Use of centrifugation to prepare a retractable seal over reagents in a reaction container

A method is provided using centrifugation to prepare a seal of solidified wax, grease or polymer mix over an aqueous reagent in a reaction container such that the reagent is separated from contact with the atmosphere. The amount of solidified wax, grease or polymer mix is not sufficient when melted to a liquid to separate the reagent from contact with the atmosphere under gravity. A reagent and solidified wax, grease or polymer mix are combined in a container. During centrifugation and heating, the solidified wax, grease or polymer mix melts to a liquid, and centrifuging causes the liquid to form over the reagent a layer that completely separates the reagent from the atmosphere. As centrifugation continues, the liquid is cooled and solidified to form the seal. Additional reagents are preferably added on top of the seal such that when the container is heated and the seal melted the upper and lower reagents mix for reaction. Preferred use of sealing reagents by this method is in polymerase chain reaction (PCR), reverse transcriptase reactions, nucleic acid sequencing, and colorimetric, fluorometric or chemiluminescent labeled immunoassays.

FIELD OF THE INVENTION 
This invention is for the preparation and use of chemical reaction 
containers or tubes preloaded with one or more reagents that are sealed 
under a retractable layer of wax that is thinner than is normally 
possible. The small amount of wax used in this invention is insufficient 
to spontaneously cover the aqueous layer. This is because when the amount 
of wax provided herein is melted over the aqueous reagent under gravity 
alone, most of the wax moves to the sides of the container. This leaves a 
central hole in the wax layer that allows contact of the reagent solution 
with the atmosphere. However, in this invention, the molten wax is 
centrifuged, forcing the wax down over the aqueous surface to form a 
complete seal as it is cooled to a solid. 
After the reagents are sealed under the wax layer they may be stored, or 
additional solutions with reagents may be added on top of the wax and 
remain separated from the sealed reagents. The sealed reagents are 
subsequently released by heating to melt the wax layer, at which time the 
reagents can mix. Since the original seal retracts and is destroyed when 
it is melted, this is a non-barrier system in that no wax barrier remains 
after the reaction is initiated. This invention is for use in various in 
vitro chemical, biochemical and immunological reactions. 
DESCRIPTION OF THE PRIOR ART 
The task of running chemical reactions is made more convenient and 
consistent through preloading the reaction containers with premeasured 
aqueous reagent. In the prior art, a convenient way to seal the aqueous 
reagent in the container is to cover it with a layer of solid wax. This 
provides protection of the reagent from the atmosphere and excessive 
evaporation, and allows for storage of the containers for future use. Such 
wax sealed, preloaded containers or tubes, are of particular importance in 
certain biochemical reactions. Some examples are nucleic acid 
hybridizations, the reverse transcriptase reaction (RTR), used to produce 
complementary DNA (cDNA) from RNA (Biochemistry 30, 7661-7666, 1991), and 
in DNA sequencing procedures. 
Wax sealed, preloaded tubes are also useful in nucleic acid amplification 
such as the polymerase chain reaction (PCR). The PCR (U.S. Pat. Nos. 
4,683,202 and 4,683,195) employs a heating and cooling cycle to drive the 
reaction. First, the reaction mixture is heated to, or above, the nucleic 
acid melting temperature (denaturization), then cooled to allow specific 
oligonucleotide primers to bind to the sample (annealing), and then heated 
to optimize the addition of complementary bases to the amplified nucleic 
acid (extension). Using heat stable, Taq DNA polymerase (U.S. Pat. No. 
4,889,818), this cycle of denaturing, annealing and extension is repeated 
as many times as needed to generate the desired product. 
The PCR has become a major tool in molecular biology, where there is a need 
for high specificity during amplification. One method for increasing 
specificity is pre-amplification heating. H. A. Erlich, et al, Science 
252, 1643-1651 (1991), and R. T. D'Aquila, et al, Nucleic Acids Res. 19, 
3749 (1991) have described this method. It requires exclusion of at least 
one essential reagent (dNTP's, Mg.sup.2+, DNA polymerase or primers), from 
the reaction until it has been heated to the desired annealing 
temperature. However, the procedure generally requires the sample tubes to 
be closed until heated, and then reopened for addition of the missing 
reagent. 
The procedure, also called "hot start" PCR, has been improved in the prior 
art by the use of a non-retractable wax barrier or seal, formed in the 
reaction tube that separates some of the reagents until the tube is heated 
to melt the barrier. Perkin Elmer Corporation, Norwalk, Conn., now sells a 
wax pellet (Ampliwax.TM.) for this purpose. Their published procedure 
includes the steps of: (1) to a sample tube containing primers, Mg.sup.2+, 
dNTP's in buffer, add one pellet of Ampliwax.TM.; (2) heat the tube to 
80.degree. C. to melt the wax; (3) cool to room temperature to form a wax 
barrier on top of the solution; (4) over the wax barrier, add the sample 
with DNA polymerase in buffer; and (5) start normal PCR cycling. 
The uses for Ampliwax.TM. and greases, to form non-retractable barriers 
between reagents in a reaction tube are more fully disclosed in U.S. Pat. 
No. 5,411,876, by Will Bloch, et al. The Bloch, et al patent requires a 
sufficient mass of wax or grease to spontaneously form a vapor barrier 
over an aqueous sample in a PCR reaction tube. Wax or grease is cast as a 
molten layer, where gravity spreads the molten layer over an aqueous 
solution. The molten layer is allowed to cool and solidify and is then 
used as a barrier between subsets of PCR reagents within the reaction 
tube. The method teaches that a sufficient mass of wax be used to form a 
vapor barrier of molten wax during the reaction as well as form a barrier 
after cooling the wax to a solid. 
Also, U.S. Pat. No. 5,576,197 by R. Arnold, discloses a PCR container with 
a mass of wax attached to the inside surface. As with the Ampliwax.TM., 
this patent also requires that the mass of wax be of sufficient mass that 
it will spontaneously cover the aqueous solution to form a non-retractable 
seal even after the reaction has been stopped. 
Arnold also describes the disadvantages of a centrifugation method called 
"phase inversion". In this method a PCR solution is added to solid wax in 
a tube and centrifuged to force the liquid phase through the solid wax. 
Apparently an excessive length of time and extremely high speeds are 
required to generate the necessary force. Arnold describes several 
problems with the centrifugation method. One problem is that some of the 
aqueous liquid can become trapped in the wax and react violently when 
heated. Also, reagents can absorb onto the solid wax, which removes them 
from the PCR. 
Contrary to Arnold, who teaches against centrifugation, the applicants have 
discovered that centrifugation can work if it is used in a different 
method. Instead of using phase inversion, the instant invention requires 
that the wax must be molten before or during centrifugation. As will be 
shown below, the use of molten wax avoids all the problems of solid wax. 
Horton, et al, Biotechniques Vol. 16, No. 1, (1994), disclose a method that 
employs petroleum jelly to form a spontaneous seal over a reagent in a 
tube that is then overlaid with oil to carry out hot start PCR. Horton, et 
al also teaches away from the use of a wax and oil mixture due to the 
"opaque slurry" that it forms. Also, the reference points out that 
freezing wax seals is not recommended in the 1991 Ampliwax.RTM. product 
insert. 
Apparently, no one in the prior art tried or even contemplated centrifuging 
molten wax. This may be true since there is no convenient way to keep wax 
molten while centrifuging it. As far as the applicants can determine, all 
laboratory centrifuges are specifically designed to cool samples off by 
circulation of air or by refrigeration. As is explained below, this was a 
difficulty that the applicants had to overcome in order to centrifuge 
molten wax. 
In the prior art of preparing solid wax layers or seals over aqueous 
solutions in a container, it is known that the molten wax, like oil, is 
normally spread by gravity over the aqueous surface. The molten wax forms 
a meniscus, so that the wax layer is noticeably thinner in the center than 
at the perimeter or sides of the container (see Bloch, et al, col. 26, 
lines 60-68). After the wax has solidified, it maintains the meniscus 
shape, and in a relatively narrow container such as a PCR reaction tube, 
the wax forms a crater-like depression. Under these conditions, an 
insufficient amount of wax will leave a central hole at the bottom of the 
solidified meniscus, leaving the aqueous solution exposed to the 
atmosphere. 
In the prior art, in order to have a wax seal form spontaneously over an 
aqueous solution under gravity, it must be non-retractable. 
Non-retractable means there must be enough wax mass to overcome the 
meniscus problem and close any potential hole at the center. Consequently, 
under normal gravity conditions that allow a deeper wax meniscus to form, 
a larger mass of wax is required. 
This is a serious problem because, as Bloch, et al have emphasized, it is 
important to keep the total mass of wax as low as possible when performing 
reactions such as the PCR. Bloch, et al describe several problems that 
result from having to use too much wax to prepare the required seals to 
form spontaneously over an aqueous reaction solution or sample (see col. 
14, line 65 through col. 15, line 8 and also col. 8, line 55 through col. 
9, line 31). First, too much wax can make it difficult to mechanically 
penetrate the wax seal in order to recover the aqueous sample. Second, too 
much wax can clog or plug the pipette tip, preventing aspiration of the 
aqueous sample. Third, excess wax requires more pressure to penetrate the 
seal, which can cause spurting of the aqueous sample during penetration 
and lead to aerosol contamination problems. And fourth, which pertains 
mostly to the PCR, the excess mass of wax must be heated and cooled along 
with the aqueous sample during the thermocycling. Since the larger mass 
requires additional time to heat and cool compared to a smaller mass, the 
procedure becomes more time consuming. Therefore, a major problem in the 
prior art of preparing wax seals over aqueous solutions has been to find 
conditions that reduce the depth of the solidified wax meniscus, which in 
turn permits the use of a smaller mass of wax. 
Through their invention and subsequent claims, Bloch, et al disclose in 
great detail, several ways to minimize the mass of wax needed when working 
under normal gravity conditions. For instance, they suggest adding a 
suitable surfactant to the wax and possibly to the aqueous reagent. 
Another remedy is to employ plastic reaction tubes with a hydrophilic 
internal surface, prepared by complicated coating procedures with 
surfactants, or possibly plasma etching the container. Yet another 
suggestion is to add certain types of buoyant plastic mesh or particles to 
the wax. 
The methods disclosed by Bloch, et al require either adding certain 
substances or materials to the wax, or modifying the physical surface of 
the container. Their methods require testing the various additives for 
purity and/or lack of interference, or complicated procedures for altering 
the container surface. There is no suggestion or teaching of solving the 
problem with centrifugation. Contrary to the teachings and suggestions of 
Bloch, et al, it has been discovered that additives to the wax and/or 
modifications to the container are not required for reducing the mass of 
wax. 
Cummins, et al, U.S. Pat. No. 5,364,591, disclose a device for performing 
PCR with beads or particles that are moved from one chamber to another. 
The chambers are separated by barriers that must be pierced. In one 
method, centrifugation may be used to force the beads through the barrier 
layer between confined chambers. This procedure uses centrifugation of a 
solid phase to destroy a barrier and teaches away from the function of the 
instant invention, where centrifugation is used to create a seal rather 
than destroy one. 
The use of centrifugation to separate various materials into zones based on 
their relative density or buoyancy is well known. For instance, E. T. 
Roginski, U.S. Pat. No. 4,927,545, has disclosed a method and apparatus 
for monitoring the separation of red blood cells from serum during 
centrifugation. In that method, as in other centrifugation methods, more 
dense materials such as red blood cells are allowed to pass through a 
separating fluid of specified density, while less dense materials, such as 
serum, are not. These procedures require passage of materials through a 
hydrophilic fluid to form a three phase system with an aqueous phase that 
remains exposed to the atmosphere. This teaches away from the function of 
the instant invention, which requires melting a solid to a liquid and 
formation of a two phase system sealed. Subsequently, the melted wax is 
cooled back to a solid during centrifugation to form a hydrophobic layer 
to seal the aqueous phase from the atmosphere. 
