U.S. patent application number 15/113786 was filed with the patent office on 2017-02-23 for purification chemistries and formats for sanger dna sequencing reactions on a micro-fluidics device.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Swati Goyal, Achim Karger, Peter Ma, S. Jeffrey Rosner, Ian Walton, Jonathan Wang, Michael Wenz.
Application Number | 20170051344 15/113786 |
Document ID | / |
Family ID | 52463184 |
Filed Date | 2017-02-23 |
United States Patent
Application |
20170051344 |
Kind Code |
A1 |
Goyal; Swati ; et
al. |
February 23, 2017 |
Purification Chemistries and Formats for Sanger DNA Sequencing
Reactions on a Micro-Fluidics Device
Abstract
According to various embodiments described herein, a
microfiuidics-chip based purification device and system for
Sanger-sequencing reactions is provided. The device and system
allow for the introduction into a sequencing system of a cartridge
containing purification technologies specific to the sequencing
contaminants or sequencing method where the simplified purification
solution of a cartridge allows automation of the sample
purification process, reduced consumption of purification reagents,
and consistency in sampling by reducing the sampling errors and
artifacts. These various embodiments therefore solve the need for a
microfiuidics-chip-based, Sanger-sequencing reaction purification
system for CE devices. The microfiuidic chips described can be used
as a PCR chip by reorganizing the on-chip reagents, reaction wells
and work flow steps.
Inventors: |
Goyal; Swati; (San Mateo,
CA) ; Karger; Achim; (Foster City, CA) ; Ma;
Peter; (Cupertino, CA) ; Rosner; S. Jeffrey;
(Palo Alto, CA) ; Walton; Ian; (Redwood City,
CA) ; Wang; Jonathan; (Mountain View, CA) ;
Wenz; Michael; (Redwood City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
52463184 |
Appl. No.: |
15/113786 |
Filed: |
January 23, 2015 |
PCT Filed: |
January 23, 2015 |
PCT NO: |
PCT/US2015/012777 |
371 Date: |
July 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61931549 |
Jan 24, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/502753 20130101;
B01L 2300/0887 20130101; B01L 2200/0631 20130101; C12Q 1/6869
20130101; B01L 2200/10 20130101; C12Q 1/6806 20130101; B01L
2200/141 20130101; B01L 2300/12 20130101; B01L 2300/16
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B01L 3/00 20060101 B01L003/00 |
Claims
1. A microfluidic sequencing reaction purification device for
reducing the number of sequencing contaminants in a single-stranded
DNA sequencing sample, the microfluidics chip-based sequencing
reaction purification device comprising, a surface; a solid-phase
extraction substrate; and silane bound to a structure selected from
the group consisting of microstructures, the surface of the
microfluidic device, a membrane, a high-surface area, convoluted
material, and combinations thereof.
2. The microfluidic sequencing reaction purification device of
claim 1, wherein the microstructure comprises microbeads, tubes, or
plates.
3. The microfluidic sequencing reaction purification device of
claim 1, wherein the microstructures comprise polystyrene, latex,
agarose, an ion-exchange resin, an immobilized metal affinity
chromatography resin, or any other substrate or resin capable of
being coated by or bound to silane.
4. The microfluidic sequencing reaction purification device of
claim 2, wherein the microstructures are nonmagnetic, magnetic, or
paramagnetic.
5. The microfluidic sequencing reaction purification device of
claim 1, wherein the high-surface area, convoluted material
comprises frit or wool.
6. The microfluidic sequencing reaction purification device of
claim 1, wherein the sequencing contaminants comprise salts, free
salts, salt ions, ions, sequencing amplification compounds, short
primers, dNTPs, enzymes, short-failed Polymerase Chain
Reaction/Capillary Electrophoresis products, salts from Polymerase
Chain Reaction/Capillary Electrophoresis products, or
unincorporated dideoxyNTPs.
7. The microfluidic based sequencing reaction purification device
of claim 1, wherein, after the sequencing reaction is completed,
the device is configured to introduce unbound silane into the DNA
sequencing sample.
