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How long is DNA stable in a freezer?

How long is DNA stable in a freezer?



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Inspired by the post about extracting pet DNA, how long would genomic DNA be stable for in a -20°C freezer? It is common practice to store DNA (double-stranded, plasmid) in a -20°C freezer in the lab, but would genomic DNA last longer in a -80°C freezer? With either method, how long would it be stable for?


If the DNA is pure, it should last quite a long time. If there are enzymes and other biological molecules in there, -80C will work much better.

I think you could keep pure DNA at -20C practically indefinitely.

Purity is the main issue there, also pH stabilized, sealed properly, etc. That makes all the difference.


You can store your DNA for 1 week at -4C and for one month at -20C but with storing DNA over these period of time there is a deduction of 10-15% of yield from your DNA sample.


What’s the Shelf-Life of DNA?

Photo by Andrew Cowie/AFP/Getty Images

The body of Richard III has been found beneath a parking lot in Leicester, England, according to experts from the University of Leicester. DNA testing was used to match the infamous king with DNA from a descendant of his sister. What’s the shelf life of DNA?

About a month to a million years, theoretically. The decay rate of DNA depends on the conditions of its storage and packaging. Above all, it depends on whether the DNA is exposed to heat, water, sunlight, and oxygen. If a body is left out in the sun and rain, its DNA will be useful for testing for only a few weeks. If it’s buried a few feet below the ground, the DNA will last about 1,000 to 10,000 years. If it’s frozen in Antarctic ice, it could last a few hundred thousand years. For best results, samples should be dried, vacuum-packed, and frozen at about -80 degrees Celsius. Even then, ambient radiation is likely to render DNA unrecognizable before it celebrates its millionth birthday.

Some scientists contend that DNA could survive beyond our current theoretical estimates. In fact, several scientists have claimed to find DNA hundreds of millions of years old. In 2009, a team of researchers reported that they had found 419-million-year-old DNA inside ancient salt deposits in the Michigan Basin. If confirmed, it would be the oldest DNA ever discovered. However, some experts who study ancient DNA are highly skeptical of these claims, noting that they usually turn out to be the product of contamination in the lab. Other scientists studying bird bones have estimated that under ideal conditions, DNA has a half-life of approximately 521 years, meaning that it would be broken down so much as to be useless after about 1 million years.

Despite what John Hammond and Mr. DNA might tell you, amber does not actually do a good job of keeping DNA fresh. While the fossilized tree sap can preserve insect skeletons for tens of millions of years, the DNA inside the insects breaks down very rapidly. When the organism dies, enzymes are released that begin breaking down the DNA almost immediately. Similarly, Egyptian mummies may look well-preserved—many of the proteins in their hair and muscles are intact—but their DNA has typically decayed rapidly in the heat. As a general rule, outward appearance is not a good indicator of whether DNA is still intact.

Probably the oldest DNA ever found was discovered in frozen mud taken from the base of an ice sheet in Greenland. It is estimated to be 450,000 to 800,000 years old. The sample contained genetic material from butterflies, pine trees, and other organisms. The frozen sludge broke the record previously held by plants frozen in ice in Siberia, which grew there 400,000 years ago. Neanderthal DNA has been found that is about 100,000 years old. When it comes to modern humans, the oldest DNA recovered so far has been only about 5,000 to 7,000 years old. In 2008, researchers used DNA samples that were thousands of years old to sequence the genome of the extinct woolly mammoth. While many have wondered whether we might be able to clone one of the creatures, such an endeavor presents mammoth challenges. While Spanish researchers successfully resurrected an extinct species of ibex in 2009, it died of breathing difficulties seven minutes later, likely because of flaws in its DNA.

In their efforts to identify Richard III, researchers used a kind of DNA called mitochondrial DNA—so-called because it’s contained in the mitochondria of the cell rather than in the nucleus. Mitochondrial DNA does not contain the complete human genome, making it not as useful for many researchers’ purposes. However, because it’s more abundant—there are often hundreds of mitochondria in a cell, and only one nucleus—the odds are better that you could find mitochondrial DNA intact than nuclear DNA.


Introduction

“My hunch is—and I’m not alone in this—that the next decade or so will see this used technically. The machine could be much smaller it could carry a much larger set of data.” In 1964, just 7 years after the discovery of the structure of DNA 1 , Wiener and Neiman discussed the potential density advantage of using nucleic acids as a form of memory storage 2 . Over half a century later, advancements in our understanding of the properties of DNA have confirmed its high theoretical information density of nearly 455 billion GB of data per gram 3 ,

6 orders of magnitude greater than even the most advanced magnetic tape storage systems 4 . DNA also provides a host of other unique potential advantages including highly parallelized computation within the storage system itself 5,6 , low energy requirements 7,8,9 , rapid high capacity transportation of data 10 , potentially longer lifetimes, and stabilities of decades or centuries compared to conventional media which are replaced every 3–5 years, as well as ease of replication 11 by molecular biology approaches to ward off degradation. While it has taken more than just the decade Wiener predicted, a large body of knowledge in molecular biology 12,13 and computer and information systems 14,15 has been developed in the intervening period that has set the foundation for the recent interest and investment in DNA-based information storage technologies.

