Instrument for monitoring polymerase chain reaction of DNA

An optical instrument for monitoring polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent primary dye that fluoresces proportionately in presence of DNA, the instrument comprising:

• a light source for emitting a source bean having at least a primary excitation frequency that causes the primary dye to fluoresce at an emission frequency;
• first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency;
• primary focusing means disposed to focus the excitation beam into each suspension such that the primary dye emits an emission beam having the emission frequency, the emission beam having an intensity representative of concentration of DNA in each suspension, the focusing means being receptive of and passing the emission beam;
• second means disposed to be receptive of the emission beam from the focusing means so as to further pass the emission beam at the emission frequency;
• emission focusing means for focusing the emission beam;
• a detector disposed to be receptive of the emission beam from the second means and the emission focusing means such that the emission beam is focused onto the detector, the detector generating primary data signals representative of the emission bean and thereby a corresponding concentration of DNA in each vial;
• and
• processing means receptive of the primary data signals for computing primary signal data and the corresponding concentration of DNA;
• wherein the block is configured to hold a plurality of vials, the focusing means comprises a corresponding plurality of vial lenses each being disposed for positioning over a vial such that the emission beam comprises individual beams each associated with a vial, and a field lens disposed cooperatively with the vial lenses to effect focusing of the excitation bean into each suspension, and to pass the individual beams to the second means, and the detector comprises an array of photoreceptors receptive of the individual beams to generate corresponding data signals such that the processing means computes concentration of DNA in each vial.

An optical instrument for monitoring polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent primary that fluoresces proportionately in the presence of DNA, the instrument comprising:

• a light source for emitting a source beam having at least a primary excitation frequency that causes the primary dye to fluoresce at an emission frequency;
• first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency;
• primary focusing means disposed to focus the excitation beam into each suspension such that the primary dye emits an emission beam having the emission frequency, the emission beam having an intensity representative of concentration of DNA in each suspension the focusing means being receptive of and passing the emission beam;
• second means disposed to be receptive of the emission beam from the focusing means so as to further pass the emission beam at the emission frequency;
• emission focusing means for focusing the emission beam;
• a detector disposed to be receptive of the emission beam from the second means and the emission focusing means such that the emission beam is focused onto the detector, the detector generating primary data signals representative of the emission beam and thereby a corresponding concentration of DNA in each vial;
• processing means receptive of the primary data signals for computing primary signal data and the corresponding concentration of DNA;
• and
• a housing containing the light source, the detector, the focusing means and a filter module, wherein the beam splitter, the excitation filter and the emission filter are affixed in the module and are associated with a selected primary dye for the suspension, and the module is removable from the housing for replacement with another module associated with another selected primary dye;
• wherein the first means further comprises an excitation filter, the second means further comprises an emission filter, and the first means and the second means together comprise a beam splitter, the excitation filter being disposed between the light source and the bean splitter, the emission filter being disposed between the beam splitter and the detector, the excitation filter passing light having the excitation frequency and substantially blocking light having the emission frequency, and the emission filter passing light having the emission frequency and substantially blocking light having the excitation frequency, the excitation filter and the beam splitter being cooperatively receptive of the source beam to effect the excitation beam, and the emission filter and the beam splitter being cooperatively receptive of the emission beam to pass the emission beam having the emission frequency to the detector.

An optical instrument for monitoring polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent primary dye that fluoresces proportionately in presence of DNA, the instrument comprising:

• a light source for emitting a source beam having at least a primary excitation frequency that causes the primary dye to fluoresce at an emission frequency;
• first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency;
• primary focusing means disposed to focus the excitation beam into each suspension such that the primary dye emits an emission beam having the emission frequency, the emission beam having an intensity representative of concentration of DNA in each suspension, the focusing means being receptive of and passing the emission beam;
• second means disposed to be receptive of the emission beam from the focusing means so as to further pass the emission beam at the emission frequency;
• emission focusing means for focusing the emission beam;
• a detector disposed to be receptive of the emission beam from the second means and the emission focusing means such that the emission beam is focused onto the detectors, the detector generating primary data signals representative of the emission beam and thereby a corresponding concentration of DNA in each vial;
• processing means receptive of the primary data signals for computing primary signal data and the corresponding concentration of DNA;
• and
• a fluorescent reference emitter that emits reference light in response to the excitation beam, the reference emitter being disposed for the emission focusing means to focus at least a portion of the reference light as a reference beam onto the detector, the detector being further receptive of the reference beam to generate a reference signal, and the processing means comprises means respective of the reference signal for computing reference data, and means for normalizing the primary data with the reference data for a chosen in the reaction replication of DNA, thereby correcting for instrument drift during the monitoring;
• wherein the reference emitter comprises a plurality of reference emitters each emitting a reference bean of different intensity in response to the excitation beam, the reference emitters being disposed for the emission focusing means to focus each reference beam onto the detector, the reference member further comprises a plastic fluorescent strip and a neutral density filter mounted on the fluorescent strip such that the reference beam and a portion of the excitation beam are attenuated by the neutral density filter, the neutral density filter having a series of densities to effect the plurality of reference emitters each emitting a reference beam, the detector being further receptive of each reference beam to generate a set of reference signals for each reference emitter and the processing means comprises means receptive of the reference signals for computing corresponding sets of reference data, means for selecting from the sets the selected reference data that has the highest signal data less than a predetermined maximum, the selected reference data being used for normalizing the primary data.

A system for replication of DNA and monitoring thereof, comprising a reaction apparatus for polymerase chain reaction replication of DNA, and an optical instrument for monitoring presence of DNA during such replication, wherein the apparatus comprises a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent dye that fluoresces proportionately in presence of DNA, and further comprises means for thermal cycling the block and thereby the suspension so as to effect the polymerase chain reaction;

• wherein the instrument comprises:
• a light source for emitting a source beam having at least an excitation frequency that causes the dye to fluoresce at an emission frequency;
• first means disposed to be receptive of the source beam to effect an excitation beam having the excitation frequency and to pass the excitation beam to a focusing means, the focusing means being for focusing the exitation beam into each suspension such that the dye emits an emission beam having the emission frequency, and for passing the emission beam to a second means, the second means being for further passing the emission beam to a detector, the detector being disposed to be receptive of the emission beam from the second means so as to generate data signals representative of the emission beam and thereby concentration of DNA;
• and
• processing means receptive of the data signals for computing and displaying the concentration of DNA;
• wherein the block is configured to hold a plurality of vials the focusing means comprises a corresponding plurality of vial lenses each being disposed for positioning over a vial such that the emission beam comprises individual beams each associated with a vial, and the detector comprises an array of photoreceptors receptive of the individual beams and capable of generating corresponding data signals such that the processing means is capable of computing a respective concentration of DNA in each vial.

A filter module for an optical instrument that monitors polymerase chain reaction replication of DNA in a reaction apparatus that includes a thermal cycler block for holding at least one vial containing a suspension of ingredients for the reaction, the ingredients including a fluorescent dye that fluoresces proportionately in presence of DNA, the instrument including a housing, a light source disposed in the housing for emitting a source beam having at least an excitation frequency that causes the dye to fluoresce at an emission frequency, focusing means disposed in the housing for focusing an excitation beam having the excitation frequency into each suspension such that the dye emits an emission beam having the emission frequency, a detector disposed in the housing to be receptive of the emission beam so as to generate data signals representative of the emission beam and thereby concentration of DNA, and processing means receptive of the data signals for computing and displaying the concentration of DNA, wherein the module comprises:

• a support frame, the instrument being receptive of the support frame into the instrument;
• a beam splitter affixed in the support frame so that, with the module inserted, the beam splitter is receptive of the source beam so as to effect the excitation beam, and receptive of the emission beam to pass the emission beam to the detector;
• an excitation filter that passes light having the excitation frequency and substantially blocks light having the emission frequency, the exitation filter being affixed in the support frame so that, with the module inserted, the excitation filter is disposed between the light source and the beam splitter;
• and
• an emission filter that passes light having the emission frequency and substantially blocks light having the excitation frequency, the emission filter being affixed in the support frame so that, with the module inserted, the emission filter is disposed between the detector and the beam splitter;
• the beam splitter, the excitation filter and the emission filter, and thereby the module, being associated with a selected dye for the suspension, and the module being removable from the housing for replacement with another module associated with another selected dye.