8.-25. (canceled)
26. The microfluidic sequencing reaction purification device of
claim 7, wherein the reagent comprises a binding buffer.
27. The microfluidic sequencing reaction purification device of
claim 26, wherein the high-surface area, convoluted material
comprises frit or wool.
28. The microfluidic sequencing reaction purification device of
claim 26, wherein the microstructures comprise microbeads, tubes,
or plates.
29. The microfluidic sequencing reaction purification device of
claim 26, wherein the microstructures comprise polystyrene, latex,
agarose, an ion-exchange resin, an immobilized metal affinity
chromatography resin, or any other substrate or resin capable of
being coated by or bound to the ion exchanger.
30. The microfluidic sequencing reaction purification device of
claim 26, wherein the sequencing contaminants comprise salts, free
salts, salt ions, ions, sequencing amplification compounds, short
primers, dNTPs, enzymes, short-failed Polymerase Chain
Reaction/Capillary Electrophoresis products, salts from Polymerase
Chain Reaction/Capillary Electrophoresis products, or
unincorporated dideoxyNTPs.
31.-32. (canceled)
33. The microfluidic sequencing reaction purification device of
claim 7, wherein the reagent comprises a hybridization-based
pull-out oligonucleotide selected to be complementary to a
sequencing contaminant.
34. The microfluidic sequencing reaction purification device of
claim 33, wherein the high-surface area, convoluted material
comprises frit or wool.
35. The microfluidic sequencing reaction purification device of
claim 33, wherein the microstructures comprise microbeads, tubes,
or plates.
36. The microfluidic sequencing reaction purification device of
claim 33, wherein the microstructures comprise polystyrene, latex,
agarose, an ion-exchange resin, an immobilized metal affinity
chromatography resin, or any other substrate or resin capable of
being coated by or bound to the ion exchanger.
37. The microfluidic sequencing reaction purification device of
claim 33, wherein the sequencing contaminants comprise salts, free
salts, salt ions, ions, sequencing amplification compounds, short
primers, dNTPs, enzymes, short-failed Polymerase Chain
Reaction/Capillary Electrophoresis products, salts from Polymerase
Chain Reaction/Capillary Electrophoresis products, or
unincorporated dideoxyNTPs.
38. The microfluidic sequencing reaction purification device of
claim 33, wherein the microstructures are nonmagnetic, magnetic, or
paramagnetic.
39. The microfluidic sequencing reaction purification device of
claim 33, wherein, after the sequencing reaction is completed, the
device is configured to introduce into the DNA sequencing sample
unbound hybridization-based pull-out oligonucleotides selected to
be complementary to the sequencing contaminants.
40. The microfluidic sequencing reaction purification device of
claim 7, wherein the reagent comprises any combination of silane,
an ion exchanger, a reagent adapted to sequester the sequencing
contaminants based on the size and charge of the sequencing
contaminants, a binding buffer, or a hybridization-based pull-out
oligonucleotide selected to be complementary to a sequencing
contaminant, as well as their unbound analogues.
41.-48. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] DNA sequencing is the process of determining the precise
order of nucleotides within a DNA molecule. Two common sequencing
methods are Sanger sequencing and "Next-Gen" sequencing, where
Sanger sequencing is a method of DNA sequencing based on the
selective incorporation of chain-terminating dideoxynucleotides (or
ddNTPs) by DNA polymerase during in vitro DNA replication. While
"Next-Gen" sequencing methods are typically used for large-scale,
automated genome analyses, Sanger sequencing is primarily used for
smaller-scale projects with the goal of obtaining especially long
contiguous DNA sequence reads (>500 nucleotides). The use of
labeled (either radioactively or fluorescently) amplification
compounds (modified nucleotides or ddNTPs) for detection in
automated sequencing machines typically results in contamination of
subsequent sequences if the systems are not properly decontaminated
and cleaned. Because avoidance of any overlap in amplification
compound detection requires precise adjustment of all amplification
products, the cleanup of, for example, fluorescence-based Sanger
sequencing reactions is a crucial sample preparation step before
subsequent sample analysis. In the context of other DNA
purification methods, sequencing reaction purification or cleanup
efforts generally focus on single-stranded DNA and generally
requires expulsion to a high degree of the sequencing reaction
contaminants, including buffering salts (de-salting), other ions,
and unincorporated dye-ddNTP (dye-terminator) such that ionic
strength is significantly reduced relative to the original
reaction. For example, a target for residual salt level after
purification could be less than or equal to 5 mM. Further, for dye
terminators, a post-purification target could be greater than
1000-fold reduction from the original dye terminator
concentration.
