(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2003/0147779 A1
`
`Azarani et al.
`(43) Pub. Date:
`Aug. 7, 2003
`
`US 20030147779A1
`
`(54) LOW VOLUNIE MICRO-PLATE AND
`VOLUME-LIMITING PLUGS
`
`Publication Classification
`
`(76)
`
`Inventors: Arezou Azarani, San Jose, CA (US);
`David J. Wright, San Mateo, CA (US);
`Kenneth Christensen, Makawao, H1
`(US); Jesse Cohen, Palo Alto, CA (US)
`
`(51)
`
`Int. Cl.7 ....................................................... B01L 3/00
`
`(52) US. Cl.
`
`.............................................. 422/99; 422/102
`
`Correspondence Address:
`Larry B. Guernsey, Esq.
`IPLO
`Intellectual Property Law Ofiices
`1901 S. Bascom Avenue Suite 660
`5
`Campbell CA 95008 (US)
`
`(21) Appl. No.1
`
`10/345,607
`
`(22)
`
`Filed:
`
`Jan. 15, 2003
`
`Related US, Application Data
`
`(60) Provisional application No. 60/349,776, filed on Jan.
`16, 2002.
`
`(57)
`
`ABSTRACT
`
`.
`.
`.
`A system (10) for reducmg volume of material used in
`laboratory processes, including a low—volume micro—plate
`~
`.
`.
`(70,80) having a number of wells (14) bound together with
`matrix material (16), where the wells include secondary
`wells (74, 84) having reduced volume, and volume limiting
`plugs (20). The volume limiting plugs (20) have a shank
`portion (30) and a tip portion (28), where the shank and tip
`portions (20,30) are configured to extends a substantial
`distance into a well (14) of the multi-well plate (70,80) to
`reduce the volume of the well. Also volume limiting plugs
`(20) for use with standard multi-well plates and low-volume
`micro-plates (70,80) for use with standard plugs.
`
`10
`
`
`
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`

`

`Patent Application Publication
`
`Aug. 7, 2003 Sheet 1 0f 9
`
`US 2003/0147779 A1
`
`FIGURE1
`
`THERMO FISHER EX. 1043
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`THERMO FISHER EX. 1043
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`

`

`Patent Application Publication
`
`Aug. 7, 2003 Sheet 2 0f 9
`
`US 2003/0147779 A1
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`Patent Application Publication
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`Patent Application Publication
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`Patent Application Publication
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`Aug. 7, 2003 Sheet 5 0f 9
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`Patent Application Publication
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`Patent Application Publication
`
`Aug. 7, 2003 Sheet 7 0f 9
`
`US 2003/0147779 A1
`
`FIGURE8
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`

`Patent Application Publication
`
`Aug. 7, 2003 Sheet 8 0f 9
`
`US 2003/0147779 A1
`
`FIGURE9
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`Patent Application Publication
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`US 2003/0147779 A1
`
`Aug. 7, 2003
`
`LOW VOLUME MICRO-PLATE AND
`VOLUME-LIMITING PLUGS
`
`[0001] The following claims priority from US. provi-
`sional application serial No. 60/349,776, filed Jan. 16, 2002,
`which has the same inventors as the present application.
`
`TECHNICAL FIELD
`
`[0002] The present invention relates generally to process-
`ing of DNA samples and more particularly to apparatus for
`DNA sequencing and amplification.
`
`BACKGROUND ART
`
`[0003] Afundamental area of interest in modern molecular
`biology is concerned with the isolation and amplification of
`DNA sequences. The genetic framework, or the genome, of
`an organism is encoded in the double—stranded sequence of
`nucleotide bases in the deoxyribonucleic acid (DNA) which
`is contained in the somatic and germ cells of the organism.
`The genetic content of a particular segment of DNA, or
`gene,
`is only manifested upon production of the protein
`which the gene ultimately encodes. There are additional
`sequences in the genome that do not encode a protein (i.e.,
`“noncoding” regions) which may serve a structural, regula-
`tory, or unknown function. Thus, the genome of an organism
`or cell is the complete collection of protein-encoding genes
`together with intervening noncoding DNA sequences.
