 is then placed adjacent to the inner end of region
18, followed by heating and/or mechanical pinching of the tube
region immediately above the support to form a completed chamber 20
which encloses first support 22. By repeating the steps of adding a
support and then heating or pinching the tube region immediately
above the added support to complete additional chambers, a flow
path passing through a plurality of supports in series can be
formed. A region defining inlet 15 can be added by joining a second
tube segment to the top of the first tube and forming a
liquid-tight connection by sonic welding, melting, etc.
Alternatively, the entire column can be formed from a single tube
and, if the inlet region is designed to have a greater outer
diameter than the main body, the outer diameter of the main body
can be made to be smaller than that of the inlet region by
heat-shrinking the chamber walls around the solid supports during
formation of the chambers of the column.
[0039] With continued reference to FIG. 1, apparatus 12 further
includes a multi-piece jacket 26 that is adapted to fit around the
exteriors of the chambers. In the embodiment shown, the jacket is
formed by joining together two opposing halves which individually
surround the different chambers. In more particular embodiments,
the jacket may include individual heating coils or electric field
generating devices, as described below.
[0040] With reference to FIG. 2, a plurality of columns 13 may be
formed within a single unit. Similarly, jackets for the parallel
columns may be provided in integral halves 26.
[0041] FIG. 3 shows another embodiment in accordance with the
present invention, wherein the support chambers are formed by
matching halves that define a plurality of columns. Each half
defines a plurality of serial chambers which are separated by
intervening segments which help seat the plurality of chambers.
Conveniently, the matching halves may be formed by injection
molding to produce monolithic pieces that can be joined together as
shown in the Figure.
[0042] FIG. 4 illustrates another column construction wherein the
flow path is defined by a cylindrical region 14. In this
embodiment, solid supports 22 are separated from each other by
separators 24. Thus, the chambers that surround the individual
supports are defined by the wall regions of region 14 that
immediately surround each support, and which are separated from
each other by separators 22. Such an arrangement can be made by
inserting alternating supports and insulators into inlet port
15.
[0043] FIG. 5 illustrates still another construction which employs
a monolithic support 23 which is adapted to fit into the column
body as shown in the figure, and which comprises a plurality of
solid supports having different capture reagents located in series
along support 23.
[0044] The solid supports used in the present invention can be made
of any material that is suitable for the purposes of the invention.
In particular, the material must be capable of forming stable
covalent or non-covalent bonds with sequence-specific capture
agents so that the agents are capable of capturing complementary
target sequences from the sample mixture. In addition, the material
should have low affinity for polynucleotides other than the target
polynucleotides which are to be bound by the capture agent. In one
embodiment, the solid supports comprise high density polystyrene,
preferably in the form of a cylindrically shaped frit. The solid
support preferably occupies a small volume and has high binding
capacity. For purposes of illustration only, supports can comprise
high density polystyrene with the following properties: pore size:
25-30 .mu.m; length: 0.365''; diameter: 0.128''; void volume:
>50%; surface area: 0.26 m.sup.2/g. In another embodiment, the
supports may comprise controlled pore glass (CPG). In yet further
embodiments, the solid support is provided in the form of beads,
powder clusters, membranes, or the like, which may be held in place
by porous frits or membranes.
[0045] The column that contains the supports can be made of any
appropriate material that is compatible with the purposes of the
invention. The column material should be chemically inert towards,
and have low binding affinity for, the polynucleotides that are to
be separated. Thus, columns can be made of plastics such as
polyethylene, polypropylene, polystyrene, polyacrylamide, poly
carbonate, or the like; metals or metal alloys such as aluminum or
stainless steel, silicates or coated silicates (glass);
polysaccharides; etc. For example, materials used in standard
electrophoresis or chromatographic DNA separation methods can be
used. Columns formed from multiple materials or components are also
contemplated.
[0046] As discussed further herein, the device of the invention is
used to bind target polynucleotides to different, sequence specific
capture agents which are located on different solid supports (or
different solid support regions). As a result, a polynucleotide
mixture can be "deconvoluted" into separate components or separate
polynucleotide populations. These separated polynucleotides can
then be released specifically from individual supports and eluted
in separate form, substantially free from contamination by other
polynucleotides in the mixture which are still bound to other
supports in the flow path. Although polynucleotide release from
selected supports can be accomplished manually, automation of this
process is preferable. Accordingly, operation of the invention is
described below with respect to configurations that allow computer
controlled automation of various steps.
[0047] For example, a control mechanism in communication with the
supports can be used to selectively alter a physical property of a
particular support, in order to denature and thereby release
captured polynucleotides from that support, so that the released
polynucleotides can be eluted separately from polynucleotides
captured on other supports. In one embodiment, during alteration of
the physical property of the particular support, the other supports
remain unchanged.
[0048] FIG. 6 illustrates an embodiment wherein the control
mechanism comprises a temperature control unit 50 which includes an
individually controllable heating/cooling element 52. Although
element 52 is shown as a heating coil in the Figure, equivalents
thereof can also be used, such as a peltier device. An element 52
is positioned adjacent to or around the outer wall 20a of each
chamber, and can be embedded within a jacket 26 such as described
above.
[0049] In this embodiment, each column is made so that the chamber
walls surrounding the solid supports have high thermal conductivity
to readily transfer heat to and from the solid supports. However,
in arrangements where the supports are selectively heated, heat
transfer between different chambers) i.e., along the length of the
column, should be low enough to avoid release of polynucleotides
from non-selected supports. For this purpose, exterior wall regions
located between serial chambers can be formed from materials having
low thermal conductivity, or may be surrounded by external
insulation layers that help maintain temperature stability in those
regions. Alternatively, or in addition, adjacent solid supports can
be separated by insulation materials that occupy a portion of the
flow path between the supports but which allow adequate fluid flow
for elution.