[0002] As a result, what is needed is a system to automate and
simplify the generation of a clean sequencing sample that, except
for the potential addition of certain compounds (such as formamide)
and certain processes (such as heat-denaturation), yields a
sequencing solution with low contaminant concentrations and is
ready for electrokinetic injection and electrophoretic separation
by capillary electrophoresis (CE).
[0003] A further need, for reasons discussed below, is the
implementation of such a system for purification on a microfluidics
chip, as known methods for purification have not been implemented
on a microfluidics chip.
[0004] Purification of double-stranded DNA on microfluidics chips
(for example, during the extraction and purification of biological
samples such as whole blood or the clean up of PCR-products) have
been largely described in the literature. However, only a limited
amount of research has been devoted to systems allowing on-chip
purification of Sanger sequencing single-stranded DNA reactions
because sequencing is a very demanding application to integrate on
a microchip, by requiring two rounds of thermocycling, with each
requiring subsequent cleanup. Most published DNA assay chips (e.g.,
Rheonix Card.RTM.) are more modest in scope, implementing simpler
workflows, or performing simple 1- or 2-step protocols like the
gDNA preparation or one thermocycling PCR reaction alone.
[0005] One of the few publications demonstrating a
microfluidics-based Sanger sequencing reaction cleanup (Mathies
group of Blazej, Kumaresan and Mathies) describes an affinity
capture/electro-elution chip functionality for the purpose of
sequencing reaction clean-up. In particular, universal capture
oligonucleotides covalently attached to the surface of a gel matrix
`capture gel` (created in one area of the chip) hybridize the
sequencing products. Although this system allows for removal of
charged impurities such as excess dye-terminator and salt by
electrophoretic-elution, the sequencing reaction cleanup method
requires the chip to be electrically connected and able to perform
electrophoresis to function.
SUMMARY OF THE INVENTION
[0006] According to various embodiments described herein, a
microfluidics-chip based purification device and system for
Sanger-sequencing reactions is provided. The device and system
allow for the introduction into a sequencing system of a cartridge
containing purification technologies specific to the sequencing
contaminants or sequencing method where the simplified purification
solution of a cartridge allows automation of the sample
purification process, reduced consumption of purification reagents,
and consistency in sampling by reducing the sampling errors and
artifacts. These various embodiments therefore solve the need for a
microfluidics-chip-based, Sanger-sequencing reaction purification
system for CE devices. Though the following focuses on
Sanger-sequencing reaction purification systems for CE, the
microfluidic chips described can be used as a PCR chip by
reorganizing the on-chip reagents, reaction wells and work flow
steps.
[0007] In an embodiment, a microfluidic sequencing reaction
purification device is provided for reducing the number of
sequencing contaminants in a single-stranded DNA sequencing sample.
The microfluidics chip-based sequencing reaction purification
device can comprising a surface, a solid-phase extraction substrate
and silane bound to a structure. The structure can be selected from
the group consisting of microstructures, the surface of the
microfluidic device, a membrane, a high-surface area, convoluted
material, and combinations thereof.
[0008] In another embodiment, a microfluidic sequencing reaction
purification device is provided for reducing the number of
sequencing contaminants in a single-stranded DNA sequencing sample.
