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IP Watch: Recent Patents Related to PCR, Nucleic Acid Amplification, and Sample Prep: Aug 3, 2011

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This article has been updated from a previous version to correct the number of US Patent No. 7,999,561.

Roche Molecular Systems has been awarded US Patent No. 7,991,562, "PCR elbow determination using curvature analysis of double sigmoid."

Ronald Kurnik and Jianmei Wang are named as inventors on the patent.

Discloses systems and methods for determining characteristic transition values such as elbow values in sigmoid or growth-type curves, such as the cycle threshold (Ct) value in PCR amplification curves. A double sigmoid function with parameters determined by a Levenberg-Marquardt regression process is used to find an approximation to a curve that fits a PCR dataset. The curve can then be normalized using one or more of the determined parameters. After normalization, the normalized curve is processed to determine its curvature at some or all points to produce a dataset or plot representing curvature versus the cycle number. The cycle number at which the maximum curvature occurs corresponds to the Ct value, which is then returned and may be displayed or otherwise used for further processing.


Roche has also been awarded US Patent No. 7,991,561, "Ct determination by cluster analysis with variable cluster endpoint."

Ronald Kurnik is named as the sole inventor on the '558 patent.

Discloses systems and methods for determining the Ct value in a kinetic PCR amplification curve. The PCR data set may be visualized in a two-dimensional plot of fluorescence intensity versus cycle number, and is transformed to produce a partition table of data points with one column including the fluorescence at cycle n and a second column including the fluorescence at cycle n+i, where i is typically 1 or greater.

In addition, a cluster analysis process is applied to the partition table data set to determine a plurality of clusters in the partition table data set. In one aspect, the clustering process used includes a k-means clustering algorithm, where the number of identified clusters, k, is greater than or equal to three. In another aspect, a partitioning around medoids algorithm is used to identify three or more clusters. Using the identified clusters, a linear slope of each of the clusters is determined … and for each cluster, a ratio of the slope of that cluster with the slope of an adjacent cluster is determined, the patent's abstract states. The ratios are then compared, with an end point of a cluster having the largest or smallest ratio representing a specific point of interest in the data curve. The data point representing the elbow or Ct value of the PCR curve is identified as an end point of one of the identified clusters, and the cycle number corresponding to this data point is returned or displayed.


Roche has also been awarded US Patent No. 7,991,558, "Systems and methods for determining real-time PCR cycle thresholds using cluster analysis."

Ronald Kurnik is also named as inventor on the '558 patent.

Discloses systems and methods for determining the elbow or Ct value in a real-time, or kinetic, PCR amplification curve data set. The PCR data set may be visualized in a two-dimensional plot of fluorescence intensity versus cycle number, and is transformed to produce a partition table of data points with one column including the fluorescence at cycle n and a second column including the fluorescence at cycle n+i, where i is typically one or greater.

In addition, a cluster analysis process is applied to the partition table data set to determine a plurality of clusters in the set. In one aspect, the clustering process includes a k-means clustering algorithm, where k is greater than or equal to three. The data point representing the elbow or Ct value of the PCR curve is identified as an end point of one of the identified clusters, and the cycle number corresponding to this data point is returned or displayed.


Roche has also been awarded US Patent No. 7,989,169, "Selective amplification of methylated nucleic acids."

Frank Bergmann and Christine Markert-Hahn are named as inventors on the patent.

Relates to a method for amplifying a methylated target nucleic acid in a sample while avoiding amplification of a non-methylated target nucleic acid by inactivating it. This is accomplished by a restriction enzyme digest after bisulfite treatment of the target nucleic acid. The invention further relates to the use of a restriction enzyme to avoid amplification of a non-methylated target nucleic acid while amplifying a methylated target nucleic acid in a sample; and kits for performing the described methods.


Roche has also been awarded US Patent No. 7,989,168, "Proofreading primer extension."

Ellen Fiss and Thomas Myers are named as inventors on the patent.

