Scientists have developed and tested a "DNA robot" that delivers payloads such as drug molecules to specific cells. The container was made using a method called "DNA origami", in which long DNA chains are folded in a prescribed way. Then, so-called aptamers - which can recognise specific cell types - were used to lock the barrel-shaped robot. In lab tests described in Science, the locks opened on contact with cancer cell proteins, releasing antibodies that halted the cells' growth. The method could find wide use in biological applications, where this kind of "specificity" is highly prized. Lead author of the research, Shawn Douglas of the Wyss Institute at Harvard University, said the result brings together several recent research strands. "We've been working on figuring out how to build different shapes using DNA over the past several years, and other researchers have used antibodies as therapeutics, in order to manipulate cell signalling, and yet others have demonstrated that aptamers can be used to target cancer cell types," Dr Douglas told BBC News. "The novel part is really integrating all those different pieces and putting them together in a single device that works." In essence, the approach co-opts a number of strategies of our immune systems, with the robots playing the role of white blood cells that hunt down problematic cells and destroy them. The team tested the robots using several cultures of cancer cells including lymphoma and leukemia, with corresponding payloads of antibodies. Because DNA is found in nearly all of our cells, Dr Douglas said the robots posed fewer problems of toxicity than many materials would have. Scientists have already discovered a large number of different aptamers that can "recognise" proteins corresponding to different diseases, so the approach could in principle be adapted to a wide range of applications. Dr Douglas said that there was still much optimisation to be done on the robots; for now the team will create a great many of them to be tested in an animal model.
The barrel shape opens and spills its payload when the "locks" come into contact with target cells
728
Web Search
Mar 5, 2012
'DNA robot' targets cancer cells
Posted by
DNAworld
at
6:01:00 AM
0
comments
Mar 4, 2012
New DNA Study Reveals Glacier Mummy's Medical Problems
European scientists say a recently completed DNA map of the Iceman - the well-preserved, 5,300-year-old mummy discovered in a melting Alpine glacier in 1991 - is yielding new details about the man's physical look, his ethnic origins and his health - including an apparent predisposition for heart disease.
The details are described in a new paper by scientists at the European Academy for Mummies and the Iceman in Bolzano, Italy, and at the Institute for Human Genetics in Germany. Among the key findings is that the Iceman, whom scientists have nicknamed Oetzi, was genetically at risk for heart disease, even though he was neither sedentary nor overweight. Researchers say that fact is significant because it shows that the cardiovascular condition existed more than 5,000 years ago, and therefore cannot be associated primarily with modern lifestyles.
In addition to his heart problems, Oetzi’s newly mapped genome reveals that he also suffered from the chronic tick-borne illness, Lyme disease, or borreliosis. The researchers say this is the earliest-known case of the bacterial infection, and provides proof that Lyme disease was present in the New Stone Age period.
Oetzi also was lactose intolerant, meaning he could not digest milk products. The scientists say this finding supports the theory that lactose intolerance - which persists today in most Asians and Africans but affects few northern Europeans - was still a common condition in Oetzi's time, even though his people were becoming increasingly involved in farming and the domestication of dairy animals.
The researchers believe Oetzi’s ancestors likely hailed from the Middle East, and migrated to Europe as agriculture and cattle breeding continued to spread. They say the dwindling populations of modern-day Europeans who share the Iceman’s genetic heritage are found mostly in geographically isolated areas, such as the Mediterranean islands of Sardinia and Corsica.
Oetzi's new DNA map also has helped researchers reconstruct the physical appearance of the ill-fated traveler. They say he had brown eyes and long, wavy brown hair, which scientists believe would have been worn loose. He stood approximately 1.6 meters tall --an average height for a man during the New Stone Age - and he weighed an estimated 50 kilograms.
The analysis of the Iceman’s complete genome is published in the journal Nature Communications.
Earlier studies concluded that Oetzi died at the venerable age of 45. However, later examinations of wounds on the mummy suggested the Iceman was murdered, fatally shot in the back with an arrow and left to die on an Alpine glacier.
Many scientists believe that the high quality of the Iceman’s clothing and items he was carrying when he was killed, such as a fine copper axe, make it likely that Oetzi and his family had considerable social standing within their community.
Oetzi the Iceman's frozen corpse was naturally mummified in the spot where he fell dead, more than 600 years before the first bricks were laid in Giza for Egypt's Great Pyramid. Entombed under a deepening layer of snow and ice, the mummy remained undisturbed until 1991, when two German hikers happened on the partially exposed corpse while trekking through the Oetztal Alps, near the Italian border.