The microfluidic sequencing reaction purification device can
comprise a surface and a reagent bound to a structure. The
structure can be selected from the group consisting of
microstructures, the surface of the microfluidic device, a
membrane, a high-surface area, convoluted material, and
combinations thereof.
[0009] In a further embodiment, a microfluidic sequencing reaction
purification system is provided for reducing the number of
sequencing contaminants in a single-stranded DNA sequencing sample
and automating the purification process. The microfluidics
chip-based sequencing reaction purification system can comprise a
DNA sequencing system, a microfluidics device configured to operate
within the DNA sequencing system. The microfluidics chip can
comprise a surface and a reagent bound to a structure selected from
the group consisting of a solid-phase extraction structure,
microstructures, the surface of the microfluidics device, a
membrane, a high-surface area, convoluted material, and
combinations thereof.
BRIEF DESCRIPTION OF DRAWINGS
[0010] FIG. 1 illustrates sequencing data from sequence purified
using ChargeSwitch.RTM. in 96 well plate.
[0011] FIG. 2 illustrates sequencing data from sequence purified
using Dynabeads.RTM. in 96 well plate.
[0012] FIG. 3 illustrates a side view of a flow-channel according
to one embodiment of the invention.
DETAILED DESCRIPTION
[0013] The following description provides embodiments of the
present invention. Such description is not intended to limit the
scope of the present invention, but merely to provide a description
of embodiments.
[0014] Several formats are possible for the implementation of
purification media on a microfluidics device. Formats include, for
example, micro-beads (for example polystyrene, latex beads or
ion-exchange resin), modified surface on the microfluidics device,
frits and membranes composed of DNA binding material and
paramagnetic beads.
[0015] Combinations of non-limiting examples of purification
methods and formats are summarized in the following table.
Selection can be based on commercial availability, ease of
implementation on a microfluidics device and least required R&D
development effort.
TABLE-US-00001 Surface coating (Non- Para- of the magnetic)
magnetic micro Frit or micro micro fluidics wool Chemistry/Format
beads beads device Membrane material Silane X ChargeSwitch .RTM. X
X BDX .RTM. X PureLink .RTM. X Hybridization X based pull-out Size
Exclusion X
[0016] Each of these exemplary formats are discussed below in
relation to exemplary purifications chemistries.
1. Purification by Solid Phase Extraction Utilizing Silane
[0017] Solid-phase extraction (SPE) is a separation process by
which compounds (solutes) dissolved or suspended in a liquid
mixture are separated from other compounds in the mixture according
to their physical and chemical properties by using the affinity of
the solutes for a solid through which the sample is passed to
separate the mixture into distinct components. In one embodiment,
the microfluidics-chip-based purification device is a solid phase
extraction cartridge utilizing silane bound to microbeads,
membranes, or microstructures within the device (e.g., plastic
tubes or plates). In one embodiment, the microfluidics device and
silane-coated microbeads is utilized in combination as a Sanger
sequencing reaction purification system. In alternative
embodiments, the silane-coated microbreads may comprise
polystyrene, latex, agarose, an ion-exchange resin, an immobilized
metal affinity chromatography (IMAC) resin, or any other substrate
or resin capable of being coated by silane and utilized in a Sanger
sequencing reaction purification system. In yet another embodiment
of the purification system utilizing silane-coated microstructures
(such as microbeads, tubes, plates, or any combination of these
structures), the microstructures may be non-magnetic, magnetic, or
paramagnetic. In yet another embodiment, the silane is bound to a
structure including, but not limited to, a frit, a wool, a membrane
or any other high-surface area, convoluted material, structure, or
compound capable of being incorporated into a microfluidics device.
The microfluidics device can be, for example, a microfluidic chip,
card or cartridge.