[0050] In FIG. 5, heating/cooling elements 52 are incorporated into
a circuit which also includes a voltage source 54 and a switch 56
for selecting which element 52 is to be adjusted at a particular
time. In the illustrated configuration, one terminal for each
element 52 is connected to a first terminal of the voltage source,
while a second terminal for each element 52 is connected to the
output terminal of switch 56. The input terminal of switch 56 is
connected to the other terminal of voltage source 54. The switch
may be manually operated or may be programmed to control the
activation and timing of activation or adjustment of each
heating/cooling element. Appropriate circuitry may also be included
in the switch to control the amount of heat generated by the
coil(s).
[0051] In one embodiment, the switch is processor-controlled.
Processor 58 may be a microprocessor which is embedded in the
switch itself or may be part of a computer system 40 which includes
other computer components such as random-access memory (RAM) 60,
read-only memory (ROM) 62, input devices 64, such as a keyboard and
mouse, output devices 66, such as a monitor and printer, and a
storage media 68 such as an internal or external hard disk.
[0052] The programming of the switch may be implemented with
software which may be fetched from RAM and executed by the
processor. The software may be stored in storage medium 68 which
may be any suitable medium, including various magnetic media such
as disks or tapes, and various optical media such as compact disks.
The software may also be conveyed to computer 40 over communication
paths throughout the electromagnetic spectrum including signals
transmitted over a network or the internet and carrier waves
encoded to transmit the software. Alternatively, the programming of
the switch may be implemented with functionally equivalent hardware
using discrete components, one or more application specific
integrated circuits (ASICs), digital signal processing circuits, or
the like. Such programmed hardware may be physically integrated
with the processor or may be a separate component which may be
embodied on a computer card that can be inserted into an available
slot in the computer. Thus, the programming of the switch may be
implemented using software, hardware, or combination thereof. It
will be apparent to one skilled in the art of programming to
implement a system to perform necessary control processes.
[0053] In another embodiment, illustrated in FIG. 7, the control
mechanism comprises a circuit including a device 70 for selectively
applying an electric field (or voltage potential) to the different
supports. Such a circuit is generally similar to the circuit shown
in FIG. 6, and like components are identified with like reference
numerals. However, as shown schematically in FIG. 7, the electric
field (or voltage potential) applying circuit includes a pair of
capacitor plates 72, or equivalents thereof, for each support,
instead of heating/cooling elements. In this embodiment, switch 56
is configured to selectively apply an electric field to a selected
one or more of the supports. Each pair of capacitor plates is
symmetrically positioned about a respective one of the support
chambers. The plates are preferably shaped to follow the contour of
the chambers.
[0054] In this embodiment, each column is constructed to have high
electrical conductivity across the chamber walls. Preferably, the
electrical conductivity in wall regions between the chambers, i.e.,
along the length of the column, should be low enough to avoid
releasing polynucleotides from non-selected supports. Thus, the
chamber walls between consecutive chambers may be impregnated with
an electrically insulating material.
[0055] In the illustrated arrangement, a plate in each capacitor
plate pair is connected to one of the terminals of the voltage
source, while the other plate is connected to the output terminal
of the switch. The input terminal of the switch is connected to the
other terminal of the voltage source.
[0056] The switch may be manually operated or may be programmed to
control the capacitor plate pair(s) to which a voltage is applied,
the magnitude of voltage applied, and the time during which a given
voltage is applied to a given capacitor plate pair. Appropriate
circuitry may also be included to apply different voltages to
different capacitor plate pairs at the same time. Such variables
may be adjusted to accommodate different electrical binding
affinities in the different groups of sequence-specific capture
agents. As described for the previous embodiment, programming of
the switch may be implemented using software, hardware, or a
combination thereof.
[0057] Processes in accordance with the invention can be automated
using apparatus as described above, to control sample loading,
content and flow rate of solvent, selective polynucleotide release,
and polynucleotide collection. A typical process begins with
introduction of the sample into the flow path through an input port
as discussed above. The physical properties (physical conditions)
of the supports are set to be in a target-binding state so that
each support is able to specifically capture polynucleotides that
contain sequences or recovery tags that are complementary to the
support-bound tag binding compounds. For supports whose binding
properties are controlled by temperature, the supports can be set
to room temperature or a somewhat higher temperature, such as
30.degree. C., 35.degree. C., or 40.degree. C., so that non-target
polynucleotides pass through the supports without binding to them,
and only target polynucleotides are bound to the appropriate
supports. The flow rate of sample through the flow path is chosen
to allow sufficient time for target polynucleotides to bind to the
binding compounds on the supports, according to known hybridization
principles and using empirical optimization if necessary.
[0058] In one embodiment, sample flow is performed continuously
during the loading step, at a continuously positive flow rate. In a
second embodiment, solvent flow is alternated between a positive
flow rate and stopped flow, so that when flow is stopped, the
polynucleotides in the sample have additional time to bind to
complementary binding compounds on the supports. In a third
embodiment, the direction of flow is reversed at least once per
support so that the sample can be passed back through each supports
to increase capture of target polynucleotides. Solvent flow may be
continued until most or all of the non-target polynucleotides have
been washed from the supports.
[0059] After binding (hybridization) is complete, bound
polynucleotides are released from selected supports into the flow
path by altering a physical property of the corresponding support.