`
`[0004] Fundamental operations conducted by molecular
`biologists include amplification and sequencing of DNA
`molecules. Sequencing includes any lab technique used to
`find out the sequence of nucleotide bases in a DNA molecule
`or fragment. Amplification involves an increase in the num-
`ber of copies of a specific DNA fragment, either in a living
`organism or in a laboratory apparatus. One of the most
`successful techniques for DNA amplification is by poly-
`merase chain reaction (PCR).
`
`[0005] Polymerase chain reaction (PCR) is a powerful
`analytical tool permitting the amplification of any desired
`specific nucleic acid sequence contained in a nucleic acid or
`mixture thereof. DNApolymerases synthesize the formation
`of DNA molecules which are complementary to a DNA
`template. The PCR method for amplifying a DNA base
`sequence uses a heat-stable polymerase and two multiple-
`base primers, one complementary to the (+)-strand at one
`end of the sequence to be amplified and the other comple—
`mentary to the (—)—strand at the other end. Because the newly
`synthesized DNA strands can subsequently serve as addi-
`tional templates for the same primer sequences, successive
`rounds of primer annealing, strand elongation, and disso-
`ciation produce rapid and highly specific amplification of the
`desired sequence. PCR also can be used to detect
`the
`existence of the defined sequence in a DNA sample.
`
`PCR permits the copying, and resulting amplifica-
`[0006]
`tion, of a target nucleic acid. Briefly, a target nucleic acid,
`e.g. DNA, is combined with a sense and antisense primers,
`dNTPs, DNA polymerase and other reaction components.
`The sense primer can anneal to the antisense strand of a
`DNA sequence of interest. The antisense primer can anneal
`to the sense strand of the DNA sequence, downstream of the
`location where the sense primer anneals to the DNA target.
`In the first round of amplification, the DNA polymerase
`extends the antisense and sense primers that are annealed to
`
`the target nucleic acid. The first strands are synthesized as
`long strands of indiscriminate length. In the second round of
`amplification, the antisense and sense primers anneal to the
`parent
`target nucleic acid and to the complementary
`sequences on the long strands. The DNA polymerase then
`extends the annealed primers to form strands of discrete
`length that are complementary to each other. The subsequent
`rounds serve to predominantly amplify the DNA molecules
`of the discrete length.
`
`[0007] This process for amplifying the target sequence
`involves introducing a molar excess of two oligonucleotide
`primers which are complementary to their respective strands
`of the double-stranded target sequence to the DNA mixture
`containing the desired target sequence. The mixture is
`denatured and then allowed to hybridize. Following hybrid-
`ization, the primers are extended with polymerase so as to
`form complementary strands. The steps of denaturation,
`hybridization, and polymerase extension can be repeated as
`often as needed, in order to obtain relatively high concen-
`trations of a segment of the desired target sequence.
`
`In laboratory operations, PCR or cycle sequencing
`[0008]
`is done in a PCR machine, usually by distributing DNA
`samples into a multi-well plate, typically having either 96 or
`384 wells. Reagents such as enzymes, primers, buffers and
`dNTP are added, and then the mixture is thermally cycled
`normally 20-30 times to enable the reaction. Heat is gener-
`ally applied from a Peltier module in the pedestal on which
`the well-plate is seated. There is also generally a heating
`plate on top of the sample to prevent sample evaporation/
`condensation. The heat lid constantly maintains higher tem-
`perature (103 degree C.) than the cycle pedestal, so that
`during the cycle reaction, the sample liquid/reagent in the
`well will not evaporate and liquid won’t condensate on the
`top of the well. This helps the concentrations of PCR
`reaction ingredients to remain unchanged since the concen-
`trations of the PCR reaction ingredients are critical.
`
`[0009] As PCR has been found to introduce sequence
`errors into the process and is limited to amplification of short
`DNA segments, another technique known as RCA for R011-
`ing Circle Amplification has also come into use. RCA also
`involves the step of heating mixtures of DNA samples and
`reagents.
`
`[0010] Both techniques involve the use of enzymes which
`are very expensive. A large portion of the cost incurred by
`DNA sequencing and amplification is due to the high cost of
`the enzyme, primers, dNTP and dye terminators used during
`cycle sequencing or PCR. The multi-well plates used in
`these operations typically hold volumes of 50 yL (50 micro-
`liter=50 ><10'6 liters) of material. Although this seems a
`minute quantity by everyday standards, it is estimated that
`enzymes in quantities and concentrations recommended by
`the supplier can cost in excess of $7.00 per reaction. Since
`some labs now reach throughputs in excess of 100,000
`samples per day, the cost of performing the cycle sequencing
`reaction alone can represent a significant amount of the total
`cost of obtaining sequencing data.