Edens, et al, U.S. Pat. No. 5,106,633, discloses live yeast cells 
immobilized within a wax coating inside a container to act as an oxygen 
scavenger. Edens, et al, is using wax to solve very different problems, 
since; (1) there is no mention of centrifugation, and (2) the invention is 
a coating on the inside surface of a container, not in the form of a seal 
caste over an aqueous reagent. None of these disclosures teach or suggest 
preparing preloaded containers or tubes using centrifugation. 
The methods and reagents disclosed in the references herein are hereby 
incorporated into this patent application by reference. As will become 
apparent with the disclosures to follow, the instant invention solves 
several problems in preparing wax seals for the PCR as well as in other 
high temperature enzymatic methods such as reverse transcriptase. 
SUMMARY OF THE INVENTION 
This invention describes novel methods for the preparation of reaction 
containers that contain premeasured, aqueous reagents sealed under a 
retractable wax layer that is thinner than is possible without 
centrifugation. Preferably, the reagents sealed in the container are used 
for reaction with other materials (such as a buffered aqueous sample), 
subsequently added to the container. The invention eliminates the problems 
in the prior art of having to add various surfactants or other materials 
to the wax, or to treat the container surface. With the distinguishing 
step of centrifuging molten wax instead of solid wax, the invention also 
eliminates the problems with the phase inversion method. For instance, 
there is no observed entrapment of aqueous liquid in the molten wax and 
there is no absorption of reagents to the liquid wax. 
In the wax barrier PCR methods of the prior art, the barrier is not 
retractable and remains in place even after melting due to the large mass. 
Therefore, it is necessary to add a solution of sufficient volume and/or 
density (i.e. use densifying agents) over the wax barrier so that when the 
wax is melted, the upper solution will push through the liquefied wax and 
mix with the lower solution (see Bloch, et al, col. 18, lines 15-35). The 
instant invention when applied to the PCR is a non-barrier system in that 
no wax barrier remains after the PCR is initiated. Therefore, the prior 
art restriction is eliminated because when the seal of the instant 
invention is melted, the barrier is destroyed. Since the barrier moves out 
of the way when melted, the upper solution is immediately in contact with 
the lower solution to facilitate mixing. 
The surprising discovery was made that centrifugation at the required speed 
will overcome the tendency of molten wax to retract to the sides of the 
container. This reduces the wax meniscus and allows the use of less wax 
than is possible in the prior art. By experimenting with the parameters of 
heat, wax mass, aqueous volume, container composition and centrifugation 
speed, it was discovered that the mass of wax required to seal over a 
given aqueous volume can be reduced to less than that which is possible 
under normal gravity conditions. Under the proper conditions of this 
invention, it is possible to use a mass of wax that is normally 
insufficient to make a seal over a given aqueous volume. 
The problems cited in the prior art of heating and cooling excess mass and 
of penetrating the wax layer after the reaction, are eliminated. The 
invention also reduces the problems of measuring and adding one or more 
reagents to a reaction container. 
After adding an aqueous sample to the preloaded container, the container is 
heated to a predetermined temperature to melt the wax layer and the 
previously sealed reagents are released into the surrounding medium, and 
become available for reaction with other substances in the medium. The 
most preferred applications for this invention are in various types of 
nucleic acid hybridizations, PCR's, RTR's, nucleic acid sequencing and 
product generating reactions such as colorimetric, fluorometric and 
chemiluminescent enzyme labeled immunoassays. 
A mixture of all the other needed reagents can be prepared in a separate 
solution. Then, the preloaded tube with entrapped reagents and the 
reaction mixture may be combined and held indefinitely and no reaction 
will occur until the medium is heated to a predetermined temperature. 
Several different reaction mixtures can be prepared and combined with the 
preloaded tubes as needed, and eventually all reactions can be initiated 
simultaneously. This will avoid the problem of adjusting for product 
differences in time-sensitive and/or kinetic reactions such as enzymatic 
production of colored, fluorescent or chemiluminescent products used in 
immunoassays and molecular biology. Preloaded tubes of certain reagents 
will afford protection from degradation during storage and are more easily 
and/or accurately dispensed.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
For the purposes of this invention, the following definitions are used. 
Seal and Sealer 
A seal is defined here as a hydrophobic layer that completely covers an 
aqueous solution within a container to form a barrier layer that 
completely separates the atmosphere from contacting the solution. The 
preferred sealer is a solid material, such as wax, grease or polymer mix, 
that does not easily flow when solidified at room temperature (i.e. 
25.degree. C.). The preferred sealer can be melted to form a free flowing 
liquid at a preferred critical temperature that has a lower density than 
water. Preferred melting temperatures are above room temperature 
especially above 30.degree. C., the upper limit depending on the heat 
tolerance of the reagents used and the type of reaction employed. The 
sealer material is for in vitro use and is essentially inert with the 
surrounding medium. The sealing material is water resistant or water 
insoluble and is not readily dissolved with acids or bases. Therefore, 
there is little or no release of the aqueous reagent sealed under the 
sealing material when exposed to an aqueous medium below the critical 
temperature. This definition is meant to exclude sealing materials such as 
hydrogels, presently used for many drug delivery systems. 
Retractable Seal 
A retractable seal is defined here as a hydrophobic layer that maintains a 
complete seal over an aqueous solution within a container while the sealer 
is solidified. However, when the retractable seal is melted, the material 
retracts toward the sidewalls of the container leaving a central hole that 
allows the atmosphere to contact the aqueous solution. A non-retractable 
seal is a hydrophobic layer that has enough sealer mass to form a complete 
seal over an aqueous solution within a container even when melted. A 
non-retractable seal is one that forms spontaneously when melted over an 
aqueous solution under gravity. 
Reaction Container 
Reaction containers are defined as any suitable container used for 
containing an in vitro reaction. The suitability of the container is 
determined by its suitability for containing a sealer such as wax and an 
aqueous reagent and for centrifuging it while heated to melt the wax. 
Reaction containers include any suitable tubes, wells, cups, plates or 
capillaries, available in various sizes, that are suitable for chemical, 
biochemical and enzymatic reactions such as PCR. They can be made from any 
suitable glass, plastic, resin or polymer including acetals, polyallomers, 
polycarbonates, polyurethanes, polyvinyls, polyethylenes, polypropylenes, 
styrenes, nylons, acrylics, rubbers, polychlorinated or polyfluorinated 
polymers and tetrafluoroethylenes. 
The reaction containers include various tubes or plates with wells at 
specific distances apart to coincide with automated equipment used in 
various biochemical and enzymatic assays such as ELISA's, and the PCR For 
example, tubes available at 9 mm center spacing and in strips of 8 or 12, 
so that they are compatible in spacing with the conventional 96 well 
microtiter plate format. Also included are plates with formats of 96 tubes 
or wells at 9 mm center spacing. Also suitable are plates with multiple or 
divided wells that provide 192 or 384, or more wells per plate. 
Reagent 
A reagent is defined as any suitable chemical or biochemical substance used 
in an in vitro reaction. Reagent suitability means that it can be 
dissolved or suspended in an aqueous medium, preloaded into a reaction 
container and sealed under a hydrophobic layer such as wax that is 
subsequently heated to melt and release the reagent into a reaction 
without excessive inactivation of the reagent. The following are examples 
of reagents for preloaded tubes. 
a. Heat Resistant Enzymes 
Heat resistant enzymes are a preferred group of reagents for sealing in 
reaction containers or tubes. For the purposes of this invention, a heat 
resistant enzyme is defined as any enzyme that retains most of its 
activity after one hour at 40.degree. C. under optimal conditions. Many 
such enzymes can be used such as those from thermophilic organisms. For 
example, various RNA polymerases such as from Thermus sp., or Q beta 
replicase from bacteriophage, also SP6, T3, T4 and T7 RNA polymerases can 
be used, among others. Most preferred are various thermophilic enzymes 
including DNA polymerases from Thermus sp. such as from Thermus aquaticus 
("Taq"), Thermus thermophilus ("Tth"), Thermus flavus ("Tfl"), and Thermus 
brokianus. Also included are enzymes from Thermoccocus sp. such as 
Thermococcus litoralis ("Tli" or "Vent.TM. New England Biolabs"); from 
Pyroccocus sp. such as Pyroccocus furiosus ("Pfu"), Pyroccocus woesei 
("Pwo"); from Thermotoga sp. such as Thermotoga maritima ("Tma"); and from 
Bacillus sp. such as Bacillus stearothermophilus ("Bst"). Also included 
are RNA and DNA ligases such as "ampligase", from Epicentre Technologies, 
and any "recombinant" enzymes (i.e. rTaq, rTth, rTfl, rTli, rPwo, rBst, 
rTma and rPfu, among others). Also, any other enzymes from thermophilic 
microorganisms and invertebrates, including forms produced by mutations or 
by recombinant DNA technology. Another preferred group of enzymes are 
reverse transcriptases, such as from avian myeloblastosis virus, "AMV", or 
from Moloney murine leukemia virus "M-MLV", especially enzymes modified by 
point mutations or deletions such as the "SuperScript.TM." reverse 
transcriptases from Life Technologies, Gaithersburg Md. Other enzymes that 
can be used are restriction endonucleases, helicases, glycosylases, 
kinases, proteases (i.e. thermophilic protease from Thermus sp. strain 
Rt41A), thioredoxins, nucleases, RNAses, DNAses, phosphatases (i.e. 
alkaline phosphatases "AP" and bacterial alkaline phosphatases "BAP"), 
peroxidases (i.e. horseradish peroxidase "HRP"), glucose oxidases, 
galactose oxidases and many others. Preferably these enzymes have 
sufficient thermally stable properties naturally (i.e. by isolation from 
thermophilic organisms), or by suitable chemical modification, mutation, 
or by genetic engineering. 
Also included are various derivatives, analogs and labeled forms of 
enzymes, such as enzymes labeled with biotin, avidin, streptavidin, 
digoxigenin, sulfur, cyclodextrins, fluorophores, and radioactive nuclides 
such as .sup.3 H, .sup.14 C, .sup.32 P, .sup.35 S, and .sup.125 I, among 
others. 
b. Enzyme Substrates 
Another useful group of reagents for sealing in reaction containers is any 
suitable substrate. For example, in the PCR, these include any labeled or 
unlabeled nucleotides and nucleotide triphosphates (dNTP's), any 
deoxynucleoside triphosphates (NTP's), any dideoxynucleoside triphosphates 
(ddNTP's) and ribonucleoside triphosphates. Some examples are; 
2'-deoxyadenosine 5'-triphosphate (dATP), 2'-deoxycytidine 5'-triphosphate 
(dCTP), 2'-deoxyguanosine 5'-triphosphate (dGTP), 2'-deoxythymidine 
5'-triphosphate (dTTP), 2'-deoxyuridine 5'-triphosphate (dUTP), 
2'-deoxyinosine 5'-triphosphate (dITP), 7-deaza-2'-deoxyguanosine 
5'-triphosphate (I-N7-dGTP), among others. Also included are members of 
this group labeled with radioactive nuclides such as 3H, 14C, 32P, 35S, 
and 125I, among others. 
Also included are various derivatives, analogs and labeled forms of NTP's, 
dNTP's and ddNTP's, such as biotin labeled, bio-4-dUTP, and bio-11-dUTP, 
also dNTP's labeled with digoxigenin (i.e. DIG-UTP, DIG-dUTP, DIG-ddUTP, 
Biotechniques 12, 104-113 (1992)), sulfur, cyclodextrins, fluorophores, 
isotopes, and amino groups such as 5-(3-aminoallyl)-2'-deoxyuridine 
5'-triphosphate (AA-dUTP). 
