U.S. patent application number 09/781655 was filed with the patent office on 2001-11-22 for method of determining the sequence of nucleic acids employing solid-phase particles carrying transponders.
This patent application is currently assigned to Pharmaseq, Inc.. Invention is credited to Mandecki, Wlodek.
Application Number | 20010044109 09/781655 |
Document ID | / |
Family ID | 24256194 |
Filed Date | 2001-11-22 |
United States Patent
Application |
20010044109 |
Kind Code |
A1 |
Mandecki, Wlodek |
November 22, 2001 |
Method of determining the sequence of nucleic acids employing
solid-phase particles carrying transponders
Abstract
A method is described for determining the sequence of nucleic
acids. The method employs small solid phase particles having
transponders, with a primary layer of an oligonucleotide of known
sequence attached to the outer surface of the particle. A
read/write scanner device is used to encode and decode data on the
transponder. The stored data includes the sequence of the
oligonucleotide immobilized on the transponder. The sequence of
sample nucleic acids is determined by detecting annealing to an
oligonucleotide bound to a particle, followed by decoding the
transponder to determine the sequence of the oligonucleotide.
Inventors: |
Mandecki, Wlodek;
(Libertyville, IL) |
Correspondence
Address: |
K. Shannon Mrksich, Ph.D., Esq.
BRINKS HOFER GILSON & LIONE
P.O. BOX 10395
CHICAGO
IL
60610
US
|
Assignee: |
Pharmaseq, Inc.
|
Family ID: |
24256194 |
Appl. No.: |
09/781655 |
Filed: |
February 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09781655 |
Feb 12, 2001 |
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09491271 |
Jan 26, 2000 |
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09491271 |
Jan 26, 2000 |
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09013114 |
Jan 26, 1998 |
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6046003 |
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09013114 |
Jan 26, 1998 |
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08564860 |
Nov 30, 1995 |
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5736332 |
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Current U.S.
Class: |
435/6.19 ;
250/200; 250/356.1; 435/287.2; 436/94 |
Current CPC
Class: |
C12Q 1/6874 20130101;
C40B 70/00 20130101; Y10S 435/81 20130101; C12Q 2563/185 20130101;
C12Q 2563/149 20130101; C12Q 1/6869 20130101; Y10S 435/973
20130101; Y10T 436/143333 20150115; B01J 2219/005 20130101; B01J
2219/00567 20130101; C12Q 1/6869 20130101; C40B 40/06 20130101;
B01J 2219/0059 20130101; B01J 2219/00596 20130101; B01J 19/0046
20130101; B01J 2219/00722 20130101 |
Class at
Publication: |
435/6 ; 436/94;
435/287.2; 250/200; 250/356.1 |
International
Class: |
C12Q 001/68; G01N
033/00; C12M 001/34 |
Claims
I claim:
1. A method of determining the sequence of a target nucleic acid
sequence in a sample, comprising the steps of: (a) providing a
solid phase comprising particles having transponders, the particles
having an oligonucleotide probe attached to a surface of the solid
phase particles, the transponders having memory elements and an
index number indicating sequence of the probe encoded on the
transponders; (c) contacting the solid phase with a sample to form
a sample mixture; (d) denaturing nucleic acids in the sample
mixture; (e) hybridizing the nucleic acids in the sample mixture,
whereby target nucleic acid sequences hybridize to complementary
probes; (f) analyzing the solid phase to detect the presence of a
label indicative of binding target nucleic acid to probes; (g)
decoding the data encoded on transponders using the dedicated
read/write scanner to identify the sequence of the probes to which
target nucleic acids are bound.
2. The method of claim 1, further comprising the step of analyzing
the sequences of probes to which target nucleic acid bound to
determine at least a portion of the sequence of the target nucleic
acid.
3. The method of claim 1 wherein the label is bound to the target
nucleic acid.
4. The method of claim 1 wherein the label is added after the
annealing step through a chain extension reaction using DNA
polymerase.
5. The method of claim 1 wherein the data comprises the sequence of
the oligonucleotide probe deposited on solid phase.
6. The method of claim 1 wherein the data comprises characteristics
of the sample.
7. A method of determining the sequence of target nucleic acid
thought to contain a plurality of subsequences, comprising the
steps of: (a) introducing into the sample at least two populations
of solid phase particles, each particle having a transponder and
having an oligonucleotide probe corresponding to one of the
subsequences attached to its surface, a first population having a
different oligonucleotide probe sequence than a second population
and the transponders in the first population being encoded with a
different identification than the transponders of the second
population; (b) denaturing the nucleic acids in the sample; (c)
hybridizing the nucleic acids in the sample, whereby target nucleic
acid sequences hybridize to the oligonucleotide probes; (c)
analyzing the particles to detect a label indicating that target
nucleic acid has bound to the probe; and (d) decoding the
transponder to determine the sequence of the probe.