The nature of Wiener’s ‘machine’ will remain in constant flux and development. Indeed, multiple types of systems may arise to address different applications, from long-term ‘write-once-read-never’ archival storage to highly dynamic and frequently accessed data storage, potentially with in-storage computational capabilities. It is important to imagine these possible types of DNA information storage systems and the different unit processes that will comprise these systems, and identify how the chemical, physical, and encoding properties of DNA will influence their design. As DNA or an analog will be the substrate of this class of polymeric systems, its stability under different environmental and process conditions will be a central design consideration, informing the nature of both physical unit processes and encoding algorithms.

An end-to-end DNA storage system is depicted in Fig. 1 with generic unit processes. As applications range from cold archival storage to frequently accessed or even dynamically manipulated data, the DNA is exposed to more manipulation such as phase changes or physical shearing through liquid handling, and to more distinct types of environmental conditions such as buffers with different salt concentrations and pHs. These present more opportunities for degradation as well as specific degradation mechanisms that may influence encoding strategies and sources for data and decoding errors (Fig. 1). Here we review what is known about the stability of DNA under each of these conditions, organized by their relevance to systems with different operating timescales. We then provide a quantitative analysis of the relative tradeoffs in density, physical redundancy, and encoding strategies that must be sacrificed to achieve increasingly sophisticated system capabilities such as increased access frequency and in-storage computation.

(Top) A generic DNA-based system showing distinct types of storage modes. (Middle) Functional and physical characteristics of each storage mode. (Bottom) Molecular mechanisms of damage most relevant to each storage mode.

It will be useful to also describe the molecular architectures common to almost all DNA storage systems proposed to date. DNA storage systems are comprised of many files, and each file consists of many distinct DNA strands that typically are

150–200 nt long as that is the current limit of phosphoramidite synthesis chemistries, but could be longer with advances in technology. All strands comprising one file share a common address sequence located on one or both ends of the strands. These addresses can be read and retrieved using PCR or transcription. Mutations within strands, or loss of strands due to breakage or degradation would lead to potential decoding errors or even complete loss of information. Therefore, error correction codes are usually applied to help compensate for errors and lost strands with the trade-off of decreasing information density.


VII. Working with E. coli

A. Small Scale Cultures

Experiments using E. coli cells should always be done on fresh cultures, either from a freshly streaked plate or from a glycerol stock. To grow a small scale E. coli culture, prepare 3-5 ml of LB (or appropriate broth - include antibiotic if the culture contains a plasmid) in two sterile 50 ml tubes. (Note: smaller tubes can be used but the culture will not be appropriately aerated and hence will not grow well and is not recommended). Inoculate one tube with a single colony from a fresh plate or a scraping from a glycerol stock. The second tube is used as a broth control. Incubate both tubes at 37 deg C, shaking vigorously overnight. Inspect the tubes the next morning. The broth control should be clear and the inoculated culture should be very turbid. Make a note of any debris found in the tubes and only incubate longer if the culture is not dense. Do not allow cells to overgrow. Use immediately. For some applications, cells can be stored at 4 deg C for short periods prior to use.

B. Permanent Storage

For every culture used, in particular, for newly constructed strains or for cells containing plasmids, a permanent glycerol stock must be prepared as soon as the construct has been confirmed and this stock must be placed in the laboratory stock collection with the appropriate documentation and location information. Failure to follow these procedures will result in serious penalties. This procedure pertains not only to E. coli but to any organism for which a deep freeze stock can be prepared. Also, all plasmid constructs, including construction intermediates, must be maintained in cells, not as naked DNA stocks. For each construct, at least 2 stocks should be made. To prepare a glycerol stock for E. coli cells, combine 1.4 ml of a freshly grown overnight culture with 0.6 ml of sterile 50% glycerol. Mix well. Transfer to two freezer vials labeled with the strain name, the date and your initials (not an eppendorf tube). Immediately place into a dry ice/ethanol bath or into a box in the -80 deg C freezer. Note the location and enter data into the strain book.

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Last updated on 3/2/2010

is designed for students interested in careers in industrial and biomedical sciences.