(Source: VOA)
Posted by
DNAworld
at
6:01:00 AM
0
comments
Jul 1, 2011
Chromosome In Situ Hybridization
A modern approach to the specific location of genes on chromosomes is a technique for the hybridization of DNA and RNA "in situ." With this procedure, specific radioactive RNA or DNA (known as probes) can be isolated (or synthesized "in vitro") and then annealed to chromosomes which have been treated in such a manner that their basic double stranded DNA has been "melted" or dissociated.
In theory, and fortunately in practice, when the DNA is allowed to re-anneal, the probe competes for the binding, but only where it mirrors a complimentary sequence. Thus, RNA will attach to the location on the chromosome where the code for its production is to be found. DNA will anneal to either RNA which is still attached to a chromosome, or to the complimentary sequence DNA strand within the chromosome. Since the probe is radioactive, it can be localized via autoradiographic techniques.
Finally, it is possible to produce an RNA probe that is synthesized directly from repetitive sequences of DNA, such as that found within the nucleolar organizer region of the genome. This RNA is known as cRNA (for copied RNA) and is a convenient source of a probe for localizing the nucleolar organizer gene within the nucleus, or on a specific chromosome.
The use of in situ hybridization begins with good cytological preparations of the cells to be studied, and the preparation of pure radioactive probes for the analysis. The details depend upon whether the hybridization is between DNA (probe) and DNA (chromosome), DNA (probe) and RNA (chromosome), or between RNA (probe) and DNA (chromosome).
Preparation of the Probe:
Produce radioactive RNA by incubating the cells to be measured in the presence of
H-uracil, a specific precursor to RNA. Subsequent to this incubation, extract rRNA from the sample and purify through differential centrifugation, column chromatography or electrophoresis. Dissolve the radioactive RNA probe in 4X Saline-Citrate containing 50% formamide to yield a sample that has 50,000 to 100,000 counts per minute, per 30 microliter sample, as determined with a scintillation counter. Add the formamide is added to prevent the aggregation of RNA.
Preparation of the Slides:
Fix the materials to be studied in either 95% ethanol or in 3:1 methanol:water, attach to pre-subbed slides (as squashes for chromosomes) and air dry.
Hybridization
Place the air dried slides into a moist chamber, usually a disposable petri dish containing filter paper and carefully place 30 microliters of RNA probe in 4X SSC-50% formamide onto the sample.
Carefully add a cover slip (as in the preparation of a wet mount), place the top on the container and place in an incubator at 37° C for 6-12 hours.
Washing:
Pick up the slides and dip into 2X SSC so that the coverglass falls off.
Place the slides in a coplin jar containing 2X SSC for 15 minutes at room temperature.
Transfer the slides to a treatment with RNase (50 microgram/ml RNase A, 100 units/ml RNase T1 in 2X SSC) at 37° C for 1 hour.
Wash twice in 2X SSC, 15 minutes each.
Wash twice in 70% ethanol, twice in 95% ethanol and air dry.
Autoradiography:
Add photographic emulsions to the slides and after a suitable exposure period, develop the slides, counterstain and add cover slips.
Analyze the slides by determining the location of the radioactive probe on the chromosomes or within the nuclei.
(Dr. William H. Heidcamp)
Posted by
DNAworld
at
4:13:00 AM
0
comments
DNA Nanoparticles Protocols
Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery
Weijun Li and Francis C. Szoka, Jr.1
Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry,
1Corresponding author (szoka@cgl.ucsf.edu)
INTRODUCTION
This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo.
Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery
Li Shyh-Dar1, Li Song2, and Huang Leaf1,3
1 Division of Molecular Pharmaceutics,
2 Center for Pharmacogenetics,
3Corresponding author (leafh@pitt.edu)
INTRODUCTION
Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. The LPD nanoparticles contain a highly condensed DNA core surrounded by lipid bilayers with an average size of ~100 nm. The nanoparticle complex is injected into mice, and expression of the transfected DNA is monitored with an appropriate assay.