8. The method of claim 7, wherein the solid phase comprises at
least three populations of solid phase particles, each particle
having a transponder and having an oligonucleotide probe
corresponding to one of the subsequences attached to its surface,
each of the three populations having a different oligonucleotide
probe sequence and each of the populations being encoded with a
different identification than the transponders of the second
population.
9. The method of claim 7 wherein the surface of the particles is
glass, latex or plastic.
10. The method of claim 7 wherein the oligonucleotide probe is
single-stranded.
11. The method of claim 7, wherein the-oligonucleotide probe is
biotinylated and the particle is coated with a layer of
streptavidin.
12. A kit for determining the sequence of target nucleic acids in a
sample, comprising: (a) at least one assay vessel, containing at
least one solid phase particle having a transponder, and an
oligonucleotide probe bound to a surface of the particle; and (b)
at least one label reagent.
13. The kit of claim 12, wherein the label reagent comprises a
reagent that labels the target nucleic acid.
14. The kit of claim 12, wherein the label reagent comprises a
labelled nucleoside for use in a chain extension reaction using DNA
polymerase.
15. The kit of claim 12, further comprising: (a) a sample diluent
buffer solution; and (b) an enzyme reaction buffer solution.
Description
BACKGROUND OF THE INVENTION
[0001] This invention relates to materials and methods for
determining the sequence of unknown or target nucleic acids, and
more specifically to materials and methods for determining the
sequence of target nucleic acids using an electronically-indexed
solid phase, with transponders associated with the solid phase
particles.
[0002] A high throughput method for ascertaining the sequence of
sample nucleic acids is sequencing by hybridization (SBH). In that
method, a large number of oligonucleotide probes is allowed to
interact with the nucleic acid molecules in a sample, and a
detection system is provided to determine whether individual
oligonucleotides have annealed to the template. Two basic designs
have been described. In one, the oligonucleotide probes are
arranged in a two-dimensional array on the surface of a membrane,
filter, VLSI chip, or the like. In the other, the array on the
membrane is formed with a large number of sample DNA sequences, and
each membrane is subjected to a series of hybridization steps with
different oligonucleotide probes. A label used to monitor the
binding, either a radioactive isotope or a fluorophore, is carried
on the sample DNA or on the oligonucleotide probe. The sequence is
derived from coordinates of spots showing a high level of the label
deposition on the two-dimensional arrays.
[0003] Conventional SBH methods are limited by difficulties related
to preparation of arrays of DNA, non-specific annealing of DNAs,
the need for special instrumentation to read the data and the
automation of the process and data analysis. In SBH, the sequence
is determined by defining the two-dimensional coordinates of
relevant dots in the array formed by the deposited DNA molecules.
In the present invention, a partial or the complete sequence of the
DNA molecule is -determined by decoding the electronic memory
elements associated with DNA probes of known sequence.
[0004] An advantage of the present invention over conventional
sequencing methods is that it is extremely fast, because the
sequence is deduced from a series of readings of digitally-stored
sequences in the transponder, rather than from a series of
measurements of a chemical or physical property of DNA, or the
location of DNA in an array. The method of this invention is
referred to hereinafter as digital sequencing.
SUMMARY OF THE INVENTION
[0005] The present invention overcomes the problems of conventional
sequencing methods by employing solid phase particles having a
transponder associated with each particle. The particles carry
oligonucleotide probes attached to their surface, and the sequence
of the oligonucleotide is encoded on a memory element on the
transponder. The oligonucleotide probes correspond to subsequences
believed to exist in the target sequence.
[0006] To determine the sequence of sample, or "target" DNA, the
target DNA of unknown sequence is labeled with a fluorophore and
combined with transponder particles carrying known oligonucleotides
under annealing conditions. The transponders are analyzed to detect
the fluorescence or color originating from a label that indicates
that target DNA has bound to the probe attached to the surface of
the transponder, and the information stored electronically in the
transponder is decoded. Dedicated sequence analysis software may
then be used to determine the complete or partial sequence of the
DNA target.
[0007] In one aspect, the present invention provides a solid phase
particle for use in determining the sequence of nucleic acids,
comprising a solid phase particle having a transponder, and an
oligonucleotide probe having a known sequence attached to an outer
surface of the particle.
[0008] In another aspect, the present invention provides a method
of determining the sequence of sample nucleic acids, comprising the
steps of employing solid phase particles with transponders.
[0009] In another aspect, the invention provides a kit for
determining the sequence of unknown nucleic acids, comprising an
assay vessel, and a set of solid phase particles having
transponders, and a different oligonucleotide attached to the
surface of the probe.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic representation of a sequencing
procedure of this invention.