`
`To reduce this cost, many laboratories have
`[0011]
`reduced the reaction volumes and diluted the stock enzyme/
`dye mix (referred to as ‘brew”). Reduced volume of material
`has natural advantages in reduction of costs, as for instance,
`a factor of 10 reduction in volume can be expected to
`produce a factor of 10 reduction in cost of material used.
`
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`US 2003/0147779 A1
`
`Aug. 7, 2003
`
`However, substantial dilution of brews can lead to degra-
`dation of sequence quality, presumably because of decreased
`nucleotide and enzyme concentrations.
`
`[0012] Unfortunately, there are technical problems limit-
`ing volume reduction as well. Reduced volume compared to
`surface area can result
`in excessive evaporation of the
`sample during heat cycling. Also, reduced volume gives
`more top space for condensation during the cool cycle, when
`the temperature typically changes from 96 to 50 degree C.
`There can also be difficulties in handling samples of such
`small volume.
`
`[0013] There have been various devices through the years
`which are used to close the openings of arrays of vials. Some
`examples are U.S. Pat. No. 5,112,574 to Horton, U.S. Pat.
`No. 5,005,721 to Jordan, U.S. Pat. No. 6,136,273 to Seguin,
`U.S. Pat. Nos. 5,544,778 and 5,702,017 to Goncalves, U.S.
`Pat. No. 4,599,314 to Shami, and U.S. Pat. No. 5,282,543 to
`Picozza. These devices are effective in sealing the contents
`of the vials inside, but are not designed to significantly
`reduce volume of the wells, nor to transfer heat to the wells
`contents.
`
`[0014] Thus, there is a need for a system that allows for
`reduced volumes of material to be processed, but without the
`disadvantages of excessive evaporation and condensation
`during cooling that have been problems typical of prior
`systems which have used reduced volume of reagents.
`
`DISCLOSURE OF INVENTION
`
`[0015] Accordingly, it is an object of the present invention
`to provide a system which allows use of volumes of material
`which are preferably reduced by as much as a factor of 40
`(from a 20,11L to a 0.5 ,uL total reaction volume).
`
`[0016] Another object of the invention is to produce a
`system in which the cost of processing is also reduced by as
`much as a factor of 40.
`
`[0017] A further object of the present invention is to
`provide a system which can be used in either 96—well or
`384-well format and provides either a new configuration of
`well structures, or which is adaptable to use with existing
`multi—well plates and processing equipment.
`
`[0018] An additional object of the present invention is to
`provide a system in which problems due to evaporation and
`condensation of materials such as variations in reactant
`concentrations is minimized.
`
`[0019] Briefly, one preferred embodiment of the present
`invention is a system for reducing volume of material used
`in laboratory processes. The system includes a low-volume
`micro-plate having a number of wells bound together with
`matrix material, where the wells include secondary wells
`having reduced volume, and volume limiting plugs. The
`volume limiting plugs have a shank portion and a tip portion,
`where the shank and tip portions are configured to extends
`a substantial distance into a well of a multi-well plate to
`reduce the volume of the well.
`
`material costs, and decreasing quantities of materials to
`which laboratory personnel are exposed.
`
`[0022] And another advantage of the present invention is
`that problems of evaporation and condensation on the tops
`of the wells of materials are reduced greatly.
`
`[0023] A further advantage of the present invention is that
`the system can utilize either 96-well or 384-well formats,
`
`[0024] A yet further advantage is that the system is adapt-
`able to use with existing multi-well plates and processing
`equipment.
