Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-01-24
2004-10-12
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024320, C536S025320, C536S026600
Reexamination Certificate
active
06803201
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of polynucleotide sequence determination, in particular, relates to determine the identity of a single nucleotide in a target polynucleotide sequence, e.g., single nucleotide polymorphism (“SNP”) analysis.
BACKGROUND
Techniques for the analysis of polynucleotide sequences have found widespread use in basic research, diagnostics, and forensics. Single nucleotide detection is applied in processes including the detection of single nucleotide polymorphisms, identification of single base changes, speciation, determination of viral load, genotyping, medical marker diagnostics, and the like.
Single nucleotide detection can be accomplished by a number of methods. Most methods rely on the use of the polymerase chain reaction (PCR) to amplify the amount of target DNA. One of the first developed PCR-dependent methods is restriction site polymorphism detection, where the PCR product is cleaved by a restriction enzyme and then analyzed by electrophoresis. Another early method is allele-specific PCR in which one of the PCR primers is designed such that it will discriminate at its 3′ end between DNA targets having a sequence that perfectly matches the primer from those targets not perfectly matching the primer.
TaqMan was the first homogenous assay capable of detecting single nucleotide polymorphisms (U.S. Pat. No. 5,723,591). In this assay, two PCR primers flank a central probe oligonucleotide. The probe oligonucleotide comprises two fluorescent moieties. During the polymerization step of the PCR process, the polymerase cleaves the probe oligonucleotide. The cleavage causes the two fluorescent moieties to become physically separated, which causes a change in the wavelength of the fluorescent emission. As more PCR product is created, the intensity of the novel wavelength increases. While TaqMan accomplishes the goal of single nucleotide detection in a homogenous assay, it has two disadvantages. The first is that each nucleotide to be detected requires a different oligonucleotide probe comprising two different fluorescent moieties. Such probes must be custom-synthesized and are thus expensive. The second disadvantage is that TaqMan probes are not very discriminating for single nucleotide differences. Thus there can be significant false-positive signals.
Molecular Beacons are an alternative to TAQMAN (U.S. Pat. Nos. 6,277,607; 6,150,097; 6,037,130). Molecular Beacons undergo a conformational change upon binding to a perfectly matched template. The conformational change of the Beacon increases the physical distance between a fluorophore moiety and a quencher moiety on the Beacon. This increase in physical distance causes the effect of the quencher to be diminished, thus increasing the signal derived from the fluorophore. Molecular Beacons are more discriminating of single nucleotide differences, as compared with TaqMan probes. However they still require the synthesis of a custom oligonucleotide (the Beacon) having two different fluorescent moieties for each target sequence being examined. Thus the technology is expensive.
There are several other fluorescent and enzymatic PCR technologies, such as SCORPIONS™, SUNRISE™ primers, and DNAzymes. Not all of these are suitable for single nucleotide detection, and most of them require the synthesis of a custom, fluorescently labeled oligonucleotide for each target nucleotide.
Hybridization to a “DNA chip” is another way of detecting single nucleotide differences (U.S. Pat. No. 5,856,104). Typically oligonucleotides that are complementary to the suspected target DNAs are synthesized on a solid surface (“chip” or “oligonucleotide array”). The target DNA is PCR amplified, labeled, and then hybridized to the oligonucleotide array. Ideally, perfectly matched PCR fragments will hybridize to the array, but mismatched fragments will not. While the technology, in theory, offers the opportunity to look at many different loci simultaneously, in practice the need to amplify the target DNA using PCR limits the degree to which the assay can be multiplexed. In addition the start-up costs for designing an oligonucleotide microarray can be very expensive. Lastly, the frequency of false-positive and false-negative spots is very high, and necessitates the use of many surface-bound oligonucleotides for each target DNA sequence.
There currently are two non-PCR based technologies capable of detecting single nucleotide changes in complex genomes. The Invader-Squared method (U.S. Pat. No. 6,001,567) utilizes a cascade of DNA cleavage reactions. While sensitive, it requires the synthesis of several long, target-specific oligonucleotides in addition to several detection oligonucleotides. The rolling circle detection method (Lizardi et al., Nature Genetics 19: 225-232) utilizes a target nucleotide-specific ligation reaction to create a circular template that is then replicated with a polymerase in rolling-circle fashion. One of the advantages is that the reaction does not require thermal cycling. One drawback is that ligation reactions are not highly specific for single nucleotide detection.
Single base extension (“SBE”; also called minisequencing) is a technology that uses dideoxy chain terminators in combination with a DNA polymerase to determine the identity of a single nucleotide in a target DNA sample that has been PCR amplified (Syvanen et al., 1990, Genetics 8:684-642; U.S. Pat. No. 5,888,819; Euoropean patent application EP 0648280 A1, each of which is incorporated herein by reference). The technology uses a DNA primer that is hybridized to a target polynucleotide in the presence of dideoxy chain terminators, but typically in the absence of deoxynucleotide triphosphates. A DNA polymerase will add a single dideoxy chain terminator to the 3′ end of a primer that is reasonably hybridized to the DNA target. The polymerase incorporates the appropriate dideoxy terminator determined by the complementary sequence in the target polynucleotide. Thus, the identity of the dideoxy terminator that is incorporated reflects the identity of the nucleotide within the target polynucleotide that is immediately adjacent to the target nucleotide that is hybridized with the 3′ nucleotide of the primer.
There are a number of patents and patent applications for SBE. In U.S. Pat. No. 6,013,431, the dideoxy chain terminators would be labeled with reporter moieties, such as fluorescent molecules, and the incorporation of a label into a primer is measured by gel electrophoresis. The method described in U.S. Pat. Nos. 6,015,675; 5,582,989; 5,578,458 relates to placing the primer on a solid surface, such as a chip. The chip is exposed to a solution containing the target polynucleotide plus fluorescently labeled dideoxy chain terminators and polymerase. When a single labeled base is added to the bound primer, the probe begins to fluoresce.
Fluorescence polarization has been used to perform SBE. With this approach the chain terminators are fluorescently labeled as with other methods. However rather than separating the labeled primers by gel electrophoresis or physical separation, the incorporated chain terminators are generated by shining polarized light on the sample, and then detecting the polarization of the emitted fluorescent light. Fluorescent light emitted by unincorporated terminators will not be polarized because these small molecules are rapidly moving in solution. However labeled terminators that have been incorporated onto the end of a primer will be moving more slowly and tend to emit polarized light. Thus the degree to which the emitted light is polarized reflects the degree to which there has been incorporation of a dideoxy chain terminator onto the end of a primer. The color of the polarized emitted light reflects the particular dideoxy terminator (A, C, G, or T) that was incorporated onto the 3′ end of the primer. The advantage to the fluorescent polarization method is that it is homogeneous (all done in a single test tube). However the input target DNA is typically a PCR fragment, and the PCR reaction ne
Arezi Bahram
Hogrefe Holly
Sorge Joseph A.
Horlick Kenneth R.
Palmer & Dodge LLP
Stratagene
Wilder Cynthia B.
Williams Kathleen M.
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