[0011] FIG. 2 is a cross-sectional view of a solid phase particle
with a transponder, and a primary layer of biomolecules bound to a
surface thereof.
[0012] FIG. 3 is a schematic diagram of the signal pathway for
encoding and decoding data on the transponders of the solid
phase.
[0013] FIG. 4 is a schematic representation of a miniature
transponder.
[0014] FIG. 5 is a plan view of a miniature transponder.
[0015] FIG. 6 is a plan view of a transport system/analytical
instrument for implementing the present invention.
[0016] FIG. 7 is a plan view of a modified flow cytometer for high
speed analysis of solid phase particles of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] FIG. 1 depicts a simple sequencing procedure of the current
invention. A solid phase particle 10 with a transponder 12 is
derivatized by attaching an oligonucleotide probe 14 of known
sequence to the outer surface 16 of the particle 10. The
transponder 12 is encoded with an index number that indicates the
sequence of the oligonucleotide probe 14. The particle 10 is
immersed in a solution containing labelled target DNA 11, and the
solution is heated to cause DNA to dissociate, then cooled to allow
the DNA to anneal, resulting in target DNA 11 annealing to the
probe 14. The transponder 12 is analyzed to detect the fluorescence
of any target DNA 11 bound to the transponder 12, and the
transponder 12 is decoded using a read/write scanner device (not
shown). In practice a group of transponders, each carrying a
different probe and each encoded with the sequence of that probe
would be used.
[0018] The target DNA is preferably pre-treated by digestion with
an appropriate restriction endonuclease, or fragmented by digesting
with DNase I, to yield relatively short (preferably 10 nt to 100
nt) preferably single-stranded DNA. Pretreatment may also involve a
conversion of DNA to RNA by cloning and in vitro transcription,
followed by a partial hydrolysis of RNA (if necessary), or a
generation of a DNA fragment by PCR, and the latter can be coupled
with labeling of DNA with a fluorophore. DNA provided for
sequencing may already be in the preferred form. If, however, the
DNA is in the form of a long double-stranded DNA molecule, a long
single-stranded DNA molecule, a closed circular DNA molecule, or a
nicked circular double-stranded DNA molecule, the DNA must be
pre-treated.
[0019] Labelling sample DNA with a fluorogenic hapten before
annealing is a practical, although not always necessary, step in
digital sequencing. Restriction fragments with the 5'-protruding
end can be labeled in a chain extension reaction with a DNA
polymerase utilizing one or more of dNTPs derivatized with a
fluorogenic hapten. 3'-ends of DNA molecules can be labeled in an
enzymatic reaction employing a terminal deoxynucleotidyl
transferase (TdT) and dNTP derivatized with a fluorogenic hapten.
In some sandwich-type applications, the fluorogenic hapten can be
replaced with any hapten (e.g. biotin) for which an antibody (or a
binding partner) is available, the goal being to detect the binding
of labeled DNA to the transponders through a sandwich configuration
involving an anti-hapten antibody and a secondary anti-antibody
antibody conjugated to an enzyme, said enzyme catalyzing a reaction
with a precipitating fluorogenic substrate.
[0020] Several types of nucleic acid can be immobilized on the
transponders. They include DNA, RNA and modifications thereof, such
as protein-nucleic acid (PNA) molecules. It is preferred that the
immobilized nucleic acids are single-stranded. The length of the
immobilized nucleic acids can vary in different implementations of
the digital sequencing, the requirement being that the length
should be sufficient to provide a desired level of binding
specificity to the target DNA. In one embodiment of this invention,
the oligonucleotide probes correspond to subsequences that are
believed to exist in the target nucleic acid sequence.
[0021] There are several methods to immobilize nucleic acids on the
solid phase particles of this invention, including the conjugation
of oligonucleotides to the transponders, or the direct chemical
synthesis of the oligonucleotide on the transponders. Combinatorial
synthesis is a preferred method of direct chemical synthesis, and
involves up to four independent condensations of a four different
nucleosides (A, C, G and T) on a large number of transponders in
four vessels. The transponders are divided into four pools, and
each pool is reacted with a different nucleoside. After each
condensation, the transponders in the four pools are encoded with a
symbol indicating the nucleoside used in the condensation. The four
transponder pools are then combined and redistributed into four
vessels, and the process is repeated as many times as necessary and
practical. The net result of combinatorial synthesis on
transponders is that the transponders are derivatized with
different oligonucleotides, and the sequence of the oligonucleotide
is encoded in the transponder.