`
`[0025] These and other objects and advantages of the
`present invention will become clear to those skilled in the art
`in view of the description of the best presently known mode
`of carrying out the invention and the industrial applicability
`of the preferred embodiment as described herein and as
`illustrated in the several figures of the drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0026] The purposes and advantages of the present inven-
`tion will be apparent from the following detailed description
`in conjunction with the appended drawings in which:
`
`[0027] FIG. 1 shows a PCR machine which uses the
`system of the present invention;
`
`[0028] FIG. 2 illustrates a cross-sectional view as taken
`through line 2-2 of FIG. 1;
`
`[0029] FIG. 3 shows a detail view as seen in detail circle
`A of FIG. 2'
`
`[0030] FIG. 4 illustrates a cross-sectional view of a stan-
`dard 50 yL multiwell plate of the prior art;
`
`[0031] FIG. 5 shows the plugs of the present invention in
`use with a standard 50 uL multiwell plate which uses
`discrete plugs;
`
`[0032] FIG. 6 shows a second embodiment of the present
`invention which includes a number of plugs bound into a
`unitary plate;
`
`[0033] FIG. 7 illustrates a third embodiment of the present
`invention in which a number of plugs have been fashioned
`from the heating plate;
`
`[0034] FIG. 8 shows a fourth embodiment of the present
`invention having reduced volume wells and a second type of
`plug;
`
`[0035] FIG. 9 shows a fifth embodiment of the present
`invention, having reduced well volume and a third type of
`plug; and
`
`[0036] FIG. 10 illustrates comparative data from a con-
`ventional plate and seal, a cycleplate and metal plugs, and a
`low-volume plate with metal plugs.
`
`BEST MODE FOR CARRYING OUT THE
`INVENTION
`
`[0020] Also disclosed are low-volume micro-plates and
`volume limiting plugs for use with the multi-well plates.
`
`invention is that
`[0021] An advantage of the present
`reduced volumes of material can be used, thus decreasing
`
`[0037] Apreferred embodiment of the present invention is
`a system for reducing the volume of material used in
`laboratory processing, including low volume micro-plates
`and volume-limiting plugs. As illustrated in the various
`drawing herein, and particularly in the view of FIG. 1, a
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`Aug. 7, 2003
`
`form of this preferred embodiment of the inventive device is
`depicted by the general reference character 10.
`
`FIGS. 1-3 illustrate a machine for processing labo-
`[0038]
`ratory samples, which in this case, will be assumed to be a
`machine 2 for Polymerase Chain Reaction (PCR) cycling. It
`should be understood that other types of machines for
`laboratory processing can be used with the present inven—
`tion, whenever the volume of samples is desired to be
`reduced, and the particular details of the processes allow.
`The PCR machine includes a cabinet 3 which has a lid 4 and
`
`an enclosure 5, which are attached by a hinge. The lid 4
`includes a heater plate 6, which is used to minimize the
`effects of evaporation and condensation in the samples and
`reagents for amplification and sequencing processes. The
`enclosure 5 includes a central cavity 7 into which a pedestal
`8 has been fashioned. The pedestal 8 is connected to a Peltier
`module 15 that heats and cools the pedestal, plates and wells.
`The pedestal 8 is a heavy metal block that can change
`temperature rapidly in response to the heating and cooling
`system in the PCR machine. There is a fan (not shown) for
`cooling under the Peltier module 15 and the module 15 is
`also wired to a heating system not shown.
`
`[0039] A multi-well plate 12 is shown, which can be a
`standard 384 well—plate with 50 ,uL (50 micro—liter=50><10‘6
`liters) capacity wells, or can be a low-volume multi-well
`plate, as discussed below. The multi-well plate 12 includes
`a number of wells 14, of which 384 wells in an array of
`16x24 wells is preferred. The wells 14 are separated by a
`matrix 16 of connecting material which maintains the wells
`in ordered spatial relation to each other. The wells 14 may
`contain samples 18 which are to be processed. Anumber of
`plugs 20 are shown for which there would actually be one
`for each well 14, but which are reduced in number here for
`ease of viewing.
`
`[0040] FIG. 2 shows the PCR machine 2 from a front
`cut-away view as seen from the line 2-2 in FIG. 1, and FIG.
`3 shows a detail View of the area seen in detail circle A of
`
`FIG. 2. The lid 4, heating plate 6, pedestal 8, Peltier module
`15, multiwell plate 12 with wells 14, matrix portion 16, and
`one plug 20 are seen. The well-plate 12 preferably includes
`a lip portion 22 which fits over the pedestal 8 to position the
`plate 12 accurately and hold it in place.
