DNA存储中的编码技术

脱氧核糖核酸(Deoxyribonucleic Acid, DNA)是一种天然的信息存储介质,具有存储密度高、存储时间长、损耗率低等特点。在传统存储方式不能满足信息增长的需求时,DNA数据存储技术逐渐成为研究热点。DNA编码是用尽可能少的碱基序列无错的存储数据信息,包括压缩(尽可能少的占用空间)、纠错(无错存储)和转换(数字信息转为碱基序列)3部分。DNA编码是DNA存储中的关键技术,它的结果直接影响存储性能的优劣和数据读写的完整。本文首先介绍DNA存储的发展历史,然后介绍DNA存储的框架,其中重点介绍DNA编码技术,最后对DNA存储中的编解码技术的未来发展方向进行讨论。     

Microbial Degradation of Forensic Samples of Biological Origin: Potential Threat to Human DNA Typing

Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.     

Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility

Extended semen doses from some boars used for AI have been shown to develop high levels of sperm DNA fragmentation during storage. Studies in other animals and humans have shown that if DNA damage is present in a certain percentage of the sperm cells the fertility potential of the semen sample is reduced. The objectives of the present study was to determine the relationship between sperm DNA fragmentation measured using the sperm chromatin structure assay (SCSA) in extended stored semen and field fertility in the boar. Three ejaculates from each of 145 boars were collected. Preparation of the semen doses included dilution with an EDTA extender and storage for up to 72 h post collection. The semen doses were assessed using flow cytometric methods for the percentage of viable sperm (PI/SYBR-14) and sperm DNA fragmentation (SCSA) at 0, 24, 48, and 72 h. A total of 3276 experimental inseminations in Danish breeding herds were conducted. The results showed that for 11 (7.6%) of the boars at least one of the three samples showed a value of DNA fragmentation index (DFI) above 20% within the storage period. Total number of piglets born (litter size) for Hampshire, Landrace and Danish Large White boars was, respectively, 0.5, 0.7 and 0.9 piglets smaller per litter when DFI values were above 2.1% as opposed to below this value. In conclusion the SCSA technique appears to be able to identify individuals with lower fertility with respect to litter size, and could in the future be implemented by the pig industry after a cost-benefit analysis.     

Prolactin storage in a clonal strain of rat pituitary tumor cells is cell-cycle dependent

GH4C1 cells (CH cells) are a clonal strain of rat pituitary tumor cells which secrete prolactin. We measured intracellular prolactin at different stages of the cell cycle using flow microfluorometry. Prolactin was stained by an indirect immunocytochemical technique using fluorescein isothiocyanate (FITC)-conjugated antiserum, and DNA was stained simultaneously with propidium iodide. We found that prolactin storage in GH cells was cell-cycle dependent; prolactin storage increased as cells passed from G1 to S to G2 + M. We have shown previously that insulin and 17 beta-estradiol act synergistically to increase intracellular prolactin three- to sevenfold and slow the rate of cell growth to approximately 70% of control cells. In this study we observed that insulin and estradiol increased prolactin storage at each stage of the cell cycle but did not affect the cell-cycle distribution of the population even though cell growth was slowed. We conclude that insulin and estradiol did not increase prolactin storage by affecting the cell-cycle distribution of the population.     

Effects of storage type and time on DNA amplification success in tropical ungulate faeces

The present study compares the effect of three storage media (silica, RNAlater®, ethanol) and time to extraction (1 week, 1 month and 3 months) on mitochondrial and nuclear marker amplification success in faecal DNA extracts from a sympatric community of small to medium‐sized Central African forest ungulates (genera Cephalophus, Tragelaphus, Hyemoschus). The effect of storage type and time on nuclear DNA concentrations, genotyping errors and percentage recovery of consensus genotypes was also examined. Regardless of storage method, mitochondrial and nuclear amplification success was high in DNA extracted within the first week after collection. Over longer storage periods, RNAlater yielded better amplification success rates in the mitochondrial assay. However, samples stored on silica showed (i) highest nuclear DNA concentrations, (ii) best microsatellite genotyping success, (iii) lowest genotyping errors, and (iv) greatest percentage recovery of the consensus genotype. The quantity of nuclear DNA was generally a good predictor of microsatellite performance with 83% amplification success or greater achieved with sample DNA concentrations of ≥ 50 pg/µL. If faecal DNA samples are to be used for nuclear microsatellite analyses, we recommend silica as the best storage method. However, for maximum mitochondrial amplification success, RNAlater appears to be the best storage medium. In contrast, ethanol appeared inferior to the other two methods examined here and should not be used to store tropical ungulate faeces. Regardless of storage method, samples should be extracted as soon as possible after collection to ensure optimal recovery of DNA.     

