Abstract: Apparatus and method for performing a nucleic acid amplification reaction and preferably a polymerase chain reaction (PCR) in a reaction mixture in at least one capillary tube. Several different embodiments are disclosed. One embodiment cycles a sample through a capillary tube loop passing through two thermostatted fluid baths. Another embodiment has the capillary tube routed alternatingly between two heat exchangers to that the sample makes only one pass through the tube. Other embodiments maintain the heat exchangers stationary and translate the samples between them. Still further embodiments maintain the samples stationary and either automatically translate or rotate the heat exchangers past the samples contained within the capillary tubes to perform the thermal cycles necessary for the amplification reaction.

Abstract: A cryogenic fluid conveying device has removable connection of a metal tube and a receptor for the tube. The receptor is formed of a housing member with an aperture therein for conveying the fluid. A tubular member of resilient PTFE has a bore size so as to be slidingly receptive of the rigid tube. A ferrule is disposed on the tubular member which is positioned in the housing member. An annular nut is threaded onto an end of the housing to constrict the ferrule and thereby constrict an annular section of the tubular member. This constriction provides a sealingly tight fit for the rigid tube such that the metal tube can be inserted into and removed from the tubular member.

Abstract: A solid support having surface-attached carboxylic acid groups is reacted with an isoxazolium salt to form an activated support having enol ester and sulfonate groups. After washing to remove residual isoxazolium salt and base, the activated support is dried. A polypeptide is covalently or non-covalently bound to the support. The resultant immobilized polypeptide can be conveniently sequenced by N- and C-terminal sequencing methods. The support can be a polyvinylidene difluoride membrane or a glass fiber membrane having surface attached carboxylic acid groups, and the enol and sulfonate groups are provided by reacting the support with 2-ethyl-5'-phenylisoxazolium sulfonate. The dry support can be stored for at least about 3 months to achieve a peptide immobilization yield that is substantially the same as the yield obtained in the absence of drying.

Abstract: An improved assay system for homogeneous assay detection and/or measuring target nucleic acid sequence replicated in a PCR amplification procedure is provided by employing a nucleic acid polymerase having 5' to 3' exonuclease activity and devoid of 3' to 5' exonuclease activity, oligonucleotide analytical probe blocked against chain extension at its 3' terminus and labeled at its 5' terminus with an energy transfer donor fluorophore, and oligonucleotide detection probe labeled at its 3' terminus with an energy transfer acceptor fluorophore, said probes being complements of each other but differing in nucleotide length so that their Tm's are at least 10.degree. C.

Abstract: In a gas chromatographic system that includes a column and an injector with a chamber, a carrier gas normally flows into the chamber at a constant rate, and a constant primary pressure is maintained at the column inlet. A sampling receptacle selectively communicates a sample vessel with a source of transfer gas or the chamber. The vessel is pressurized with the transfer gas to a starting pressure greater than the primary pressure. Analyte sample in transfer gas is then tranferred from the vessel to the chamber by the vessel pressure while the vessel pressure remains greater than the primary pressure. Carrier gas flow is discontinued during transfer, so that analyte sample transfers through the chamber into the column by the primary pressure linearly with time. Standby and venting states are provided.

Abstract: A two-piece plastic holder for loosely holding microliter sample tubes of a preselected design, each tube having a cylindrically shaped upper section open at its top end, a closed, tapered lower section extending downwardly therefrom, and a circumferential shoulder extending outwardly from the upper section at a position on the upper section spaced from the open end thereof. The holder includes a one-piece tray member and a one-piece retainer. The one-piece tray member including a flat, horizontal plate section containing holes in a rectangular array, a first vertical tray sidewall section around the plate extending upwardly to a height greater than the height of a tube resting in one of the holes, and a second vertical tray sidewall section around the plate extending downwardly approximately to the bottom of the upper section of a tube resting in one of the holes. The one-piece retainer is releasably engageable inside the tray over any sample tubes resting in the tray.

Abstract: Avian nucleotide sequences for use as sex specific markers are provided. Probes or primers derived from the sequences find use in defining the sex of a bird, by producing hybridization patterns or amplification products that are sex-specific. For a given avian species the probes may hybridize to both Z and W chromosomes so as to differentiate between the two chromosomes on the basis of restriction fragment length polymorphisms. Alternatively, the probes may hybridize exclusively to one of the two sex chromosomes in some species. The sequences taught are also useful for the isolation of other nucleotide sequences that are sex specific markers.