`
`[0041] As shown in FIG. 4 (Prior Art), the usual operation
`of the PCR machine typically involved placing a standard
`well—plate 9, 12 into the box 5, and covering it with a plastic
`seal 24, which helps to decrease evaporation. This seal is not
`limited to plastic, but may be of other materials, and may
`include a film of mineral oil. The heating plate 6 then
`engages the seal 24 and the tops of the wells 14 and the
`Peltier module 15 heats and cools the samples 18 through the
`necessary cycles.
`
`[0042] FIG. 5 shows a first embodiment 40 of the system
`of the present invention. In this embodiment, a standard
`multi-well plate 9, 12 is placed in the box 5, and plugs 20 are
`inserted into the wells 14. The plugs 20 are metal or
`preferably plastic, and have head 26 which are in contact
`with two heating plate 6 and tips 28 which are connected by
`a shank portion 30. The volume of the wells 14 has been
`effectively reduced to 2 ML, the sample material 18 has been
`drastically reduced, and may be as little as 0.8 yL, a
`reduction in volume, and cost in materials, of a factor of 25.
`Of course, it would have been possible in the prior art to
`
`simply reduce the amount of material placed in the wells.
`However, the plugs 20 greatly reduce evaporation over the
`seals 24 seen in FIG. 4, as the available surface area of the
`wells 14 is also greatly reduced. The tips 28 confine vapors
`to a small area, and the plugs 20 additionally provide
`thermal conduction to the vicinity of the sample material 18.
`This reduces the evaporation and condensation of materials
`in the wells and helps the concentrations of PCR reactants to
`remain more constant, which is very critical to success of the
`operations.
`
`[0043] FIG. 6 shows a second embodiment 50 in which
`the individual plugs 20 have been bound together in a single
`continuous plate 52 with multiple shanks 30 and tips 28 that
`protrude into the wells 14 of the multi-well plate 12. The
`same benefits of improved heat transfer, reduced volumes
`and reduced evaporation/condensation also apply. This
`embodiment 50 has the added benefit that all pins 20 can be
`installed simultaneously, thus reducing processing time fur-
`ther.
`
`[0044] The Society of Biomolecular Screening (SBS) has
`established standard XY dimensions for multi-well micro-
`
`plates. Two of the most commonly used standardized con-
`figurations are 96 well microplates with 9 mm center—to
`center spacing, and 384 well microplates with 4.5 mm
`spacing. This has allowed many processes to be automated.
`The spacing of the plugs 20 and the reduced volume wells,
`as discussed below, also preferably conform to this same
`center-to-center spacing.
`
`[0045] FIG. 7 shows a third embodiment 60, in which the
`heating plate 6 of the PCR machine 2 has been formed to
`include plugs 20, whose shafts 30 and tips 28 also protrude
`into the wells 14 of the well-plates 12, thus allowing reduced
`volume of materials 18 to as little as 0.8 yL.
`
`[0046] FIG. 8 illustrates a fourth embodiment 70 in which
`the actual capacity of the wells 74 have been reduced,
`preferably to 2 ML. A secondary well 76 with reduced
`volume is introduced, and a second type of plug 72 is used
`to seal the top of the secondary well 76. The 2 71L well has
`then been filled to only a fraction of its capacity, perhaps to
`as little as 0.5 ,uL, from which good sequencing results have
`still been obtained.
`
`[0047] FIG. 9 shows a fifth embodiment 80 in which the
`capacity of the wells 84 have also been reduced, again
`preferably to 2ML. Asecondary well 86 with reduced volume
`is configured differently, and a third type of plug 82 is used
`to seal the top of the secondary well 86. Once again, good
`sequencing results have still been obtained when the 2 ,uL
`well has been filled to only a fraction of its capacity, perhaps
`to as little as 0.5 ML.