For example, this may be performed by selectively heating the
supports. That is, the heating element associated with a support on
which a specific polynucleotide population (i.e., the i.sup.th
population) resides, is activated to denature the polynucleotides
in that population. Concurrently with, or after, the denaturing, a
suitable elution solvent is introduced into the flow path to elute
the released i.sup.th population of polynucleotides through the
flow path for separate collection.
[0060] If there are other populations of polynucleotides to be
eluted, variable i can be increased by 1, and the process returns
to the previous step in which another population of polynucleotides
is released by activation of the support on which that population
resides. The steps of release and elution of specific populations
of polynucleotides are repeated until all of the populations have
been individually collected.
Methods
[0061] The present invention is useful for separating and isolating
different-sequence polynucleotides. These polynucleotides can be
from any source or produced by any appropriate method. Preferably,
the polynucleotides contain unique recovery tags that permit
localized capture on different, tag-specific binding compounds that
are immobilized on a series of solid supports in the flow path.
[0062] In one embodiment, the polynucleotides comprise a plurality
of sequencing ladders derived from different templates. In another
exemplary embodiment, the polynucleotides comprise a plurality of
different-sequence PCR products, which may be prepared by methods
described in PCT Pub. WO 94/21820 (Wallace), for example. In
another embodiment, the polynucleotides are products of
template-dependent probe ligation or gap-filling ligation, as
described for example in U.S. Pat. No. 4,988,617 (Landegren) and
U.S. Pat. No. 5,242,794 (Whiteley), EP 320308 (Backman), EP 439182
(Backman), PCT Pub. WO 90/01069 (Segev), EP 336731 (Wallace), and
PCT Pub. WO 97/31256 (Barany).
[0063] In yet another embodiment, the polynucleotides comprise a
plurality of primers extended by a single-base, as described for
example WO 93/25563 (Wallace). In other embodiments, the
polynucleotide mixture is produced by 3SR (Guatelli et al, Proc.
Natl. Aca. Sci. USA 87:1874-1878 (1990), or nucleic acid
sequence-based amplification (NASB) (van Gemen et al., J. Virol.
Methods 45:177-188 (1993). Polynucleotide mixtures produced by any
other methods are also contemplated.
[0064] For convenience, operation of the invention is discussed
below mainly with reference to separation and isolation of
sequencing ladders and PCR products. However, it will be apparent
to one of skill in the art how the invention can be used with other
polynucleotide mixtures.
[0065] Thus, one embodiment relates to methods for simultaneously
generating a plurality of polynucleotide sequencing ladders,
typically in a single reaction vessel (or in a plurality of vessels
whose products are combined after amplification), and analyzing the
sequence information derived from the simultaneously generated
sequencing ladders. Each sequencing ladder is formed from a
recoverable primer having a unique recovery tag. Each
polynucleotide member of a polynucleotide set that constitutes a
sequencing ladder is labeled with essentially the same recovery tag
(or a functional equivalent of a recovery tag). After the
simultaneous generation of multiple sequencing ladders, the
different polynucleotide sequencing ladders are separated from one
another by binding of the recovery tags (or functional equivalents
of recovery tags) to recovery tag binding compounds that are
immobilized on the solid supports in the apparatus discussed above.
Protocols for forming sequencing ladders are well known to persons
of ordinary skill in the art. Chain termination sequencing is a
preferred method of sequencing ladder generation.
[0066] Numerous protocols for chain termination sequencing of
polynucleotides have been published. Such protocols may be used for
simultaneously generating a plurality of polynucleotide sequencing
ladders (and separating the ladders generated) so as to realize
significant savings with respect to costly reagents such as
thermostable enzymes, fluorescently labeled primers, and
fluorescently labeled terminators. Conventional polynucleotide
sequencing techniques usually employ at least 8 to 12 units of Taq
DNA polymerase for each sequence ladder generated. The term "unit"
as used herein with respect to the thermostable polymerase sold
under the name AmpliTaq DNA polymerase (Perkin Elmer, Applied
Biosystems Division, Foster City, Calif.) is defined as the amount
of enzyme that will incorporate 10 nmol dNTP's into acid insoluble
polynucleotide material in 30 minutes at 74.quadrature.C; this
definition may be used to determine corresponding amounts of other
thermostable DNA polymerases. It will be appreciated by those
skilled in the art that the foregoing definition of "unit" may be
applied to many DNA polymerases and is not limited to AmpliTaq DNA
polymerase. Thus, by employing the methods of the invention,
sequencing ladders may be produced by using approximately 4 to 6
units (for two-fold multiplexing), or less, of DNA polymerase for
each sequencing ladder generated. It will also be appreciated by
those skilled in the art that a variety of different DNA
polymerases, both thermostable and heat-labile, may be used for
sequence ladder generation and that similar degrees of reductions
in reagent usage can be achieved with different DNA polymerases.
Numerous different DNA polymerases or mixtures of DNA polymerases
may be used for sequence ladder generation. When the sequence
ladders are generated through cycle sequencing, the DNA polymerase
used is preferably a thermostable DNA polymerase. Examples of
suitable thermostable DNA polymerases include Taq.TM.
(Perkin-Elmer, Norwalk Conn.), Vent.TM. (New England Biolabs,
Beverly Mass.), Deep Vent.TM. (New England Biolabs, Beverly Mass.),
Pyrococcus furiosus DNA polymerase (Stratagene, La Jolla Calif.),
Thermotaga maritima DNA polymerase, and AmpliTaq DNA polymerase,
FS.TM. polymerase, and Ampli DNA polymerase, Taq FS DNA polymerase.