[0022] The derivatized transponders and the target DNA are kept in
a single vessel in an appropriate buffer. The volume of the buffer
must be sufficient to completely immerse the transponders in the
buffer. An appropriate buffer for the annealing reaction is
phosphate-buffered saline (PBS), but many other buffers are
suitable, as is well-known by those of ordinary skill in this art.
The vessel is heated to a temperature typically in the range of
60.degree.-100.degree. C. The temperature should be sufficient to
allow for melting of double-stranded DNA that might exist in the
vessel into the single-stranded form. The vessel is then slowly
cooled to a temperature below the melting temperature for the
sequences immobilized on the transponder, which typically is in the
range of 0.degree.-60.degree. C., and is often room temperature.
The transponders are then washed thoroughly several times to remove
unbound target DNA.
[0023] If the target DNA was fluorescence-labeled before annealing,
no post-annealing treatment is needed, and the transponders can be
subjected to decoding, and the fluorescence of the surface can be
measured. Alternatively, if the target DNA was not labeled,
fluorescence-labeling must be done after annealing. Either target
DNA (associated with the oligonucleotide bound to the transponder)
can be labeled, or the oligonucleotide probe on the transponder
(but only those that are associated with the target DNA) can be
labeled. A method suitable for both approaches is chain extension
utilizing a DNA polymerase.
[0024] Chain extension labeling procedures can differ with regard
to: (a) the type of the fluorophore-tagged nucleotide used for the
labeling, or (b) the number of different fluorescent nucleotides
used in the extension reaction (choice of the adenine, cytosine,
guanine or thymine derivatives). As to the type of nucleotide used,
the nucleotide can be either of the deoxy type, or the dideoxy
type. More than one labeled deoxy nucleotide can be incorporated
into the extended portion of the chain, but the incorporation of
only one dideoxy nucleotide is attainable, since it prevents
further chain extension.
[0025] The labeling can be combined with a fluorescence detection
method for transponders which allows for distinguishing the type of
the fluorescence label used by identifying the maximum emission
wavelength of the fluorophore, similar to the implementation in
automated sequencing on Applied Biosystems sequencers. The use of
dideoxy nucleotides in digital sequencing, therefore, offers the
ability to identify the residue immediately proximal to the 3' end
of either target DNA, or the oligonucleotide on the transponder,
depending on the approach used.
[0026] In a preferred embodiment of the labeling procedure in the
presence of four fluorophore-labeled dideoxy nucleotide
triphosphates (ddATP, ddCTP, ddGTP and ddTTP) the primers attached
to the transponder that annealed to the target are extended by one
nucleotide residue by a DNA polymerase. The type of the
incorporated residue (A, C, G or T) is determined by the sequence
of the target. Therefore, the wavelength at which the maximum
intensity of fluorescence is observed indicates the residue type.
After the extension step, the particles are passed through a
fluorometer capable of discriminating between four wavelengths of
emitted light. In this approach, more information is obtained than
in the basic implementation of the method. As previously, the
presence of the fluorescence is an indication that the annealing
took place, but now the wavelength of the fluorescence additionally
identifies the target residue that is immediately downstream from
the primer (i.e. close to the 3' end of the primer).
[0027] In an alternative version of the above procedure, only one
fluorophore is used in the reaction, but four separate extension
reactions are performed in four separate vessels employing one of
four ddNTPs in each vessel. After the reaction and appropriate
washes, the particles from the four vessels are mixed together, and
the remainder of the procedure is carried through as presented
above. In this case, however, an additional encoding step of the
particle's memory is needed to provide the information about the
type of nucleotide used in the extension reaction for the given
transponder.
[0028] FIG. 2 depicts a solid phase particle for use in this
invention. The particle 10 is derivatized by attaching an
oligonucleotide probe 14 of known sequence to a surface 16 of the
particle 10. A transponder 12 is associated with the particle 10.
The transponder 12 is encoded with an index number that indicates
the sequence of the probe 14. The transponder may be pre-programmed
by the manufacturer, or it may be encoded by the user, using a
scanner read/write device.
[0029] A transponder is a radio transmitter-receiver activated for
transmission of data by reception of a predetermined signal, and
may also be referred to as a microtransponder, radiotransponder,
radio tag, transceiver, etc. The signal comes from a dedicated
scanner, which also receives and processes the data sent by the
transponder in response to the signal. The scanner function can be
combined with the write function, i.e. the process of encoding the
data on the transponder. Such a combination instrument is called a
scanner read/write device. An advantage of the transponder-scanner
system stems from the fact the two units are not physically
connected by wire, but are coupled inductively, i.e. by the use of
electromagnetic radiation, typically in the range from 5-1,000 kHz,
but also up to 1 GHz and higher.