How long is DNA stable in a freezer? - Biology

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U.S. DEPARTMENT OF AGRICULTURE

Agricultural Genetic Resources Preservation Research: Fort Collins, CO

General preservation questions:

What are genetic resources? Genetic resources are living material such as crops, livestock, related species, rare and endangered varieties and breeds that include genes, genetic combinations (a.k.a. genotypes) or genetic frequencies that give diversity to future varieties or breeds. In agriculture, genetic resources are used by breeders to increase yields and stress tolerance, improve nutrition, and add value, beauty, flavor, and adaptability.

What is germplasm? Germplasm is a set of propagules that carries the desired genetic resource (i.e. genes, genetic combinations or gene frequencies).

What is an accession? An accession is a genetically unique plant sample from a particular geographic location. At NLGRP, an accession may be a bag of seeds, plant tissue cultures, or buds from twigs of fruit crops. NLGRP sometimes stores more than one sample of a particular accession. When samples are regrown or reproduced, the subsequent generation has the same accession number as the parent sample. The new sample has an inventory number that identifies the generation number.

What is a propagule? A propagule is a tissue, organ or plant part that can be regenerated into a whole plant (i.e. seeds, cuttings and budwood).

What is genetic diversity? Genetic diversity is variation in life that is heritable. Modern crops or livestock breeds may have a low amount of diversity because of repeated selection for desirable traits and widespread use of few individuals or varieties. Populations become diverse through the processes of mutation and sexual reproduction. Natural selection acts on this diversity to favor the changes that enable the best survival in a particular environment. Populations that have a lot of genetic diversity can survive dynamic environments better than populations that have low genetic diversity. Genetic diversity is what enables breeders to improve plant varieties and animal breeds.

What is a clone? A clone is a group of genetically identical cells descended from a single common ancestor one or more organisms descended asexually from a single ancestor one that is an exact replica of another (Webster II. The Riverside Publishing Company, 1984).

What is cryopreservation? Cryopreservation is a process of cooling cells or various kinds of tissues to temperatures below zero to slow down effectively life processes without damage to the material. Plant tissues or plant cells are usually cryopreserved at liquid nitrogen (-196 o C) or vapor of liquid nitrogen (ca. -156 o C).

What are plant tissue cultures? Plant tissue cultures are plant tissue, cells or plant organs maintained or propagated in vitro under aseptic conditions on sterile culture medium. This method of plant propagation is referred often as micropropagation. Plants derived from one original propagule (plant, tissue or cell) are clones.

What is a recalcitrant seed? A recalcitrant seed, in contrast to most crop seeds, is a seed that cannot survive drying and so cannot survive in the freezer. Preservation of recalcitrant seeds requires a procedure that prevents damage by drying or freezing. This has been accomplished in several species by excising the growing part of the seed, optimizing the water content, and cooling very rapidly. Recalcitrant seeds are frequently produced by temperate-zone forest trees, riparian species, and plants from the tropics. Examples of recalcitrant seeds are oak seeds, wild rice, and citrus.

Why is the NLGRP located in Fort Collins, Colorado? The dry climate was a primary reason that the USDA National Seed Storage Laboratory (NSSL) was located in Fort Collins, CO. With an average relative humidity of about 30%, little effort was needed to adjust seed moisture content to the optimum level needed for long-term storage. In the 1950s a Colorado State University professor, Dr. D.W. (Scotty) Robertson, was very active in promoting germplasm preservation. A barley breeder, Dr. Robertson, argued that a base collection for genetic resources should be established and worked to have the Laboratory built on the Colorado State University campus. The Beet Sugar Development Foundation also supported these efforts.

What is the NLGRP capacity? The NLGRP has capacity to store between one and 1.5 million accessions, depending on the size of the propagule, in storage vaults cooled to -18 o C (conventional storage). In addition, the NCGRP has a 220 cryotank capacity, with each cryotank holding about 3,000 seed and 70,000 semen accessions.

How safe are the collections? The facility has protected access to all work areas in the building. The storage areas , including the seed cold vaults and cryogenic room, are further restricted by added security with limited access of only a few individuals, additional locked doors, security cameras, and coded information on sample bags. The vault area is fortified against tornadoes and floods and there are backup mechanical systems.

Can anyone get seeds from NLGRP? Distributions are made for research purposes from the NPGS sites located around the US.

Where does the germplasm come from and who donates it? Germplasm comes from all over the world and it is donated by collectors, breeders or experts in systematics who locate material with unusual or interesting traits that may eventually be useful in agriculture. For example, a collector may find an apple with unusual flavor or a wheat landrace that is resistant to aphids. Most of the germplasm for agricultural crops comes from the area where that crop evolved. This area, known as the Center for Diversity, is believed to have the highest genetic variability in the smallest geographic area.

Does NLGRP save endangered plant species? In collaboration with conservation groups, we store seeds of endangered species. This activity can preserve the remaining genetic diversity of an endangered species until it is reintroduced into native habitats.

