Multiplex assay for nucleic acids employing transponders

Disclosed are materials and methods for performing multiplex assays for nucleic acids, in which a transponder is associated with the bead(s) forming the solid phase used in the assay, nucleic acid probes are bound to the surface of the particles, and data concerning the assay is encoded on the transponder. A dedicated read/write device is used to remotely encode or read the data.

BACKGROUND OF THE INVENTION 
This invention relates to materials and methods for detecting nucleic acids 
in samples and, more particularly, to solid phase assays wherein 
transponders are associated with the beads constituting the solid phase, 
nucleic acid probes are bound to the surface of the particles and data 
concerning the assay is encoded on the transponders. 
Solid phase assays have been used to determine the presence of nucleic 
acids, including deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and 
their modified forms. Solid-phase assays can be applied to nucleic acids 
either in simple buffers, or in biological fluids, such as blood, serum, 
plasma, saliva, urine, tissue homogenates, and many others. 
In solid phase assays, small beads, or microparticles, are used as the 
solid phase to capture the analyte. Solid-phase microparticles can be made 
of different materials, such as glass, plastic, latex, depending on the 
particular application. Some beads are made of ferromagnetic materials to 
facilitate their separation from complex suspensions or mixtures. 
In conventional solid-phase assays, the solid phase mainly aids in 
separating molecules that bind to the solid phase from molecules that do 
not bind to the solid phase. Separation can be facilitated by gravity, 
centrifugation, filtration, magnetism, immobilization of molecules onto 
the surface of the vessel, etc. The separation may be performed either in 
a single step in the assay or, more often, in multiple steps. 
Often, there is a need to perform two or more different assays on the same 
sample, most of the time in a single vessel and at about the same time. 
Such assays are known in the art as multiplex assays. Multiplex assays are 
performed to determine simultaneously the presence or concentration of 
more than one molecule in the sample being analyzed, or alternatively, 
several characteristics of a single molecule, such as, the presence of 
several epitopes on a single protein molecule. 
One problem with conventional multiplex assays is that they typically 
cannot detect more than about five analytes simultaneously, because of 
difficulties with simultaneous detection and differentiation of more than 
about five analytes. In other words, the number of different analytes that 
may be assayed in a single solid phase assay is limited by the solid 
phase. 
SUMMARY OF THE INVENTION 
The present invention overcomes many of these problems by employing 
transponders associated with the solid phase beads to index the particles 
constituting the solid phase. Thus, each individual solid phase particle 
can be assigned a unique index number electronically encoded inside the 
particle, that can be retrieved at any time, e.g., at one time during the 
assay, at multiple times during the assay, or continuously during the 
assay. The index number may define the nucleotide sequence of the 
oligonucleotide deposited on the surface of the particle, the catalog 
number of a DNA fragment deposited on the particle, index numbers of 
chemical steps which were involved in the chemical synthesis of an 
oligonucleotide bound to the particle, or some other relevant 
characteristics of the deposited molecules. 
In an electronically-indexed multiplex assay of this invention, two or more 
classes of transponders, each encoded with a different index number and 
constructed to bind a different nucleic acid sequence, are incubated with 
the sample in a single vessel. After necessary washes, incubations and 
additions are performed, the solid phase is analyzed to detect a label 
indicative of binding of nucleic acid in the sample to the oligonucleotide 
on the transponder, such as fluorescence, color, radioactivity or the 
like. Solid phase analysis is either preceded or followed by decoding of 
the index numbers programmed on the transponders. 
Determination of the label and decoding of the memory of the transponder 
can be done manually on two different instruments, such as a fluorometer 
and a dedicated scanner, although a single automated instrument that would 
perform both functions may be used. Such an instrument can be a modified 
fluorometer in which the scanner is mounted in the proximity of the 
fluorometer readout window, and reading the sample fluorescence and 
decoding the transponder are coordinated by a central computer. In 
addition, such an instrument can be equipped with an automated transport 
system for transponders. 