`
`It will be obvious to one skilled in the art that many
`[0048]
`different variations in shape and configuration of the wells
`and plugs are possible. For example,
`there may be no
`secondary wells as such, but only a reduced volume well
`which is plugged in a similar manner to that shown in the
`first embodiment 10 (see FIG. 5). Also, it will be obvious
`that the use of a continuous plate 52 in the manner of the
`second embodiment 50 (see FIG. 6) or a configured heating
`plate 62 as in the third embodiment 60 (see FIG. 7) can be
`used with either or any of these reduced volume wells 74, 84
`of the fourth embodiment 70 and fifth embodiment 80. It is
`also obvious that volume limiting plugs 20 of the present
`
`THERMO FISHER EX. 1043
`
`THERMO FISHER EX. 1043
`
`

`

`US 2003/0147779 A1
`
`Aug. 7, 2003
`
`invention may be used with standard multi-well plates and
`that low-volume micro-plates 70,80 of the present invention
`may be used with standard plugs. In both cases the amount
`of material will be reduced, with comparable cost savings.
`
`[0049] Recently, a base calling program called “Phred”
`has been developed for DNA sequence traces, which is
`capable of generating base-specific quality scores. These
`Phred scores have become widely accepted as a way to
`characterize the quality of sequences, in order to compare
`different sequencing reactions. Quality scores are logarith-
`mically linked to error probabilities, so that a Phred quality
`score of 10 has a probability that a base is called wrong of
`1m 10, or 90% accuracy, a Phred of 20 has 99% accuracy,
`a Phred of 30 has 99.9% accuracy, etc.
`
`indicator of the quality of the
`[0050] Another useful
`results is found in regard to the “Read length” which is a
`measure of the number of DNA sequence bascs scientists
`can read from the sequencing reaction. Coupling the length
`of the sequence with the Phred numbers gives a measure of
`the “Read Length Phred>20”, meaning the length of a
`sequence for which 99% accuracy is obtained.
`
`[0051] A simple way to look at a “PHRED analysis” is to
`treat it as a measurement of the quality of one sequence
`reaction. The more the PHRED read length is, the more
`DNA sequence bases scientists can read from the sequencing
`reaction, so the more information scientists can gather from
`one reaction. Typically an “Excellent” sequencing reaction
`will yield more than 700 bases of “Read Length Phred>20”.
`A “Good” sequencing reaction will give us about 400-
`700“Read Length Phred >20”. If a reaction totally doesn’t
`work, than the Read Length Phred is 0 (i.e. one can not get
`any information from the reaction).
`
`[0052] Thus, these numbers are useful in comparing the
`quality of results obtained by standard methods using 50 ,uL
`well, first with a standard cycleplate and plugs, as discussed
`above with regard to the first through third embodiments 40,
`50, 60, shown in FIGS. 5-7, and secondly as discussed with
`regard to the prototype low-volume plate and plugs, as in the
`fourth and fifth embodiments 70 and 80, shown in FIGS. 8
`and 9.
`
`[0053] FIG. 10 shows tables of comparison of Read
`length Phred>20 data between the conventional plate and
`seal, the column indicated by the element number 100, with
`data obtained from a cycleplate with metal lid, or the plugs
`discussed above, column indicated by element number 200,
`and prototype low-volume plate with prototype metal lid,
`also meaning plugs, and indicated in column marked 300.
`
`[0054] The reaction did not even occur for volumes less
`than 2.5 ,uL in column 100, or for volumes less than 0.8 ,uL
`in column 200, whereas data was obtained for volumes as
`low as 0.5 ML in column 300, which still produces results
`considered to be well within the “Good” range of Phred
`400-700, and produced “Excellent” results in volumes as
`low as 1.0 ,uL. Thus the present invention provides a way to
`produce results that are good to excellent, while greatly
`reducing the cost of laboratory procedures by reducing the
`required amounts of materials. Additional Phred data is
`shown for elements labclcd 200 and 300, corresponding to
`the cycleplate with lid of metal or other materials such as
`plastic, and prototype low-volume plate with prototype lid
`of metal or other materials such as plastic, respectively.
`
`[0055] As referred to above, the present invention is useful
`in a variety of laboratory operations, including amplification
`by Polymerase Chain Reaction (PCR), DNA sequencing,
`Rolling Circle Amplification (RCA) and in fact any labora-
`tory operation using multi-well plates where the volume of
`materials is desired to be reduced.
`
`[0056] While various embodiments have been described
`above, it should be understood that they have been presented
`by way of example only, and not limitation.