Frank Alexis, Jieming Zeng, and Wang Shu1,2
Institute of Bioengineering and Nanotechnology,
1Department of Biological Sciences, National
2Corresponding author (swang@ibn.a-star.edu.sg)
INTRODUCTION
This protocol describes the preparation of polyethylenimine (
Preparation of Gold Nanoparticle–DNA Conjugates
T. Andrew Taton1
1University of
Publication Name:
Current Protocols in Nucleic Acid Chemistry
Unit Number: UNIT 12.2
DOI: 10.1002/0471142700.nc1202s09
Online Posting Date: August, 2002
ABSTRACT
This unit describes the preparation of conjugates between nanometer-scale gold particles and synthetic oligonucleotides. Oligonucleotide-functionalized gold nanoparticles are finding increased use in both the construction of complex, tailored nanostructures and the optimization of DNA sequence analysis. The protocols in this unit outline the synthesis, purification, and characterization of nanoparticle-DNA conjugates for applications in nanotechnology and biotechnology. Separate procedures are presented for nanoparticles functionalized with just one or a few oligonucleotide strands and for nanoparticles functionalized with a dense layer of oligonucleotide strands. The different physical and chemical properties of these two types of conjugates are discussed, as are their stability and utility in different environments.
Chitosan-plasmid DNA nanoparticles used for contraceptive vaccine.
Publication: Immunotherapy Weekly
Publish date: July 21, 2004
2004 JUL 21 - (NewsRx.com & NewsRx.net) -- Researchers have prepared chitosan-plasmid DNA nanoparticles encoding zona pellucida glycoprotein-3 alpha and characterized its expression in the mouse.
"In the present study, the porcine zona pellucida (ZP)-3alpha eukaryotic expression vector pVAX1-pZP3alpha was constructed by genetic recombinant technology, then the recombinant plasmid was encapsulated in nanoparticles with chitosan, and the imaging of chitosan/pVAX1-pZP3alpha nanoparticles by Atomic Force Microscope (AFM) was processed. Feeding mouse with those microencapsulation by gastric larvae, and after five days, detecting its expression in mouse intestine by RT-PCR …
Posted by
DNAworld
at
4:12:00 AM
1 comments
DNA and Evidence Collection Principles
Robert E. Kramer
The dawn of a new age has arrived in law enforcement in the form of DNA research and testing. We in law enforcement, especially those of us working the crime scenes need to be aware of what we can do "in the field" to assure that proper evidence collection techniques are followed. Only then will the groundwork for successful evidence examinations be in place when we submit the case to a forensic laboratory for analysis.
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is the DNA evidence analysis technique which is being practiced at the state laboratory at the Division of Criminal Investigation in
Impact and Exchange
It is widely embraced within the law enforcement forensic field that, to at least some degree, the process of impact and exchange occurs at every crime scene. For example, a "run" vehicle impacts the accident scene and exchange occurs with the transfer of paint to the victim vehicle; a burglar impacts a scene with the approach of the area, and exchange occurs when footwear impressions are left behind. As law enforcement officers and crime scene specialists, it is our job to collect and preserve evidence at the scene - evidence which may not only connect the suspect to the scene - but connect the suspect to the incident itself.
Swab Method
The collection and preservation of evidence which will be subjected to DNA analysis is best accomplished by the seizure and submission of the original item. For example, it would be desirable to collect and submit undergarments worn after an incident involving suspected sexual assault rather than cutting or swabbing the specimen. Sometimes, however, the submission of the original item is impossible or impractical. Imagine a shooting or stabbing scene where there is evidence of considerable blood loss on a tile or linoleum floor. The practice of swabbing for the evidence is then practical for collection of possible DNA evidence.
It is preferred that swabs to be submitted to the D.C.I. Lab in
Slightly moisten a cotton tip swab with clean water.
concenrate the stain as much as possible.
avoid potential sample-to-sample contamination during the process.
avoid contamination by the collector (wear protective clothing).
if cotton balls are chosen as the collection medium, forceps used (if applicable) need to be cleaned thoroughly after each specimen.
(2) Air dry - NEVER use a hair drier.
(3) Package separately in paper (no plastic containers).
(4) Keep out of direct sunlight.
Eliminating the chances of cross, sample-to-sample, or collector contamination cannot be stressed enough. There are steps which can be taken in advance which will both: a) make the job easier, and b) reduce and possible eliminate that chance of evidence contamination.