Another group of enzyme substrates preferred for sealing in reaction 
containers is any phosphorylated enzyme substrate that produces a colored, 
fluorescent or chemiluminescent product when dephosphorylated, as with AP, 
such as 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and nitro blue 
tetrazolium (NBT); 4-methylumbelliferyl phosphate, and any phosphorylated 
dioxetanes 
(3-(2'-spiroadamantanane)-4-methoxy-4-(3"phosphoryloxy)phenyl-1,2-dioxetan 
e (AMPPD)) and HMPPD, among others. Also preferred for sealing in reaction 
containers are any substrates for peroxidases such as o-phenylenediamine 
(OPD), 3,3'-diaminobenzidine tetrahydrochloride dihydrate (DAB), and 
3,3',5,5'-tetramethylbenzidine (TMB), among others. 
c. Metal Salts 
Another group of reagents for sealing in reaction containers is various 
salts (i.e. chlorides or sulfates), of metals such as Mg, Mn, Fe, Co, Cu, 
Zn, Sn, etc. 
d. Nucleic Acids 
Another important group for sealing in reaction containers is nucleic 
acids. A nucleic acid is defined as any nucleic acid sequence from any 
source that is suitable for use in the instant invention. Said nucleic 
acid includes all types of RNA, all types of DNA, and oligonucleotides 
including primers used in the polymerase chain reaction (PCR) or DNA 
sequencing, and other genetic materials including synthetic nucleic acid 
polymers. Also included are DNA and/or RNA fragments, and derivatives from 
any tissue, cells, nuclei, chromosomes, cytoplasm, mitochondria, 
ribosomes, and other cellular sources. Also included are modified and 
derivatized nucleic acid sequences including those that are coupled to or 
associated with other substances such as proteins, lectins, histones, 
polypeptides, carbohydrates, lipids, resins, steroids and hormones. 
An especially important group of nucleic acids for sealing in reaction 
containers includes any suitable labeled or unlabeled oligonucleotides for 
use as hybridization probes or primers. For instance, in the PCR, any 
appropriate antisense (reverse) primers and sense (forward) primers can be 
used including those labeled with any suitable label such as biotin, AP, 
digoxigenin, sulfur, cyclodextrins, fluorophores, isotopes, and proteins. 
Also included are members of this group labeled with radioactive nuclides 
such as .sup.3 H, .sup.14 C, .sup.32 P, .sup.35 S, and .sup.125 I, among 
others. 
e. Antibodies 
Another preferred group of reagents for sealing in reaction containers are 
any antibodies, antibody fractions and monoclonal antibodies, including 
derivatives thereof. 
f. Antigens 
Another preferred group of reagents for sealing in reaction containers are 
antigens or derivatives thereof, defined as substances capable of 
stimulating an immunologic reaction such as the formation of antibodies in 
an organism or in a biological system. 
g. Avidins and Streptavidins 
Also included are any avidins and streptavidins including derivatives, 
labeled forms, fractions and avidins produced by recombinant DNA 
technology. 
h. Biotins 
Also included are any biotins, including derivatized biotins such as 
amino-biotins, sulfo-biotins and photobiotins. 
i. Cyclodextrins 
Another preferred group of reagents for sealing in reaction containers are 
cyclodextrins. A cyclodextrin (CD) is an oligosaccharide composed of 
glucose monomers coupled together to form a conical, hollow molecule with 
a hydrophobic interior or cavity. Said cyclodextrins (CD's), of the 
instant invention can be any suitable cyclodextrin, including alpha-, 
beta-, and gamma-cyclodextrins, and their combinations, analogs, isomers, 
and derivatives. Also included are altered forms, such as crown ether-like 
compounds prepared by Kandra, L., et al, J. Inclus. Phenom. 2, 869-875 
(1984), and higher homologues of cyclodextrins, such as those prepared by 
Pulley, et al, Biochem. Biophys. Res. Comm. 5, 11 (1961), and soluble 
dimers, trimers and polymers. Some recent reviews on cyclodextrins are: 
Atwood J. E. D., et al, Eds., "Inclusion Compounds", vols. 2 & 3, Academic 
Press, New York (1984); Bender, M. L., et al, "Cyclodextrin Chemistry", 
Springer-Verlag, Berlin, (1978) and Szejtli, J., "Cyclodextrins and Their 
Inclusion Complexes", Akademiai Kiado, Budapest, Hungary (1982). 
Relative Centrifugal Force or R.C.F. 
Relative centrifugal force (g) is calculated from the following formula: 
EQU g=1.12.times.10.sup.-5 (r)(rpm).sup.2, 
where r=radius in centimeters, and rpm=revolutions per minute. 
Wax For Sealing Containers 
Wax is defined as an essentially water insoluble, hydrophobic hydrocarbon 
that is solid or semisolid at room temperature, but can be melted above 
room temperature to form a dispersible liquid that has a lower density 
than water. Wax includes any naturally occurring and synthetic waxes, and 
wax esters that have the desired melting temperature. Waxes suitable for 
this invention are less dense than the aqueous reagent they are used with 
so that they form a layer over the aqueous reagent. Also, suitable waxes 
have little or no adverse reactivity with the aqueous reagent. 
The most preferred waxes are many natural or synthetic long chain 
hydrocarbons such as white waxes, paraffins, silicon waxes, polychorinated 
or polyfluorinated hydrocarbons, polyether waxes and polyester waxes. Some 
examples of paraffins and approximate melting point (m.p.), that can be 
used in this invention are; hexacosane (56.4.degree. C.), hentriacosane 
(59.degree. C.), octacosane (61.4.degree. C.), nonacosane (62.7.degree. 
C.), triacontane (65.6.degree. C.), hentriacontane (67.6.degree. C.), 
dotriacontane (69.5.degree. C.), tetratriacontane (72.5.degree. C.), 
pentatriacontane (74.4.degree. C.), hexatriacontane (75.7.degree. C.), 
including others with shorter or longer carbon chains. One suitable source 
of paraffin waxes for use in this invention are those from Fluka Chemical 
Corp., St. Louis, Mo., with m.p.'s of; 44-46.degree. C., 50-52.degree. C., 
54-56.degree. C., 58-60.degree. C., and 68-74.degree. C. 
Other useful waxes are various plant derived waxes, including carnauba wax, 
ouricuri wax, candelilla wax, raphia wax, apple, cotton and cactus waxes; 
waxes produced by bacteria (i.e. cetyl stearate); fungi, protozoa and 
algae; various invertebrate waxes including insect waxes such as beeswaxes 
(i.e. triacontyl palmitate, palmatyl palmitate), and Coccus sp. derived 
waxes (i.e. lac, cochineal and Chinese insect). Also included are suitably 
modified derivatives and combinations of waxes, including various waxes 
produced by recombinant DNA technology. 
Depending on the desired properties, such as melting point, inertness, 
solubility, buoyancy, etc., any of the waxes described here can be 
combined with polymers such as polyethylenes and polypropylenes in various 
proportions to give the desired result. Useful waxes can also be suitably 
chlorinated or fluorinated. 
NON-WAX SEALING MATERIALS 
It has been discovered that certain non-wax materials that are suitably 
hydrophobic and inert in many biochemical reactions such as PCR, can be 
used as sealing materials in the instant invention. They include certain 
greases, long-chain alcohols, long-chain acids, long-chain surfactants and 
polymers mixed with oil, that are solids at RT that can be melted to a 
liquid that has a lower density than water. 
Greases For Sealing Containers 
Preferred greases are solid or semi-solid hydrocarbons or silicones that 
are soft at RT and melt at about 30-80.degree. C. to form a liquid that 
has a lower density than water. Some examples of preferred greases are 
white petrolatum (i.e. Vaseline.RTM.), low density silicone grease. 
Alcohols For Sealing Containers 
Certain long-chain (fatty) alcohols with even or odd numbers of carbons, 
have wax-like properties of melting temperature, density and inertness, 
and are suitable sealers. Some examples of preferred fatty alcohols (and 
approximate melting point), that can be used in this invention are listed 
below. 
______________________________________ 
Alcohol Name M.P. .degree. C. 
Formula 
______________________________________ 
carnaubyl 68-69 
lauryl (dodecanol) 48 C 
.sub.12 H.sub.26 O 
myristyl (1-tetradecanol) 38 C.sub.14 H.sub.30 O 
palmityl (1-hexadecano1) 63-64 C.sub.16 H.sub.34 O 
margaryl (1-heptadecanol) 59.3 C.sub.17 H.sub.36 O 
stearyl (1-octadecanol) 59-60 C.sub.18 H.sub.38 O 
arachidyl (1-eicosanol) C.sub.20 H.sub.42 O 
behenyl (1-docosanol) C.sub.22 H.sub.46 O 
lignoceryl (1-tetracosanol) C.sub.24 H.sub.50 O 
ceryl (1-hexacosanol) 80 C.sub.27 H.sub.54 O 
melissyl (1-triacontanol) 88 C.sub.3O H.sub.62 O 
octadecyl 59 
______________________________________ 
Acids For Sealing Containers 
Some examples of saturated fatty acids (and approximate melting point), 
that can be used in this invention are listed below. 
______________________________________ 
Saturated Acid Name 
M.P. .degree. C. 
Formula 
______________________________________ 
capric 31.3 
lauric (dodecanioc) 48 C 
.sub.12 H.sub.24 O.sub.2 
myristic (tetradecanioc) 58 C.sub.14 H.sub.28 O.sub.2 
palmitic (hexadecanioc) 63-64 C.sub.16 
H.sub.32 O.sub.2 
margaric (heptadecanioc) 61 C.sub.17 H.sub.34 O.sub.2 
stearic (octadecanioc) 70.5-71.5 
C.sub.18 H.sub.36 O.sub.2 
arachidic 76-77 
behenic (docosanoic) 81-82 C.sub.22 H.sub.44 O.sub.2 
tetra-cosanic 84.5-85.5 
lignoceric 75-80 
cerotic 78 
melissic 91 
______________________________________ 
Some examples of unsaturated fatty acids (and approximate melting point), 
that can be used in this invention are listed below. 
______________________________________ 
Unsaturated Acid Name 
M.P. .degree. C. 
Formula 
______________________________________ 
tiglic 64-65 
hypogaeic 33 
-49 
gaidic 39 
physetoleic 30 
elaidic 44-45 
oleic (cis-9-octadecanoic) 58-59 C.sub.18 H.sub.34 O.sub.2 
isooleic 44-45 
erudic 33-34 
brassidic 65 
isoerudic 54-56 
______________________________________ 
Some examples of esters that can be used in this invention are listed 
below. 
______________________________________ 
Ester Name Formula 
______________________________________ 
behenic acid stearyl ester (stearyl behenate) 
C.sub.40 H.sub.80 O.sub.2 
stearic acid lauryl ester (lauryl stearate) C.sub.30 H.sub.60 O.sub.2 
______________________________________ 
Non-wax Polymers For Sealing Containers 
The surprising discovery was made that certain polymers can be combined 
with oils, long-chain alcohols and long-chain acids to form non-wax 
polymer mixes useful as sealing materials. Some examples of polymers are 
polyethylenes, polypropylenes, certain gums and rubbers. Most preferred 
polymers are low and medium density polyethylenes and polypropylenes 
(Scientific Polymer Products, Ontario N.Y.), with melting points below 
120.degree. C. Also included are synthetic rubbers such as isoprene 
polymers, hydrogenated rubber, butadiene polymers, chloroprene polymers 
and butyl polymers. 
Coloring Compounds 
Also, under suitable conditions, the wax and other sealing materials can 
have coloring added to it in the form of a colored or fluorescent dye, 
preferably any suitable oil or fat soluble dye can be used. Some examples 
are, Sudan I, Sudan III, Sudan IV, Sudan Black B, Solvent Red 27 and 
Solvent Blue 14. 