In one aspect, the present invention provides an electronically-indexed 
solid phase particle for use in solid phase assays for nucleic acids, 
comprising a transponder and a nucleic acid sequence attached to the 
transponder. 
In another aspect, the present invention provides a method for detecting 
nucleic acids in a sample, using solid phase particles having 
transponders. 
In another aspect, the present invention provides kits for detecting 
nucleic acids in samples, comprising assay vessels, at least one 
transponder having a nucleic acid probe bound to the transponder, and a 
labelled reagent to detect binding of sample nucleic acids to the probe.

DETAILED DESCRIPTION OF THE INVENTION 
FIG. 1 depicts a simple assay of the invention. A solid phase particle 10, 
with a transponder 12 is derivatized by attaching an oligonucleotide probe 
11 to the outer surface 16 of the particle 10. Information concerning the 
assay, e.g., an index number identifying the patient, is encoded on the 
transponder, either by the manufacturer of transponder, or by the user 
with a remote read/write scanner device (not shown). Sample containing 
target nucleic acid 13 is treated to label all of the nucleic acid 
therein. The derivatized particle 10 is placed in a sample, and the sample 
is heated to cause nucleic acids to dissociate. The sample is then cooled 
under controlled conditions to cause the nucleic acids to anneal. Target 
nucleic acids 13 complementary to the oligonucleotide probe 11 anneal to 
the probe 11. The particle 10 is thoroughly washed to remove unbound 
components. The labelled target nucleic acid 13 bound to the probe 11 is 
detected with a fluorometer to identify those transponders 12 that have 
target nucleic acid 13 bound thereto, and the transponder 12 is decoded 
using the scanner device (not shown) to retrieve the information encoded 
thereon. 
The detection and decoding steps may be done separately or may be done 
simultaneously. Alternatively, the particles of many samples may be pooled 
into a vessel in no particular order with mixing allowed, and passed 
through a reader (not shown) that determines and records the fluorescence 
and, at the same time, decodes the index number recorded in the 
transponder 12. It is important to note that when encoding or reading data 
on a transponder, other transponders must be shielded by a metal barrier 
or other means to prevent the electromagnetic radiation from reaching such 
"non-target" transponders. 
In an alternative labelling technique, depicted in FIG. 1A, a second 
fluorescent-labelled oligonucleotide probe 15 complementary to a second 
sequence of the target nucleic acid 13 is added to the sample mixture, to 
specifically label transponders 12 to which target nucleic acids 13 have 
bound. 
A multiplex assay according to this invention is conducted in a similar 
manner, as depicted in FIG. 2, with two or more transponders 12 in each 
assay vessel (not shown) to detect more than one target nucleic acid 13 
simultaneously. The transponders 12 are divided into two or more classes 
12 and 12', each class having a distinct index number identifying the 
class, and each class having a different oligonucleotide probe 11 and 11' 
bound to the surface 16 of the particle 10 and 10'. Using each class of 
transponder 12, 12' is separately encoded, either by the manufacturer or 
by the user with a read/write scanner device (not shown), with an index 
number to identify, e.g., the sequence of the probe 11 bound to the 
surface 16 of the particle 10. Again, it is necessary to shield other, 
non-target transponders during the encoding process. The transponders 12, 
12' are added to a sample, and the sample is heated to cause nucleic acids 
to dissociate. The sample is then cooled under controlled conditions to 
cause the nucleic acids to re-anneal. Target nucleic acid 13, 13' 
complementary to the respective probes 11, 11' anneals to the probes 11, 
11'. The transponders 12, 12' are then washed thoroughly to remove unbound 
sample components and reagents. The labelled probes 15, 15' are detected 
with a fluorometer to identify those transponders 12, 12' that have target 
nucleic acids 13, 13' bound thereto, and the transponder 12, 12' is 
decoded using the scanner device (not shown) to retrieve the information 
encoded thereon. The detection and decoding steps may be done separately 
or may be done simultaneously. Alternatively, the particles 10, 10' may be 
pooled into a vessel in no particular order with mixing allowed, and 
passed through a reader (not shown) that determines and records the 
fluorescence and, at the same time, decodes the index number recorded in 
the transponder 12, 12'. 