How large is the NLGRP plant collection? There are more than 500,000 accessions in the collection. Each accession contains about 3,000 to 5,000 seeds, depending on the reproductive biology of the species.

How many species are in the NLGRP plant collection? About 12,000. Changing needs for US agriculture and landscapes will lead to an inevitable increase in the numbers of species collected and stored at NLGRP.

How do I request germplasm? Search and request germplasm using GRIN-Global.

What is the best way to store seeds? Allow seeds to dry for a few weeks in a place with about 20% relative humidity. Then store seeds in vapor-proof containers such as a glass jar or sealed moisture-proof bag in a cold place like a home freezer.

How long can seeds survive in storage? Seed longevity depends on storage conditions and seed quality. We expect most undamaged seeds that are properly dried to survive about a hundred years in conventional storage (-18C) and about a thousand years under cryogenic (liquid nitrogen) conditions.

What is the oldest living seed? The most reliable studies show some seeds in soil at archeological sites surviving for 100 to 1,700 years. (e.g. Odum 1965. Germination of ancient seeds: floristical observations and experiments with archaeological dated soil samples. Dan. Bot, Arkiv 24(2):1-70 Shen-Miller, J., Mudgett, M.B., Schopf, J.W., Clarke, S., and Berger, R. 1995. Exceptional seed longevity and robust growth: Ancient sacred lotus from China. American Journal of Botany).

How long does DNA last? DNA, the genetic code, found in all life, is a very stable molecule. Fragments that are thousands of years old have been found in archeological artifacts, especially if the artifacts have been kept dry or free from microbes that cause decay.

Can I plant seeds from an apple I really enjoyed? You can plant seeds from that apple and get a tree in about 5-10 years, but the fruit from the new tree will not be the same as the apple that you enjoyed. This is because fruit quality is specific to the mother plant, and the mother tree and the offspring tree are genetically different. Most fruit crops must cross-pollinate to produce seeds. For fruit crops, the same genetic line is usually maintained by grafting budwood from the mother plant onto a rootstock or rooting stems that are cut from the mother plant.


Ensure your sample quality is maintained

Stability of your biological samples is important to ensure that your data and results are accurate and not biased. The best way to ensure your samples remain stable during transport and storage is to make sure you a have a reliable sample collection method that can effectively stabilize your DNA over long periods of time at ambient and elevated temperatures.

If you are interested to learn more about our Oragene® and ORAcollect® products, send us an email at [email protected] or click the button below to request trial kits for evaluation.

Related Blogs:

References:

[1] Hansen et al. Collection of blood, saliva and buccal cell samples in a pilot study on the Danish nurse cohort comparison of the response rate and quality of genomic DNA. Cancer Epidermiol Biomarkers & Prevention. 2072-6 (2007).

[2] Galbete C et al. Lifestyle factors modify obesity risk linked to PPARG2 and FTO variants in an elderly population: a cross-sectional analysis in the SUN project. Genes Nutr. 8(1):61-67 (2013).

[3] Anthonappa et al. Evaluation of long-term storage stability if saliva as a source of human DNA. Clin Oral Invest 17:1719-1725 (2013).

[4] Davis R et al. Specimen collection within the CRN: a critical appraisal. CRN (2010).

[8] Karni et al. Thermal degradation of DNA. DNA and cell biology. 32. (2013) 10.1089/dna.2013.2056.


How Long Does DNA Last?

Even minimal exposure to forensic science on shows like CSI and NCIS will impress upon a viewer what a whopping big deal DNA analysis is. It’s the opposite of circumstantial evidence: undeniable proof of someone’s identity that is impossible to fake, short of swapping out one sample for another. The technique may be applied to murder victims or long-dead English kings or illegitimate children and their custody-dodging fathers—any subject from which intact genetic information can be extracted—and that's what makes DNA as valuable a tool in anthropological study as it is in police investigations. For a long-dead subject, DNA has an expiration date, but when exactly is it?

The entire formula for human life is encoded in the sub-microscopic molecules of deoxyribonucleic acid, and has been throughout all stages of evolution. Like fingerprints, genetic code is particular to an individual, which makes it a unique identifier in the absence of other information, like modern dental records. DNA, however, is fragile, and breaks down over time. How long the decomposition process takes will vary with the circumstances under which it is found. Take, for example, if DNA is exposed to the elements: Like the human body itself, DNA decays with increasing rapidity in the presence of heat, water, sunlight, and oxygen. Those essential conditions of life also speed the process of death, potentially rendering DNA useless for analysis in a matter of weeks.