Mercantile Kits for Preloaded Containers with Wax Seals 
A mercantile kit is defined as a collection of materials such as 
containers, reagents, buffer solutions, instructions for use, packaging, 
and the like, prepared as a package for sale. Any of the preloaded 
containers or tubes disclosed in the instant invention can readily be 
incorporated into a mercantile kit. 
Preloaded tubes of the instant invention are very advantageous for selling 
in mercantile kits. For example, reagents would be sealed under wax in 
tubes or plates and are covered with a plastic sheet or slipped into a 
suitable plastic sleeve or bag to form packages which would minimize 
handling. 
The following applications illustrate the great diversity of uses for the 
instant invention. With suitable modification by one skilled in the art, 
this invention can be applied to any of the following applications. 
APPLICATIONS 
A. PCR Using DNA Polymerase Enzyme Sealed In Reaction Containers 
The DNA sample to be amplified is in a final volume of 0.02-0.1 ml 
containing; 0.5 mM each of dATP, dCTP, dGTP, and dTTP (or any other 
suitable labeled or unlabeled dNTP's), 0.002 mM each of any appropriate 
antisense (reverse) primers and sense (forward) primers, in a buffer of 
2.5 mM MgCl.sub.2, 500 mM KCl, 100 mM Tris-HCl, 0.1% w/v gelatin, pH 8.3. 
Also included is any heat stable DNA polymerase in a suitable container 
preloaded under a wax seal as described herein. In this case, 
approximately 1-3 units of Taq DNA polymerase in about 1-20 .mu.l of 
aqueous reagent entrapped under a wax seal that melts at a specific 
temperature (preferred range 50-100.degree. C.). The PCR reaction is 
initiated when the wax seal over the DNA polymerase enzyme is melted to 
release the polymerase into solution. 
Amplification is performed by sequential immersion in water baths or in any 
suitable thermal cycling machine. A "hot start" method is initiated when 
the sample is heated to the critical releasing temperature of the wax 
seal. Samples are optionally denatured at 98.degree. C. for 30 seconds 
followed by 20-40 cycles of; 94.degree. C. for 20-60 seconds, 65.degree. 
C. for 20-60 seconds, 72.degree. C. for 60-120 seconds. 
B. High Temperature RTR 
The instant invention is useful in high temperature reverse trancriptase 
reactions such those reported by A. L. Schaffer, et al, Anal. Biochem. 
190, 292-296 (1990) and T. W. Myers, et al, Biochem. 30, 7661-7666 (1991). 
The procedure is modified in that one or more essential enzymes, such as 
Taq DNA polymerase, or Tth DNA polymerase, or reverse transcriptases 
(preferably thermostable), are first preloaded in a reaction container and 
sealed under a sealing material (i.e. wax), that melts at or near the 
desired reaction temperature (i.e. 60-80.degree. C.). The sample 
containing the RNA to be transcribed in suitable aqueous solution is added 
over the sealing layer in the container. Then, the reaction is initiated 
when the container has been heated and has reached the required 
temperature to melt the seal and release the reagent(s). The high 
temperature RTR is reported to improve product specificity and destabilize 
many secondary structures in the template RNA, to allow more complete 
transcription. 
C. High Temperature Nucleic Acid Sequencing 
This method is based on the procedures of F. Sanger, et al, Proc. Natl. 
Acad. Sci. USA 74, 5463-5467 (1977), S. Tabor, et al, Proc. Natl. Acad. 
Sci. USA 84, 4767-4771 (1987) and M. A. Innis, et al, Proc. Natl. Acad. 
Sci. USA 85, 9436-9440 (1988). This method utilizes the three basic steps 
of annealing, labeling and extension/termination used in previous 
dideoxynucleotide methods (Sanger) except that the need for reopening and 
transferring of reaction products is reduced. For instance, the annealing 
and labeling reactions can be combined by preparing the template DNA in a 
mixture with the needed primer(s) in a suitable aqueous buffer. 
A suitable sequencing enzyme such as Taq DNA polymerase and labeling 
mixture of dNTP's are preloaded in a reaction container and sealed under a 
sealing material (i.e. wax), that melts at or near the desired reaction 
temperature (i.e. 50-100.degree. C.). The sample containing the DNA to be 
sequenced is added over the sealing layer in the container. Then, the 
reaction is initiated when the container has been heated and has reached 
the required temperature to melt the seal, denature the DNA and release 
the reagent(s). 
D. Coupled RTR and PCR with Two Enzymes and Antibody 
This invention can be used in another method where the RTR and PCR are 
coupled using two enzymes and an optional antibody. The first enzyme, used 
for the reverse transcriptase reaction, is any suitable reverse 
transcriptase enzyme such as from AMV, M-MLV or a thermal stable reverse 
transcriptase. 
The second enzyme is any suitable thermal stable DNA polymerase used to 
perform the PCR. The optional antibody is any suitable antibody specific 
for inactivating the reverse transcriptase enzyme being used, and blocking 
it from subsequent interference with the PCR. For instance, the antibody 
could be monoclonal antibody specific for binding to the active binding 
site of the reverse transcriptase enzyme. 
The DNA polymerase is first preloaded in a reaction container and sealed 
under a sealing material (i.e. wax), that melts at or near the desired 
reaction temperature (i.e. 60-80.degree. C.). The optional antibody can be 
included with the sealed reagent. 
In one method, the sample containing the RNA to be transcribed with the RT 
enzyme in suitable buffer containing dNTP's, primers, etc., is added over 
the sealing layer in the container. The RT reaction is done first, to 
synthesize cDNA from the sample RNA, keeping the temperature below the 
melting point of the seal material. 
Then, the PCR is initiated when the container has been heated and has 
reached the required temperature to melt the seal and release the sealed 
reagent(s). The temperature is raised enough to denature the cDNA and to 
melt the seal. If included, the released antibody can then inactivate the 
RT enzyme and when the sample is thermocycled for the PCR, the DNA 
polymerase will amplify the cDNA under PCR conditions. 
E. Other Types of PCR 
With suitable modification by one skilled in the art, PCR reagents sealed 
in containers by the methods of this invention can be applied to a wide 
variety of PCR methods. These include asymmetric PCR, inverse PCR and 
arbitrarily primed PCR (APPCR), among others. For example, this invention 
is adaptable for applications to in situ PCR, described by G. J. Nuovo, et 
al, Amer. J. Pathol. 139, 847-854 and 1239-1244 (1991). 
The enzymes of this invention are also applicable to inverted PCR (IPCR) as 
described by S. Takagi, et al, in Biotechniques 13, 176-178 (1992), and 
especially heat-soaked PCR (HS-PCR), as described by G. Ruano, et al, in 
Biotechniques 13, 266-274 (1992), but suitably modified so that a bolus of 
enzyme would be used that had been sealed in a reaction container of this 
invention. 
F. Specific Binding Assays Using Reagents Sealed in a Reaction Container 
It was discovered that the centrifugation method of the instant invention 
now makes it possible to prepare very thin seals in reaction containers 
(i.e. wells), with relatively large diameters (i.e. 6.6 mm). Some examples 
are 96 well "microtiter" plates of polypropylene or polystyrene. Using the 
centrifugation method of the instant invention, it was found that only 
about 24 mg of wax was required to make a seal over a 150 .mu.l aqueous 
volume in polystyrene flat bottom Removawells.RTM. (0.4 ml), from Dynatech 
Laboratories, Chantilly, Va. However, without centrifugation, the same 
aqueous volume required about twice as much wax (i.e. 48 mg) to make a 
seal. 
This type of container is widely used for binding assays in which an 
oligonucleotide probe, PCR primer, avidin, streptavidin, antibody, or 
antigen is bound or attached to the inside surface of the reaction well 
and used to bind other specific substances for ultimate detection. After 
the reaction is completed, measurements are conveniently made by directly 
reading the amount of light absorbance, or fluorescence, or luminescence 
in the sample that is in the well. 
1. Sandwich Immunoassay 
In one example, the instant invention can be used in a "sandwich 
immunoassay" where an antibody specific for an antigen to be detected 
(i.e. protein, virus, etc.), is bound (i.e. by various coating methods 
known in immunoassays), to the inside surface of the well (preferably 
covalently bound). To the bottom of the well is added about 100 .mu.l of 
buffered solution containing a second antibody specific for the same 
antigen (preferably specific for a different epitope on the antigen than 
the first antibody). The second antibody may also be suitably labeled such 
as with alkaline phosphatase (AP) or peroxidase enzyme, or with a 
fluorescent or radioactive label. These reagents are then sealed in the 
container by the method of the instant invention. For example, about 24 mg 
of paraffin wax (i.e. mp 44-46.degree. C.), is added and then melted and 
solidified during centrifugation at about 350-500 R.C.F. 
A sample to be tested for the antigen in question is then added to the 
sealed container in a suitable volume (i.e. 100-200 .mu.l) of buffered 
solution. The specific binding reaction is initiated by briefly heating 
the well to melt the wax seal, which allows the sample and sealed reagent 
to combine. During a suitable incubation period, antigen that is present 
in the sample will bind to the antibody on the well surface and will also 
be bound by the labeled second antibody to form a sandwich. 
If, for instance the second antibody is labeled with AP, the excess unbound 
antibody and unbound sample materials are removed by washing the well with 
buffer (i.e. as in a standard ELISA procedure). The presence of the AP 
label is detected by combining the enzyme in a suitable buffer such as AP 
buffer (i.e. composed of 0.1 M Tris HCl, 0.2 M NaCl, 0.01 M MgCl.sub.2, pH 
9.5-10), with a suitable substrate that generates a detectable product. In 
this case, the substrate is a mixture of 5-bromo-4-chloro-3-indoyl 
phosphate (BCIP) and nitro blue tetrazolium (NBT), at approximately 0.17 
mg/ml BCIP and 0.33 mg/ml NBT. The sample is incubated at RT to produce a 
colored product that is measured by absorption. Alternatively, the 
substrate can be one that produces a chemiluminescent or fluorescent 
product when dephosphorylated. 
Where the label on the antibody is any suitable peroxidase such as 
horseradish peroxidase, the preferred buffer is more acidic such as 0.1 M 
sodium citrate, pH 5.0. With peroxidase, a suitable substrate is about 
0.1% o-phenylenediamine (OPD), which produces a colored product that is 
measured by absorption at 490 nm. 
There is another application of the instant invention to this type of 
container. For instance, after coating specific antibodies or antigen to 
the inside surface of the well, it would be useful to dry the well and 
seal the bottom surface under a thin wax layer. For example, about 24 mg 
of paraffin wax (i.e. mp 44-46.degree. C.), is added to the coated well(s) 
and then melted and solidified during centrifugation at about 350-500 
R.C.F. This would provide a removable, protective layer to the reagents 
bound to the well surface. 
2. Hybridization Assay 
The instant invention can also be used in a hybridization assay. In this 
example an oligonucleotide is used that has a complementary region 
specific for a known sequence of DNA or RNA that is to be detected (i.e. 
from a virus, bacterium, etc.), so that it can hybridize with it. The 
oligonucleotide is bound to the inside surface of the well (preferably 
covalently). To the bottom of the well is added about 100 .mu.l of 
buffered solution containing a second oligonucleotide that can hybridize 
to another region of the DNA or RNA to be detected. The second 
oligonucleotide may also be suitably labeled such as biotin or with a 
fluorescent or radioactive label. These reagents are then sealed in the 
container by the method of the instant invention. For example, about 24 mg 
of paraffin wax (i.e. mp 44-46.degree. C.), is added and then melted and 
solidified during centrifugation at about 350-500 R.C.F. 