[0030] FIG. 3 is a flow chart illustrating the communication
between the transponder 12 and a remote scanner read/write device
18. The transponder 12 associated with the solid phase beads 10 is
encoded with data sent by electromagnetic waves from a remote
scanner read/write device 18. After the assay steps are completed,
the beads 10 are analyzed to detect the presence of a label
indicative of binding of analyte and those that show the presence
of the label are decoded. The scanner 18 sends a signal to the
transponder 12. In response to the signal, the transponder 12
transmits the encoded data to the scanner 18.
[0031] Some transponders similar to those used in this invention
are available commercially. Bio Medic Data Systems Inc. (BMDS, 255
West Spring Valley Ave., Maywood, N.J.) manufactures a programmable
transponder for use in laboratory animal identification. The
transponder is implanted in the body of an animal, such as a mouse,
and is glass-encapsulated to protect the electronics inside the
transponder from the environment. One of the transponders
manufactured by this corporation, model# IPTT-100, has dimensions
of 14.times.2.2.times.2.2 mm and weighs 120 mg. The transponder is
user-programmable with up to 16 alphanumeric characters, the 16th
letter programmable independently of the other 15 letters, and has
a built-in temperature sensor as well. The electronic animal
monitoring system (ELAMS) includes also a scanner read/write system
to encode or read data on/from the transponder. The construction of
the transponder and scanner is described in U.S. Pat. Nos.
5,250,944, 5,252,962 and 5,262,772, the disclosures of which are
incorporated herein by reference. Other similar transponder-scanner
systems include a multi-memory electronic identification tag (U.S.
Pat. No. 5,257,011) manufactured by AVID Corporation (Norco,
Calif.) and a system made by TEMIC-Telefunken (Eching, Germany).
AVID's transponder has dimensions of 1 mm.times.1 mm.times.11 mm,
and can encode 96 bits of information, programmed by the user. The
present invention can be practiced with different transponders,
which might be of different dimensions and have different
electronic memory capacity.
[0032] The commercially available transponders are relatively large
in size. The speed at which the transponders may be decoded is
limited by the carrier frequency and the method of transmitting the
data. In typical signal transmission schemes, the data are encoded
by modulating either the amplitude, frequency or phase of the
carrier. Depending on the modulation method chosen, compression
schemes, transmission environment, noise and other factors, the
rate of the signal transmission is within two orders of magnitude
of the carrier frequency. For example, a carrier frequency of 1,000
Hz corresponds to rates of 10 to 100,000 bits per second (bps). At
the rate 10,000 bps the transmission of 100 bits will take 0.01
sec. The carrier frequency can be several orders of magnitude
higher than 1,000 Hz, so the transmission rates can be
proportionally higher as well.
[0033] Therefore, the limiting factor in the screening process is
the speed at which the transport mechanism carries the transponders
through the read window of the fluorometer/scanner device. The rate
of movement of small particles or cells is 10.sup.4-10.sup.5 per
second in state-of-the-art flow cytometers. A flow cytometer may be
used to practice the present invention, if two conditions are met:
(1) the transponders are small enough to pass through the flow
chamber, and (2) the design of the flow chamber of the flow
cytometer is modified to include an antenna for collecting the
electromagnetic radiation emitted by transponders.
[0034] A miniature transponder is depicted in FIGS. 4 and 5. The
source of the electrical power for the transponder 12a is at least
one photovoltaic cell 40 within the transponder 12a, illuminated by
light, preferably from a laser (not shown). The same light also
induces the fluorescence of the fluorogenic molecules immobilized
on the surface of the transponder 12a. The transponder 12a includes
a memory element 42 that may be of the EEPROM type. The contents of
the memory is converted from the digital form to the analog form by
a Digital-to-Analog converter 44 mounted on the transponder 12a.
The signal is amplified by an amplifier 46, mixed with the carrier
signal produced by an oscillator 48, and emitted to the outside of
the transponder 12a by an antenna 50.
[0035] The contents of the transponder memory can be permanently
encoded during the manufacturing process of the transponder,
different batches of transponders being differently encoded.
Preferably, the memory of the transponder is user-programmable, and
is encoded by the user just before, during, or just after the
biological material is deposited on the surface of the transponder.
A user-programmable transponder 12a must have the "write" feature
enabled by the antenna 50, amplifier 44 and the Analog-to-Digital
converter 46 manufactured on the transponder 12a, as well as the
dedicated scanner read/write device 27.
[0036] The advantages of the transponder of FIGS. 4 and 5 are
several-fold. First, the dimension of the transponder is reduced,
since most of the volume of current transponders is occupied by the
solenoid. The design discussed above will enable the production of
cubic transponders on the order of 0.01 to 1.0 mm along a side,
preferably 0.05 to 0.2 mm.
