Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-30
2002-10-29
Benzion, Gary (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C436S094000, C536S025320
Reexamination Certificate
active
06472156
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and devices for analyzing nucleic acid samples for sequence variations at multiple loci. More particularly, the invention relates to the use of differential fluorescent emission and differential hybridization melting temperature of fluorescently labeled nucleotide probes to identify nucleic acid samples, including PC and LCR products, which have multiple potential sequences at multiple loci. The invention also relates to devices which allow simultaneous analysis of three or more fluorescent labels having different emission spectra.
BACKGROUND OF THE INVENTION
The continued discovery of novel genes provides a resource of genetic material for studying the association between genotype and disease. As databases for polymorphic markers and disease causing mutations continue to grow, there is an increasing need for procedures that can screen nucleic acid sequences for the presence of known polymorphisms and mutations. Optimally, the procedure should be capable of analyzing multiple DNA sites simultaneously (including nucleic acid loci that are physically separated by great distances) for the presence of mutations or polymorphisms.
Current methods for determining the genetic constitution of individuals (genotyping) include oligonucleotide ligation, allele-specific oligonucleotide hybridization, and PCR-restriction fragment length analysis. All these methods require time consuming multiple manual steps. One alternative method of genotyping uses the melting temperature of fluorescent hybridization probes that hybridize to a PCR-amplified targeted region of genome
ucleic acid sequence to identify mutations and polymorphisms.
The polymerase chain reaction (PCR) is a technique of synthesizing large quantities of a preselected DNA segment. The technique is fundamental to molecular biology and is the first practical molecular technique for the clinical laboratory. PCR is achieved by separating the DNA into its two complementary strands, binding a primer to each single strand at the end of the given DNA segment where synthesis will start, and adding a DNA polymerase to synthesize the complementary strand on each single strand having a primer bound thereto. The process is repeated until a sufficient number of copies of the selected DNA segment have been synthesized. During a typical PCR reaction, double stranded DNA is separated into its single strands by raising the temperature of the DNA containing sample to a denaturing temperature where the two DNA strands separate (i.e., the “melting temperature of the DNA”) and then the sample is cooled to a lower temperature that allows the specific primers to attach (anneal), and replication to occur (extend). Currently preferred methods utilize a thermostable polymerase in the polymerase chain reaction. A preferred thermostable DNA polymerase for use in the PCR reaction is the Taq DNA Polymerase and derivatives thereof, including the Stoffel fragment of Taq DNA polymerase and KlenTaql polymerase (a 5′-exonuclease 1 deficient variant of Taq polymerase (see U.S. Pat. No. 5,436,149).
Other nucleic acid amplification procedures are also widely practiced. For example, the self-sustained sequence replication (3SR) reaction utilizes three enzymes. The 3SR method is described in Guatelli et al., PNAS (USA) 87:1874-1878 (1990) and Mueller et al., Histochem. Cell Biol. 108(4-5):431-437 (1997). A similar method is described in U.S. Pat. No. 5,399,491. Strand displacement amplification (SDA) is another method of isothermal nucleic acid amplification. SDA relies on primer-directed nicking activity of a restriction enzyme and strand replacement activity of a polymerase which is exonuclease-deficient. SDA is described in Walker et al., PNAS (USA) 89:392-396 (1992); Walker et al., Nucleic Acids Res. 20(7):1691-1696 (1992); Nadeau et al., Anal. Biochem. 276(2):177-187 (1999) and in U.S. Pat. Nos. 5,270,184, 5,422,252, 5,455,166, and 5,470,723. Yet another method, rolling-circle amplification (RCA) utilizes DNA polymerase to replicate circularized oligonucleotides. RCA is described in Lizardi et al., Nat. Genet. 19(3):225-232 (1998) and U.S. Pat. No. 5,854,033. Each of the above-cited references is incorporated herein in its entirety.
Thermal cycling may be carried out using standard techniques known to those skilled in the art, including the use of rapid cycling PCR. Rapid cycling techniques are made possible by the use of high surface area-to-volume sample containers having relatively high thermal conductivity. The use of high surface area-to-volume sample containers allows for a rapid temperature response and temperature homogeneity throughout the biological sample. Improved temperature homogeneity also increases the precision of any analytical technique used to monitor PCR during amplification.
In a method compatible with the present invention, amplification of a nucleic acid sequence is conducted by thermal cycling the nucleic acid sequence in the presence of a thermostable DNA polymerase. The method comprises the steps of placing a biological sample comprising the nucleic acid sequence in a PCR vessel, raising the temperature of the biological sample from a first temperature to a second temperature, wherein the second temperature is at least 15° C. higher than the first temperature, holding the biological sample at the second temperature for a predetermined amount of time, lowering the temperature of the biological sample from the second temperature to at least the first temperature and holding the biological sample at a temperature at least as low as the first temperature for a pre-determined length of time. The temperature of the biological sample is then raised back to the second temperature, and thermal cycling of the biological sample is repeated a predetermined number of times. In one embodiment, the method of amplifying a DNA sequence comprises a two temperature cycle wherein the samples are cycled through a denaturation temperature and an annealing temperature for a predetermined number of repetitions. However the PCR reaction can also be conducted using a three temperature cycle wherein the samples are cycled through a denaturation temperature, an annealing temperature and an elongation temperature for a predetermined number of repetitions.
In the state of the art for PCR, each temperature cycle of the PCR reaction is completed in approximately 60 seconds or less. Rapid cycling times can be achieved using the device and techniques described in U.S. Pat. No. 5,455,175, the disclosure of which is expressly incorporated herein.
PCR amplification of one or more targeted regions of a DNA sample has been conducted in the presence of fluorescently labeled hybridization probes, wherein the probes are synthesized to hybridize to a specific locus present in a target amplified region of the DNA. Many different probes are available for monitoring PCR each cycle. Dyes like ethidium bromide or SYBR Green I, which preferentially bind to double-stranded DNA, can be used in any amplification and a re inexpensive. Although not sequence specific, product specificity can be increased by analysis of melting curves (Ririe et al.,
Anal. Biochem
. 245:154-160 (1997)), or by acquiring fluorescence at a high temperature where nonspecific products have melted (Morrison et al.,
BioTechniques
24(6):954-958, 960, 962 (1998)). However, multiplexing by color is not possible.
Multiplexing by color is possible with dual-labeled oligonucleotides, including hairpin primers (Sunrise™ primers), hairpin probes (Molecular Beacons™), and exonuclease probes (TaqMan™ probes). Hairpin primers include one fluorophore and one quencher (Nazarenko et al.,
Nucl. Acids Res
. 25:2516-2521 (1997)). Hairpin probes hybridize internal to the primers and are sequence specific (Tyagi et al.,
Nature Biotechnology
14:303-308 (1996)). Exonuclease probes are cleaved during polymerase extension by 5′-exonuclease activity (Livak et al.,
PCR Meth. Appl
. 4:357-362 (1995)). All these dual-labeled probes require careful design and are
Herrmann Mark G.
Wittwer Carl T.
Benzion Gary
Dorsey & Whitney LLP
The University of Utah
Trecartin Richard F.
Tung J.
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