A sample to be tested for the DNA or RNA in question is then added to the 
sealed container in a suitable volume (i.e. 100-200 .mu.l) of buffered 
solution. The hybridization reaction is initiated by briefly heating the 
well to melt the wax seal, which allows the sample and sealed reagent to 
combine. During a suitable incubation period, any complementary regions on 
the DNA or RNA that is present in the sample will bind by hybridization to 
the oligonucleotide on the well surface and will similarly be bound by the 
labeled second oligonucleotide to form a sandwich. 
The excess unbound oligonucleotide and unbound sample materials are removed 
by washing the well with buffer (i.e. as in a standard hybridization 
procedure). If the label used is fluorescent, it is detected by adding a 
suitable buffer to the well and measuring fluorescence emission when the 
label is activated by a suitable U.V. light. Other label systems are 
detected by the appropriate methods. 
PREATION METHODS FOR RETRACTABLE SEALS IN REACTION CONTAINERS 
The following examples show preparation methods and use of this invention. 
Unless otherwise stated, the materials and equipment used were from the 
sources given below. The centrifuge used for all examples with or without 
modification was a table top centrifuge (IEC Model HN-SII), from 
International Equipment Co., Needham Hts, Mass., using a swinging bucket 
rotor (IEC #215). The polymerase chain reaction (PCR), was performed in a 
model PTC-100 thermocycler (M. J. Research, Watertown, Mass.). 
A "PCR" pipettor refers to a positive displacement pipettor (Finnpipette 
P.C.R., 0.5-25 .mu.l), from Labsystems, Helsinki, Finland with a 
disposable polypropylene tip and plunger. 
A "standard" pipettor is an air displacement pipettor (Oxford Benchmate 
40-200 .mu.l), from Sherwood Medical, St. Louis, Mo., with a disposable 
polypropylene tip. The polypropylene, 0.2 ml PCR tubes were from Robbins 
Scientific Inc., Sunnyvale, Calif., and the polycarbonate, PCR plates were 
from Corning Costar Corp., Cambridge, Mass. 
Paraffin wax was from Fluka Chemical Co., Ronkonkoma, N.Y.; buffer salts 
and additives, dyes, DNA, primers and dNTP's were from Sigma Chemical Co., 
St. Louis, Mo.; PCR enzymes and reagents were from Promega Corp., Madison 
Wis., Perkin-Elmer Corp., Norwalk, Conn., or Boehringer Mannheim Corp., 
Indianapolis, Ind. 
EXAMPLE I 
Preparation of Retractable Seals in Reaction Tubes Preloaded with 
MgCl.sub.2 and Food Color 
(Exper. MK164) A few grams of paraffin wax, melting point 58-60.degree. C., 
(Fluka), was melted in an aluminum weighing boat. Into the bottom of each 
of 12, 0.2 ml PCR tubes (bobbins), was dispensed 2 .mu.l (about 2.5 mg), 
of the melted paraffin wax using a "PCR" positive displacement pipettor 
(Labsystems) with a disposable polypropylene tip and plunger. The 2.5 mg 
aliquots of wax were allowed to cool and solidify. 
A loading solution was prepared, composed of 60 mM MgCl.sub.2 in water, 
with about 1% of FD&C blue #1 food color (McCormick & Co., Baltimore Md.). 
To each of the above tubes was added 2 .mu.l of the loading solution, 
using a standard air displacement pipettor (Oxford Benchmate, Sherwood 
Medical, St. Louis, Mo.), with a disposable polypropylene tip. 
A table top centrifuge (IEC), using a swinging bucket rotor (IEC #215), 
with trunnion rings and metal shields (IEC #320), was adapted for this 
procedure. In order to hold the 0.2 ml polypropylene PCR tubes upright in 
the 50 ml shields for centrifugation, a "tube assembly" holding system was 
devised. 
To prepared the tube assembly, the PCR tubes were first stacked in groups 
of 6. The conical bottom of each upper tube in the stack, protruded part 
way into the tube below it, leaving room for the solution and wax in the 
bottom of each tube. Each stack of 6 PCR tubes was supported in a 1.5 ml 
polypropylene centrifuge tube, which was then placed in a Pyrex.TM. glass 
tube, 16.times.100 mm. Finally, the glass tube containing the PCR tubes 
was placed in a 50 ml, conical bottomed, polypropylene centrifuge tube, 
which would then fit properly into the shield. 
The heating method consisted of preheating the empty metal shields on a hot 
plate to about 150.degree. C., quickly placing them into the trunnions in 
the centrifuge, and then immediately placing the tube assemblies 
containing the loaded PCR tubes previously described, into the shields. It 
was determined empirically that under these conditions, the internal 
temperature of the tube assembly reached about 94.degree. C. in 4 minutes 
to melt the wax, and remained there for at least 3 more minutes before 
slowly cooling. 
Immediately after placing the loaded PCR tube assembly into the heated 
shields, the centrifuge was turned on to 2500 rpm for 10 minutes. The 
heated shields heated the wax in the tubes above the melting point, while 
the centrifugation forced the melted wax to form a thin layer over the 
aqueous reagent in the bottom of the tube. As centrifugation continued, 
the tubes cooled to room temperature and the thin wax layer solidified 
forming a seal over the aqueous reagent. 
Testing the Wax Seal 
The PCR tubes were removed from the tube assembly and inspected. A thin, 
very flat wax layer with little or no visible meniscus, completely covered 
the colored aqueous reagent in each tube. The integrity of the wax layers 
was tested by adding a top layer of 0.1 ml of deionized water to each tube 
and letting the tubes sit at room temperature. After about 3.5 hours, the 
top layer of water in each tube was visually inspected for color. The 
result was that none of the tubes showed visible color in the upper water 
layer indicating that the wax seal was complete. 
Releasing the Sealed Reagent 
The 0.1 ml of water added previously was removed and 0.05 ml of deionized 
water was added to each tube, over the wax seal. The tubes were heated for 
2 minutes at 94.degree. C. and cooled back to room temperature. A uniform 
blue color was observed in all of the tubes, indicating that the seals had 
retracted and mixing had occurred between the originally sealed blue 
reagent in the bottom and the deionized water above. 
The magnesium concentration in the water was determined for each tube. 
Indicator solution (2.times.) was prepared by combining 1 part of 0.1% 
calmagite (Sigma Chemical Co.) in water with 5 parts of 0.5 M Tris base, 
pH 9.0, in water. Sample was combined 1:1 with Indicator Solution. The 
absorbance was read at 515-520 nm using a spectrophotometer. Sample 
absorbance is compared to a standard curve prepared from known Mg 
concentrations. One hundred percent of the tubes released Mg into the 
water above, with absorbances ranging from 1.894 to 2.054. The calculated 
mean Mg concentration released was 0.055 mM with a coefficient of 
variation of about 6.4%. 
EXAMPLE II 
A Thermocycling Centrifuge and Procedure for the Preparation of Retractable 
Seals in Reaction Containers 
A thermocycling centrifuge was devised (Exper. MK166). A table top 
centrifuge (IEC Model HN-SII, described previously), using a swinging 
bucket rotor (IEC #215), was modified for rapid heating and cooling by 
lining the inside walls with a 5/16 inch layer of aluminized "bubble wrap" 
insulation (Reflectix, Inc., Markleville, Ind.) and replacing the metal 
lid with 2 inch thick insulated lid of polystyrene foam. The foam lid had 
about 2 inch diameter holes cut into it for forcing air in and out of the 
centrifuge chamber. A thermometer was placed over the exit hole to monitor 
the inside chamber temperature. 
During the heating cycle, hot air was blown into one of the holes in the 
lid using an 1875 watt heat gun (Model RV406, Revlon), set on high heat 
and blowing. The temperature was monitored with the thermometer and 
regulated by turning the heater off and on. For cooling, the heater 
element was switched off and the blower left on to blow room temperature 
air. 
Two polypropylene "swinging" holders for centrifuging 0.2 ml PCR reaction 
tubes were prepared from disposable tip racks. The holders had 3/16 inch 
diameter holes that held the conical bottomed PCR tubes upright. The 
holders were mounted on opposite positions on the rotor using two loops of 
stainless steel wire as bales. The bales were threaded through the rack 
and over the rotor hooks normally used for the trunnions, so that the 
holders would swing out horizontally when being centrifuged. The 
horizontal position is preferred so that the centrifugal force applied to 
the tubes is nearly parallel with the sides of the container. The 
centrifugal force thereby forces the reagent to the bottom of the 
container with the lighter, melted wax on top. The centrifugal force tends 
to flatten the liquid wax over the aqueous reagent, leaving the exposed 
surface at about a 60-90 degree angle to the walls. 
For the procedure, a sufficient amount of wax was melted in a suitable 
container, such as a 2-6 inch diameter aluminum or glass pan. The desired 
mass of melted wax (i.e. 1.0 or more mg), was dispensed into the bottom of 
a suitable container for making wax seals, such as a 0.2-1.0 ml centrifuge 
tube, PCR tube or 96 well plastic plate. The aliquots of dispensed wax 
were allowed to cool and solidify. 
An aqueous loading solution was prepared containing the desired reagents to 
be sealed under the wax. Each of the above containers that received a mass 
of wax, was loaded with a suitable volume of the loading solution by 
dispensing an aliquot of the loading solution into the bottom of each 
container using a standard pipettor. 
The loaded containers were divided evenly and placed in opposing holders in 
the centrifuge apparatus and closed with the polystyrene insulated lid. 
The containers were centrifuged at about 500-2000 revolutions per minute 
(rpm), for 1-5 minutes, then the chamber was heated with hot air blown 
into the chamber while continuing to centrifuge. 
Based on the combined radius of the rotor and holders, the calculated 
relative centrifugal force (R.C.F.), was about 34 R.C.F.'s at 500 rpm and 
about 302 R.C.F.'s at 1500 rpm. 
The chamber temperature was maintained 10-50.degree. C. above the melting 
point of the wax, for a sufficient time (i.e. about 2-5 minutes), to allow 
the wax to melt and form a flattened layer over the aqueous loading 
solution. Then, while still centrifuging, the chamber was cooled to room 
temperature to solidify the wax, by blowing in room temperature air with 
the heater off. 
EXAMPLE III 
Comparison of the Instant Invention with the Prior Art Method 
(Exper. MK173) The purpose of this example is to show that the instant 
invention makes it possible to seal aqueous reagent under a minimal layer 
of wax that is not possible with the same amount of wax using methods in 
the prior art. Without centrifugation, liquid wax has a tendency to 
retract and collect more at the inside walls so that there is much less 
wax in the center than at the edges of the aqueous surface. In addition, 
as the liquid wax cools and solidifies, it tends to contract further. The 
end result is that during solidification in the prior art, the wax seems 
to pull away from the center and leave a hole in the center of the 
solidified wax layer. 
A total of 12 PCR tubes were prepared in duplicates of various ratios of 
wax to aqueous reagent. The wax used was paraffin, m.p. 58-60.degree. C. 
(Fluka). The aqueous reagent consisted of 4 ml of 50% glycerol in water 
and 0.1 ml of 10% bromocresol green in water. The wax was melted in an 
aluminum weighing dish at 95.degree. C., dispensed with a PCR pipettor 
into the bottoms of the tubes and allowed to cool to a solid. The aqueous 
reagent was dispensed using a standard pipettor. 
The Method of the Instant Invention 
Tubes numbered 1A, 2A, 3A, 4A, 5A and 6A were centrifuged at about 1500 rpm 
at RT for about 2 minutes. Then, while still centrifuging, the chamber was 
heated with hot air and maintained from 65-75.6.degree. C. for about 2 
minutes, then cooled down to rt in about 3 more minutes. Inspection of the 
tubes showed that all 6 had a flattened wax seal over the aqueous reagent. 
The Method of the Prior Art 
Tubes numbered 1B, 2B, 3B, 4B, 5B and 6B were placed upright in the 
previously described thermocycler and heated for 5 minutes at 80.degree. 