Taq.TM. FS DNA polymerase (Perkin-Elmer, Norwalk Conn.) is
particularly preferred for use in cycle sequencing.
[0067] Multiplex sequencing involves the simultaneous generation of
a plurality of polynucleotide sequencing ladders in the same
solution. The method comprises the step of mixing a plurality of
recoverable sequencing primers with one or more sequencing
templates. The mixing may take place in a single reaction vessel.
The act of mixing comprises placing the recoverable primers and the
templates into the same solution, thereby permitting the primers to
anneal at specific sites on the template (or templates) so that the
primers may be extended. Optionally, the reaction vessels may be
agitated to improve mixing of the solution components. The reaction
vessel serves as a container for the primers, templates, enzymes,
dNTPs, chain terminators, and other reagents required for sequence
ladder generation. The reaction vessel may take on any of a variety
of shapes and sizes that would be known to a person of ordinary
skill in the art, such forms include, but are not limited to,
Eppendorf tubes, sealed capillary tubes, covered multi-well plates,
and the like. After or concurrently with the mixing step, the
recoverable sequencing primers are subjected to conditions that
permit the recoverable primers to hybridize (anneal) to their
respective templates. The plurality of templates used in the
subject methods may be present on the same or different
polynucleotide molecules. For example, a single chromosome or
plasmid may comprise a plurality of sequencing templates if the
recoverable primers are selected so as to anneal to multiple
regions of the same DNA molecule. Alternatively, individual
recoverable primers may be designed to hybridize to a plurality of
templates that are present as separate DNA molecules. Recoverable
primers designed for sequencing may be used to prime both strands
of the same polynucleotide sequence during the same sequence
generating reaction. Examples of sequencing templates include
chromosomal DNA, cDNA, RNA, or DNA inserted into cloning vectors,
and the like. Optionally, the templates for sequencing may be
polynucleotides generated by nucleotide amplification reactions
such as PCR (polymerase chain reaction).
[0068] Preferably, sequencing ladders are formed by
cycle-sequencing A description of cycle sequencing can be found,
among other places, in Murray V., Nucl. Acid. Res., 17:8889 (1989).
Typically, cycle-sequencing is a sequencing ladder generating
technique comprising the following steps: (a) hybridization of an
oligonucleotide primer to a template for sequencing so as to form a
primed template, (b) extending the primer with a DNA polymerase,
(c) ending the extension reaction with a chain terminator (e.g., a
dideoxynucleotide terminator), (d) denaturing the primed template,
(e) repeating steps (a) to (d) for multiple cycles. Increasing the
number of cycles may be used to increase the amount of labeled
polynucleotide produced, thereby compensating for relatively small
amounts of starting material.
[0069] In embodiments of the invention in which the recoverable
primers anneal to the same strand of the same template, the
annealing sites on the templates may be sufficiently close to one
another so that interactions between the two sites during
sequencing ladder generation may be detected. For example, a first
sequencing primer and second sequencing primer may be selected to
anneal to the same chromosome such that the first primer anneals
about 400 bases 5' with respect to the annealing site of the second
primer. The intensity of the sequence ladder signal, i.e., the
quantity of polynucleotide constituents of the sequencing ladder,
produced from the first primer falls off abruptly (though not to
undetectable levels) when the sequence ladders extends through the
annealing site of the second primer. This decrease in intensity may
be used to determine when the sequence information obtained from
two primers is contiguous.
[0070] Any of a variety of chain terminator sequencing may be used
to obtain sequence information from a given template. The different
methods may involve variations in parameters such as the site of
labeling (on the primer or on the chain terminator); the identity
of the labels employed, the number of different labels employed,
and the like.
[0071] For labeled terminator sequencing, sequence information may
be obtained from a given template and a single recoverable primer
by using four chain terminators, each chain terminator
corresponding to a different nucleotide base and labeled with a
distinctive fluorescent label.
[0072] For labeled primer sequencing, the recoverable primers are
labeled and four distinct recoverable primers, each annealing to
the same template site, but having a distinctive fluorescent label,
are used in four separate reaction vessels to obtain the sequence
of each template in the multiplexed sequencing reaction. For
example, a first set of four labelled recoverable primers are
prepared to prime at the same location on a given template. Each of
these primers in the set is labeled with a different detectable
label (four spectrally distinct labels are used). The recovery tag
on each of the primers in a set is labeled with the same or
different recovery tags (preferably the same recovery tag is used
for each member of the set). Additional four primer sets are
prepared for each template to be sequenced. The different members
of each set of primers are then distributed between four reaction
vessels, such that each vessel contains multiple primers (and
templates) but only one primer from each primer set. A sequencing
reaction is then prepared in each vessel, using a single type of
chain terminating dideoxynucleotide in each vessel (A, G, C, or T).
Each primer can be labeled with the same label or with or different
labels. Alternatively, labels can be introduced to sequencing
products using labeled terminators, for example.