[0037] Second, a large number of transponders can be manufactured
on a single silicon wafer, and no further assembly would be
required to attach the solenoid to the VLSI chip. As depicted
schematically in FIG. 5, a silicon wafer 60 is simply cut to yield
active transponders 12a. Third, the transponder, according the new
design, will not need the glass capsule as an enclosure, further
reducing the size of the transponder. Silicone dioxide (SiO.sub.2)
would constitute a significant portion of the surface of the
transponder, and SiO.sub.2 has chemical properties which are very
similar to glass in terms of the feasibility of derivatization or
immobilization of biomolecules. Alternatively, microtransponders
may be coated with a variety of materials, including plastic, latex
and the like.
[0038] Finally, most important, the narrow focus of the beam of the
laser light would enable only one transponder to be active at a
time, significantly reducing the noise level. Advanced
user-programmability is desirable as well, various memory registers
need to be addressable independently (writing in one register
should not erase the contents of other registers).
[0039] FIG. 6 shows the analytical instrumentation and transport
system used in an embodiment of the present invention. A quartz
tube 20 is mounted in the readout window 22 of a fluorometer 24.
The quartz tube 20 is connected to a metal funnel 26. The length of
the quartz tube 20 is similar to the dimensions of the transponder
12. Transponders 12 are fed into the metal funnel 26, and pass from
the funnel 26 into the quartz tube 20, where the fluorescence is
read by the fluorometer 24 and the transponder 12 is decoded by the
scanner 27, and then exit through a metal tube 28 and are conducted
to a collection vessel (not shown). The metal funnel 26 and metal
tube 28 are made of metal shield transponders 12 outside of the
read window 22 by shielding from the electromagnetic signal from
the scanner 27. This shielding prevents the scanner signal from
reaching more than one transponder 12, causing multiple
transponders 12 to be decoded.
[0040] Minimal modification of the fluorometer 24 would be needed
in the vicinity of the location that the tube occupies at the
readout moment to allow for positioning of the transponder reading
device. To assure compatibility with existing assays, the glass
surrounding the transponder could be coated with plastic currently
used to manufacture beads.
[0041] In a preferred design, depicted in FIG. 4, a metal coil
antenna 30 is wrapped around the flow cell 32 of a flow cytometer
29. The transponders 12 pass through the flow cell 32, and are
decoded by the scanner device 27. The signal carrying the data sent
from the transponders 12 is amplified by a first amplifier 34 and
processed by the scanning device 27. As the transponders 12 are
decoded, fluorescence from the transponders 12 is detected and
analyzed by the flow cytometer 29.
[0042] In one embodiment of the present invention, multiple passes
of the same set of transponders through the detector are
implemented. The temperature is incrementally increased at each
pass, the purpose being to gradually increase the stringency of
binding, and to reduce effects of non-specific binding of the
target to the probes on the transponders. After the detection step
is completed, the transponders can be reconditioned for further use
in another experiment by stripping the biomolecular coating
chemically, and clearing the memory. If the target DNA is labeled
with a fluorophore, an alternative to chemical stripping is the
extensive wash at high temperature to dissociate and remove
non-covalently bound target DNA.
[0043] Upon completion of the analysis step and decoding of the
transponders, the data is analyzed to determine the sequence by
correlating the sequence of the oligonucleotide probe immobilized
on the transponder and the fluorescence readout of the transponder
carrying the probe. The type of data related to the fluorescence
readout may vary depending of the procedures used. If the target
DNA is labeled with the fluorophore prior to annealing, or if the
label is a single fluorophore, introduced in a chain extension
reaction with a DNA polymerase after annealing, the fluorescence
readout is simply the intensity of the fluorescence. Alternatively,
if four ddNTPs are used with the purpose of identifying the
nucleotide residue proximal to the 3' end of the probe or target
DNA (depending on the design of the experiment), the fluorescence
readout is both the intensity and the information about the
wavelength of maximal fluorescence. The form of the data set is
analogous to that yielded by sequencing by hybridization
methods(SBH), and algorithms previously developed for SBH can also
be used for digital sequencing (Drmanac R. et al., 1993, DNA
Sequence Determination By Hybridization: A Strategy For Efficient
Large-Scale Sequencing. Science 260, 1649-1652).
EXAMPLE 1
Immobilization of DNA Probes on Transponders Using the Chemical
Synthesis of DNA
[0044] Nucleic acids can be covalently linked to glass by direct
chemical synthesis on a glass support. To prepare the support,
5'-dimethoxytrityl thymidine is reacted with one equivalent of
tolylene-2,6-diisocyanate in the presence of one equivalent of
N-ethyldiisopropylamine as a catalyst in
pyridine/1,2-dichloroethane to generate the monoisocyanate. The
monoisocyanate is not isolated, but is added directly to the
alkylamine glass support, i.e. the
aminopropyltriethoxysilane-derivatized glass surface of the
transponders. The procedure is described in detail in B. S. Sproat
and D. M. Brown, A New Linkage For Solid Phase Synthesis Of
Oligodeoxyribonucleotides, Nucleic Acids Res. 13, 2979-2987, 1985.