Detection of Ostreid Herpesvirus 1 (OsHV-1) DNA in seawater by PCR: influence of water parameters in bioassays

Since 1991, herpesvirus infections have been reported among larvae and juveniles of various bivalves. Most of the studies focused on detection of viral infections of economically important species. However, the persistence of bivalve herpesviruses in the marine environment is poorly documented. The present study concerns the role of seawater parameters in Ostreid Herpesvirus 1 (OsHV-1) detection by polymerase chain reaction (PCR). Viral DNA extracted from purified particles or virions present in infected oyster larvae were detected by PCR after storage in different media at different temperatures. The lowest detection threshold was found using distilled water or Tris EDTA buffer. In seawater, the threshold was higher. The use of sterile media permitted detection of viral DNA stored over a longer period. Storage temperature also had a significant influence on detection, with lower temperatures promoting DNA detection over a longer period. In summary, water parameters such as temperature influenced detection of OsHV-1 DNA by PCR. However, the PCR technique may also be successfully applied to samples in natural seawater. Indeed, the PCR technique permitted detection of naked viral DNA at 100 ng l(-1) in seawater in bioassays.     

Evaluation of different sources of DNA for use in genome wide studies and forensic application

In the field of epidemiology, Genome-Wide Association Studies (GWAS) are commonly used to identify genetic predispositions of many human diseases. Large repositories housing biological specimens for clinical and genetic investigations have been established to store material and data for these studies. The logistics of specimen collection and sample storage can be onerous, and new strategies have to be explored. This study examines three different DNA sources (namely, degraded genomic DNA, amplified degraded genomic DNA and amplified extracted DNA from FTA card) for GWAS using the Illumina platform. No significant difference in call rate was detected between amplified degraded genomic DNA extracted from whole blood and amplified DNA retrieved from FTA™ cards. However, using unamplified–degraded genomic DNA reduced the call rate to a mean of 42.6% compared to amplified DNA extracted from FTA card (mean of 96.6%). This study establishes the utility of FTA™ cards as a viable storage matrix for cells from which DNA can be extracted to perform GWAS analysis.     

A survey of DNA diagnostic laboratories regarding DNA banking.

This article reports the findings of a survey of 148 academically based and commercial DNA diagnostic labs regarding DNA banking (defined as the storage of individual DNA samples in some form with identifiers for later retrieval). The population surveyed consisted of all laboratories listed with HELIX, a national directory of DNA diagnostic labs that includes a fairly comprehensive listing of clinical service labs as well as a large number of research labs. The survey was concerned primarily with the legal and ethical issues that the long-term storage of DNA may raise. The survey inquired into the respondents' policies and procedures concerning (1) the extent of DNA banking and of interest in developing DNA banking in academia and industry and (2) the degree to which DNA banks had developed written internal policies and/or a written depositor's agreement (a signed document defining the rights and obligations of the person from whom the sample was taken and the bank) designed to anticipate or prevent some of the ethical and legal problems that can arise from the long-term retention of DNA. Our research suggests that (1) the activity of DNA banking is growing, particularly in the academic setting, and (2) most academically based DNA banks lack written internal policies, written depositor's agreements, or other relevant documentation regarding important aspects of this activity.     