[0073] Suitable fluorescent labels which may be used in practicing
the invention include, but are not limited to, 6-carboxyfluorescein
(6-FAM), 5-carboxyfluorescein (5-FAM),
6-carboxy-4,7,2',7'-tetrachlorofluorescein (TET),
6-carboxy-4,7,2',4',5',7'-hexachlorofluorescein (HEX), 5-(and
6)carboxy-4',5'-dichloro-2'7'-dimethoxyfluorescein (JOE), and
5-carboxy-2',4',5',7'-tetrachlorofluorescein (ZOE),
tetramethylrhodamine (TAMRA), 4,7-diclorotetramethyl rhodamine
(DTAMRA), rhodamine X (ROX), rhodamine 6G (R6G), rhodamine 110
(R110), and the like. Descriptions of suitable fluorescent labels
can be found, for example, in U.S. Pat. No. 5,366,860 (Bergot),
U.S. Pat. No. 5,188,934 (Menchen), U.S. Pat. No. 5,654,442
(Menchen), U.S. Pat. No. 6,020,481 (Benson), U.S. Pat. No.
5,863,727 (Lee), U.S. Pat. No. 5847162 (Lee), U.S. Pat. No.
6,008,379 (Benson), and U.S. Pat. No. 5,936,087 (Benson), for
example.
[0074] Non-fluorescent labels may also be used, such as enzymatic
labels, radioactive labels, chemiluminescent labels, etc.
[0075] As discussed above, the recovery tag binding compounds are
located on distinct solid supports (or distinct regions of a
monolithic support) which are located in series in a flow path. The
recovery tag binding compounds are attached to the solid support in
a manner so as to permit the recovery tag binding compounds to
interact with their respective recovery binding tags. The recovery
tag binding compounds may be attached to the support through either
direct or indirect linkages. The term "direct linkage" refers to
the covalent binding of the recovery tag binding compound to the
solid support, including covalent bonding through a linker (and
optionally a spacer arm). The term "indirect linkage" refers the
binding of the of the recovery tag binding compound to the solid
support through a specific binding pair, e.g., biotin-avidin (or
streptavidin) pairs or antigen-hapten, wherein one member of the
pair is joined to the recovery tag binding compound and the other
member of the pair is joined to the solid support.
[0076] A variety of techniques may be used to immobilize the
recovery tag binding compounds on the solid supports. The specific
techniques selected will depend upon the choice of recovery tag
binding compounds and solid support materials. Techniques for
immobilizing proteins and polynucleotides are well known to persons
of ordinary skill in the art of molecular biology. For example,
proteins may be conjugated to solid supports through formaldehyde,
DMS (dimethyl suberimidate), and reductive amination.
Polynucleotides may be conjugated to solid supports through agents
such as 1,3-diaminopropane, 3,3'-iminobisproplyamine, EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), SPDP
(N-succinimidyl 3-(2 pyridyldithio propionate)), and SATA
(N-succinimidyl S-acetylthioacetate). Examples of moieties for
linking oligonucleotides to solid supports can be found in Pon et
al, Biotechniques, 6-768-775 (1988); U.S. Pat. No. 4,659,774
(Webb); WO 92/04384 (Barany et al.); Brown et al, J. Chem. Soc
Commun., 1989:891-893; Dahma et al, Nucleic Acids Res. 18:3813-3821
(1990); Beattie et al, Clinical Chemistry, 39:719-722 (1993); and
Maskos and Southern, Nucleic Acids Res. 20:1679-1684 (1992).
[0077] The strength of binding between a binding compound and its
complementary recovery tag can be characterized by a melting
temperature, Tm, which defines a temperature at which 50% of either
the tag or the binding compound is bound to the other. In a
preferred embodiment, the recovery tags and recovery tag binding
compounds are polynucleotides. For polynucleotides, melting
temperatures are a function of several factors, such as sequence
composition, length of complementary regions, salt concentration,
pH, solvent composition (aqueous vs organic), and concentration of
binding partners (tag and complementary binding compound) and can
be calculated using any of a variety of predictive methods.
Exemplary methods can be found in Breslauer et al., Proc. Natl.
Acad. Sci. 83:3746-3750 (1986); Rychlik et al., Nucleic Acids Res.
17:8543-8551 (1989) and 18:6409-6412 (1990); Wetmur, Crit. Rev.
Biochem. Mol. Biol. 26:227-259 (1991); Osborne, CABIOS 8:83 (1991);
and Montpetit et al., J. Virol. Methods 36:119-128 (1992)).
Typically, complementary polynucleotide sequences are between 15
and 36 nucleotides in length. Complementary sequence lengths of 18
to 24 nucleotides are preferred because such polynucleotides tend
to be very sequence-specific when the annealing temperature is set
within a few degrees of an oligonucleotide melting temperature
(Dieffenbach, supra). Binding interactions can be further optimized
by empirical methods, if desired.
[0078] After a polynucleotide mixture has been prepared (e.g., a
sequencing ladder), the polynucleotide mixture is introduced into
the inlet of the flow path of the apparatus in any appropriate way,
such as by pumping, gravity flow, or vacuum loading. As the sample
mixture passes through the series of supports, polynucleotides that
are complementary to the binding compounds on the first support are
captured thereon by specific binding interactions, while the other
polynucleotides in the mixture continue moving to the next support,
until the mixture has passed through all of the supports. Sample
loading is preferably performed under conditions such that binding
of nontarget polynucleotides to the capture compounds is minimal.
Thus, loading should be performed at a temperature that is (1) well
below (e.g., at least 10.degree. C. below) the lowest Tm of the
capture compounds for binding to their complementary tag sequences,
and (2) well above (e.g., at least 10.degree. C. above) the highest
Tm value of the capture compounds for binding non-complementary
sequences that may be present in the sample. By binding the
recovery tags to their cognate recovery tag binding compounds, the
sequencing ladders (or any other fragments that are to be captured)
are separated from one another and purified.