In an alternative labelling technique, depicted in FIG. 2A, second 
fluorescent-labelled oligonucleotide probes 15, 15' that bind to second 
sequences of the target nucleic acids 13, 13' are added to the sample 
vessel to bind to the target nucleic acids 13, 13'. Alternatively, the 
label may be a radioisotope, such as .sup.32 P, .sup.35 S, .sup.125 I, and 
the like. The label may also be a chemiluminescent label, such as a 
luminol derivative or an acridinium ester, that emits light upon oxidation 
of a substrate. The label may be an enzyme, such as alkaline phosphatase, 
catalyzing a reaction employing a precipitating fluorogenic substrate, 
e.g., attophos (JBL Scientific, San Luis Obispo, Calif.), a precipitating 
chromogenic substrate, e.g., 5-bromo-4-chloro-3-indolyl phosphate), or a 
chemiluminescent substrate, e.g., adamantyl 1,2-dioxetane phosphate 
(Tropix, New Bedford, Mass.). Finally, the label may be a biolumnescent 
enzyme such as luciferin. 
The present multiplex assay can be applied to different types of nucleic 
acids, DNA, RNA, modified nucleic acids and analogs of nucleic acids (in 
particular protein-nucleic acids, PNAs). The analyte can be a complex of 
biomolecules, such as a virus particle, a nucleic acid-protein complex, or 
a nucleic acid-hapten complex. It is also evident that the target nucleic 
acid analyte, which is being monitored, can be present in a variety of 
forms, such as a solution in a simple buffer, but also in a complex 
biological fluid, such as blood, serum, urine, saliva, and many others. 
The target nucleic acid can be mixed with many other analytes that are 
simultaneously being assayed in the multiplex format. The purity of the 
nucleic acid deposited as a primary layer on the surface of the 
transponder can vary as well, from unpurified, partially purified to pure 
compounds. 
The nucleic acids, their complexes and aggregates can be deposited as a 
primary layer on the surface of the transponder by a variety of means, 
including chemical conjugation to an active group on the support, direct 
chemical synthesis, combinatorial synthesis, adhesion or non-specific 
binding through hydrophobic interactions. The nucleic acid deposited as a 
primary layer on the surface of the transponder can be made in vivo, in an 
enzymatic reaction in vitro, or chemically synthesized. A preferred 
example of a product of an enzymatic reaction in vitro is the nucleic acid 
obtained from the polymerase chain reaction (PCR). 
FIG. 3 depicts a solid phase particle 10 of the present invention, having a 
transponder 12, and a primary layer 14 of an oligonucleotide probe 
attached to the outer surface 16 of the particle 10. 
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 that also receives and processes the 
data sent by the transponder. 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 referred to as a scanner 
read/write device. An advantage of the transponder-scanner systems is that 
the two units are not electrically 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. 
FIG. 4 is a flow chart illustrating the communication between the 
transponder 12 and a remote scanner read/write device 18. The transponder 
12 is encoded and/or decoded with data sent by electromagnetic waves from 
a remote scanner read/write device 18, unless the transponders 12 have 
been encoded by the manufacturer. After the assay steps are completed, the 
beads 10 are analyzed to detect the presence of a label indicative of 
binding of analyte and the transponders 12 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. 
Some transponders similar to those used in the present invention are 
available commercially. BioMedic 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. The transponder 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 weight of 
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, such as the DAS-501 console 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) 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, programmable by the user. The present invention 
can be practiced with different transponders, which might be of different 
dimensions and have different electronic memory capacity. 
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 of 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. 
