Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-04-24
2003-09-09
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091200, C536S026600
Reexamination Certificate
active
06617136
ABSTRACT:
BACKGROUND
Many different chemical, biochemical, and other reactions include thermal cycling. Examples of thermal processes in the area of genetic amplification include, but are not limited to, Polymerase Chain Reaction (PCR), Sanger sequencing, etc. The reactions may be enhanced or inhibited based on the temperatures of the materials involved. Although it may be possible to process samples individually and obtain accurate sample-to-sample results, individual processing can be time-consuming and expensive.
One approach to reducing the time and cost of thermally processing multiple samples is to use a device including multiple chambers in which different portions of one sample or different samples can be processed simultaneously. When multiple reactions are performed in different chambers, however, one significant problem can be accurate control of chamber-to-chamber temperature uniformity. The need for accurate temperature control may manifest itself as the need to maintain a desired temperature in each of the chambers, or it may involve a change in temperature, e.g., raising or lowering the temperature in each of the chambers to a desired setpoint. In reactions involving a change in temperature, the speed or rate at which the temperature changes in each of the chambers may also pose a problem. For example, slow temperature transitions may be problematic if unwanted side reactions occur at intermediate temperatures. Alternatively, temperature transitions that are too rapid may cause other problems. As a result, another problem that may be encountered is comparable chamber-to-chamber temperature transition rate.
Another problem that may be encountered in those reactions in which thermal cycling is required is overall speed of the entire process. For example, multiple transitions between upper and lower temperatures may be required. Alternatively, a variety of transitions (upward and/or downward) between three or more desired temperatures may be required. In some reactions, e.g., polymerase chain reaction (PCR), thermal cycling must be repeated up to thirty or more times. Typical thermal cycling devices and methods that attempt to address the problems of chamber-to-chamber temperature uniformity and comparable chamber-to-chamber temperature transition rates, however, typically suffer from a lack of overall speed—resulting in extended processing times that ultimately raise the cost of the procedures.
One or more of the above problems may be implicated in a variety of chemical, biochemical and other processes. Examples of some reactions that may require accurate chamber-to-chamber temperature control, comparable temperature transition rates, and/or rapid transitions between temperatures include, e.g., the manipulation of nucleic acid samples to assist in the deciphering of the genetic code. See, e.g., J. Sambrook and D. W. Russell,
Molecular Cloning, A Laboratory Manual
3
rd
edition,
Cold Spring Harbor Laboratory (2001). Nucleic acid manipulation techniques include amplification methods such as polymerase chain reaction (PCR); target polynucleotide amplification methods, such as self-sustained sequence replication (3SR); methods based on amplification of probe DNA, such as ligase chain reaction (LCR) and QB replicase amplification (QBR); transcription-based methods, such as ligation activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA); and various other amplification methods, such as repair chain reaction (RCR) and cycling probe reaction (CPR).
One common example of a reaction in which all of the problems discussed above may be implicated is PCR amplification. Traditional thermal cycling equipment for conducting PCR uses polymeric microcuvettes that are individually inserted into bores in a metal block. The sample temperatures are then cycled between low and high temperatures, e.g., 55° C. and 95° C. for PCR processes. When using the traditional equipment according to the traditional methods, the high thermal mass of the thermal cycling equipment (which typically includes the metal block and a heated cover block) and the relatively low thermal conductivity of the polymeric materials used for the microcuvettes result in processes that can require two, three, or more hours to complete for a typical PCR amplification.
Another problem experienced in the preparation of finished samples (e.g., isolated or purified samples of, e.g., nucleic acid materials such as DNA, RNA, etc.) of human, animal, plant, or bacterial origin from raw sample materials (e.g., blood, tissue, etc.) is the number of thermal processing steps and other methods that must be performed to obtain the desired end product (e.g., purified nucleic acid materials). In some cases, a number of different thermal processes must be performed, in addition to filtering and other process steps, to obtain the desired finished samples.
One example is in the preparation of a finished sample (e.g., purified nucleic acid materials) from a starting sample (e.g., a raw sample such as blood, bacterial lysate, etc.). To obtain a purified sample of the desired materials in high concentrations, the starting sample must be prepared for, e.g., PCR, after which the PCR process is performed to obtain a desired PCR product. The PCR product is then subject to further manipulation such as sequencing, ligation, electrophoretic analysis, etc.
One method of improving conventional thermal cycling processes involves the use of electromagnetic radiation and energy absorbing pigments and dyes to absorb the radiation and convert it to thermal energy. The use of electromagnetic radiation absorbers such as pigments and dyes can interfere with reactions that involve the use of an enzyme. The enzyme can be deactivated, thereby preventing the formation of the desired products, e.g., PCR amplification products. Thus, there is a need for methods that allow for the use of electromagnetic radiation and absorbers such as dyes without adverse affects on the formation of the desired reaction products.
SUMMARY OF THE INVENTION
The present invention provides various compositions and methods that involve the use of an enzyme and a dye, such as a near-infrared (near-IR or NIR) dye. Such compositions and methods are preferably used for processing sample mixtures that include biological materials. Preferred methods involve the use of thermal cycling of a sample material that includes a biological material and an enzyme through the application of electromagnetic energy. A dye is used to convert the electromagnetic energy into thermal energy and a surfactant is used to inhibit (i.e., reduce, prevent, and/or reverse) interaction between the enzyme and the dye. As used herein, inhibiting interaction between the enzyme and the dye involves reducing the interaction compared to the same system when the surfactant is not present. Preferably, inhibiting interaction between the enzyme and the dye involves preventing the interaction from occurring and/or substantially completely reversing such interaction.
The present invention provides a composition that includes a near-IR dye and greater than about 0.5 wt-% of a surfactant selected from the group of a nonionic surfactant, a zwitterionic surfactant, and a mixture thereof, wherein the composition is stable in a thermal cycling process that includes cycling (preferably, at least about 10 cycles, and more preferably at least about 40 cycles) between about 50° C. and about 95° C. Preferably, the composition also includes an enzyme, which is a polymerase or a ligase.
The present invention also provides a composition that includes a near-IR dye, at least about 1 wt-% of a surfactant selected from the group of a nonionic surfactant, a zwitterionic surfactant, and a mixture thereof, a polymerase enzyme, and a triphosphate (e.g., a dNTP), wherein the composition is stable in a thermal cycling process that includes cycling (preferably, at least about 10 cycles, more preferably, at least about 40 cycles) between about 50° C. and about 95° C. Preferably, the near-IR dye is a cyanine dye or a diimminium dye.
A
Bedingham William
Dirksen Michael D.
Larson Christopher J.
Menon Vinod P.
Parthasarathy Ranjani V.
3M Innovative Properties Company
Busse Paul W.
Gram Christopher D.
Riley Jezia
Sprague Robert W.
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