[0018] In an embodiment, silane-coated microbeads (e.g.,
paramagnetic beads) bind single stranded DNA (ssDNA) and RNA. DNA
can be bound in very low copy numbers. For example, as little as 10
copies of M13 single-stranded DNA could be captured and eluted from
the paramagnetic beads. In a typical silane-coated bead
purification protocol, 2 mg are used in a 400 .mu.l bind reaction
volume to capture approximately 5 .mu.g of genomic double stranded
DNA onto the surface of the paramagnetic micro beads. The
conditions chosen for these experiments were such that the standard
(tube)-scale amounts of beads (2 mg per assay) were used to extract
the ssDNA in a 400 .mu.l volume. Moreover, since the beads are
paramagnetic, they can be easily immobilized in a microfluidic
device (e.g., cartridge) format.
2. Purification by Reversible Ion-Exchange Binding of DNA
[0019] Reversible ion-exchange binding of DNA is a purification,
separation, or decontamination process by which ions are exchanged
between two electrolytes or between an electrolyte solution and a
complex. The process typically involves solid polymeric or
mineralic ion exchangers (e.g., ion exchange resins (such as
functionalized porous or gel polymers), zeolites, and
montmorillonite, clay, or soil humus). Ion exchangers can also
include, for example, ionizable (or switchable) ion exchangers. In
an embodiment of an ion-exchanger, the ion-exchanger comprises a
surface ligand whose surface charge is a function of pH. The
ion-exchanger surface ligand, for example, can be positively
charged at low pH, and neutral at pH 8.5, to bind and elute
plasmid.
[0020] In an alternative embodiment of the purification system, a
microfluidics device comprising microstructures (such as
microbeads, tubes, or plates) coated in at least one ion-exchanger
is utilized as a Sanger sequencing reaction purification system.
For example, the surface of the microfluidics device can be coated
in at least one ion-exchanger. In another example, the
microfluidics device can comprise a membrane coated with at least
one ion-exchanger. In yet another example, the ion-exchanger can be
bound to a structure including, but not limited to, a frit, a wool,
a membrane or any other high-surface area, convoluted material,
structure, or compound capable of being incorporated into a
microfluidics device. In a further example, one or any combination
of the microstructures, the device surface, a high-surface area
structure, or a membrane can be coated in an ion-exchanger as
described above. In another embodiment of the purification system
utilizing ion-exchanger-coated microstructures (such as microbeads,
tubes, plates, or any combination of these structures), the
microstructures may be, for example, non-magnetic, magnetic, or
paramagnetic. The microfluidics device can be, for example, a
microfluidic chip, card or cartridge.
3. Purification by Size Exclusions and Ion-Exchange (SEW)
[0021] Size exclusion and ion-exchange (SEIE) is a process in which
molecules in solution are separated by their size (e.g., molecular
weight) and charge. In an alternative embodiment of the
purification system, a Sanger sequencing reaction purification
system utilizes a microfluidics device and reagents specifically
chosen to sequester reaction components based on the components
charge and size. These reagents can be utilized by the
microfluidics device to capture unincorporated dye exterminators,
dNTPs, free salts, or salt ions generated during the sequencing
reaction. The reagents can be bound to microstructures, where the
microstructures may comprise microbeads, membranes, or structures
within the chip (e.g., plastic tubes or plates). The reagents can
be bound to microstructures (such as microbeads, tubes, plates, or
any combination of these structures) that may be, for example,
non-magnetic, magnetic, or paramagnetic. Alternatively, the
reagents can be bound to the surface of the microfluidics device,
or can also be bound to a structure including, but not limited to,
a frit, a wool, a membrane or any other high-surface area,
convoluted material, structure, or compound capable of being
incorporated into a microfluidics device. Finally, the
size-exclusion and ion-exchange reagents can be bound to one or any
combination of microstructures, the surface of the microfluidics
device, or a high-surface area, convoluted structure within the
microfluidics device as described above. The microfluidics device
can be, for example, a microfluidic chip, card or cartridge.
[0022] In contrast to other cleanup chemistry (bind-wash-elute
style), size exclusion beads work by binding the known impurities.