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 and a 
read/write scanner device for collecting the electromagnetic radiation 
emitted by transponders. 
A miniature transponder is depicted in FIGS. 5 and 6. 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 
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, or the ROM 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 
45, mixed with the carrier signal produced by an oscillator 48, and 
emitted to the outside of the transponder 12a by an antenna 50. 
In an alternative embodiment, the signal from the scanner (not shown) is 
transmitted to the transponder 12a by modulating the intensity of the 
light illuminating the transponder 12a, which also serves to actuate the 
photovoltaic cell power source 40. 
The contents of the miniature 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/write device 27. 
The advantages of the transponder of FIGS. 5 and 6 are several-fold. First, 
the transponder dimensions are reduced relative to a conventional 
transponder, because most of the volume of a conventional transponder 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 as 
measured along a side of the cube, preferably 0.05 to 0.2 mm. 
Second, a large number of transponders can be manufactured on a single 
silicon wafer. As depicted schematically in FIG. 6, 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 like glass in terms that allow 
derivatization or immobilization of biomolecules. Alternatively, the 
transponder may be coated with a variety of materials, including plastic, 
latex and the like. 
Finally, most importantly, the narrow focus of the beam of the laser light 
would enable only one transponder to be active at a time during the 
decoding step, significantly reducing noise level. Advanced 
user-programmability is desirable as well, and preferably, various memory 
registers are addressable independently, i.e., writing in one register 
does not erase the contents of other registers. 
FIG. 7 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. 
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 or replaced with the type of plastic currently used to manufacture 
beads. 
In a preferred design, depicted in FIG. 8, 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 
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. 
EXAMPLE 1 
Multiples DNA-Based Assay on Transponders Employing DNA Synthesized on the 
Solid Support 
The glass outer surface of the transponders is first derivatized by an 
aminoalkylsilane treatment. The transponders (e.g., IPTT-100, BMDS) are 
cleaned by washing with xylene, followed by a 70% ethanol rinse and air 
drying. The transponders are then submerged for about 30 seconds in a 2% 
solution of aminopropyltriethoxysilane (Cat.# A3648, Sigma, St. Louis, 
Mo.) in dry acetone. The transponders are then sequentially rinsed with 
dry acetone and distilled water, and then air dried. This procedure is 
described in the Pierce catalog (pp. T314-T315 of the 1994 catalog, 
Pierce, Rockford, Ill.). 
Nucleic acid probes are then covalently linked to the 
aminoalkylsilane-treated glass by direct chemical synthesis on the glass 
support. A thymidine-derivatized support containing a stable 
nucleoside-urethane linkage is prepared, in which 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 reacted directly with 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. 
The thymidine-derivatized support containing a stable nucleoside-urethane 
linkage is used directly for the chemical synthesis of 
oligodeoxynucleotides by manual synthesis on sintered funnels using 
standard phosphoramidite-based DNA synthesis reagents, as described in 
Caruthers, M. H. et al., Deoxyoligonucleotide Synthesis Via The 
Phosphoramidite Method, Gene Amplification and Analysis, Vol. III (T.S. 
Papas et al., Eds., Elsevier/North Holland, Amsterdam). The 
thymidine-urethane linker is resistant to cleavage with base during 
deprotection, and the resulting product is the deprotected oligonucleotide 
attached to the glass surface of the transponder through the 
urethane-thymidilate linker. 