Such thymidine-derivatized support containing the stable
nucleoside-urethane linkage is directly used for the chemical
synthesis of oligodeoxynucleotides using a manual synthesis
protocol on sintered funnels as described before (Caruthers, M. H.
et al., Deoxyoligonucleotide synthesis via the phosphoramidite
method. In: Gene Amplification and Analysis, Vol III (T. S. Papas
et al., Eds.) Elsevier/North Holland, Amsterdam), using standard
phosphoramidite-based DNA synthesis reagent. The thymidine-urethane
linker is resistant to cleavage with base during the deprotection,
and the resulting product is the deprotected oligonucleotide
attached to the glass surface of the transponder through the
urethane-thymidilate linker.
EXAMPLE 2
Determination of the Sequence of a Three Nucleotide Region Using
AVID Transponders
[0045] The target DNA is a single-stranded 50-residue long
oligodeoxynucleotide. It is obtained from a PCR reaction using a
5'-fluoresceinated oligonucleotide as one of the primers, after
linearly amplifying DNA. DNA used in the experiment is purified
after PCR. Most of the sequence of 47 nucleotides in the 50 nt
target is known, except for a region consisting of three residues,
residue numbers 18-20, marked as NNN in the figure below:
1 transponder-linker-GGTACTGCXXXACCTTCCA-3'
.vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline.
.vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline.
3'-ACGTTAAGCCCAGTATGCCATACCATGACGNNN- TGGAAGGTAGAGATACT-5'-fluor 50
40 30 20 10 1
[0046] The symbol ".linevert split." denotes the complementarity of
nucleotides in the two sequences. There are exactly 64 possible
sequences for 5'-GGTACTGCXXXACCTTCCA, where X can be any nucleotide
residue (A, C, G or T). 64 sets of AVID transponders are prepared,
each set having a different oligonucleotide probe (upper sequence
in the figure above) synthesized on the surface of the transponder
using standard phosphoramidite chemistry as described in Example 1.
The linker has the (dT).sub.10 sequence to facilitate annealing.
The transponders are electronically encoded with a number 1 through
64, the number corresponding to the sequence of the oligonucleotide
bound to the transponder. 64 transponders, each from a different
set, are put into a 10 ml vessel. A solution containing the target
(5 ml volume) is then added to the vessel, the contents of the
vessel is heated to 95.degree. C., cooled slowly over the period of
10 min to the experimentally predetermined wash temperature
T.sub.wash (T.sub.wash can range from room temperature to
60.degree. C., data not shown). After the annealing step, the
transponders were extensively washed at temperature T.sub.wash, and
then stored in a buffer at room temperature.
[0047] Each of the 64 transponders was subjected to both
fluorescence measurement and electronic decoding of the tag. As a
result, each of 64 possible sequences was associated with a number,
fluorescence readout, that indicated whether the template had
annealed to the oligonucleotide on the transponder, thus providing
the information about the sequence of the 3 nt region.
EXAMPLE 3
Determination of the Sequence of a Two Nucleotide Region Using
Combinatorial Oligonucleotide Synthesis on BMDS Transponders
[0048] The target was a single-stranded 50-residue long
oligonucleotide. It was obtained from a PCR reaction using a
5'-fluoresceinated oligonucleotide primer, after linearly
amplifying DNA. DNA used in the experiment is purified after PCR.
Most of the sequence of 48 nucleotides in the 50 nt template was
known, except for the region of two residues, residue numbers
18-19, marked as NN in the figure below:
2 transponder-linker-GGTACTGCAXXACCTTCCA-3'
.vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline.
.vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline.