Discloses primer extension reactions, including polymerase chain reactions, in which a polymerase having 3'-to-5' exonuclease activity edits a primer that is not fully complementary, thereby allowing for amplification and detection of target nucleic acids that may have variability in their sequences.


Invitrogen Dynal (Life Technologies) has been awarded US Patent No. 7,989,614, "Isolation of nucleic acid."

Arne Deggerdal and Frank Larsen are named as inventors on the patent.

Provides a method of isolating nucleic acid from a sample, said method comprising contacting the sample with a detergent and a solid support, whereby soluble nucleic acid in the sample is bound to the support and separated from the sample. Whereas the method of the invention is used to isolate DNA, it may be coupled with a further step to isolate RNA from the same sample, according to the patent's abstract.


Stanford University has been awarded US Patent No. 7,989,185, "Rapid, informative diagnostic assay for influenza viruses including H5N1."

Nader Pourmand, Lisa Diamond, Jochen Kumm, and Ronald Davis are named as inventors on the patent.

Describes a rapid diagnostic assay for influenza virus, particularly avian influenza and more particularly H5N1. The assay is based on amplifying a significant portion of the hemagglutinin (HA) gene and sequencing several loci within the HA gene using techniques that can obtain real-time sequence information from multiple sites of a target DNA, in particular pyrosequencing and bioluminescence regenerative cycle. The assay contemplates the use of information-rich subsequences within the HA gene, e.g., a glycosylation sequon; receptor binding site; and HA1/HA2 cleavage site. Other subsequences for sequencing include strain and clade markers, which vary among H5N1 strains.


Medical Diagnostic Laboratories has been awarded US Patent No. 7,989,186, "Compositions and methods for detecting Cryptococcus neoformans."

Melanie Feola, Martin Adelson, Eli Mordechai, and John Entwistle are named as inventors on the patent.

Disclosed are oligonucleotides useful in methods for determining whether a sample contains Cryptococcus neoformans, a causative agent for human cryptococcosis. These oligonucleotides, which have nucleotide sequences derived from a coding segment of the gene encoding the fungal specific transcription factor gene in Cryptococcus neoformans, are useful as forward and reverse primers for a polymerase chain reaction using nucleic acids from a biological sample as templates, and as probes for detecting any resultant amplicon. Detection of an amplicon indicates the sample contains Cryptococcus neoformans. The patent also discloses real-time PCR and detection using florescence resonance energy transfer.


MicroFluidic Systems (now part of PositiveID) has been awarded US Patent No. 7,988,935, "Handheld and portable microfluidic device to automatically prepare nucleic acids for analysis."

Bob Yuan and Nima Aflaatooni are named as inventors on the patent.

Describes a handheld and portable extraction device based on a microfluidic-based system to be used in the field to extract and purify an analyte, preferably a nucleic acid, from a fluid-based sample, preferably water-based. The fluid-based sample can also be a biological fluid sample. The device includes a syringe-like component coupled to a purification chip, which is preferably included within a chip block that is removable from the remaining portion of the device. The analyte collected within the purification chip can be later removed and collected for analysis.


STMicroelectronics of Agrate Brianza, Italy, has been awarded US Patent No. 7,988,841, "Treatment of biological samples using dielectrophoresis."

Mario Scurati, Torsten Mueller, and Thomas Schnelle are listed as inventors on the patent.

Describes a plurality of planar electrodes in a microchannel used for separation, lysis, and PCR in a chip. Cells from a sample are brought to the electrodes and, depending on the properties of the sample, phase pattern, frequency, and voltage of the electrodes and flow velocity are chosen to trap target cells using dielectrophoresis while the majority of unwanted cells are flushed through. After separation, the target cells are lysed while still trapped by applying radio frequency pulses and/or thermally so as to change the dielectric properties of the trapped cells. After lysis, the target cells are amplified within the microchannel so as to obtain separation, lysis, and PCR on same chip, the patent's abstract states.

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