Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-11-13
2003-12-23
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S103000, C435S100000, C435S183000
Reexamination Certificate
active
06667165
ABSTRACT:
FIELD OF THE INVENTION
This application relates generally to methods and compositions for improving the sensitivity and specificity of polynucleotide synthesis and more particularly to methods and compositions using non-nucleic acid polyanions for reversibly inhibiting thermostable polymerase, in a temperature dependent manner, during polynucleotide synthesis.
BACKGROUND OF THE INVENTION
Polynucleotide synthesis techniques, and polymerase chain reaction (PCR) in particular, include some of the most important biotechnological innovations in the fields of molecular and cell biology and biomedical research. Polynucleotide synthesis involves the synthesis of a complementary polynucleotide strand from a template polynucleotide strand, so for example, the information in the template polynucleotide strand directly guides the formation of a complementary polynucleotide strand from its own sequence. In its more complicated state, polynucleotide synthesis, for example PCR, can be used to amplify specific segments of RNA or DNA in a rapid and highly reproducible manner. Saiki, et al. (1988)
Science
239:487-491. Applications for PCR have continued to expand from its inception, for example, PCR is now being used to clone from genomic DNA or cDNA, to perform in vitro mutagenesis and engineering of DNA, to genetically fingerprint forensic samples, to detect pathogenic agents like hepatitis C in blood samples, and to perform direct nucleotide sequencing on genomic DNA.
PCR is a rapid procedure for the in vitro enzymatic amplification of target polynucleotides in an exponential manner. Three nucleic acid segments are required to practice a PCR reaction: a double-stranded polynucleotide containing the target nucleic acid sequence for amplification, and a pair of single-stranded oligonucleotide primers that flank that target sequence. An enzyme (thermostable polymerase—functional at elevated temperatures) and the appropriate deoxyribonucleoside triphosphates (dNTPs), as well as a buffer make up the reaction mixture. In use, the primers are mixed with a buffered solution containing the template polynucleotide, the thermostable polymerase, and the dNTPs for all four deoxynucleotides. The solution is heated to a temperature sufficient to denature the double-stranded template polynucleotide, and abruptly cooled to a temperature sufficient to allow the primers to anneal to the sequences flanking the target sequence on the template polynucleotide. The thermostable polymerase recognizes and binds to the primer-template complexes and the temperature is cycled upward to a temperature at which the thermostable polymerase has optimum activity for polynucleotide synthesis. The thermostable polymerase forms a complementary strand to the template polynucleotide and the process of temperature cycling is repeated. Numerous cycles, providing up to millions/billions of the target sequence, can be performed without altering the reaction mixture.
Polynucleotide synthesis at the elevated temperatures used in PCR tends to prevent the non-specific annealing of primers to non-target polynucleotides and thus improves the specificity and sensitivity of the PCR reaction. In order to operate at these elevated temperatures, thermostable polymerases have been isolated from a number of thermophilic bacterium that live at elevated temperatures, for example, in hot springs, next to underwater volcanic vents, etc. Because these enzymes normally operate at high temperatures, their use eliminates the necessity of repetitively adding temperature sensitive polymerases to the PCR reaction after each temperature cycle.
However, although the performance of PCR at elevated temperatures has reduced the level of non-specific annealing of primers to polynucleotide sequences in the reaction mixture, especially at the elevated temperatures required for optimum thermostable polymerase activity, non-specific primer interactions with polynucleotide sequences, and some level of corresponding primer elongation by the thermostable polymerase, does occurs at lower temperatures. The non-specific interactions and activity of the thermophilic polymerase tends to occur even at temperatures as low as 25° C., i.e., during the set-up of the PCR reaction mixture at room temperature, especially when a large number of reactions are handled simultaneously. The activity of Taq DNA polymerase, the most frequently used thermostable DNA polymerase, at 30° C. is still 12-15% of its full activity at 70° C. This problem is especially prevalent in PCR applications having a small number of target polynucleotide sequences in a milieu containing an excess of non-target, i.e., non-specific, DNA and/or RNA. Several approaches have been advanced within the art to minimize these inherent shortcomings in PCR, the most prevalent of which is termed “hot start PCR.”
The overall approach to hot start PCR reactions is to physically, chemically or biochemically block the polymerization reaction until the reaction reaches a temperature above the optimal annealing temperature of the primers. In this manner the thermostable polymerase is unable to elongate primer-template polynucleotides at temperatures where non-specific primer-template DNA interactions can exist. With regard to physical hot start PCR, the thermostable polymerase, or one of the other critical reaction components, e.g., dNTPS or magnesium ions, is withheld from the reaction until the reaction reaches temperatures in the range of 85° C. to 95° C. This temperature is sufficiently high enough to not permit even partial hybridization of the primers to the template polynucleotide, i.e., substantially no non-specific primer annealing to polynucleotides. A number of physical blocks can be used to partition the reaction in a heat dependent manner, including, a wax barrier or wax beads with embedded reaction components, which melts at around 55° C. to 65° C. However, a shortcoming to using these wax barriers/beads is that the melted material remains in the reaction for the duration of the PCR, forming a potential inhibitor for some PCR applications as well as being incompatible with some potential downstream applications of the amplified product. In some cases the barrier can be physically removed from the reaction to accommodate later uses, but the removal increases the risk of sample-to-sample contamination and requires time and energy to accomplish. A second physical hot start PCR technique utilizes a compartmentalized tube in a temperature regulated centrifuge. The components of the PCR reaction are compartmentalized within the tube from a critical component of the PCR reaction, where the components are all brought together by rupturing the compartments of the tube at a certain g force that corresponds to the specific annealing temperatures of the primer-template polynucleotide. This is accomplished by a dedicated centrifuge that regulates g force with rotor temperature. However, this technique requires expensive equipment—compartmentalized tubes for each PCR reaction and a specialized centrifuge—each factor limiting the number of reactions that can be run at one time and effecting the cost of each reaction.
Another way to implement hot start PCR is to use a thermostable polymerase that has been reversibly inactivated by a chemical modification, such as AMPLITAQ GOLD™ DNA polymerase (Birch et al., 1998, U.S. Pat. No. 5,773,258; Ivanov et al., 2001, U.S. Pat. No. 6,183,998). These techniques are generally referred to as chemical hot start PCR. In the most common type of chemical hot start PCR, the thermostable polymerase, mainly Taq DNA polymerase, has been chemically cross-linked to inactivate the enzyme. The cross-linked thermostable polymerase is reactivated by heating the polymerase prior to the reaction for a predetermined amount of time at 95° C. and at a specific pH. Moretti et al. (1998)
Biotechniques
25:716-725. The optimal pH for the destruction of the cross-links at 95° C. is adjusted by using reaction buffers, which have a pH of 8.0 at 25° C. However, this buffer pH is suboptimal for the activity of the th
Dorsey & Whitney LLP
Eppendorf AG
Siew Jeffrey
Tung Joyce
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