Preparation is the key word when it comes to DNA evidence collection. You wouldn't wait until the night of a multi-thousand dollar safe burglary to order footwear casting and fingerprint supplies from the manufacturer would you? Some very low cost supplies can be obtained in advance which will "keep" for a considerable period of time. Paper, plastic, or wooden shafted swabs all work fine, but the durability of the wooden shaft swabs should be considered. A styrofoam block should be obtained and kept in your evidence collection kit. The wooden shaft swabs can be placed, shaft end down, in the block and allowed to dry. Prior to doing so, you may want to affix a piece of double side sticky tape on the bottom of the block to prevent it from tipping over as the swabs are attached to it. Small adhesive labels can be purchased and attached to the swab (prior to the sample being collected) which can be used to identify the swab. When the swabs are dry they should be placed in separate paper envelopes for preservation. Plain letter envelopes work well, although manilla or glycine envelopes are equally suitable.
The cotton swabs, a small glass jar (with a secure lid) of water, latex gloves, envelopes, stickers, a marker, and the styrofoam can be packaged neatly in a tackle box. (I prefer a $1.49 plastic pencil case purchased at the local discount department store). NOTE: In the interest of really saving time at the crime scene, package the swabs in individual envelopes in advance. This reduces handling the swab at the scene - and if using manilla envelopes, place the swab tip-side-down so that it may be removed from the envelope without handling the cotton end.
Tape Lift
Dried blood samples can be conveniently lifted from non-porous surfaces with conventional fingerprint tape. This process is beneficial in that the very size and shape of the stain may in fact be preserved on the lift. Of course, the lift should be placed sticky side down on a piece of plain white typing paper. It is suggested that paper be used (in lieu of plastic or fingerprint backing material) due to the fact that the paper will allow the specimen to "breath" As with the swab, the lift should be packaged in a separate envelope.
Control Samples
When conducting DNA analysis, the criminalist needs to have a "control" sample to compare with the suspect swab/evidence. For this reason, the crime scene examiner needs to document, collect, and preserve a control sample with the same care that the suspect sample is treated. For obvious reasons, it is suggested that the control sample be collected prior to the suspect sample. By collecting the control sample first, there is less chance of contaminating it with the blood or other biological fluid as the subsequent samples are being collected. Also make sure the same water is used to collect both the control and evidence samples. Finally, if cuttings of a suspected sample are being submitted for analysis (ie: a couch) it is preferred that the control samples also be cuttings, rather than swabs or merely fibers.
Other Evidence
Known biological specimens can be collected from both living and deceased persons easily, and we have been doing so for years in the form of sexual assault kit supplies. Known blood in quantity should be collected and preserved in one of the three following tubes:
Grey NaF (blood alcohol)
Purple (EDTA)
Yellow (ACD) sexual assault kit
Red top (plain) or green top (heparin) tubes SHOULD NOT BE USED.
Cheek swabs can be collected from individual and may in fact result in the discovery of some of the most highly concentrated DNA cells. The cheeks swab is non-threatening, in that the individual feels less intimidated by the process. The procedure is quick and simple:
A cotton tipped swab is scrubbed on the inside of the cheek.
No food or drink prior to twenty minutes of the collection.
Preferred that the technique no be used if the mouth is bleeding.
Deceased Individuals
Common sense and knowledge of previously approved practices seem to be the rule when deceased individuals are concerned, particularly when severe decomposition is present and blood work not practical. If hairs are to be submitted, make sure the collector obtain pulled hairs. The tissue associated with the hair root is needed in the DNA analysis. Other samples which may be suitable for DNA analysis include: bones (rib or long bones preferred), teeth, muscle tissue, and associated property which may be found with the body (hairbrush, toothbrush, etc.)
Questioned Evidence
The following are being submitted as miscellaneous tips which should be considered prior to and during the DNA evidence collection process....
positive considerations
saliva: cigarette butts, ski masks, envelopes, stamps.
seminal fluid: oral, rectal, vaginal swabs, clothing.
blood: (if the stain is visible - DNA results are likely)
hair
negative considerations
urine and feces.
biological samples contaminated with soil.
some substrates (jeans - denim) have proven to compromise DNA analysis.
Wear protective outer clothing, as well as the standard latex gloves. Since the crime scene examiner is subjected to exposure to elements, it is recommended that the outer clothing be changed upon returning to the scene after leaving.
Finally - maintain the samples at ambient conditions or cooler. Room temperature is acceptable, refrigeration is desirable, and freezing is preferred.
REMEMBER - practice common sense. Don't let the collection of biological evidence be intimidating. If sound procedures are followed, successful and thorough crime scene work can be accomplished in a safe manner, with valuable evidence in hand.
Posted by
DNAworld
at
4:11:00 AM
0
comments
Hop DNA Extraction Protocol
1. Obtain an adequate amount (~ 1g) of fresh hop leaves and crush them with liquid Nitrogen and a small amount of Carborundum powder (fine 320 grit).
