Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-04
2001-04-24
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06221603
ABSTRACT:
FIELD OF INVENTION
The present invention relates to analysis of nucleic acids and specifically to the detection of nucleic acid variants.
BACKGROUND OF INVENTION
There is a significant need for the rapid, low-cost analysis of nucleic acid sequences. The extensive size and variability of nucleic acid samples have presented challenges for the analysis of genetic variation. The human genome contains over three billion base pairs, which contain the sequences for tens of thousands of genes. Variation at individual nucleotides is common. Single nucleotide polymorphisms, a set of single nucleotide variants at a genomic loci, are distributed throughout a genome. In the human genome, such single nucleotide variation occurs relatively frequently, about once in every 200-1000 bases, resulting in millions of single nucleotide polymorphisms in the human genome. To determine the identity of all single nucleotide polymorphism variants within a population would require a very large number of reactions for a very large number of individuals. If a single nucleotide polymorphism exists at a locus with a gene, the variant may result in a phenotypic difference. In addition to the analysis of genomic DNA, information on gene expression or alternate splice forms of mRNA may be gathered from the analysis of mRNA or cDNA. Analysis of DNA often requires screening DNA libraries, the analysis of genomic segments stored in an array of plasmid, cosmid or viral vectors. Bacterial plasmids and viral genomes are also sources of nucleic acid sequences that often require analysis.
The rapid advances in sequencing have produced a flood of known sequences and known polymorphisms. These advances potentially have diagnostic, therapeutic and research applications. However, such applications require the ability to rapidly analyze the genotype of a large number of samples. With this new sequence information has come a greater need for the ability to rapidly and accurately assay genetic variation in nucleic acid sequences.
Presently there are a number of assays for detection of nucleic acid sequences. These assays are sensitive and can produce detectable results with 100 target molecules or fewer. These assays are also specific, allowing accurate detection of specific sequences. The polymerase chain reaction (PCR) is one such method for amplification and detection of a specific sequence. This method consists of repeated cycles of denaturing a template strand of DNA, annealing matched primer pairs to the DNA, and extending the DNA from the primer using a DNA polymerase. A matched set of primers is used to amplify the sequence between the locations where the primers anneal. After each cycle, the resulting copy may act as a template for additional copying, allowing exponential amplification. Following the amplification of a DNA sequence, the sequence can be analyzed by sequencing or by restriction fragment analysis.
Ligase chain reaction (LCR) is another genetic analysis procedure. In this method, sequence-specific ligation is effected to determine the presence of a genetic variant. The sequence-specific ligation is preformed in cycles. In each cycle following hybridization and ligation, the joined fragments are subjected to a heat cycle to separate the ligated fragments from the parent strand. The fragments can serve as a template for further sequence specific ligation. Like PCR, LCR provides the exponential amplification of a target sequence.
Both PCR and LCR require temperature cycling to effect the reaction. Thermal cycling requires time for each cycle and requires dedicated instruments to generate the thermal cycles.
To generate the probes and primers for PCR and LCR, circularized DNA amplification has been used. In PCT filing WO 99/09216, Kool discloses a method of using rolling circle amplification to generate concatameric DNA. A single stranded, circularized template is combined with a polymerase and nucleotide triphosphates to yield a concatamer comprised of repeating oligonucleotide sequences. If the template has a site complementary to a restriction enzyme site, the concatamer may be digested into oligonucleotides of a single length. Either the concatamer or the digested oligonucleotides may be subsequently used, for example as probes.
One alternative for the detection of nucleic acid polymorphisms is the use of rolling circle amplification systems. In such a system, a probe is hybridized to a target nucleic acid sequence at a specific genomic locus. If the nucleic acid sequence targeted is present, the ends of the probe will hybridize with the target nucleic acid in such a way that the ends may be ligated together. This probe would then become an amplification target circle which could serve as the template for the generation of tandem-sequence DNA. Included as part of the probe is a primer complementary region. A primer added to the reaction mixture would hybridize onto the probe. If the amplification target circle is formed, a DNA polymerase which begins to produce a DNA transcript of the circle would then produce repeated copies of the amplification target circle.
In U.S. Pat. No. 5,871,921, Landegren et al. describes one method in which rolling circle amplification may be used for detection of genomic variants. In the assay, a detectable nucleic acid probe is hybridized to a single stranded nucleic acid target. The probe will hybridize with the target nucleic acid only if the targeted sequence is present. The hybridized probe ends are then covalently connected to form a continuous loop of probe nucleic acid. Following the formation of the continuous loop, the probe/target is subjected to conditions that would remove probes that did not form a continuous circuit, such as denaturing the probe/target hybrid or subjecting the probe to exonuclease activity to remove the non-cyclized probes. The target molecule may then be detected by determination of the presence of the interlocking catenated probe. Analysis of the reaction product requires separation of target DNA that does not have a tethered ligated probe from target DNA that does have the tethered ligated probe.
An alternative method of using the rolling circle amplification process is disclosed in U.S. Pat. No. 5,648,245 to Fire et al. The reference describes a four-step process for generating a concatamer library. In the procedure, the first step is to generate an amplification target circle by annealing ends of a padlock probe to a target nucleic acid sequence followed by ligation of the ends of the padlock probe to form a continuous loop. Once the amplification target circle is formed, the second step is to create a single stranded tandem-sequence DNA by rolling circle amplification of the amplification target circle. The third step requires converting the single stranded tandem-sequence DNA to double stranded tandem-sequence DNA. Finally, the double stranded tandem-sequence DNA is cloned or used for in vitro selection.
U.S. Pat. No. 5,866,377 to Schon uses rolling circle amplification as a method to detect variants in a nucleic acid sequence. In this method, a padlock probe hybridizes to a single stranded nucleic acid such that the ends are adjacent to each other. A ligase then joins the ends of the probe. The ligation reaction will be carried out only if the target nucleic acid contains a specific variant base at the locus near the end base of one of the probe ends. Detection of the presence of the catenated probe on the target nucleic acid indicates the presence of the specific variant. U.S. Pat. No. 5,854,033 to Lizardi describes a similar assay where the catenated probe is used to produce tandem-sequence DNA by rolling circle amplification. The tandem sequence is detected to determine the amount of target sequence present.
The use of rolling circle amplification presents certain advantages. The ligation creates a unique circularized template. The nature of the reaction is highly specific to the target nucleic acid of interest. The two probe hybridization regions assure that the hybridization to the target will be highly specific. In addition, roll
Molecular Dynamics, Inc.
Schneck David M.
Schneck Thomas
Whisenant Ethan
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