C. to melt the wax, then cooled to RT. Inspection of the tubes showed the 
wax in tubes 1-5B had solidified over the aqueous reagent in a ring that 
covered the outermost edges of the solution and some of the tube wall, but 
left an opening or hole in the center where aqueous reagent was exposed to 
the atmosphere. Tube 6B had little or no wax over the aqueous reagent 
since the wax failed to rise to the top when melted. Apparently, the 
affinity between the wax and the polypropylene tube was enough to keep 
this small mass of wax below the surface. 
Testing for a Wax Seal 
To each of the prepared tubes 1A-6A and 1B-5B was added an upper layer of 
0.1 ml of deionized water. In tubes numbered 1B, 2B, 3B, 4B and 5B, the 
bromocresol green dye immediately began mixing into the upper layer of 
water, showing that none of those tubes had a seal. 
In contrast, the dye in tubes 1A, 2A, 3A, 4A, 5A and 6A remained below the 
wax layer, with no visible mixing with the upper water layer. Tubes 1A-6A 
were inspected the next day, and again after 3 days and there was still no 
mixing, showing that they were completely sealed. 
Summary of Results 
The following table shows the results with various ratios of wax to aqueous 
reagent in duplicate tubes and compares the centrifugation method of the 
instant invention versus no centrifugation used in the prior art gravity 
method. 
__________________________________________________________________________ 
Tube # 1A 1B 
2A 2B 
3A 3B 
4A 4B 5A 5B 
6A 6B 
__________________________________________________________________________ 
Mg of Wax 
2 2 4 4 4 6 
Ml of Reagent .004 .006 .010 .020 .050 .050 
Centrifuged 
Yes 
No 
Yes No 
Yes 
No 
Yes 
No Yes 
No 
Yes 
No 
Seal Formed Yes No Yes No Yes No Yes No Yes No Yes No 
__________________________________________________________________________ 
EXAMPLE IV 
Comparison of Centrifigal Force Needed in Polypropylene vs. Polycarbonate 
Containers 
(Exper. MK212) The purpose was to determine how much relative centrifugal 
force (R.C.F.), is needed to provide a retractable wax seal in 
polypropylene vs. polycarbonate containers using the method of the instant 
invention. 
The polypropylene containers were 0.2 ml PCR tube strips described 
previously. The polycarbonate containers were sections of the 0.2 ml wells 
cut from a PCR "thermowell plate" (#6510 from Corning Costar). Paraffin 
wax (m.p. 58-60.degree. C.), was melted in an aluminum weighing dish at 
95.degree. C., dispensed with a PCR pipettor into the bottoms of the 
containers and allowed to cool to a solid. 
The aqueous reagent consisted of 50% glycerol, 245 mM MgCl.sub.2, and about 
1.25% blue food color in water. Into the bottom of each container, 2 .mu.l 
of the aqueous reagent was dispensed. 
In a series of experiments, the containers were divided into groups of 6 
polypropylene and 6 of polycarbonate containers. Each group was 
centrifuged at either 1750 rpm, 500 rpm or 250 rpm, while heated for about 
2-3 minutes to about 70-82.degree. C., then quickly cooled to about 
37.degree. C. before removing from the centrifuge. 
In the containers centrifuged at 1750 rpm, the aqueous reagent was sealed 
under a good coating of wax that had no visible meniscus or differences in 
wax thickness between the sides and the centers. However, there was a 
visible meniscus in the wax layer in all of the other groups, indicated by 
more wax against the inside walls than in the center. There may also have 
been some evaporation from the aqueous phase in the groups centrifuged at 
lower rpm's. 
In the containers centrifuged at 500 rpm, the aqueous reagent was sealed 
under a complete wax layer or coating even though it was thinner in the 
center. The containers centrifuged at 250 rpm had very poor seals (very 
thin), or no seals, with holes in the wax layer at the center of the 
meniscus. The presence of a hole in the wax was verified by gently probing 
the wax layers with a suitable length of 30 gauge Teflon tubing, to see if 
it could pass into the aqueous solution below the wax. The results are 
presented in the following table. 
______________________________________ 
Centrifuged 
Approximate 
6 Polypropylene 
6 Polycarbonate 
rpm R.C.F. Containers Containers 
______________________________________ 
1750 412 sealed, no holes 
sealed, no holes 
500 34 sealed, no holes sealed, no holes 
250 8.4 no seal, with holes very poor seal 
______________________________________ 
These results show that there is a minimum requirement of centrifugal force 
to obtain a retractable wax seal. For the ratio of wax to aqueous reagent 
tested, the minimum R.C.F. is between about 8.4 and 34. 
To compare results with the prior art method of not centrifuging, the 
identical groups of 6 polypropylene and 6 polycarbonate containers that 
had been sealed when centrifuged at 1750 rpm were again melted upright in 
a thermocycler at 70.degree. C. for 30 seconds without centrifugation, and 
cooled to RT. These groups were then designated as "0 rpm", and compared 
to the 500 rpm groups, prepared above, in a leakage test. 
The leakage test is designed to detect any holes in the wax layer not 
detectable by the naked eye or with probing. It consisted of adding 0.02 
ml of water to each container to make an over lay on the wax, and allowing 
them to soak at RT. Then, the containers were inspected at various soaking 
times to see if any of the blue colored reagent below the wax had leaked 
through the wax into the upper water layer. The results are shown in the 
table below. 
______________________________________ 
Centri- 
fuged Approx. Minutes 6 Polypropylene 6 Polycarbonate 
rpm R.C.F. Soaked 
Containers Containers 
______________________________________ 
500 34 10 minutes 0 leaked 0 leaked 
0 1 10 minutes 0 leaked 4 leaked 
500 34 40 minutes 0 leaked 0 leaked 
0 1 40 minutes 6 leaked 6 leaked 
500 34 16 hours 0 leaked 0 leaked 
0 1 16 hours 6 leaked 6 leaked 
______________________________________ 
In the 0 rpm groups, the wax in 4 polycarbonate containers leaked within 10 
minutes, and all of the 0 rpm containers showed leakage by 40 minutes. In 
contrast, none of the 500 rpm groups leaked at 10 or 40 minutes or even 
after 16 hour of soaking. These results demonstrate that the instant 
invention method is capable of producing a retractable wax seal in 
polypropylene or polycarbonate containers under conditions where a seal is 
not possible with the prior art method of not centrifuging. 
EXAMPLE V 
Comparison of Light Absorbance Through Retractable Wax Seals Prepared in 
Polypropylene vs. Polycarbonate Containers 
(Exper. MK212) The purpose was to provide an objective measurement of the 
comparative flatness of the meniscus in wax layers or seals that results 
from variations in relative centrifugal force (R.C.F.). Also, a comparison 
is made between polypropylene and polycarbonate containers using the 
method of the instant invention. 
In the preparation of wax seals, the prior art has solved the problem of 
mining the mass of wax needed by adding surfactants that cause the melted 
wax to spread out more and thereby flatten the wax meniscus. In the 
instant invention, it was observed that flattening the meniscus of a given 
mass of wax using centrifugation also increases the relative thickness of 
the wax seal at the center. The relative thickness of wax at the center 
can be measured as an increase in the absorbance of light passed through 
the center area. 
The polypropylene containers were 0.2 ml PCR tube strips and the 
polycarbonate containers were sections of the 0.2 ml wells cut from a PCR 
"thermowell plate" (#6510 from Corning Costar), as described and/or 
prepared in the previous example. Paraffin wax (m.p. 58-60.degree. C.), 
was melted in an aluminum weighing dish at 95.degree. C., dispensed with a 
PCR pipettor into the bottoms of the containers and allowed to cool to a 
solid. The aqueous reagent consisted of 50% glycerol, 245 mM MgCl.sub.2, 
and about 1.25% blue food color in water. Into the bottom of each 
container, 2 .mu.l of the aqueous reagent was dispensed. The containers 
were divided into groups of 6 polypropylene and 6 of polycarbonate 
containers. Each group was centrifuged at 500-1750 rpm's as listed in the 
table below, while heated for about 2-3 minutes to about 64-82.degree. C., 
then quickly cooled to about 37.degree. C. before removing from the 
centrifuge. 
The result was that all centrifuged groups from 500-1750 rpm formed a 
retractable wax seal over the aqueous reagent. As was observed previously, 
the 1750 rpm groups had no visible meniscus while those prepared at lower 
rpm (i.e. 500 rpm), did. 
For reading absorbance through the central area of the wax layers, the 
containers were positioned upright in polystyrene RemovaWell.RTM. strips 
(#11-010-6301, Dynatech Laboratories, Inc., Chantilly, Va.). The 
absorbance was measured from a light beam directed vertically through the 
middle of each tube. The spectrophotometer used was a Microwell Strip 
Reader, model EL301 (Bio-Tek Instruments Inc., Winooski, Vt.), with a 515 
nm filter. 
For a baseline, the 1750 rpm groups (from Example IV), were read for 
absorbance and then remelted upright in a thermocycler at 70.degree. C. 
for 30 seconds without centrifugation, and cooled to RT. This procedure 
produced holes in the wax of these containers, which were then read again, 
and the absorbance values used for the "0 rpm" groups. All absorbance 
readings were taken before subsequent probing or leakage tests. The 
readings of 6 tubes in each group were averaged and presented in the table 
below. 
______________________________________ 
Centrifuged 
Approx. Mean Absorbance 
Mean Absorbance 
rpm R.C.F. Polypropylene Polycarbonate 
______________________________________ 
0 1 0.921 0.727 
500 34 0.952 0.863 
750 76 0.985 0.878 
1000 134 1.020 0.953 
1500 302 1.090 1.010 
1750 412 1.100 0.986 
______________________________________ 
The results demonstrate an increase in absorbance through the center of the 
wax seal with increasing centrifugal force. The absorbance was highest at 
about 1000 rpm for polypropylene and about 1500 rpm for polycarbonate. 
These absorbance levels also correlate with the observation that the 
higher rpm groups had flatter wax layers. 
EXAMPLE VI 
Comparing The Mass of Wax Vs Grease Required For a Retractable Seal 
The following tables show the minimum mass of wax vs petrolatum grease 
required to form a retractable seal over a given volume of aqueous reagent 
in various plastic containers, comparing the invention vs the prior art. 
The wax used was paraffin (m.p. 58-60.degree. C.), from Fluka, and the 
petrolatum (m.p. 56-58.degree. C.), was white petrolatum, USP 
(Vaseline.RTM.), from Chesebrough-Ponds Co., Greenwich, Conn. The aqueous 
reagents used consisted of water that usually contained about 1% food 
color and, in some cases, 50% glycerol, and salts. The seals were 
determined as complete by the lack of any observed movement or diffusion 
of color through the seal between the reagent and water, after soaking 
several hours. 
The containers used were; polystyrene flat bottom Removawells.RTM. (0.4 
ml), from Dynatech Laboratories, Chantilly, Va.; polypropylene conical 
(V-bottom) strip tubes (0.2 ml), from Robbins Scientific Corp., Sunnyvale, 
Calif.; polypropylene conical microfuge tubes (0.5 ml), from Fisher 
Scientific, Pittsburgh, Pa.; and polycarbonate plate wells (V-bottom, 0.2 
ml), from Stratagene, La Jolla, Calif. Seals were prepared in the 
containers comparing the centrifugation method of the invention vs the 
gravity method of the prior art. For the centrifuge method, sealer was 
melted over the aqueous reagent, and centrifuged at about 340 g (R.C.F.) 
while it is cooled to a solid layer. For the prior art method, sealer was 
melted and solidified over the aqueous reagent at 1 g (no centrifuging). 
The data are summarized from several experiments and are mean values from 
groups of 2, 4 or 6 containers. 
