Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

• This invention relates to the storage on a solid matrix of genetic material, in particular DNA that has been purified prior to the application to the solid matrix.
• nucleic acids particularly DNA
• storage of nucleic acids is typically accomplished in solution using refrigeration either at 4°C for up to several days or -20°C or even lower temperatures for longer periods. This is costly and space consuming for static storage. It presents greater issues when nucleic acid samples require transportation and samples are often shipped in dry ice.
• Burgoyne (WO 90/03959 ) described a method whereby biological samples, usually blood, could be applied to a solid matrix which combined reagents which lysed the cells. The released DNA was retained on the solid matrix. These samples could be stored for long periods at room temperature.
• Figure 1 shows the struture of inulin.
• PEG is prepared in a variety of molecuar weights defined by the average number of subunits per molecule. Polymers of MW 400, 1000 and 3350 were evaluated. It was observed that PEG 1000 produced the best recovery of applied DNA results (data not shown) and was used in the remainder of the tests but is referred to as PEG. At concentration of PEG at about 25% the results varied between experiments; this may imply small inconsistencies in the coating process of the solid matrix at these concentrations.
• Inulin is a naturally occuring polysaccharide found in many plants. Its structure is given in Figure 1. it is anticpated that simple modifications of inulin eg esterification would be possible and still achieve the improved elution of the applied purified DNA.
• the purified DNA can be applied to the solid matrix that has been treated with inulin in buffers that are routinely used in nucleic acid chemistry. Up to 10% PEG can also be included in the application buffer.
• the DNA prior to application to the solid matrix can be purified by a variety of standard laboratory techniques.
• the solid matrix was FTA Elute 903 matrix from Whatman.
• a 4 M stock of guanidine thiocyanate was prepared and diluted to 2 M using various concentrations of test reagents.
• 903 matrix (2 1 ⁇ 4" x 2 1 ⁇ 4") was placed into trays containing guanidine thiocyanate/ test reagent mixes and agitated gently for 10 seconds.
• Matrices were stored at room temperature in a desiccator until use.
• Microfuge tubes were placed in a heat block for 30 min at 98°C. 4) Microfuge tubes were pulse vortexed for 60 sec and then briefly centrifuged.
• Quantification of DNA in eluates was performed by QPCR using a 7900HT Thermal Cycler (Applied Biosystems). Reactions were set up using an RNase P assay and TAQMAN ® Universal PCR Master Mix (Applied Biosystems). A four point standard curve was prepared using a serial dilution from 10 to 0.01 ng/ ⁇ of the same Roche DNA used for experiments. Early QPCR quantifications were performed in 96-well plates and were set up manually. Following the introduction and validation of a liquid handling robot, later quantifications were performed using 384-well plates. Both 96 and 384-well plates were validated and also tested against each other to check for consistency between methods.
• the matrix containing 20 % inulin showed the highest % recovery of applied purified DNA. Since PEG was also identified as a possible additive to matrix
• Results were also obtained from experiments where the discs with applied purified DNA had been stored in a dessicator at room temperature. The results showed that the discs could be stored for at least twenty-three days before DNA elution with similar increased recovery of DNA when the matrix had been treated with inulin.
• Results also showed that the amount of DNA applied and recovered from the test matrix could be as low as lng and as high as ⁇ g (the maximum tested) and the increased effect on inulin treatment was still observed.

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• Polysaccharides And Polysaccharide Derivatives (AREA)
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• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)

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• 2010
  • 2010-12-21 JP JP2012546416A patent/JP5899118B2/ja active Active
  • 2010-12-21 AU AU2010338349A patent/AU2010338349B2/en active Active
  • 2010-12-21 CA CA2785913A patent/CA2785913C/en active Active
  • 2010-12-21 CN CN201080059859.XA patent/CN102725406B/zh active Active
  • 2010-12-21 WO PCT/EP2010/070389 patent/WO2011080160A1/en not_active Ceased
  • 2010-12-21 EP EP10796044.5A patent/EP2519631B1/en active Active
  • 2010-12-21 US US13/519,391 patent/US20120289690A1/en not_active Abandoned
• 2020
  • 2020-12-15 US US17/122,606 patent/US11725201B2/en active Active
• 2023
  • 2023-06-23 US US18/340,609 patent/US12247199B2/en active Active

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