Abstract: Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In the method, a mixture of sequence-specific probes are reacted with the target polynucleotide under hybridization conditions, and the hybridized probes are treated to selectively modify those probes which are bound to the target polynucleotide in a base-specific manner. The resulting labeled probes include a polymer chain which imparts to each different-sequence probe, a distinctive ratio of charge/translational frictional drag, and a detectable label. The labeled probes are fractionated by electrophoresis in a non-sieving matrix, and the presence of one or more selected sequences in the target polynucleotide are detected according to the observed electrophoretic migration rates of the labeled probes in a non-sieving medium.

Abstract: An oligonucleotide probe is disclosed, the probe including an oligonucleotide, a fluorescer molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide. The probe is rendered impervious to digestion by the 5'.fwdarw.3' exonuclease activity of a polymerase and the 3'.fwdarw.5' extension of by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the above method further includes the addition of a strand displacer to facilitate amplification. Additional similar combined PCR hybridization methods are disclosed, such methods not requiring probes having their 5' ends protected, wherein (i) the polymerase lacks 5'.fwdarw.3' exonuclease activity, (ii) a 5'.

Abstract: A complete in situ PCR system for amplification of nucleic acids contained in a prepared cell or tissue sample. The containment system for the PCR sample comprises a glass microscope slide, a specimen sample containing the target nucleic acid sequence mounted on the slide, a flexible plastic cover over the sample, and a retaining assembly fastened to the slide and to the cover to retain and seal a reaction mixture against the sample during thermal cycling. The retaining assembly includes a rigid ring on a rim portion of the cover, a cross beam having spaced parallel rails joined by opposite flat ends, and a pair of clips which are pressed over the ends and opposite sides of the slide to fasten the cross beam and cover to the slide.

Abstract: A complete in situ PCR system for amplification of nucleic acids contained in a prepared cell or tissue sample. The containment system for the PCR sample comprises a glass microscope slide, a specimen sample containing the target nucleic acid sequence mounted on the slide, a flexible plastic cover over the sample, and a retaining assembly fastened to the slide and to the cover to retain and seal a reaction mixture against the sample during thermal cycling.The retaining assembly includes a rigid ring on a rim portion of the cover, a cross beam having spaced parallel rails joined by opposite flat ends, and a pair of clips which are pressed over the ends and opposite sides of the slide to fasten the cross beam and cover to the slide.

Abstract: A Control circuit for a differential scanning calorimeter which includes a sample heater, a reference heater, a system for generating a sample heater DC voltage and a system for generating a reference heater DC voltage. A balanced operational amplifier arrangement is provided which includes a circuit for controlling the differential heater voltage and a circuit for independently controlling the average heater voltage, so that the average heater power and the differential heater power are independently controlled.

Abstract: An apparatus for controlling level of cryogenic liquid in a vessel includes a transfer duct to a pressure supply tank. Pressure pulses are applied to the tank at one rate when the level is below a primary sensor in the vessel, and at a lower rate when the level is above the sensor, so as to compensate for intrinsic declination of the level. At startup a constant pressure is applied to the tank until the level reaches a lower sensor, and a relief valve over the tank is opened when the level overshoots to an upper sensor. Turbulence in the vessel is reduced by a pair of coaxial baffles at the liquid inlet to the vessel, and by a housing with tubular extensions enveloping the sensors.

Abstract: A method of forming a thiohydantoin from an N-protected amino acid is described. The method employs a phosphate compound selected from the group consisting of(R.sub.1 O)(R.sub.2 O)P(.dbd.O)X and(R.sub.1 O)(R.sub.2 O)P(.dbd.O)--O--P(.dbd.O)(OR.sub.3)(OR.sub.4)to form acylphosphate moieties from the carboxyl groups of internal aspartic acid and glutamic acid residues and an acylphosphate moiety at a C-terminal carboxyl. The later acylphosphate, unlike the internal acylphosphates, spontaneously cyclizes to an oxazolone, which is less reactive with nucleophilic reagents. R.sub.1 and R.sub.2 are each alkyl, aryl, or alkaryl groups which are the same or different and which may be covalently linked to each other; R.sub.3 and R.sub.4 are each alkyl, aryl, or alkaryl groups which are the same or different and which may be covalently linked to each other; and X is a leaving group, such as chlorine or bromine, which is substantially unreactive towards thiohydantoins.