TABLE VIa 
__________________________________________________________________________ 
Retractable Wax Seals 
Seal Seal 
Volume 
Seal Mass per 
Preparation Diameter Mass Covered Surface Area Exper. 
Container Method in mm. 
in mg in .mu.l in mg/mm.sup.2 
__________________________________________________________________________ 
# 
Polystyrene 
centrifuged 
6.6 24 150 0.70 mk216 
0.4 ml @ 340 g 
flat well 
Polypropylene centrifuged 4.1 2 50 0.15 
mk224 
0.2 ml PCR @ 340 g 
strip tube 
Polypropylene centrifuged 4.3 2 50 0.14 
mk218 
0.5 ml @ 340 g 
microtube 
Polycarbonate centrifuged 4.1 2 50 0.15 
mk224 
0.2 ml PCR @ 340 g 
plate tube 
__________________________________________________________________________ 
TABLE VIb 
__________________________________________________________________________ 
Non-Retractable Wax Seals 
Seal Seal 
Volume 
Seal Mass per 
Preparation Diameter Mass Covered Surface Area Exper. 
Container Method in mm. 
in mg in .mu.l in mg/mm.sup.2 
__________________________________________________________________________ 
# 
Polystyrene 
gravity 
6.6 48 150 1.40 mk216 
0.4 ml 
flat well 
Polypropylene gravity 2.8 9 10 1.46 mk218 
0.2 ml PCR 
strip tube 
Polypropylene gravity 4.3 22 50 1.52 mk224 
0.5 ml 
microtube 
Polycarbonate gravity 4.1 20 50 1.52 mk224 
0.2 ml PCR 
plate tube 
__________________________________________________________________________ 
The preceding tables (VIa and VIb), show that several times more wax is 
required for nonretractable seals compared to retractable seals in the 
same containers over comparable aqueous volumes. 
TABLE VIc 
__________________________________________________________________________ 
Retractable Grease Seals 
Seal Seal 
Volume 
Seal Mass per 
Preparation Diameter Mass Covered Surface Area Exper. 
Container Method in mm. in 
mg in .mu.l in mg/mm.sup.2 # 
__________________________________________________________________________ 
Polypropylene 
centrifuged 
4.1 0.5 50 0.04 mk224 
0.2 ml PCR @ 340 g 
strip tube 
Polypropylene centrifuged 4.3 1 50 0.07 mk224 
0.5 ml @ 340 g 
microtube 
Polycarbonate centrifuged 4.1 1 50 0.08 mk224 
0.2 ml PCR @ 340 g 
plate tube 
__________________________________________________________________________ 
TABLE VId 
__________________________________________________________________________ 
Non-Retractable Grease Seals 
Seal Seal 
Volume 
Seal Mass per 
Preparation Diameter Mass Covered Surface Area Exper. 
Container Method in mm. 
in mg in .mu.l in mg/mm.sup.2 
__________________________________________________________________________ 
# 
Polypropylene 
gravity 
4.1 16 50 1.21. mk224 
0.2 ml PCR 
strip tube 
Polypropylene gravity 4.3 20 50 1.38 
mk224 
0.5 ml 
microtube 
Polycarbonate gravity 4.1 14 50 1.06 
mk224 
0.2 ml PCR 
plate tube 
__________________________________________________________________________ 
The preceding tables (VIc and VId), show that several times more grease is 
required for nonretractable seals compared to retractable seals in the 
same containers over comparable aqueous volumes. Also, in tables VIa and 
VIc, comparing retractable wax seals to retractable grease seals, less 
mass of grease is required in the same containers. 
EXAMPLE VII 
Retractable Wax Seals in PCR Reaction Tubes Preloaded with Taq DNA 
Polymerase and/or dNTP's 
(Exper. MK211) Paraffin wax, melting point 58-60.degree. C., (Fluka, 
Milwaukee, Wis.), was melted at about 100-105.degree. C. in an aluminum 
weighing boat. The containers were 0.2 ml PCR tubes connected in a strip 
of 12 tubes per strip (Robbins Scientific Inc., Sunnyvale, Calif.). The 
tubes were divided into three groups of 36 tubes, labeled A, B and C. Into 
each tube was dispensed about 2.1 mg of the melted paraffin wax using a 
PCR pipettor. 
An aqueous loading solution containing PCR reagent was prepared for each 
group. Each solution contained either Taq DNA polymerase (Taq), at about 
200 units/ml, and/or dATP, dCTP, dGTP and dTTP (collectively dNTP's), at 
about 1.5 mM of each. The Taq reagent (Promega Corp., Madison Wis.), 
and/or dNTP's reagent (Amresco Inc., Solon, Ohio), for each group was 
prepared in a dilution buffer composed of 100 mM KCl, 20 mM Tris-HCl (pH 
9.0 at 25.degree. C.), 0.2% Triton.RTM. X-100, 2.0 mM dithiothreitol and 
50% glycerol in water. 
The concentrations of the reagents in each loading solution were adjusted 
so that when 2 .mu.l of loaded solution is released into a final volume of 
about 15 .mu.l, the concentrations will be suitable for a polymerase chain 
reaction. A total of 2 microliters of concentrated aqueous reagent in 
loading solution was dispensed (loaded) into each group of tubes according 
to the table that follows. 
______________________________________ 
Sealed Final Conc. 
Tube Reagent After Release 
Group in 2 .mu.l in 15 .mu.l 
______________________________________ 
A Taq/dNTP's 0.4 units/0.2 mM 
B Taq 0.4 units 
C dNTP's 0.2 mM 
______________________________________ 
The loading solution can include any suitable carrier substances or 
additives such as a carbohydrate, for instance glucose, sucrose, 
trehalose, cyclodextrins or Ficoll.RTM. (Pharmacia Inc.), or any suitable 
gel (i.e. agarose), protein (i.e. albumin, gelatin) or polymer (i.e. 
polyethylene glycol). 
The strips of tubes were divided evenly and placed in opposing holders in 
the centrifuge and the centrifuge was closed. While centrifuging the tubes 
at about 1500 rpm (about 300 R.C.F.), they were held at RT for about 5 
minutes, then hot air was blown into the chamber while still spinning. 
Turning the heat off and on regulated the temperature so that the chamber 
temperature stayed above the m.p. of the wax (at about 66-77.degree. C.), 
for about 3 minutes. Then, while still centrifuging, the chamber was 
cooled to about 37.degree. C. to solidify the wax. Cooling was done by 
blowing in room temperature air with the heater off. The tubes were 
removed and stored for 4 days at 4.degree. C. 
The wax seals that were formed over the PCR reagents in the tubes were then 
tested using PCR for release of the reagent when melted. Previous tests 
have shown that because of the insufficient mass of wax, the centrifuged 
wax seal is a non-barrier system, which retracts and leaves the aqueous 
reagent exposed to the atmosphere as soon as it is melted. Since the PCR 
machine used did not have a heated lid to prevent condensation inside the 
tubes, a 28 mg paraffin wax bead (Lot #1119, Lumitekk, SLC Utah), was 
added to each tube to provide a vapor barrier during the PCR 
The preloaded PCR reagents were tested in the PCR using commercially 
available "PCR buffer" (Promega), containing DNA template and PCR primers. 
The PCR buffer was composed of 50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 
25.degree. C.), 0.1% Triton.RTM. X-100, in water. The DNA template to be 
amplified was pBR322 plasmid DNA from E. coli (Sigma Chemical Corp). The 
16 base oligonucleotide primers were pBR322 EcoR I (clockwise) and pBR322 
BamH I (counter-clockwise), (both Promega), which complement the ends of a 
393 base pair segment on the pBR322 DNA. 
A PCR "master mix" was prepared in PCR buffer without any dNTP's, or Taq. 
The master mix contained 2.5 mM MgCl.sub.2, about 0.002 micrograms of 
pBR322 DNA, and about 0.04 nanomoles of each primer in PCR buffer, for 
each reaction volume of 0.015 ml. After dispensing 0.015 ml of master mix 
into each of three sealed tubes from each group, either buffer only, 0.4 
units of Taq or dNTP's (about 0.2 mM final), were added in buffer as 
needed. Tubes of group A received buffer only, group B received dNTP's, 
and group C received Taq. 
For the PCR, the samples were thermocycled for 35 cycles of; 60 seconds @ 
94.degree. C., 30 seconds @ 55.degree. C., and 60 seconds @ 72.degree. C. 
The PCR products were analyzed by a standard method of agarose gel 
electrophoresis (AGE). The triplicate samples A, B and C were diluted 
about 2/3 in gel loading solution (Sigma Cat# G2526). About 8 .mu.l of 
each diluted sample was loaded into corresponding wells in a 3% agarose 
gel in TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA, pH 8), containing 
0.5 micrograms/ml of ethidium bromide stain. The AGE was run horizontally 
for about 1 hour at 100 volts and 50 milliamps, then subilluminated with 
U.V. light and photographed with Polaroid.RTM. #665 instant film. 
With U.V. illumination, PCR product was visible as fluorescent bands in the 
gel for all three groups. These results indicate that the paraffin sealed 
PCR reagents were released from under the seal during the PCR and were 
active enough after 4 days storage at 4.degree. C. to produce amplified 
pBR322 DNA. 
EXAMPLE VIII 
Properties of Various Non-Wax Polymer Mixes for Sealing Reaction Containers 
(Exper. KK188, 209, 211) The following is a list of polymers tested for 
their physical properties and for use as sealing materials in reaction 
containers. 
Atactic polypropylene (APP), stiff, sticky, translucent grease-like paste, 
softening point 20.degree. C., density 0.8544, Cat #783, Scientific 
Polymer Products (SPP), Ontario, N.Y. 
Low density polyethylene (LDPE), stiff translucent grease-like paste, mp 
92-117.degree. C., SPP Cat #535. 
Medium density polyethylene (MDPE), white opaque powder, mp 109-111.degree. 
C., Cat #33,211-9, Aldrich Chemical Co. 
Stearyl alcohol (1-octadecanol), white opaque powdery flakes, mp 59.degree. 
C., Sigma Chemical Co. 
Various polymer mixes were prepared, combining certain polymers with long 
chained alcohol or with heavy mineral oil (U.S.P. grade), or combinations 
of polymer, alcohol and oil. The mixtures were prepared by melting them 
over a hotplate in 20 ml glass scintillation vials and stirring with a 
stainless steel wire or a glass rod until homogeneous. The ratios are by 
weight. The melting points were estimated by placing a few milligrams of 
each material on the inside of a polypropylene reaction tube in a 
thermocycling machine. As the machine was heating, the temperature range 
was noted when the mixture began flowing and appeared liquefied. 
Various mixtures of the non-wax polymers were tested for their ability to 
form non-retractable (spontaneously with gravity) seals and/or retractable 
seals over an aqueous solution. The tests for seals with gravity were 
performed by adding about 25 .mu.l of molten material to 50 or 100 .mu.l 
of blue colored water in a 0.2 ml polypropylene PCR tube. After melting at 
about 95.degree. C. and cooling to solidify, the presence of a complete 
seal was determined by the lack of a visible hole in the layer of mix 
material. All of the mixtures described are capable of forming suitable 
seals over an aqueous reagent. Most preferred are mixtures B through P. 
Mixture A is a 1:1 ratio of APP+Oil. This formed a clear liquid with a 
slight yellow cast when melted and a very thick, foggy syrupy gel when 
cooled to room temperature (RT). 
Mixture B is a 1:1 ratio of LDPE+Oil. This formed a clear colorless liquid 
when melted and a moderately stiff foggy solid mass when at RT, mp about 
70-72.degree. C. Two out of three tubes formed good barriers. 