Scientists have estimated that under the most ideal conditions, DNA can theoretically survive for a maximum of one million years. Although a team of researchers recently claimed to have discovered 419 million-year-old genetic material belonging to prehistoric bacteria in the Michigan Basin, others in the field have loudly contested the claim, especially in light of an earlier sample thought to be 250 million years old, but later proven contaminated by the presence of modern DNA. The oldest actual DNA samples hail from Greenland (the icy one, as opposed to Iceland, the green one), extracted from beneath a mile of ice, a “perfect, natural freezer” for DNA preservation. The 450,000 to 800,000-year-old samples provide evidence of green life on the now largely barren landmass.

As far as human genetic material goes, the record for oldest Neanderthal DNA is held by a 100,000-year-old sample found in a Belgian cave. The longest-lasting sample of human DNA was discovered in northeastern Spain, and boasts a survival age of 7000 years. In both cases, techniques pioneered by Dr. Rhonda Roby allowed researchers to use mitochondrial DNA rather than the type found in the cell nucleus although mitochondrial DNA only contains only partial genetic information, it provides sufficient evidence for identification and is present in greater abundance than nuclear DNA, increasing its odds of surviving.

How long does DNA last? The short answer is that it’s complicated, and determined by a number of unpredictable factors such as weather and the organism’s final resting place. Your DNA may be the molecular legacy you leave behind, but once you’re dead, there’s not really much you can do about it.


Contents

The double-helix model of DNA structure was first published in the journal Nature by James Watson and Francis Crick in 1953, [5] (X,Y,Z coordinates in 1954 [6] ) based on the work of Rosalind Franklin and her student Raymond Gosling, who took the crucial X-ray diffraction image of DNA labeled as "Photo 51", [7] [8] and Maurice Wilkins, Alexander Stokes, and Herbert Wilson, [9] and base-pairing chemical and biochemical information by Erwin Chargaff. [10] [11] [12] [13] [14] [15] The prior model was triple-stranded DNA. [16]

The realization that the structure of DNA is that of a double-helix elucidated the mechanism of base pairing by which genetic information is stored and copied in living organisms and is widely considered one of the most important scientific discoveries of the 20th century. Crick, Wilkins, and Watson each received one third of the 1962 Nobel Prize in Physiology or Medicine for their contributions to the discovery. [17]

Hybridization is the process of complementary base pairs binding to form a double helix. Melting is the process by which the interactions between the strands of the double helix are broken, separating the two nucleic acid strands. These bonds are weak, easily separated by gentle heating, enzymes, or mechanical force. Melting occurs preferentially at certain points in the nucleic acid. [18] T and A rich regions are more easily melted than C and G rich regions. Some base steps (pairs) are also susceptible to DNA melting, such as T A and T G. [19] These mechanical features are reflected by the use of sequences such as TATA at the start of many genes to assist RNA polymerase in melting the DNA for transcription.

Strand separation by gentle heating, as used in polymerase chain reaction (PCR), is simple, providing the molecules have fewer than about 10,000 base pairs (10 kilobase pairs, or 10 kbp). The intertwining of the DNA strands makes long segments difficult to separate. The cell avoids this problem by allowing its DNA-melting enzymes (helicases) to work concurrently with topoisomerases, which can chemically cleave the phosphate backbone of one of the strands so that it can swivel around the other. Helicases unwind the strands to facilitate the advance of sequence-reading enzymes such as DNA polymerase.

The geometry of a base, or base pair step can be characterized by 6 coordinates: shift, slide, rise, tilt, roll, and twist. These values precisely define the location and orientation in space of every base or base pair in a nucleic acid molecule relative to its predecessor along the axis of the helix. Together, they characterize the helical structure of the molecule. In regions of DNA or RNA where the normal structure is disrupted, the change in these values can be used to describe such disruption.

For each base pair, considered relative to its predecessor, there are the following base pair geometries to consider: [20] [21] [22]

  • Shear
  • Stretch
  • Stagger
  • Buckle
  • Propeller: rotation of one base with respect to the other in the same base pair.
  • Opening
  • Shift: displacement along an axis in the base-pair plane perpendicular to the first, directed from the minor to the major groove.
  • Slide: displacement along an axis in the plane of the base pair directed from one strand to the other.
  • Rise: displacement along the helix axis.
  • Tilt: rotation around the shift axis.
  • Roll: rotation around the slide axis.
  • Twist: rotation around the rise axis.
  • x-displacement
  • y-displacement
  • inclination
  • tip
  • pitch: the height per complete turn of the helix.

Rise and twist determine the handedness and pitch of the helix. The other coordinates, by contrast, can be zero. Slide and shift are typically small in B-DNA, but are substantial in A- and Z-DNA. Roll and tilt make successive base pairs less parallel, and are typically small.

