Article

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

Université de Bordeaux-plateforme Génomique Fonctionnelle, Institut Bergonié-INSERM U916 VINCO, Bordeaux, France.
Nucleic Acids Research (Impact Factor: 9.11). 12/2009; 38(5):1531-46. DOI: 10.1093/nar/gkp1060
Source: PubMed

ABSTRACT

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.

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    • "Collection, preservation and storage of biological evidence have a fundamental impact on the quality of the sample and the resultant DNA profile. If the sample quality is damaged by poor practices it can undermine the potential for it to be used as evidence [1]. These initial steps must be undertaken carefully, and the most reliable and reproducible protocols should be used for the crime scene collection and preservation of the biological materials before they reach the laboratory. "
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    ABSTRACT: A B S T R A C T The main focus in forensic genetics for two decades has been to improve the extraction of DNA from a wide variety of evidence and to make the profiling technology more sensitive and robust. In contrast, the recovery methods for biological material have seen little development. This study aims to improve the efficacy of the collection and storage processes, from crime scene to receipt at the laboratory. This study compared the use of ultrapure water as a wetting agent when collecting biological evidence using swabs with a detergent-based buffer. The results show that the stability post-collection greatly improved by using a newly developed buffer. When ultrapure water is used, DNA degradation was seen after 6 h at room temperature. However, the detergent-based buffer stabilized DNA for up to 48 h, even when the temperature was increased to 50 C. The impact of these findings may be limited where crime scene evidence can be refrigerated until it reaches the laboratory. However, there are many situations/contexts where sample refrigeration is not possible and there is scope to improve the preservation of the genetic forensic evidence before it reaches the laboratory.
    Full-text · Conference Paper · Oct 2015
    • "Expected PCR amplification targets were always detected in DNAshell-recovered material. This pilot study, as well as feedback data from 8 additional laboratories confirm previous studies on DNAshells [9] [10] [14] and therefore validates Imagene encapsulation process as a reliable room temperature storage method for preserving reference DNA material. Following the positive outcome of this pilot study, DNAshells® are now available to the 36 French laboratories that perform Biomed 2-based lymphoid clonality analyses. "
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    ABSTRACT: Performance of methods used for molecular diagnostics must be closely controlled by regular analysis of internal quality controls. However, conditioning, shipping and long lasting storage of nucleic acid controls remain problematic. Therefore, we evaluated the minicapsules-based innovative process developed by Imagene (Evry, France) for implementing DNA and RNA controls designed for clonality assessment of lymphoproliferations and BCR-ABL1 mRNA quantification, respectively. DNA samples were extracted from 12 cell lines selected for giving specific amplifications with most BIOMED-2 PCR tubes. RNA samples were extracted from 8 cell line mixtures expressing various BCR-ABL1 transcript levels. DNA and RNA were encapsulated by Imagene and shipped at room temperature to participating laboratories. Biologists were asked to report quality data of recovered nucleic acids as well as PCR results. Encapsulated nucleic acids samples were easily and efficiently recovered from minicapsules. The expected rearrangements at immunoglobulin, T-cell receptor and BCL2 loci were detected in DNA samples by all laboratories. Quality of RNA was consistent between laboratories and met the criteria requested for quantification of BCR-ABL1 transcripts. Expression levels measured by the 5 laboratories were within±2 fold interval from the corresponding pre-encapsulation reference value. Moreover aging studies of encapsulated RNA simulating up to 100years storage at room temperature show no bias in quantitative outcome. Therefore, Imagene minicapsules are suitable for storage and distribution at room temperature of genetic material designed for proficiency control of molecular diagnostic methods based on end point or real-time quantitative PCR. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Apr 2015 · Clinical biochemistry
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    • "However, others reported that gDNA is stable for at least 11 years at ambient tropical conditions [37]. It is well documented that there are several factors that may compromise sample integrity which includes high humidity, temperature, persistence of nucleases and other chemical agents as well as other sub-optimal conditions that may occur not only during transport, but also within storage facilities [38]. Dry storage of nucleic acids has been recommended to eliminate the need for cold storage based on the assumption that nucleic acids are stable when dry. However there are numerous examples where degradation occurs during storage, in the cold or at ambient conditions, that can irreversibly damage samples in solution or even those that are dehydrated [39]. "
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    ABSTRACT: Genotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis. All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 mul) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort. Both whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples. gDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies.
    Full-text · Article · Oct 2013 · BMC Genetics
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