`
`INDUSTRIAL APPLICABILITY
`
`[0057] The volume reducing system 10 of the present
`invention having volume reducing plugs 20 which are bound
`into a plate 52, or included in a configured heating plate 62,
`as well as reduced volume wells 74, 84, is designed to be
`used for many applications involving the testing and analy-
`sis of chemical compounds on a micro scale. The many
`advantages of doing work on a micro-scale include the
`reduced costs of reagents, solvents and materials due to the
`reduced amounts needed, and the generation of less waste
`materials which may be environmentally damaging and
`costly to dispose of. The present invention 10 is expected to
`be especially useful for the amplification and sequencing of
`DNA such as Polymerase Chain Reactions (PCR) and Roll-
`ing Circle Amplification (RCA).
`
`[0058] Both PCR and RCA and sequencing techniques
`involve the use of enzymes which are very expensive. A
`large portion of the cost incurred by DNA sequencing and
`amplification is due to the high cost of the enzyme, primers,
`and dye terminators used during cycle sequencing or PCR.
`The multi-well plates used in these operations typically hold
`volumes of 20 ,uL of material, which can cost in excess of
`$7.00 per reaction. Since some labs now reach throughputs
`in excess of 100,000 samples per day, the cost of performing
`the cycle sequencing reaction alone can represent a signifi-
`cant amount of the total cost of obtaining sequencing data.
`
`To reduce this cost, many laboratories have
`[0059]
`reduced the reaction volumes and diluted the stock enzyme/
`dye terminator mix (referred to as ‘brew”). However, sub-
`stantial dilution of brews can lead to lowering of sequence
`quality, and there are technical problems limiting volume
`reduction as well, such as excessive evaporation of the
`sample during heat cycling. Also, reduced volume gives
`more top space for condensation during the cool cycle, and
`there can also be difficulties in handling samples of such
`small volume.
`
`In a first embodiment 40 of the system of the
`[0060]
`present invention, volume-reducing plugs 20 are inserted
`into the wells 14. The plugs 20 are metal or preferably
`plastic, and have heads 26 which are in contact with the
`heating plate 6 and tips 28 which are connected by a shank
`portion 30. By using the plugs 20, the volume of the wells
`14 has been eifectively reduced from 50 ML to 2 ML, and the
`sample material 18 has been drastically reduced, and may be
`as little as 0.8 yL, a reduction in volume, and cost
`in
`materials, of a factor of 25. The plugs 20 greatly reduce
`evaporation, as the available surface area of the wells 14 is
`also greatly reduced. The tips 28 confinc vapors to a small
`area, and the plugs 20 additionally provide thermal conduc-
`tion to the vicinity of the sample material 18. This reduces
`the evaporation and condensation of materials in the wells
`
`THERMO FISHER EX. 1043
`
`THERMO FISHER EX. 1043
`
`

`

`US 2003/0147779 A1
`
`Aug. 7, 2003
`
`and helps the concentrations of PCR reactants to remain
`more constant, which is very critical to success of tie
`operations.
`
`
`
`[0061] A second embodiment 50 of the system of tie
`present invention 10 uses individual pins 20 which have
`been bound together in a single continuous plate 52 with
`multiple shanks 30 and tips 28 that protrude into the we ls
`14 of the multi-well plate 12. The same benefits of improved
`heat transfer, reduced volumes and reduced evaporation/
`condensation are obtained. This embodiment 50 has tie
`added benefit
`that all pins 20 can be installed simulta-
`neously, thus reducing processing time further.
`
`[0062] Athird embodiment 60 uses a heating plate 6 oftie
`PCR machine 2 which has been formed to include pins 20,
`whose shafts 30 and tips 28 also protrude into the wells 14
`of the well-plates 12,
`thus allowing reduced volume of
`materials 18 to as little as 0.8 yL.
`
`[0063] A fourth embodiment 70 has wells 74 in which the
`actual capacity of each well has been reduced, preferably to
`2 [ML by providing a secondary well 76 with reduced volume.
`A second type of plug 72 is used to seal the top of the
`secondary well 76. The 2 ,uL well has then been filled to only
`a fraction of its capacity, perhaps to as little as 0.5 M, from
`which good sequencing results have still been obtained.
`
`[0064] A fifth embodiment 80 has wells 84 in which the
`capacity of each well has also been reduced, again prefer-
`ably to 2 ,uL. A secondary well 86 with reduced volume is
`configured differently, and a third type of plug 82 is used to