Note that "tilt" has often been used differently in the scientific literature, referring to the deviation of the first, inter-strand base-pair axis from perpendicularity to the helix axis. This corresponds to slide between a succession of base pairs, and in helix-based coordinates is properly termed "inclination".

At least three DNA conformations are believed to be found in nature, A-DNA, B-DNA, and Z-DNA. The B form described by James Watson and Francis Crick is believed to predominate in cells. [23] It is 23.7 Å wide and extends 34 Å per 10 bp of sequence. The double helix makes one complete turn about its axis every 10.4–10.5 base pairs in solution. This frequency of twist (termed the helical pitch) depends largely on stacking forces that each base exerts on its neighbours in the chain. The absolute configuration of the bases determines the direction of the helical curve for a given conformation.

A-DNA and Z-DNA differ significantly in their geometry and dimensions to B-DNA, although still form helical structures. It was long thought that the A form only occurs in dehydrated samples of DNA in the laboratory, such as those used in crystallographic experiments, and in hybrid pairings of DNA and RNA strands, but DNA dehydration does occur in vivo, and A-DNA is now known to have biological functions. Segments of DNA that cells have methylated for regulatory purposes may adopt the Z geometry, in which the strands turn about the helical axis the opposite way to A-DNA and B-DNA. There is also evidence of protein-DNA complexes forming Z-DNA structures.

Other conformations are possible A-DNA, B-DNA, C-DNA, E-DNA, [24] L-DNA (the enantiomeric form of D-DNA), [25] P-DNA, [26] S-DNA, Z-DNA, etc. have been described so far. [27] In fact, only the letters F, Q, U, V, and Y are now [update] available to describe any new DNA structure that may appear in the future. [28] [29] However, most of these forms have been created synthetically and have not been observed in naturally occurring biological systems. [ citation needed ] There are also triple-stranded DNA forms and quadruplex forms such as the G-quadruplex and the i-motif.

Structural features of the three major forms of DNA [30] [31] [32]
Geometry attribute A-DNA B-DNA Z-DNA
Helix sense right-handed right-handed left-handed
Repeating unit 1 bp 1 bp 2 bp
Rotation/bp 32.7° 34.3° 60°/2
bp/turn 11 10.5 12
Inclination of bp to axis +19° −1.2° −9°
Rise/bp along axis 2.3 Å (0.23 nm) 3.32 Å (0.332 nm) 3.8 Å (0.38 nm)
Pitch/turn of helix 28.2 Å (2.82 nm) 33.2 Å (3.32 nm) 45.6 Å (4.56 nm)
Mean propeller twist +18° +16°
Glycosyl angle anti anti C: anti,
G: syn
Sugar pucker C3'-endo C2'-endo C: C2'-endo,
G: C2'-exo
Diameter 23 Å (2.3 nm) 20 Å (2.0 nm) 18 Å (1.8 nm)

Grooves Edit

Twin helical strands form the DNA backbone. Another double helix may be found by tracing the spaces, or grooves, between the strands. These voids are adjacent to the base pairs and may provide a binding site. As the strands are not directly opposite each other, the grooves are unequally sized. One groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide. [33] The narrowness of the minor groove means that the edges of the bases are more accessible in the major groove. As a result, proteins like transcription factors that can bind to specific sequences in double-stranded DNA usually make contacts to the sides of the bases exposed in the major groove. [4] This situation varies in unusual conformations of DNA within the cell (see below), but the major and minor grooves are always named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form.

Non-double helical forms Edit

Alternative non-helical models were briefly considered in the late 1970s as a potential solution to problems in DNA replication in plasmids and chromatin. However, the models were set aside in favor of the double-helical model due to subsequent experimental advances such as X-ray crystallography of DNA duplexes and later the nucleosome core particle, and the discovery of topoisomerases. Also, the non-double-helical models are not currently accepted by the mainstream scientific community. [34] [35]

DNA is a relatively rigid polymer, typically modelled as a worm-like chain. It has three significant degrees of freedom bending, twisting, and compression, each of which cause certain limits on what is possible with DNA within a cell. Twisting-torsional stiffness is important for the circularisation of DNA and the orientation of DNA bound proteins relative to each other and bending-axial stiffness is important for DNA wrapping and circularisation and protein interactions. Compression-extension is relatively unimportant in the absence of high tension.

Persistence length, axial stiffness Edit

DNA in solution does not take a rigid structure but is continually changing conformation due to thermal vibration and collisions with water molecules, which makes classical measures of rigidity impossible to apply. Hence, the bending stiffness of DNA is measured by the persistence length, defined as:

The length of DNA over which the time-averaged orientation of the polymer becomes uncorrelated by a factor of e. [ citation needed ]

This value may be directly measured using an atomic force microscope to directly image DNA molecules of various lengths. In an aqueous solution, the average persistence length is 46–50 nm or 140–150 base pairs (the diameter of DNA is 2 nm), although can vary significantly. This makes DNA a moderately stiff molecule.