[0079] Prior to passage through the supports, the polynucleotide
mixture may be modified by adding reagents that enhance binding
between the recovery tags and the recovery tag binding compounds
(e.g., to alter pH, ionic strength, or salt concentration of the
mixture). Binding between the recovery tags and the recovery tag
binding compounds may also be modified by the addition of DNA
binding proteins or other DNA binding compounds. Contact between
the immobilized recovery tag binding compounds and the recovery
tags should be for a period of time sufficient to permit the
binding of a detectable amount of polynucleotide to the immobilized
recovery tag binding compounds. The amount of time (or flow rate)
that permits sufficient binding of the recovery tags may vary,
depending on the specific recovery tag and recovery tag binding
compounds that are used, concentration, etc. The kinetics of
nucleic acid hybridization and denaturation are well understood and
may be used to calculate the time and conditions required for
binding and release. Information on hybridization kinetics can be
found in U.S. Pat. No. 5,935,793 (Wong) and references cited
therein, Berger and Kimmel, Guide to Molecular Cloning Techniques,
Academic Press, San Diego (1987), Cantor and Schimmel, Biophysical
Chemistry, Part III: The Behavior of Biological Macromolecules,
W.H. Freeman, NY (1980), and Saenger, Principles of Nucleic Acid
Structure, Springer-Verlag, New York (1989), for example. See also
WO 98/14610 and counterpart U.S. Pat. No. 6,124,092, both
incorporated herein by reference, which, among other things,
contain examples of experimental conditions that can be used in
performing parts of the present invention. Successful results can
be obtained without binding all (or even a substantial portion) of
the polynucleotides. After the sample has passed through the last
serial support, the column of supports can be washed with
additional solvent or a washing solution to remove non-specifically
bound materials, if necessary.
[0080] After loading has been completed, selected polynucleotides
or polynucleotide populations can be released from selected,
individual supports and eluted from the column for collection. As
discussed above, release of bound polynucleotides can be
accomplished by altering a physical property of the support, such
as temperature or electrical potential, such that the support is
changed from a tag-binding state to a target-non-binding state. The
released polynucleotides can then be eluted from the column by
solvent flow, until the released polynucleotides have passed, in
separated form, through the column outlet for collection.
[0081] The bound polynucleotides can be released from the different
supports in any order. For example, for a pumping arrangement that
pumps solvent unidirectionally through the column, the supports can
be activated for polynucleotide release starting with the support
farthest from the column outlet, followed sequentially by the
adjacent support closer to the outlet, and so on, until all
supports have been cleared of their bound polynucleotide. In an
alternative approach, the supports can be activated starting with
the support closest to the column outlet, followed sequentially by
the adjacent support which is farther from the outlet, until all
supports have been cleared. This latter approach may be preferable
to reduce premature elution of polynucleotides from downstream
supports that may occur when polynucleotides are released and
eluted from an upstream support. Different patterns of support
activation can also be used.
[0082] Sets of recoverable primers that have recovery tags capable
of being released under the same or similar denaturation or
releasing conditions are referred to herein as "integrated" sets of
recoverable primers. One advantage of using integrated sets of
primers is that polynucleotide capture on the different supports
can be performed under approximately the same conditions, so that
control of the conditions of the support can be simplified. In
order to provide an integrated set of primers, the recovery tags on
the primers are selected so as to have Tm's that are within
15.degree. C. of each other, preferably, within 10.degree. C., and
more preferably within 5.degree. C. of each other. The Tm is the
denaturation temperature as measured between the immobilized
recovery tag binding compound and the recovery tag. The Tm may be
determined either empirically or by reference to empirically
determined formulae for Tm calculation. Examples of such formulae
can be found among other places in Berger and Kimmel, Guide to
Molecular Cloning Techniques, Academic Press, San Diego, (1987),
Cantor and Schimmel, Biophysical Chemistry, Part III: The Behavior
of Biological Macromolecules, W.H. Freeman, NY, (1980), Saenger,
Principles of Nucleic Acid Structure, Springer-Verlag, New York
(1989), and the like.
[0083] A releasing step is performed to provide for the analysis of
the released polynucleotides. The releasing step should be
performed in such a manner so as to maintain the separation of the
different sequencing ladders that was introduced during the binding
of the recovery tags to the immobilized recovery tag binding
compounds.
[0084] When the recovery tags and recovery tag binding compounds
are both polynucleotides, release is preferably achieved by
denaturation of the duplex (or possibly triple helix) formed
between the recovery tags and their corresponding recovery tag
binding compounds. Factors influencing the denaturation temperature
of multi-stranded polynucleotides, e.g., cation concentration, are
well known to persons of ordinary skill in the art of molecular
biology. Accordingly, release of the polynucleotide recovery tags
may be achieved by subjecting the bound polynucleotides to elevated
temperatures or the addition of denaturing agents such as urea or
formamide in appropriate concentrations.
[0085] Collection of the released polynucleotide sequencing ladders
(or any other class of polynucleotides that is being isolated) may
be achieved by numerous different techniques and configurations of
devices used to collect the released polynucleotides. For example,
the released sequencing ladders can be collected separately in
individual collection vessels, such as tubes or microtiter dish
wells, and stored for loading onto a polynucleotide separation
device. Such devices are commercially available from a variety of
sources, such as an ABI 377, ABI 310, 3700, or 3100 instruments
available from Applied Biosystems, Foster City, Calif. Descriptions
of automated sequencing apparatus can be found, for example, in
U.S. Pat. Nos. 4,232,769, 4,603,114, 4,704,256, 4,811,218,
5,277,780, 5,290,419, 5,307,148, 5,366,608, 5,384,024, and
5,543,026.