Sephadex.RTM. beads, for example are very cost-effective
commercially available size exclusion beads that have the ability
to extract terminators and salt while leaving the products of the
sequencing reaction in solution. These beads advantageously have a
relatively low cost. Moreover, they have been used routinely for
sequencing reaction cleanup in conjunction with 96-well filter
plates (for example, MultiScreen.RTM. 96w plates (Durapore.RTM. or
Ultracell.RTM.-10 filter) from Millipore).
4. Purification by Membrane
[0023] Purification by membrane is a mechanical separation process
in which undesirable sequencing solution reaction compounds are
removed from the system using binding buffers and a porous physical
structure (such as a membrane) through which the bound reaction
compounds cannot pass. In one embodiment, the system uses at least
one binding buffer and at least one porous structure configured to
remove reaction compounds such as short primers, dNTPs, enzymes,
short-failed PCR/CE products, salts from PCR/CE products, or any
combination thereof. In an alternative embodiment of the
purification system, a Sanger sequencing reaction purification
system utilizes a microfluidics device comprising at least one
binding buffer and at least one membrane. The membrane may be any
structure including, but not limited to, a frit, a wool, a
membrane, or any porous, high-surface structure through which a
solution may pass. The microfluidics device can be, for example, a
microfluidic chip, card or cartridge. An example of purification by
membrane is PureLink.RTM., manufactured by Life Technologies.
5. Purification by Hybridization-Based Pull-Out
[0024] Hybridization-based pull-out is based on
hybridization-binding of the sequencing reaction products to a
complementary oligonucleotide that is attached or bound to a
microstructure or the surface of the microfluidics device. In an
embodiment of the purification system, a Sanger sequencing reaction
purification system utilizes a microfluidics device comprising at
least one hybridization-based pull-out oligonucleotide selected to
be complementary to a sequencing reaction product. In an
alternative embodiment, the hybridization-based pull-out
oligonucleotide is bound to a microstructure or the surface of the
microfluidics device. In another embodiment of the purification
system utilizing hybridization-based pull-out oligonucleotides
bound to microstructures (such as microbeads, tubes, plates, or any
combination of these structures), the microstructures may be, for
example, non-magnetic, magnetic, or paramagnetic. The
hybridization-based pull-out oligonucleotide can be bound to a
structure including, but not limited to, a frit, a wool, a membrane
or any other high-surface area, convoluted material, structure, or
compound capable of being incorporated into a microfluidics device.
The hybridization-based pull-out oligonucleotide can also be bound
to one or any combination of microstructures, the surface of the
microfluidics device, or a high-surface-area, convoluted structure
as described above. The microfluidics device can be, for example, a
microfluidic chip, card or cartridge.
[0025] Hybridization beads, for example, with capturing
oligonucleotide attached to its surface, can be made by numerous
manufacturing processes including, for example, the process used to
manufacture Anti-miRNA Bead Capture (ABC) beads. The ABC kit is a
commercialized product for capturing specific miRNA from a
biological sample (e.g., blood) directly. It can use a
complementary oligonucleotide on a magnetic bead to hybridized and
capture specific miRNA. The bead-oligo linkage is covalent and
permanent using the well-known carboxy (on bead), NH-ester (on
oligo) standard chemistry.
6. Purification Combining Multiple Purification Methods
[0026] In another embodiment, the microfluidics device comprises
multiple technologies, including, but not limited to, solid-phase
extraction technology utilizing silane, reversible ion exchange
binding of DNA, size exclusion and ion-exchange technology,
membrane technology, and hybridization-based pull-out
technology.
Examples
[0027] ChargeSwitch.RTM. (product of Life Technologies)
Purification example of reversible IE binding of DNA:
Polymer Preparation:
[0028] Bis-Tris is reacted with Polyacrylic Acid in the presence of
EDC to yield a polymer containing bound Bis-Tris. [0029] Polymer
bound Bis-Tris can be protonated by acid (H+) in acidic conditions
producing a positive charged surface. The positively charged
polymer selectively binds to DNA. [0030] At higher pH (>8), the
polymer should still be water soluble and have no charge;
effectively enabling elution of bound DNA
Protocol to Coat Polymer on Solid Surface:
[0030] [0031] 1. Polymer can be diluted in 1% PB buffer. [0032] 2.