The persistence length of a section of DNA is somewhat dependent on its sequence, and this can cause significant variation. The variation is largely due to base stacking energies and the residues which extend into the minor and major grooves.

Models for DNA bending Edit

Stacking stability of base steps (B DNA) [36]
Step Stacking ΔG
/kcal mol −1
T A -0.19
T G or C A -0.55
C G -0.91
A G or C T -1.06
A A or T T -1.11
A T -1.34
G A or T C -1.43
C C or G G -1.44
A C or G T -1.81
G C -2.17

At length-scales larger than the persistence length, the entropic flexibility of DNA is remarkably consistent with standard polymer physics models, such as the Kratky-Porod worm-like chain model. [37] Consistent with the worm-like chain model is the observation that bending DNA is also described by Hooke's law at very small (sub-piconewton) forces. For DNA segments less than the persistence length, the bending force is approximately constant and behaviour deviates from the worm-like chain predictions.

This effect results in unusual ease in circularising small DNA molecules and a higher probability of finding highly bent sections of DNA. [38]

Bending preference Edit

DNA molecules often have a preferred direction to bend, i.e., anisotropic bending. This is, again, due to the properties of the bases which make up the DNA sequence - a random sequence will have no preferred bend direction, i.e., isotropic bending.

Preferred DNA bend direction is determined by the stability of stacking each base on top of the next. If unstable base stacking steps are always found on one side of the DNA helix then the DNA will preferentially bend away from that direction. As bend angle increases then steric hindrances and ability to roll the residues relative to each other also play a role, especially in the minor groove. A and T residues will be preferentially be found in the minor grooves on the inside of bends. This effect is particularly seen in DNA-protein binding where tight DNA bending is induced, such as in nucleosome particles. See base step distortions above.

DNA molecules with exceptional bending preference can become intrinsically bent. This was first observed in trypanosomatid kinetoplast DNA. Typical sequences which cause this contain stretches of 4-6 T and A residues separated by G and C rich sections which keep the A and T residues in phase with the minor groove on one side of the molecule. For example:

¦ ¦ ¦ ¦ ¦ ¦
G A T T C C C A A A A A T G T C A A A A A A T A G G C A A A A A A T G C C A A A A A A T C C C A A A C

The intrinsically bent structure is induced by the 'propeller twist' of base pairs relative to each other allowing unusual bifurcated Hydrogen-bonds between base steps. At higher temperatures this structure is denatured, and so the intrinsic bend is lost.

All DNA which bends anisotropically has, on average, a longer persistence length and greater axial stiffness. This increased rigidity is required to prevent random bending which would make the molecule act isotropically.

Circularization Edit

DNA circularization depends on both the axial (bending) stiffness and torsional (rotational) stiffness of the molecule. For a DNA molecule to successfully circularize it must be long enough to easily bend into the full circle and must have the correct number of bases so the ends are in the correct rotation to allow bonding to occur. The optimum length for circularization of DNA is around 400 base pairs (136 nm) [ citation needed ] , with an integral number of turns of the DNA helix, i.e., multiples of 10.4 base pairs. Having a non integral number of turns presents a significant energy barrier for circularization, for example a 10.4 x 30 = 312 base pair molecule will circularize hundreds of times faster than 10.4 x 30.5 ≈ 317 base pair molecule. [39]

The bending of short circularized DNA segments is non-uniform. Rather, for circularized DNA segments less than the persistence length, DNA bending is localised to 1-2 kinks that form preferentially in AT-rich segments. If a nick is present, bending will be localised to the nick site. [38]

Elastic stretching regime Edit

Longer stretches of DNA are entropically elastic under tension. When DNA is in solution, it undergoes continuous structural variations due to the energy available in the thermal bath of the solvent. This is due to the thermal vibration of the molecule combined with continual collisions with water molecules. For entropic reasons, more compact relaxed states are thermally accessible than stretched out states, and so DNA molecules are almost universally found in a tangled relaxed layouts. For this reason, one molecule of DNA will stretch under a force, straightening it out. Using optical tweezers, the entropic stretching behavior of DNA has been studied and analyzed from a polymer physics perspective, and it has been found that DNA behaves largely like the Kratky-Porod worm-like chain model under physiologically accessible energy scales.

Phase transitions under stretching Edit

Under sufficient tension and positive torque, DNA is thought to undergo a phase transition with the bases splaying outwards and the phosphates moving to the middle. This proposed structure for overstretched DNA has been called P-form DNA, in honor of Linus Pauling who originally presented it as a possible structure of DNA. [26]

Evidence from mechanical stretching of DNA in the absence of imposed torque points to a transition or transitions leading to further structures which are generally referred to as S-form DNA. These structures have not yet been definitively characterised due to the difficulty of carrying out atomic-resolution imaging in solution while under applied force although many computer simulation studies have been made (for example, [40] [41] ).