[0086] Preferably, collection of the released polynucleotides, such
as sequencing ladders, is integrated directly with a polynucleotide
separation device, e.g., a multicapillary electrophoresis device
for analysis of the polynucleotides.
[0087] The invention permits the multiplexing of sequencing
reactions, amplification reactions, other types of primer extension
reactions, and other polynucleotide mixtures to varying degrees.
The sequencing reactions may be multiplexed by a factor of two or
more. Typically, multiplexing will be by a factor of between 2 and
20. For example, in one embodiment, the factor is 5, 10, 15, or 20.
In another embodiment, the factor is 5 or less, 10 or less, 15 or
less, or 20 or less. However, the invention also includes
embodiments in which multiplexing is by a factor greater than
twenty.
[0088] Although the foregoing discussion has been primarily
concerned with multiplex methods of sequencing, it will be readily
appreciated by persons skilled in the art that the general
principles of the invention can readily be adapted to virtually any
molecular biology technique involving probe ligation, probe
cleavage, or primer extension. For example, by using a plurality of
recoverable primers, each having a unique recovery tag (or
functional equivalent thereof, multiple primer extension reactions
may be performed simultaneously and the reaction products
subsequently separated on the basis of binding to immobilized
recovery binding tag compounds. These numerous multiplexed methods
of primer extension reactions used are considered to be embodiments
of the subject invention. Chain termination sequencing (Sanger
method) and PCR are examples of primer extension reactions.
[0089] Thus, in another embodiment, the invention also provides
methods for separating a plurality of simultaneously generated
polynucleotide amplification products. This can be accomplished by
modifying the above discussion regarding sequencing ladders so as
to generate recoverable polynucleotide amplification products
rather than recoverable sequencing ladders. Methods for
polynucleotide amplification are well known to persons of ordinary
skill in the art. Detailed protocols for polynucleotide
amplification can be found in, among other places, Dieffenbach and
Dveksler, PCR Primer, A Laboratory Manual, Coldspring Harbor Press,
Coldspring Harbor, N.Y. (1995), McPherson et. al, PCR A Practical
Approach, Vol 1, IRL Press Oxford, England (1991), McPherson et.
al, PCR A Practical Approach, Vol 2, IRL Press Oxford, England
(1995), U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,683,195, and U.S.
Pat. No. 4,965,188. Furthermore, detailed protocols for multiplex
PCR can be found in, among other places, Shuber et al, Genome
Research, 5:488-493 (1995), Eggerding, PCR Methods and
Applications, 4:337-345 (1995), Cuppens et al, Molecular and
Cellular Probes, 6:33-39 (1992), and U.S. Pat. No. 5,582,489.
[0090] The methods of separating a plurality of simultaneously
generated polynucleotide amplification products involve performing
polynucleotide amplification reactions, wherein at least one member
of a pair of amplification primers is a recoverable amplification
primer. When both members of a pair of amplification primers are
recoverable primers, the amplification products produced will have
two recovery tags. When both members of a pair of amplification
primers are recoverable primers, the recovery tags may be the same
or different from one another. The invention also includes
embodiments in which recovery tags and recovery tag binding
compounds may be selected so as to provide for the isolation of
selected sets of nucleic acid amplification fragments rather than
the isolation of individual amplification fragments. Generally, the
subject methods of separating a plurality of simultaneously
generated polynucleotide amplification products (through multiplex
PCR or similar amplification techniques) include the steps of
mixing a plurality of recoverable amplification primers having
recovery tags with a plurality of amplification templates. After
the mixing step, the amplification templates are amplified using at
least one recoverable primer so as to form a plurality of
amplification products, each product having a recovery tag, wherein
the amplification reaction is in a single reaction vessel. Next,
the recovery tags, and hence the amplification products, are
permitted to bind to recovery tag binding compounds that have been
immobilized on a solid support in a spatially addressable manner.
Subsequently, the bound amplification products are released from
the solid supports and individually collected.
[0091] The invention may also utilize recoverable primers having
oligonucleotide recovery tags that cannot be replicated during a
nucleic acid amplification reaction. Thus, when such primers are
employed in polynucleotide amplification reactions, an extension
product complementary to the recovery tag is not generated. These
recoverable primers are referred to herein as "hinged primers."
Hinged primers are particularly useful in multiplex polynucleotide
amplifications as described herein because there is no need to
denature (or prevent from renaturing) a double-stranded
polynucleotide comprising the recovery tag so that the recovery tag
may bind to a recovery tag binding compound that is a complementary
oligonucleotide. Recoverable primers that comprise a recovery tag
that can be replicated during nucleic acid amplification generate a
polynucleotide sequence complementary to the recovery tag sequence
during the process of polynucleotide amplification. This
complementary sequence can significantly compete with the binding
of a recovery tag to an immobilized recovery tag binding compound
(e.g., an immobilized complementary oligonucleotide). Accordingly,
hinged primers may be advantageously employed in many of the
methods of the invention where it is desirable to efficiently
recover the amplification products.
[0092] There are many different oligonucleotides that may be used
as recovery tags that cannot be replicated during a nucleic acid
amplification reaction. In one embodiment of hinged primers, the
recovery tag is an oligonucleotide analog that is not capable of
being replicated by the DNA polymerase used in the amplification
reaction. Examples of such non-replicable oligonucleotide analogs
include, but are not limited to peptide-nucleic acids (PNAs) and
the like. PNAs synthesis and structure is described in, among other
places, Egholm et al, J. Am. Chem. Soc. 114:1895-1897 (1992),
Kosynkina et al, Tet. Lett. 35.5173-5176, Dueholm et al, J. Org.
