Epistem Limited

Genedrive Diagnostics Ltd

Ben Cobb

Shaun Ainsworth

Gino Miele

The invention provides a method and kit for preventing inhibition of a thermal cycling reaction by protein coagulation in a sample.

Use of anticoagulants in the polymerase chain reaction

A method and an oligonucleotide probe are described for determining the presence or absence of mutant alleles in a genomic locus. The probe binds to different alleles of a target sequence with different melting temperatures (Tm). The method determines the Tm of the probe when it is hybridized to the target sequence to establish whether a variant nucleic acid such as a mutant allele is present or absent in the target sequence. There may be variants in a target sequence that are not of interest, for example phenotypically silent mutations. To ensure that these variants do not influence the Tm of the probe, the probe contains universal base sites where such variants of no interest occur.

Mutation analysis

Described is a method for quantifying nucleic acid in a nucleic acid amplification reaction, in which an asymmetric nucleic acid amplification reaction is performed on a sample such that double stranded nucleic acid product is generated in an initial stage of the reaction, and single stranded nucleic acid product is generated in a subsequent stage of the reaction once a limiting primer is exhausted. Relative amounts of double stranded nucleic acid product and single stranded nucleic acid product produced are then detected by means of a melt curve analysis. The ratio of double stranded product peak height to single stranded product peak height may then be used to quantify the amount of starting template based on the ratio of double stranded product peak height to single stranded product peak height.

Quantification methods to determine initial template concentration by means of asymmetric amplification

A reaction vessel assembly for use with thermal cyclers is described. The assembly includes a reaction vessel and a casing defining a cavity. In a first configuration, the casing receives the reaction vessel within the cavity, to act as a protective casing for the reaction vessel. In a second configuration, the casing engages with a mouth of the reaction vessel, to close the vessel. In this configuration, the casing may also act as a handle. In preferred embodiments, the reaction vessel is in the form of a capillary tube, and/or may include an integrated collimating lens. Certain embodiments also include an RFID tag.

Reaction vessel

An assay for mutations in JAK2 is described. The assay uses selective amplification of mutant alleles with a blocker probe which preferentially hybridizes to wild type alleles. The same probe is then used to detect presence or absence of wild type sequences. It is not necessary to know the specific mutant sequence beforehand.

Mutational analysis of JAK2

A method for analysing genetic mutations, and in particular single nucleotide polymorphisms (SNPs) and/or somatic mutations, is described, as well as methods for preferentially amplifying one allelic form compared with another form. The methods use an oligonucleotide probe which hybridises to a first allele with a lower melting temperature (Tm) than that with which it hybridises to a second allele, together with amplification primers which flank the oligonucleotide probe binding site and which bind to the sample with a higher Tm than that of the probe and the first allele. An amplification reaction may be carried out at a temperature such that the probe is preferentially hybridised to the second allele, thereby amplifying the first allele. The amplified sequences may be detected using the same probe as acted as the blocking probe during amplification.

Mutational analysis

We describe a thermal cycler comprising a Peltier-type thermoelectric element used for cooling a sample block, and a non-Peltier-type heating device for heating the sample block. The cycler also includes a heat sink connected to the Peltier-type element by a heat pipe, which permits thermal energy to transfer from the Peltier-type element to the heat sink. This configuration operates more efficiently than conventional thermal cyclers which use Peltier-type elements for heating and cooling, and allows a more rapid cycling time as well as operation in a wider range of ambient temperatures. Certain embodiments utilise the Peltier-type element as a thermal gate to reduce thermal loss during heating when the Peltier-type element is switched off.

Epistem genedrive

The present invention provides a method of operating a thermal cycler using readable tags (for example, Radio Frequency Identification (RFID)) to simplify the operation and to reduce user interaction requirements. The RFID tag is used to program the thermal cycler unit. This automates process flow and allows single button operation. In general terms, the invention uses RFID tags provided on reaction vessels to identify a particular vessel; while a readable program card contains data associating reaction vessel identities with specific operations (eg, thermal cycling program, detection steps) to be performed on that vessel. The thermal cycler detects the reaction vessel RFID tag, and selects an appropriate operation to perform.

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Manchester, United Kingdom

Ben Cobb