100 .mu.l of 10-100% polymer is added to the solid surface to be
coated. [0033] 3. Wait 15 min and discard polymer from the surface
[0034] 4. Add 100 .mu.l of 1% PB buffer, wait 2 min and remove the
buffer [0035] 5. Step 4 is repeated again [0036] 6. Air dry solid
surface for at least 2 hours
Protocol to Purify Sequencing Reactions
[0037] a. With polymer coated on solid surface. [0038] 1. Add 10
.mu.l of sample to the coated surface. [0039] 2. Bind the sample by
adding and equivalent volume (10 .mu.l) of DCB to it. [0040] 3.
Incubate for 15 min, remove the liquid completely. [0041] 4. Wash
the sample by adding 150 .mu.l of DCBW to the plate location [0042]
5. Incubate for 1 min, remove the liquid completely [0043] 6. A
final wash of sample is done by flowing 150-200 .mu.l of
nuclease-free-water over the surface [0044] (If possible, minimize
the time from adding the water to removing it in step 11 as much as
possible to prevent possible elution of the bound sample.) [0045]
7. Elute the sample by adding 10 .mu.l of HDF or Tris HCl (pH 8.5)
[0046] 8. Incubate for 5 min and then collect the supernatant
[0047] b. With polymer coated on magnetic beads. [0048] 1. To 10
.mu.l of sample, add 10 .mu.l DCB42 buffer and 2 .mu.l of beads
(stable at RT) [0049] 2. Mix and incubate at RT for 7 min [0050] 3.
Magnetize beads and discard supernatant [0051] 4. Demagnetize and
add 150 .mu.l of DCBW6 wash buffer and mix [0052] 5. Magnetize
beads and discard supernatant [0053] 6. Repeat step 4 & 5
[0054] 7. Demagnetize and add 10 .mu.l of elution buffer [0055] 8.
Mix and incubate for 5 min [0056] 9. Magnetize beads and collect
supernatant to sample outlet
[0057] FIG. 1 illustrates sequencing data from sequence purified
using ChargeSwitch.RTM. in 96 well plate.
[0058] DynaBeads.RTM. Purification example of SPE:
Bead Preparation:
[0059] Dynabeads.RTM. MyOne.TM. SILANE (product of Life
Technologies) are supplied at a concentration of 40 mg/ml. Prior to
use, the beads should be transferred to the appropriate binding
solution as follows: [0060] 1. Re-suspend the Dynabeads.RTM.
MyOne.TM. SILANE completely (e.g. vortex) to a homogenous
suspension prior to use. Leave on a roller until use. [0061] 2.
Transfer 400 .mu.l of re-suspended Dynabeads.RTM. MyOne.TM. SILANE
to a fresh tube. Place the tube on the magnet until the supernatant
is clear, then remove and discard the supernatant. [0062] 3.
Re-suspend in 13.33 .mu.l of 40% TEG (tetraethlyene glycol) [0063]
4. Add 240 .mu.l 100% ethanol and mix thoroughly. [0064] 5. The
final bead-solution used in the isolation protocol described below
should contain Dynabeads.RTM. MyOne.TM. SILANE at 1.5 mg/ml in 2%
TEG/90% ethanol.
Protocol for Sequence Clean-Up
[0064] [0065] 6. Add 20 .mu.l (30 .mu.g) Dynabeads.RTM. MyOne.TM.
SILANE (supplied in TEG and ethanol, see above) to 10 .mu.l
sequencing reaction mix. [0066] 7. Mix and incubate for 10 minutes
at room temperature. [0067] 8. Magnetize the beads and remove the
supernatant completely. [0068] 9. Demagnetize and re-suspend the
Dynabead.RTM. MyOne.TM. SILANE in 30 .mu.l 55% ethanol. [0069] 10.