The following oligodeoxynucleotide reagents are prepared. Sequence 1 and 
sequence 2 do not exhibit self-complementarity, are 15 nt long, and are 
linked to the transponders through a spacer, which is an oligonucleotide 
having the (dT).sub.10 sequence. Oligonucleotides C and D are derivatized 
at the 5'-end with fluorescein. The sequences are as follows: 
transponder-oligonucleotide A: 5'-spacer-sequence1 
transponder-oligonucleotide B: 5'-spacer-sequence2 
oligonucleotide C: 5'-fluorescein-sequence1complement 
oligonucleotide D: 5'-fluorescein-sequence2complement 
Four assay tubes are prepared and labeled 1, 2, 3 and 4, each assay tube to 
accommodate two transponders, one transponder carrying oligonucleotide A 
and the second transponder carrying oligonucleotide B. The transponders 
are electronically encoded with two alphanumeric characters, namely 
A1,A2,A3,A4 and B1,B2,B3,B4, where the letter corresponded to the 
oligonucleotide used to derivatize the transponder, and the digit gave the 
test tube number into which the given transponder is placed. Thus tube 1 
contains transponders A1 and B1; tube 2-A2 and B2; tube 3, A3 and B3; and 
tube 4, A4 and B4, all immersed in 50 mM Tris-HCl buffer (pH 7.5). Four 
analytes, X,Y,Z and W, are prepared, as follows. Analyte X contains 
oligonucleotide C and oligonucleotide D; Y contains oligonucleotide C 
only, Z contained oligonucleotide D only, and analyte W does not contain 
any oligonucleotides. The analyte solutions are prepared in 50 mM Tris-HCl 
(pH 7.5). The concentration of each given oligonucleotide in the analytes 
X, Y and Z is 10 nM to 10 .mu.M. After the four tubes are emptied of 
buffer, but retain the transponders, 2 mls of X,Y,Z and W analyte are 
added to tubes 1, 2, 3 and 4, respectively. The tubes are heated to 
90.degree. C., and slowly cooled to room temperature. Then the 
transponders are rinsed three times with the buffer. The fluorescence of 
each transponder is measured on a FluorImager instrument (Molecular 
Dynamics). 
EXAMPLE 2 
Multiplex DNA-Based Assay on Transponders Employing Conjugation of 
Oligonucleotides to Solid Support 
Precleaned transponders (IPTT-100, BMDS) are immersed in a 1% 
3-aminopropyltrimethoxysilane solution (Aldrich Chemical, Milwaukee, Wis.) 
in 95% acetone/water for 2 minutes, washed extensively with acetone (10 
washes, 5 minutes each) and dried (110.degree. C. for 45 minutes). The 
transponders are then treated for 2 hours with 1,4-phenylene 
diisothiocyanate (Aldrich) (PDC, 0.2% solution in 10% pyridine/dimethyl 
formamide). The transponders are washed with methanol and acetone and 
stored at 4.degree. C. in an anhydrous environment. The 5'-amino-modified 
oligonucleotides to be immobilized on the glass support are dissolved in 
100 mM sodium carbonate/bicarbonate buffer (pH 9.0) at a concentration of 
2 mM, and a 2 .mu.l aliquot is applied directly to the PDC-derivatized 
transponders and incubated at 37.degree. C. in a closed vessel for 2 
hours. The transponders are then washed with NH.sub.4 OH, three times with 
water and air dried at room temperature. This derivatization procedure is 
based on a protocol described in Guo et al. (Direct Fluorescence Analysis 
Of Genetic Polymorphism By Hybridization With Oligonucleotide Arrays On 
Glass Support. Nucleic Acids Res. 22, 5456-5465, 1994). 