Abstract: A thermally insulated calorimeter includes a vessel with a chamber containing liquid nitrogen. The vessel is formed of four walls arranged proximately in parallel, each pair of adjacent walls delimiting a space. An inner wall forms the chamber, two partition walls are between the inner wall and an outer wall which is exposed to atmosphere. Each space contains a gas at atmospheric pressure. Each spacing is selected to minimize heat transfer from the atmosphere to the inner chamber, by establishing a maximum spacing for which the air is stagnant. A cover for the vessel has a set of walls with spacings similar to those of the vessel. Sealing for such spacing is provided for a removable main cover and a removable top cover for the calorimeter sample block.

Abstract: There is disclosed herein a machine for performing nucleic acid amplification under computer control. The machine utilizes any one of a number of heating and cooling systems under control of a host computer which directs the heating and cooling systems to heat and cool a reaction-chamber-containing heat exchanger at appropriate times in the process. The reaction chambers are pre-loaded with the nucleic acid(s) to be amplified, a thermostable enzyme to catalyze polymerization, specific oligonucleotide primers, and four different nucleotide triphosphates. Also disclosed is the process for the amplification chain reaction implemented by the machine, which utilizes a thermostable enzyme.

Abstract: Long wavelength, narrow emission bandwidth fluorecein dyes are provided for detecting spacially overlapping target substances. The dyes comprise 4,7-dichlorofluoresceins, and particularly 2',4',5',7'-tetrachloro-4,7-dichloro-5- (and 6-) carboxyfluoresceins. Methods and kits for using the dyes in DNA analysis are provided.

Abstract: A method for synthesizing a polynucleotide having a nitrogen atom at the 3'-end is disclosed. The methods include the following steps. A solid support reagent is provided having the structure shown below: ##STR1## where T is an acid-cleavable hydroxyl protecting group, e.g., 4,4'-dimethoxytritil; Q is a linker connecting the nitrogen and oxygen, e.g., n-hexyl; R.sub.1 is a nitrogen substituent, e.g., hydrogen; R.sub.2 through R.sub.4 are separately hydrogen or lower alkyl; Y is an atom which is electronegative with respect to carbon, e.g., oxygen or sulfur; X.sub.1 is an atom which is electronegative with respect to carbon, e.g., sulfone; Z is a bond or spacer arm, e.g., ethylsuccinate; and W is a derivatized solid synthesis support capable of linking to Z, e.g., an amino-dirivitized controlled pore glass. The solid support is treated with acid to remove the acid-cleavable hydroxyl protecting group.

Abstract: Method and composition for detecting one or more selected polynucleotide regions in a target polynucleotide. In the method, a mixture of sequence-specific probes are reacted with the target polynucleotide under hybridization conditions, and the hybridized probes are treated to selectively modify those probes which are bound to the target polynucleotide in a base-specific manner. The resulting labeled probes include a polymer chain which imparts to each different-sequence probe, a distinctive ratio of charge/translational frictional drag, and a detectable label. The labeled probes are fractionated by electrophoresis in a non-sieving matrix, and the presence of one or more selected sequences in the target polynucleotide are detected according to the observed electrophoretic migration rates of the labeled probes in a non-sieving medium.

Abstract: An instrument for performing highly accurate PCR employing a sample block in microtiter tray format. The sample block has local balance and local symmetry. A three zone film heater controlled by a computer and ramp cooling solenoid valves also controlled by the computer for gating coolant flow through the block controls the block temperature. Constant bias cooling is used for small changes. Sample temperature is calculated instead of measured. A platen deforms plastic caps to apply a minimum acceptable threshold force for seating the tubes and thermally isolates them. A cover isolates the block. The control software includes diagnostics. An install program tests and characterizes the instrument. A new user interface is used. Disposable, multipiece plastic microtiter trays to give individual freedom to sample tubes are taught.