Magnetize the beads and remove the supernatant completely. [0070]
11. While still on the magnet, let the Dynabeads.RTM. MyOne.TM.
SILANE pellet air-dry for 5 minutes at room temperature. [0071] 12.
Demagnetize and re-suspend the Dynabeads.RTM. MyOne.TM. SILANE in
10 .mu.l water. [0072] 13. Incubate for 3 minutes at room
temperature. [0073] 14. Magnetize beads again and transfer the
supernatant containing the sequencing products for sequencing
readout.
[0074] FIG. 2 illustrates sequencing data from sequence purified
using Dynabeads.RTM. in 96 well plate.
[0075] BigDye XTerminator.RTM. example of SEIE purification. BigDye
XTerminator.RTM. (BDX) (product of Life Technologies) is used for
sequencing purification in 96 or 384 well-based plates. BDX beads
capture the left over waste products from the BDX reactions. The
BDX beads are two bead types. The first is an ion exchange bead
designed to capture negative charged items. These beads are also
coated with a surface that will prevent large negative charged
sample fragments from binding. The second bead-type is an ion
exchange bead that captures positive charged moieties.
Procedure to Purify Sequencing Product on Chip Using BDX
[0076] 1. Add 45 .mu.l of SAM solution and 5 .mu.l of beads
solution to 10 .mu.l of sample at RT [0077] 2. Mix the liquid (by
moving back and forth or as appropriate) for 20 min [0078] 3. The
supernatant containing the sequencing reaction can be separated
from beads using various methods: [0079] a. Magnetization can be
used if BDX beads are coated on magnetic core [0080] b. Micro pore
filters can be used to retain the beads. [0081] c. A flow-channel
10 can be designed to retain beads when liquid passes through it,
as illustrated in FIG. 3. Inlet 20 accepts a mix of beads and
liquid with trapped beads 30 allowing clear liquid 40 to pass
through the channel. [0082] a. The beads can be staged such that
the first beads capture the positive charged moieties and the
second stage captures the negative charged moieties. The BDX beads
also have a size exclusion coating that prevents the longer DNA
sample fragments from being captured. The beads can be staged so
that the sample does not become clogged. This may require multiple
stages of beads. [0083] d. Mixing can be used to promote efficient
waste capture in the beads. Magnetic beads can aid mixing. The
beads can transit between multiple chambers or regions in single
chamber with magnets that oscillate between positions. [0084] e. If
beads are captured in a frit then the sample can oscillate back and
forth in the beads. Alternatively, a fluid path could be created
where the sample is circulated through bead region. After a given
number of passes, a valve is opened and the clean sample is allowed
to pass to the next stage. [0085] f. The beads and sample can be
heated to increase the reaction rate of the waste to the beads.
Heating can also help when charge switch or silane beads are used.
[0086] g. Membranes with the same ion exchange and size exclusion
properties can replace the BDX beads. One membrane would capture
negative ions and a second the positive ions. Mixing would be
achieved as for beads in a frit. [0087] 4. Collect the liquid for
sequencing readout.
[0088] The preceding descriptions of various implementations of the
present teachings have been presented for purposes of illustration
and description. It is not exhaustive and does not limit the
present teachings to the precise form disclosed. Modifications and
variations are possible in light of the above teachings or may be
acquired from practicing of the present teachings. Additionally,
the described implementation includes software but the present
teachings may be implemented as a combination of hardware and
software or in hardware alone. The present teachings may be
implemented with both object-oriented and non-object-oriented
programming systems.
Sequence CWU 1
1
2175DNAHomo sapiens 1gtggctgcag cctggttatg attactgtta atgttgctac
tactgctgac aatgctgctg 60ctgcttctcc tcact 752111DNAHomo Sapiens
2ccctggagga gaacaaaggc ttacttagtt ccctcatagg agaatgaaca gcaacaggga
60aattattaaa tgcttaattc ggtaatgaca tacagtgtaa cagtgtgtca g 111
* * * * *