The following oligodeoxynucleotide reagents are prepared. Sequence1 and 
sequence2 are 15 nt long, and are linked to the transponders through an 
oligonucleotide spacer having the (dT).sub.10 sequence. Oligonucleotides C 
and D are derivatized at the 5'-end with fluorescein. The sequences are as 
follows: 
transponder-oligonucleotide A: 5'-spacer-sequence1 
transponder-oligonucleotide B: 5'-spacer-sequence2 
oligonucleotide C: 5'-fluorescein-sequence1complement 
oligonucleotide D: 5'-fluorescein-sequence2complement 
Four assay tubes are prepared and labeled 1, 2, 3 and 4, each tube to 
accommodate two transponders, one transponder carrying oligonucleotide A 
and the second transponder carrying oligonucleotide B. The transponders 
are electronically encoded with two alphanumeric characters, namely 
A1,A2,A3,A4 and B1,B2,B3,B4, where the letter corresponded to the 
oligonucleotide used to derivatize the transponder, and the digit gave the 
test tube number into which the given transponder is placed. Thus tube 1 
contains transponders A1 and B1; tube 2-A2 and B2; tube 3, A3 and B3; and 
tube 4, A4 and B4, all immersed in 50 mM Tris-HCl buffer (pH 7.5). Four 
analytes, X,Y,Z and W, are prepared, as follows. Analyte X contains 
oligonucleotide C and oligonucleotide D; Y contains oligonucleotide C 
only, Z contained oligonucleotide D only, and analyte W does not contain 
any oligonucleotides. The buffer is 50 mM Tris-HCl (pH 7.5). The 
concentration of each given oligonucleotide in the analytes X, Y and Z is 
10 .mu.M. After the four tubes are emptied of buffer, but retain the 
transponders, 2 mls of X,Y,Z and W analyte are added to tube 1,2,3 and 4, 
respectively. The tubes are heated to 90.degree. C., and slowly cooled to 
room temperature. Then the transponders are rinsed three times with the 
buffer. The fluorescence of each transponder is measured on a Fluorimager 
(Molecular Dynamics). 
EXAMPLE 3 
Conjugation Of Streptavidin To The Glass Surface Of Transponders 
The outside glass surface of transponders (IPTT-100, BMDS) is derivatized 
through the aminoalkylsilane treatment outlined above, and a linker is 
attached to the aminoalkylsilane-treated glass. A variety of methods can 
be used, as reviewed in Enzyme Immunodiagnostics, E. Kurstak, Academic 
Press, New York, 1986, pp. 13-22. This procedure a homobifunctional 
NHS-ester cross-linker, BS.sup.3, bis(sulfosuccinimidyl)suberate (Pierce 
Cat.# 21579, described on p. T159 of the 1994 Pierce catalog). 
The transponders are immersed in the 10 mM solution of BS.sup.3 in 100 mM 
phosphate buffer (pH 7.0-7.4) for 5 to 60 minutes at room temperature, and 
the transponders are rinsed with water. A 10-100 .mu.M streptavidin 
solution in 100 mM phosphate buffer (pH 7.4-8.0) is prepared. The 
transponders are submerged in the streptavidin solution and incubated at 
room temperature for 2-3 hours. The transponders are rinsed three times 
with 100 mM phosphate buffer (pH 7.4-8.0). The unreacted sites on the 
glass are blocked by incubating in Blocker BLOTTO in PBS 
(phosphate-buffered saline) (Pierce, Cat.# 37526) for 2 hrs. The 
transponders are rinsed three times with 100 mM phosphate buffer (pH 
7.4-8.0), and stored in this buffer at 4.degree. C. 
EXAMPLE 4 
Detection of a Point Mutation in the N-ras Gene 
Point mutations in the N-ras gene are frequently observed in various 
hematological and solid tumors. A well-characterized mutation is a G -&gt;C 
mutation in the first position of the 12th codon of the N-ras gene. The 
present example provides a method to detect this mutation implementing 
transponders. 
The sequence of the first exon of the N-ras gene is given in Table 1. The 
glass surface of transponders (IPTT-100, BMDS) used in this example is 
derivatized with streptavidin using the conjugation method described in 
Example 3. The following oligodeoxynucleotides are chemically synthesized: 
(1) GACTGAGTACAAACTGGTGG, corresponding to residues 3-22 of exon 1; 
(2) CTCTATGGTGGGATCATATT-biotin, corresponding to residues 111 91; 
(3) AACTGGTGGTGGTTGGAGCA, corresponding to residues 14-33, 
Oligonucleotide (2) is biotinylated at the 5' end. These sequences were 
previously used to perform mini-sequencing using scintillating microplates 
by Ihalainen et al. (BioTechniques, 16, 938-943, 1994). Cellular DNA from 
patient samples is purified using the standard Blin and Safford procedure 
(Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd 
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). 