3'-ACGTTAAGCCCAGTATGCCATACC- ATGACGTNNTGGAAGGTAGAGATACT-5'-fluor 50
40 30 20 10 1
[0049] The symbol ".linevert split." denotes the complementarity of
nucleotides in the two sequences. The linker has the (dT).sub.10
sequence to facilitate annealing. There are exactly 16 possible
sequences of oligonucleotides 5'-GGTACTGCAXXACCTTCCA, where X can
be any nucleotide residue (A, C, G or T). These sequences are
synthesized in a combinatorial fashion on 200 BMDS transponders
(14.times.2.times.2 mm, volume of each transponder is about 50
.mu.l) as follows. First, the underivatized glass surface of the
transponders is treated as described in Example 1 to introduce the
functional hydroxy groups on the surface of transponders. Then, the
transponders are subjected to 18 rounds of solid phase
oligonucleotide synthesis using standard phosphoramidite chemistry
to sequentially synthesize the following residues:
TTTTTTTTTTGGTACTGCA-3'. The transponders are split into four groups
of approximately equal numbers (i.e. 50). Each of the four groups
is subjected to a single cycle of solid phase oligonucleotide
synthesis with a different nucleotide phosphoramidite. That is,
transponders from group 1-are derivatized with adenosine
phosphoramidite (A), group 2-cytosine phosphoramidite (C), group
3-guanosine phosphoramidite (G), and group 4-thymidine
phosphoramidite (T). The volume of each of the four condensations
is about 4 ml. After completing the first cycle, the 4 groups are
subjected to electronic encoding with the residue abbreviations
given above. Thus, the first alphanumeric character indicates which
condensation takes place in the first cycle of combinatorial
synthesis. Then, all the transponders are pooled together and mixed
thoroughly, and then split into new four groups having
approximately the same number of transponders. These four new
groups are subjected to the second combinatorial coupling step. The
scheme for the second coupling is identical to that of the first
coupling. Electronic encoding after the second cycle adds a second
alphanumeric character, corresponding to the nucleoside coupled in
the second synthesis cycle. As a result, each transponder is
encoded with two alphanumeric characters which identify the
sequence of two nucleotide residues at the 3' end of the
oligonucleotide attached to the surface of the transponder. The
transponders having oligonucleotides coupled to their surfaces are
immersed in a 20 ml vessel in the solution containing the template,
the contents of the vessel is heated to 95.degree. C., cooled
slowly over the period of 10 min to the experimentally
predetermined annealing temperature T.sub.ann (T.sub.ann can range
from room temperature to 60.degree. C., data not shown). After the
annealing step, the transponders are extensively washed at
temperature T.sub.ann. The fluorescence of individual transponders
is recorded and the transponder memories are decoded. Strong
fluorescence indicates that the complement of the dinucleotide
sequence bound to the transponder is present in the target.
EXAMPLE 4
Determination of the Sequence of a Three Nucleotide Region Using a
Single-Nucleotide Extension by DNA Polymerase
[0050] The target was a single-stranded 50-residue long
oligonucleotide obtained from a PCR reaction after linearly
amplifying DNA. The target DNA does not carry any fluorophore
label. Most of the sequence of 47 nucleotides in the 50 nt target
is known, except for the region of three residues, residue numbers
18-20, marked as NNN in the figure below:
3 5' 3' transponder-linker-GTATGGTACTGCAA oligo 9
...-GTATGGTACTGCAC 8 ...-GTATGGTACTGCAG 7 ...-GTATGGTACTGCAT 6
...-GGTATGGTACTGCA 5 ...-GGTATGGTACTGCC 4 ...-GGTATGGTACTGCG 3
...-GGTATGGTACTGCT 2 ...-CGGTATGGTACTGC 1
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline.
3'-ACGTTAAGCCCAGTATGCCATACCATGACGNNNTGGAAGGTAGAGATACT-5' 50 40 30
20 10 1
[0051] The symbol ".linevert split." denotes the complementarity of
nucleotides in the two sequences. The linker has the (dT).sub.10
sequence to facilitate annealing. Nine groups of five AVID
transponders are derivatized by the chemical synthesis of
oligonucleotides 1 through 9 as shown in the figure above, and
electronically encoded with numbers 1 through 9, respectively. The
transponders are placed in one 5 ml vessel, and 2 mls of the
solution containing the target (final concentration 10 nM to 10
.mu.M) is added to the vessel. The contents of the vessel are
heated to 95.degree. C., cooled slowly over the period of 10 min to
the experimentally predetermined extension temperature Tex (Tex can
range from room temperature to 60.degree. C., data not shown).
After the annealing step, the transponders are extensively washed
at temperature T.sub.ex. DNA polymerase and four dideoxynucleotide
triphosphates, ddATP, ddCTP, ddGTP and ddTTP (obtained from Applied
Biosystems) derivatized with four different fluorophores, such as
those used in Applied Biosystems automated sequencers are then
added to the vessel, and the vessel is incubated at T.sub.ex for 15
minutes. Subsequently, the transponders are extensively washed at
the wash temperature T.sub.wash. A set of data points, each in the
form of (oligonucleotide_sequence, fluorescence_intensity,
wavelength_of_maximum_fluorescence), is obtained. For highly
fluorescent transponders, the wavelength of maximum fluorescence
indicates the residue type, and the number encoded on the
transponder indicates the position of the residue in the chain,
thus defining the sequence of the 3 nt region of the target.
* * * * *