Chem. 59:5767-5773 (1994). In another embodiment of hinged primers,
the recovery tag may be an oligonucleotide that could otherwise be
replicated by a DNA polymerase, but is blocked by a non-replicable
linker joining the recovery tag to template-annealing sequence
portion of the recoverable primer. Such non-replicable linker may
be oligonucleotide analogs. Alternatively, the non-replicable
linkers have little or no structural similarity to naturally
occurring polynucleotides. Examples of non-replicable linkers that
are oligonucleotide analogs include poly-5' to 3'-deoxyribose
(i.e., DNA without nucleoside bases), peptide nucleic acids and the
like. Examples of non-replicable linkers that are not
oligonucleotide analogs include polyethylene glycol, hydrocarbons,
and the like. Methods of conjugating linkers to polynucleotides are
well know to those of ordinary skill in the art, examples of such
conjugation techniques can be found in Hermanson, Bioconjuqate
Techniques, supra. Typically, the non-replicable linker is located
at the 5' end of the template-annealing region of the hinged
primer, thereby minimizing interference with the activity of the
DNA polymerase catalyzing the extension reaction. Alternatively,
the recovery tag of a hinged primer may be rendered non-replicable
in an amplification reaction by virtue of the site of attachment
(or orientation) of the recovery tag to the primer, e.g., at a
position other than the 5' end primer.
[0093] Although the foregoing discussion is focused primarily on
using recoverable primers for multiplexed sequencing ladders or
polynucleotide amplification, it will be appreciated that the
methods may be readily adapted for use without recoverable primers.
Oligonucleotide primers without recovery tags may be used to
generate recoverable sequencing ladders or recoverable
polynucleotide amplification products; these methods are referred
to as "non-recovery tag multiplex methods." Non-recovery tag
multiplex methods employ the functional equivalents of recovery
tags. In non-recovery tag based embodiments, primers without
recovery tags may be substituted for recoverable primers by using
recovery tag binding compounds that are polynucleotides comprising
a polynucleotide sequence capable of specifically hybridizing to a
polynucleotide sequence that is newly formed during either
sequencing ladder generation or the process of polynucleotide
generation. These newly generated polynucleotide sequences are the
portions of the polynucleotide ladder or amplicon other than the
primer sequence. Suitable recovery tag binding compounds for use
with such primers may specifically bind to either a newly
synthesized polynucleotide region or to a combination of a newly
synthesized polynucleotide region and a primer polynucteotide
region that is immediately adjacent to the 3' end of the
primer.
[0094] The recovery tag binding compounds may be designed to bind
to newly generated polynucleotide sequences that are on the
polynucleotide strand complementary to the polynucleotide strand
comprising the primer. For example, a recovery tag binding compound
may be a polynucleotide complementary to the polynucleotide
sequence in an amplicon that forms a duplex with one of the
amplification primers. In order to design recovery tag binding
compounds for use in the aforementioned embodiments, sequence
information about a portion of the newly generated sequence must
either be known or conjectured. Non-recovery tag based multiplex
methods are of particular interest because they permit primers with
"universal" template-annealing sequences to be used in the
multiplexed sequencing and nucleic acid amplification methods of
the invention. The term "universal" is used to indicate that a
given template-annealing region of a primer may used with a wide
range of templates because the region of the template that the
primer anneals to is common to multiple templates.
[0095] The present invention provides numerous features that are
advantageous when compared to earlier methods of polynucleotide
analysis. A significant advantageous aspect of the invention is
that increased amounts of sequence information may be obtained from
the same or similar amounts of reagents, thereby significantly
lowering the costs associated with producing a given unit of
sequence information. Another significant aspect is that multiple
sequencing ladders may be formed simultaneously in the same
reaction vessel. By simultaneously generating a plurality of
sequencing ladders in the same reaction vessel, the number of
sample handling manipulations is reduced. The invention also
reduces the number or manipulations required for other primer
extension reactions. Reducing the number of sample manipulations
increases the speed with which sequence ladders can be generated
and reduces the opportunities for sample handling errors. Other
aspects of the invention that make it superior to other multiplex
sequencing methods, e.g., the method of Church et al. (U.S. Pat.
No. 5,149,625), include the absence of a need for a membrane
transfer (blotting) step and the absence of a need for subcloning
the polynucleotides for sequencing into special vectors. Other
advantages of the invention are that sequencing ladders, amplicons
(polynucleotide amplification products), or other primer extension
products may be purified, separated, or concentrated with a minimal
amount of manipulations.
[0096] The degree of reduction in reagent consumption achieved by
the methods of the invention is determined, in large part, by the
degree of multiplexing. For example, a sequencing reaction that has
been multiplexed two-fold, i.e., two sequencing ladders are
generated simultaneously in a single reaction vessel, may reduce
the requirement of some sequencing reagents up to two-fold.
Similarly, a sequencing reaction that has been multiplexed
eight-fold, i.e., eight sequencing ladders are generated
simultaneously in a single reaction vessel, may reduce the
requirements for some reagents up to eight-fold. Thus the invention
exploits the "excess" polynucleotide synthetic potential in a
single sequence ladder generation reaction.
[0097] All documents cited herein are incorporated herein b
reference to the same extent as if each individual document was
specifically and individually indicated to be incorporated by
reference. While particular embodiments of the invention are
described herein, it will be apparent to those skilled in the art
that alternatives, modifications and variations can be made without
departing from the scope of the invention.
* * * * *