PCR amplification of DNA using primers (1) and (2) is done on the 
Perkin-Elmer Cycler 9600, employing 50 cycles of amplification. Each cycle 
involved a 1 minute denaturation at 94.degree. C., 1 minute annealing at 
55.degree. C. and 1 minute chain extension at 72.degree. C. in a final 
volume of 100 .mu.l. The single DNA strand carrying biotin is captured on 
two transponders conjugated to streptavidin by incubating the product of 
the PCR reaction with the transponders in a buffer containing 150 mM NaCl, 
20 mM sodium phosphate (pH 7.4) and 0.1% Tween-20 at 37.degree. C. with 
gentle shaking for 90 minutes. The bound PCR product was denatured with 50 
mM NaOH for 5 minutes at room temperature. The transponders are then 
washed extensively 3-5 times with a buffer (40 mM Tris-HCl, pH 8.8, 1 mM 
EDTA, 50 mM NaCl, 0.1% Tween-20). The patient name, consisting of six 
alphanumeric characters, is encoded on the two transponders using a 
dedicated read-write scanner. 
The diagnostic chain extension reaction is configured for one transponder 
as follows. The primer, oligonucleotide (3) is at a final concentration of 
0.4M, .sup.3 H dCTP or .sup.3 H dGTP (Amersham) at 0.2 .mu.M, and 4 units 
of Taq polymerase, in a final volume of 1 ml of a buffer containing 50 mM 
KCl, 10 mM Tris-HCl (pH 9.0 at 25.degree. C.), 0.1% Triton X-100, 4 mM 
MgCl. The final volume and the test tube type are adjusted depending on 
the number of transponders so that the whole surface of the transponders 
is covered with buffer. The reaction is incubated at 55.degree. C. for 10 
minutes with gentle shaking. 
To determine whether the mutation is present, transponders are used in two 
DNA chain extension reactions. The first reaction contains .sup.3 H dCTP 
and no other dNTPs, the second one contains .sup.3 H dGTP and no other 
dNTPs. Since the transponders are individually encoded with the patient's 
name, several transponders can be placed in the vessel where the reaction 
takes place. 
After the reactions are completed, the transponders are washed 3 times as 
described above, and dried for 60 minutes at room temperature. The 
transponders are subjected to the electronic decoding, which is followed 
by counting of the radioactivity associated with the transponders in a 
scintillation counter, with or without scintillation fluid. Radioactivity 
associated with the reaction employing .sup.3 H dCTP indicates the 
presence of the mutation in the sample DNA. 
TABLE 1 
__________________________________________________________________________ 
Sequence of exon 1 of the N-ras gene 
__________________________________________________________________________ 
* 
ATGACTGAGTACAAACTGGTGGTGGTTGGAGCAGGTGGTGTTGGGAAAAG 50 
TACTGACTCATGTTTGACCACCACCAACCTCGTCCACCACAACCCTTTTC 
MetThrGluTyrLysLeuValValValGlyAlaGlyGlyValGlyLysSe 
CGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATGATC 100 
GCGTGACTGTTAGGTCGATTAGGTCTTGGTGAAACATCTACTTATACTAG 
rAlaLeuThrIleGlnLeuIleGlnAsnHisPheValAspGluTyrAspP 
CCACCATAGAGgtgaggccc 120 
GGTGGTATCTCcactccggg 
roThrIleGlu 
__________________________________________________________________________ 
Legend to Table 1: 
Bold: Oligonucleotides (1) and (2); 
Underlined: oligonucleotide primer (3); 
Asterisk - indicates the position of the mutation G-&gt;C at codon 12. The 
sequence is from GenBank 86, entry HNSRAS1.