Effects of cold storage on plasma membrane, DNA integrity and fertilizing ability of feline testicular spermatozoa

This study examined the effects of cold storage on plasma membrane, DNA integrity, and fertilizing ability of domestic cat spermatozoa. Intact cat testes were stored at 4°C in Dulbecco's phosphate buffered saline (DPBS) for 7 days. Membrane integrity (experiment 1) and DNA integrity (experiment 2) of extracted spermatozoa were assessed over time during storage. Testicular spermatozoa were also tested for their fertilizing ability via intracytoplasmic sperm injection (ICSI) in term of gamete activation and early embryonic development at 18 h (experiment 3). The membrane integrity of testicular spermatozoa was well preserved in DPBS for 4 days compared to non-preserved control (Day 0) (P<0.05). The incidence of testicular sperm DNA fragmentation was <1% after 7 days of cold storage and was not significantly affected by the duration of cold storage (P>0.05). Finally, testicular spermatozoa could form pronuclei and sustain embryo development following ICSI regardless of the storage time (P>0.05). In conclusion, cat testicular spermatozoa can be preserved at 4°C for up to 7 days without severely compromising of plasma membrane and DNA integrity while retaining a normal fertilizing ability.     

DNA数据存储技术原理及其研究进展

信息生产与数据存储能力之间的差距日益扩大,急需分子数据存储等高密度持久性信息保存替代方案,基于脱氧核糖核酸(DNA)的数据存储因在信息保留时间、物理密度和体积编码容量等方面优于多数传统存储介质,而广受关注.本文概述了DNA数据存储技术的基本原理,总结了体外DNA存储数据库与体内分子存储器系统的研究进展,讨论了基于DNA分子的数据存储系统所涉及的各种影响因素以及面临的挑战.     

Mechanisms of degradation of DNA standards for calibration function during storage

Establishment of molecular diagnostics offering quantitative technology is directly associated with real-time polymerase chain reaction (PCR). This rapid, accurate and sensitive method requires careful execution, including reliable calibration standards. The storage of such standards is crucial to prevent nucleic acid decay and to ensure stable results using real-time PCR. In this study, a broad investigation of possible causes of DNA degradation during storage was performed, including GC-content of the fragments, long-term storage, rapid freeze-and-thaw experiments, genomic DNA and short DNA fragments of different species, the influence of shear stress and the effect of nuclease remaining after DNA isolation. Several known chemical DNA degradation mechanisms have been matched with the experimental data through a process of elimination. Protocols for practical application, as well as a theoretical model describing the underlying mechanisms of deviation of real-time PCR results due to decay of standard DNA, have been developed. Primary amines in the buffer composition, which enhance depurination of the DNA helix, and shear stress due to ice crystal formation, could be identified as major sources of interaction. This results in degradation of the standard DNA, as well as in the probability of occurrence of mismatches affecting real-time PCR performance.     

Assessment of genetic and epigenetic changes following cryopreservation in papaya

A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.     

DECREASE IN NUCLEAR FEULGEN-POSITIVE MATERIAL (DNA) UPON AGING IN IN VITRO STORAGE OF BOVINE SPERMATOZOA

The Feulgen-DNA content of sperm cells from 5 bulls was studied by means of microspectrophotometry after storage at 5°C for 2, 3, 5, and 10 days in a yolk-citrate diluent permitting slow aerobic metabolism. A subsample of sperm cells from each bull was subjected to the Feulgen technique on each of the storage days selected. The cells sampled on each of these days received a standard 12 minute, 60°C hydrolysis. Absorption measurements at 546 mµof the individual cells indicated a marked progressive decrease in the Feulgen-DNA content of the stored spermatozoa. The loss of 30 per cent of the initial DNA at the end of 5 days' storage was highly significant statistically. This decrease approximately parallels the known decrease in fertility of stored sperm cells, as well as the increase in apparent embryonic mortality resulting from the use of similarly aged spermatozoa for artificial insemination.     

PCR products generated from unpurified Salmonella DNA are degraded by thermostable nuclease activity

Oligonucleotide primers designed from repetitive extragenic palindromic (REP) sequences were used to PCR-amplify Salmonella DNA. Unpurified template DNA, present in crude cell extracts, yielded an essentially identical banding pattern to that arising from the use of purified DNA. However, the PCR product derived from the crude preparations did not survive storage at 4°C. This post-PCR DNA degradation, attributed to endogenous Salmonella nucleases, was inhibited by the addition of EDTA. or storage at -20°C.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

DNA fragmentation in N-diazoacetylglycine amide-treated cells determined, by the rate of strand separation in alkali with hydroxylapatite chromatography in batch

DNA damage induced in mammalian cells (CHO-K1) by one hour treatment with several concentrations of N-diazoacetylglycine amide (DGA) was evaluated by the method of DNA denaturation in alkali and successive neutralization followed by separation of single from double stranded DNA with the recently described technique of hydroxylapatite chromatography performed in batch. This latter technique does not need complex apparatus and simplifies the simultaneous handling of large number of samples; it also appears as sensitive and reliable as the DNA alkaline elution on filter, to which it can be regarded as both alternative and complementary.     

