Chain and conformation stability of solid-state DNA: implications for room temperature storage

There is currently wide interest in room temperature storage of dehydrated DNA. However, there is insufficient knowledge about its chemical and structural stability. Here, we show that solid-state DNA degradation is greatly affected by atmospheric water and oxygen at room temperature. In these conditions DNA can even be lost by aggregation. These are major concerns since laboratory plastic ware is not airtight. Chain-breaking rates measured between 70°C and 140°C seemed to follow Arrhenius’ law. Extrapolation to 25°C gave a degradation rate of about 1–40 cuts/105 nucleotides/century. However, these figures are to be taken as very tentative since they depend on the validity of the extrapolation and the positive or negative effect of contaminants, buffers or additives. Regarding the secondary structure, denaturation experiments showed that DNA secondary structure could be preserved or fully restored upon rehydration, except possibly for small fragments. Indeed, below about 500 bp, DNA fragments underwent a very slow evolution (almost suppressed in the presence of trehalose) which could end in an irreversible denaturation. Thus, this work validates using room temperature for storage of DNA if completely protected from water and oxygen

Stabilité chimique et conformationnelle de l ADN à l état sec et à température ambiante

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Modélisation de la dégradation de l'ADN pour la détermination du nombre de copies restantes et de la probabilité de PCR positive

International audienceIl n'existe pas de méthode avérée d'analyse d'ADN dégradé pour la quantification de la dégradation. Nous proposons et validons un modèle basé sur une fragmentation aléatoire à partir de deux analyses qPCR sur deux cibles de tailles différentes. Le modèle permet de calculer de nombreuses quantités intéressant les biologistes et en particulier la probabilité de PCR positive ou la dégradation maximale pour obtenir une PCR avec une probabilité donnée

Simultaneous assessment of average fragment size and amount in minute samples of degraded DNA

International audienc

Modélisation de la dégradation de l'ADN pour la détermination du nombre de copies restantes et de la probabilité de PCR positive

Il n'existe pas de méthode avérée d'analyse d'ADN dégradé pour la quantification de la dégradation. Nous proposons et validons un modèle basé sur une fragmentation aléatoire à partir de deux analyses qPCR sur deux cibles de tailles différentes. Le modèle permet de calculer de nombreuses quantités intéressant les biologistes et en particulier la probabilité de PCR positive ou la dégradation maximale pour obtenir une PCR avec une probabilité donnée

DNA degradation in heated encapsulated WBC.

<p>Representative electrophoresis gels: DNA samples extracted from white blood cells stored in Imagene minicapsules at the indicated temperatures (130°C, 110°C, 90°C, 70°C) and times were heat-denatured 5 min at 95°C to reveal internal cuts (single strand DNA size) and run on non-denaturating agarose gels. L_max values representing the average fragment size of DNA are indicated at the bottom of each lane. M: DNA molecular weight ladder; min: minute; h: hour.</p

High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere - Fig 2

<p><b>Ambient temperature stability of DNA in encapsulated WBC (A) and BC (B).</b> Minicapsules of WBC and BC were stored for 15 months or 18 months, respectively, in the laboratory. At each time point, minicapsules were opened and samples rehydrated. Then, DNA was extracted and analyzed on non-denaturing AGE. DNA extracted from WBC were analyzed in two conditions: with or without heat-denaturation before AGE. DNA sizes are all > 20 kb. (A): extracted DNA from WBC after (left) or before (right) heat-denaturation, (B): extracted DNA from BC after heat-denaturation. M: double stranded DNA molecular weight marker.</p

Adverse atmosphere effect on DNA stability of encapsulated white blood cells and buffy coat.

<p>(A) Opened and closed (standard Imagene conditions) BC minicapsules were left at ambient temperature for 36 months, (B) After a 24 months incubation at ambient temperature in Imagene standard condition, some minicapsules were opened. Both 2 years-old opened and closed WBC minicapsules were incubated for 7 and 15 days at 70°C under 50% relative humidity. Both WBC and BC samples (triplicates) were rehydrated, DNA extracted, heat-denatured to reveal internal cuts and analyzed on AGE.</p

qPCR Amplification efficiencies for DNA extracted from heated WBC and BC minicapsules.

<p>qPCR Amplification efficiencies for DNA extracted from heated WBC and BC minicapsules.</p