Mixture BB is a 2:1 ratio of LDPE+Oil. This formed a stiff foggy solid mass 
when at RT, mp about 80.degree. C. All three tubes formed a barrier with 
25 mg over 50 .mu.l of water. 
Mixture C is a 1:1 ratio of MDPE+Oil. At RT this formed a soft, opaque waxy 
solid mass, mp greater than 95.degree. C. 
Mixture D is a 1:1 ratio of APP+Oil. At RT this formed a stiff, pale yellow 
opaque solid mass. 
Mixture E is a 2:1 ratio of APP+Oil. At RT this formed a flowable, pale 
yellow solid mass. 
Mixture F is a 1:2 ratio of LDPE+Oil. At RT this formed a soft, foggy white 
solid mass, mp about 70-75.degree. C. All three tubes formed barriers with 
about 25 .mu.l material over 100 .mu.l water. 
Mixture G is a 1:6:1 ratio of LDPE+1-Octadecanol+Oil. At RT this formed a 
hard, waxy white opaque solid, mp greater than 60.degree. C. 
Mixture H is a 1:9:2 ratio of LDPE+1-Octadecanol+Oil. At RT this formed a 
hard, waxy white opaque solid, mp greater than 60.degree. C. 
Mixture I is a 1:3 ratio of LDPE+1-Octadecanol. At RT this formed a hard, 
waxy white opaque solid, mp greater than 60.degree. C. 
Mixture J is a 1:1 ratio of 1-Octadecanol+Oil. At RT this formed a 
semi-hard, oily white opaque solid, mp about 58.degree. C. 
Mixture K is a 1:2:1 ratio of APP+1-Octadecanol+Oil. At RT this formed a 
soft, oily and flaky material, mp 60-65.degree. C. 
Mixture L is a 1:3:2 ratio of LDPE+1-Octadecanol+Oil. At RT this formed a 
slightly brittle, slightly oily white opaque solid, mp about 60.degree. C. 
Mixture M is a 1:4:1 ratio of LDPE+1-Octadecanol+Oil. At RT this formed a 
brittle, white opaque solid, mp about 63-70.degree. C. 
Mixture N is a 1:1:1 ratio of LDPE+1-Octadecanol+Oil. At RT this formed a 
stiff, sticky, white opaque, grease-like solid mass, mp about 
70-75.degree. C. 
Mixture O is a 1:2:2 ratio of LDPE+1-Octadecanol+Oil. At RT this formed a 
slightly brittle, slightly oily white opaque solid. 
Mixture P is a 1:3 ratio of LDPE+Oil. At RT this formed a soft, foggy, 
grease-like solid mass, mp about 60-70.degree. C. This material works well 
in forming spontaneous seals and in forming retractable seals when 
centrifuged at 60-70.degree. C. over an aqueous reagent and cooled to RT 
during centrifugation. 
The retractable sealing characteristics were compared between mixture P, 
paraffin wax (mp 58-60.degree. C.) and petroleum jelly (Vaseline.RTM.). 
About 2.5 mg of each material was melted and added to 0.2 ml PCR tubes (24 
per group). Then 5 .mu.l of blue colored 1.times. PCR buffer was added to 
each tube. Retractable seals were formed over the aqueous reagent by 
centrifuging at about 1500 rpm (about 300 R.C.F.), and heating the tubes 
to about 76.degree. C. for about 1 minute. While still centrifuging, the 
tubes were cooled to RT to solidify the layers. 
All of the materials formed thin, flat, retractable seals over the aqueous 
reagent. The tubes were weighed and then subjected to 5 cycles of freezing 
at -85.degree. C. and thawing at RT (25.degree. C.). Inspection of the 
seals showed that the seals using the non-wax mixture P were unaffected. 
However, the wax seals appeared slightly tilted and the Vaseline.RTM. 
seals were slightly more tilted, indicating that some dislodging of those 
seals occurred during the freezing and thawing. Also, when the tubes were 
stored at RT for 1 week and reweighed, there was less change in weight 
(indicating less evaporation), with the non-wax seals. The weight data are 
shown below. 
______________________________________ 
Type Of Seal 
Gm Before Freezing 
Gm After Freezing 
Gm Lost 
______________________________________ 
Non-Wax 3.0094 2.9944 0.0150 
Wax 3.0008 2.9650 0.0358 
Vaseline .RTM. 3.0082 
2.9512 0.0570 
______________________________________ 
These results indicate that the non-wax seal is more resistant to 
freeze-thaw conditions. Using the non-wax mixture of 1:3 ratio of 
LDPE+Oil, it was found that the addition of solvent red #27 caused an 
increase in viscosity. This property has some advantages such as 
increasing the stability of the non-wax seal. 
EXAMPLE IX 
A Retractable Non-wax Polymer Seal in PCR Reaction Tubes Containing Taq DNA 
Polymerase and dNTP's 
(Exper. MK242, 243) A non-wax sealer material (called PE sealer), was 
prepared from a mixture of low density polyethylene (LDPE), and heavy 
mineral oil at a ratio of about 1:4. In a glass beaker, 5.6 gm of LDPE was 
combined with 22.4 gm of heavy mineral oil and 1.4 gm of heavy mineral oil 
containing 0.1% solvent red #27 (oil red O). The mixture was heated to 
about 90-100.degree. C. and mixed until homogeneous. At RT this formed a 
red colored, soft, gel-like solid mass. 
Into each of 80 PCR reaction tubes (0.2 ml polypropylene, Robbins 
Scientific, Sunnyvale Calif.), was dispensed about 3.5 .mu.l (4.4 mg) of 
PE sealer that had been heated to about 103.degree. C. in an aluminum pan. 
The dispenser used was an 8 tipped, multichannel pipettor (Labsystems Oy, 
Helsinki, Finland). 
A PCR reagent mix was prepared at about 4.5.times. concentration 
(4.5.times. PCR mix), so that a 5.5 .mu.l aliquot contained the desired 
amount of Taq DNA polymerase (Taq), dATP, dCTP, dGTP and dTTP (dNTP's), 
MgCl.sub.2 and 10.times. PCR buffer (500 mM KCl, 1% Triton.RTM. X-100, 100 
mM Tris-HCl, pH 9.0 @ 25.degree. C.), to perform PCR when DNA template and 
primers were added and the final volume was increased to 25 .mu.l of 
water. The 4.5.times. PCR mix consisted of aqueous solutions of 0.13 .mu.l 
of 5 units/.mu.l Taq (Promega Corp., Madison Wis.), 0.2 .mu.l of pooled 
dNTP's @ 25 mM each (Promega), 1.5 .mu.l of 25 mM MgCl.sub.2, 2.5 .mu.l 
10.times. PCR buffer and 1.2 .mu.l of water per 5.5 .mu.l. 
A 5.5 .mu.l aliquot of 4.5.times. PCR mix was dispensed into each reaction 
tube containing about 4.4 mg of PE sealer. Retractable non-wax seals were 
then prepared by melting and centrifugation. The sealing procedure was to 
centrifuge the tubes at about 1500 rpm (about 300 R.C.F.), while heating 
them with a hot air blower to about 77.degree. C. for about 0.5-1 minute 
to melt the sealing material, then turn off the heat to quickly cool them 
below the mp (about RT), to solidify the sealer while still centrifuging. 
The entire cycle took about 4.5 minutes. 
The resulting product (called a ReadyWell.TM.), is a PCR reaction tube 
containing about 5.5 .mu.l of PCR reagent sealed under a layer that is 
thinner than is possible using prior art methods. The containers were 
stored at -20.degree. C. After 3 weeks, the seals were inspected and found 
complete as indicated by no observable loss of liquid under the layers. 
They were then tested using PCR for release of the reagent when melted. 
The preloaded and PE sealed PCR reagents were tested in the PCR using water 
containing DNA template and PCR primers. The DNA template to be amplified 
was pBR322 plasmid DNA from E. coli (Promega). The 16 base oligonucleotide 
primers were pBR322 EcoR I (clockwise) and pBR322 BamH I 
(counter-clockwise), (both Promega), which complement the ends of a 393 
base pair segment on the pBR322 DNA template. 
A PCR "sample mix" was prepared in water without any dNTP's, or Taq. The 
sample mix contained about 0.004 micrograms of pBR322 DNA, and about 0.04 
nanomoles of each primer per 0.020 ml of water. Into each of 6 PE sealed 
tubes was dispensed 0.020 ml of sample mix so that the final reaction 
volume will be 0.025 ml when the PE seal is melted. 
Previous tests have shown that due to insufficient mass of material, the PE 
seal is a non-barrier system, which retracts and leaves the aqueous 
reagent exposed to the atmosphere as soon as it is melted. Since the PCR 
machine used did not have a heated lid to prevent condensation inside the 
tubes, a 13 mg parafin wax bead was added to each tube to provide a vapor 
barrier during the PCR. 
For the PCR, the samples were thermocycled for 35 cycles of; 60 seconds @ 
94.degree. C., 30 seconds @ 55.degree. C., and 60 seconds @ 72.degree. C. 
The PCR products were analyzed by agarose gel electrophoresis (AGE). 
Agarose (NuSieve.TM. 3:1, FMC Bioproducts, Rockland Me.), was prepared as 
a 3% gel in TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA, pH 8), 
containing 0.5 micrograms/ml of ethidium bromide stain. About 60 ml of 
melted gel solution was cast into a 10 mm deep plastic tray of about 
13.times.8 mm. 
The tray had two plastic combs suspended about 1 mm from the bottom to form 
two parallel rows of sample wells in the gel. With one of the longer edges 
oriented as the "top" of the gel, one comb was about 5 mm inside of, or 
"below", the top edge and the other comb was positioned about 3.5 mm below 
the first. The combs had 28 teeth separated by 2 mm wide spaces, 10 mm 
long. The teeth themselves were 1.5 mm thick and 2.5 mm wide so that the 
distance between the center of each tooth was 4.5 mm, which is one half 
the 9 mm distance between well centers of a standard 96 well microplate. 
Each comb is positioned so that the teeth project down into the melted gel 
in the tray and are removed when the gel is cooled and solidified. This 
leaves a row of sample wells that can be conveniently loaded using 
standard multichannel pipettors. 
The PCR samples were diluted about 2/3 in gel loading solution (17% 
glycerol, 1.3% Ficoll@, 0.07% SDS, 0.013% bromophenol blue in TBE buffer). 
About 8 .mu.l of each diluted sample was loaded into corresponding wells 
in the gel. The AGE was run horizontally for about 1 hour at 100 volts and 
50 milliamps, then subilluminated with U.V. light and photographed with 
Polaroid.RTM. #66 5 instant film. 
With U.V. illumination, PCR product was visible as fluorescent bands in the 
gel for all 6 tubes. These results indicate that the PE sealed PCR 
reagents were released from under the seal during the PCR and were active 
enough after 3 weeks storage at -20.degree. C. to produce amplified pBR322 
DNA. 
Using the same preparation method used for polypropylene tubes, 5.5 .mu.l 
aliquots of MgCl.sub.2 solution (0.75 ml 10.times. buffer, 2.25 ml 25 mM 
MgCl.sub.2, 4.5 ml water and 0.01 ml blue food color #1), were each sealed 
under about 3.2 mg of PE sealer in a polycarbonate container. The 
polycarbonate container was a PCR reaction plate with 96 individual wells 
of 0.2 ml volume (Stratagene Inc., La Jolla Calif.). These seals were 
found to be complete and free of any leakage as indicated by no observed 
leaching of the blue color into 30 .mu.l of water overlaid on top of the 
seals for 24 hours. Also, the seals showed no observable damage after 4 
cycles of freezing in dry ice (-70.degree. C.), and thawing at RT. 
While the invention has been described with references to certain specific 
embodiments, it is understood that changes may be made by one skilled in 
the art and it would not thereby depart from the spirit and scope of the 
invention which is limited only by the claims appended hereto.