Application of group specific amplified rDNA restriction analysis to characterize swine fecal and manure storage pit samples

Group specific amplified ribosomal-DNA restriction analysis was evaluated as a method to rapidly assess microbial community structure in swine fecal and manure storage pit samples. PCR primer sequences were evaluated for their specificity to ribosomal DNA from selected bacterial groups by optimizing annealing temperatures and determining specificity using a set of primer target and non-target organisms. A number of primer sets were identified targeting the following groups: Bacteroides-Prevotella, clostridial clusters I and II, clostridial clusters IX and XI, clostridial clusters XIVa and XIVb, Lactobacillus, Desulfovibrionaceae and Streptococcus-Lactococcus, as well as an universal primer set to represent total populations. Each bacterial group was digested with at least three restriction enzymes. We applied the group specific amplified ribosomal-DNA restriction analysis to swine fecal and manure storage pit samples obtained on two separate occasions. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. Results were consistent with 16S ribosomal DNA sequencing data from bacterial isolates and clones obtained from swine feces and manure storage pit. The group specific amplified ribosomal-DNA restriction analysis technique was able to rapid detect gross bacterial community differences among swine fecal and manure storage pit samples and determine groups of interest for more detailed examination.     

In vivo ischemia and storage injury cause alterations in the activity of poly(adenosine diphosphate ribose) synthetase

The objective of this study was to determine if DNA damage caused by ischemic insult (blood depletion) causes an alteration in the activity of endogenous mouse kidney poly(ADP-ribose) synthetase. The results show that kidneys made nonviable by warm (37 degrees C) in vitro ischemia (organ storage to study the effects of blood loss at normal body temperature) and in vivo ischemia (surgical depletion of the blood supply by arterial clamping) exhibit decreased levels of enzyme activity. Kidneys made nonviable by cold (0 degrees C) storage injury (organ storage as utilized for transplantation), however, possess elevated levels of enzyme activity. The DNA isolated from ischemic kidneys was shown to have a stimulatory effect upon exogenous calf thymus poly(ADP-ribose) synthetase. Also, electron microscopy analysis of DNA from ischemic kidneys showed that cold storage injury leads to the formation of large (average size = 500 bases) single-stranded regions. The results suggest that the activities of both endogenous and exogenous poly(ADP-ribose) synthetase are related to the nature of DNA damage resulting from ischemic insult.     

Membrane biofouling characterization: effects of sample preparation procedures on biofilm structure and the microbial community

Ensuring the quality and reproducibility of results from biofilm structure and microbial community analysis is essential to membrane biofouling studies. This study evaluated the impacts of three sample preparation factors (ie number of buffer rinses, storage time at 4°C, and DNA extraction method) on the downstream analysis of nitrifying biofilms grown on ultrafiltration membranes. Both rinse and storage affected biofilm structure, as suggested by their strong correlation with total biovolume, biofilm thickness, roughness and the spatial distribution of EPS. Significant variations in DNA yields and microbial community diversity were also observed among samples treated by different rinses, storage and DNA extraction methods. For the tested biofilms, two rinses, no storage and DNA extraction with both mechanical and chemical cell lysis from attached biofilm were the optimal sample preparation procedures for obtaining accurate information about biofilm structure, EPS distribution and the microbial community.     

The use of comet assay to assess DNA integrity of boar spermatozoa following liquid preservation at 5 degrees C and 16 degrees C

The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis), to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5 degrees C and 16 degrees C. In this comet assay protocol we used 2% beta-mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G) were diluted with a standard semen extender, Kortowo-3 (K-3), which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh) or ostrich egg yolk (LPFo). Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5 degrees C and 16 degrees C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5 degrees C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5 degrees C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional test of sperm function that can have diagnostic value in practice.