Proposed S-DNA structures include those which preserve base-pair stacking and hydrogen bonding (GC-rich), while releasing extension by tilting, as well as structures in which partial melting of the base-stack takes place, while base-base association is nonetheless overall preserved (AT-rich).

Periodic fracture of the base-pair stack with a break occurring once per three bp (therefore one out of every three bp-bp steps) has been proposed as a regular structure which preserves planarity of the base-stacking and releases the appropriate amount of extension, [42] with the term "Σ-DNA" introduced as a mnemonic, with the three right-facing points of the Sigma character serving as a reminder of the three grouped base pairs. The Σ form has been shown to have a sequence preference for GNC motifs which are believed under the GNC hypothesis to be of evolutionary importance. [43]

The B form of the DNA helix twists 360° per 10.4-10.5 bp in the absence of torsional strain. But many molecular biological processes can induce torsional strain. A DNA segment with excess or insufficient helical twisting is referred to, respectively, as positively or negatively supercoiled. DNA in vivo is typically negatively supercoiled, which facilitates the unwinding (melting) of the double-helix required for RNA transcription.

Within the cell most DNA is topologically restricted. DNA is typically found in closed loops (such as plasmids in prokaryotes) which are topologically closed, or as very long molecules whose diffusion coefficients produce effectively topologically closed domains. Linear sections of DNA are also commonly bound to proteins or physical structures (such as membranes) to form closed topological loops.

Francis Crick was one of the first to propose the importance of linking numbers when considering DNA supercoils. In a paper published in 1976, Crick outlined the problem as follows:

In considering supercoils formed by closed double-stranded molecules of DNA certain mathematical concepts, such as the linking number and the twist, are needed. The meaning of these for a closed ribbon is explained and also that of the writhing number of a closed curve. Some simple examples are given, some of which may be relevant to the structure of chromatin. [44]

Analysis of DNA topology uses three values:

  • L = linking number - the number of times one DNA strand wraps around the other. It is an integer for a closed loop and constant for a closed topological domain.
  • T = twist - total number of turns in the double stranded DNA helix. This will normally tend to approach the number of turns that a topologically open double stranded DNA helix makes free in solution: number of bases/10.5, assuming there are no intercalating agents (e.g., ethidium bromide) or other elements modifying the stiffness of the DNA.
  • W = writhe - number of turns of the double stranded DNA helix around the superhelical axis
  • L = T + W and ΔL = ΔT + ΔW

Any change of T in a closed topological domain must be balanced by a change in W, and vice versa. This results in higher order structure of DNA. A circular DNA molecule with a writhe of 0 will be circular. If the twist of this molecule is subsequently increased or decreased by supercoiling then the writhe will be appropriately altered, making the molecule undergo plectonemic or toroidal superhelical coiling.

When the ends of a piece of double stranded helical DNA are joined so that it forms a circle the strands are topologically knotted. This means the single strands cannot be separated any process that does not involve breaking a strand (such as heating). The task of un-knotting topologically linked strands of DNA falls to enzymes termed topoisomerases. These enzymes are dedicated to un-knotting circular DNA by cleaving one or both strands so that another double or single stranded segment can pass through. This un-knotting is required for the replication of circular DNA and various types of recombination in linear DNA which have similar topological constraints.

The linking number paradox Edit

For many years, the origin of residual supercoiling in eukaryotic genomes remained unclear. This topological puzzle was referred to by some as the "linking number paradox". [45] However, when experimentally determined structures of the nucleosome displayed an over-twisted left-handed wrap of DNA around the histone octamer, [46] [47] this paradox was considered to be solved by the scientific community.


Epidemiology

Adenovirus infections are widely distributed in human populations. The highest susceptibility is found among children from 6 months to 2 years of age and extends to the group of 5 to 9 year old children. Types 2, 1, 3, 5, 7, and 6 (in that order) are most frequently isolated from adenovirus-infected children, with types 1 and 2 constituting some 60 percent of all isolates. Nevertheless, adenovirus infections are responsible for only 2 to 5 percent of acute respiratory infections in children.

Adenovirus also infects military recruits in the United States, where this infection has been studied well, and most likely in other countries as well. Adenovirus types 4, 7, and 3 cause acute respiratory diseases, including pneumonia, in this population.

Adenoviruses have been isolated from severely immunocompromised patients, such as those with acquired immune deficiency syndrome (AIDS). Many of these isolates, including the adenovirus types 42 to 47, are found in the urine of AIDS patients.


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