2. Assume 90% of mass is water weight.
3. Add 3.3 ml of buffer per gram of wet (16 ml per gram of dried) hop leaves, and incubate for 1 to 4 hours at 60-65°C.
4. Transfer 900 μl into fresh tube
5. add 600 μl of 24:1 CHCl3:octanol and invert gently (do NOT vortex!).
6. Centrifuge at 5000g for 10 minutes.
7. Transfer supernatant (800 μl) into new 2-ml tube.
8. Add 5μl of RNAase and incubate at 37°C for 30 minutes (or more).
9. Add 0.6 volumes Isopropanol and mix gently by inverting the tubes. Check for DNA precipitation.
10. Spin down for 10 min. at RT.
11. Add 500 μl wash buffer and incubate 10 min. at RT.
12. Carefully remove wash buffer. Don't lose DNA pellet!
13. Briefly centrifuge to collect pellet at bottom of tube - remove any remaining wash buffer.
14. Dry pellet at RT or 50°C to speed up.
15. Add 100 μl ddH2O to dissolve DNA.
16. Store at -20°C until needed.
17. Run electrophoresis for analysis.
Buffer: 100 ml: 50 mM Tris/HCl (ph 8.0), 1.8 M NaCl, 50 mM EDTA. Then add 10 mg/ml of CTAB ( 200 mg per 20 ml buffer, final conc. = 1%) and 1 μl/ml 2-mercaptoethanol (20 μl to 20 ml buffer; final conc. = 0.1%).
Wash buffer 100 ml: 200 μl 5M NH4OAc (final conc. = 10 mM), 76.0 ml abs. ethanol (final conc. = 76%), and 23.8 ml of sterilized water.
Bioprotocols: Hop DNA Extraction Protocol
25 Dec 2010 ... Hop DNA Extraction Protocol. 1. Obtain an adequate amount (~ 1g) of fresh hop leaves and crush them with liquid Nitrogen and a small amount ...
bio888.blogspot.com/2010/12/hop-dna-extraction-protocol.html
Robust CTAB-activated charcoal protocol for plant DNA extraction
author:M KRIŽMAN - 2006
Dried hop cones were obtained from the experimental fields (yield 2005) .... Modification of a CTAB DNA extraction protocol for plants ...
fp.unud.ac.id/biotek/wp-content/uploads/biologisel/ekstraksi-dna.pdf
Isolation of plant DNA: A fast, inexpensive, and reliable method
author:P Guillemaut - 1992
Protocol. Isolation of plant DNA. DNA can be isolated from fresh, frozen, dried or lyophilized material without pretreatment of tissue. The procedure ...
www.springerlink.com/index/2656658377412530.pdf
Posted by
DNAworld
at
4:10:00 AM
0
comments
Transmission electron microscopy DNA sequencing
Transmission electron microscopy DNA sequencing is an emerging third-generation, single-molecule sequencing technology that uses transmission electron microscopy techniques. DNA is visible under the electron microscope; however, it must be labeled with heavy atoms so that the DNA bases can be clearly visualized. In addition, specialized imaging techniques and aberration corrected optics are beneficial for obtaining the resolution required to image the labeled DNA molecule. Transmission electron microscopy DNA sequencing advantageously may provide extremely long read lengths, but it is not yet commercially available.
History
Only a few years aft
er James Watson and Francis Crick deduced the structure of DNA, and nearly two decades before Frederick Sanger published the first method for rapid DNA sequencing, Richard Feynman, an American physicist, envisioned the electron microscope as the tool that would one day allow biologists to “see the order of bases in the DNA chain”. Feynman believed that if the electron microscope could be made powerful enough, then it would become possible to visualize the atomic structure of any and all chemical compounds, including DNA. To this day, despite the invention of a multitude of chemical and fluorescent sequencing technologies, microscopy is still being explored as a means of performing single-molecule DNA sequencing. Two biotechnology companies have conceived of methods for high throughput, direct detection of DNA bases by transmission electron microscopy; however, these studies are still in their infancy and are far from being commercially available. The following progress in these technologies has been reported: 1970 Albert Crewe developed the high-angle annular dark-field imaging (HAADF) imaging technique in a scanning transmission electron microscope. Using this technique, he visualized individual heavy atoms on thin amorphous carbon films. April 2008: ZS Genetics presented its plans for development of a transmission electron microscopy-based single-molecule sequencing platform at the
March 2010: Krivanek and colleagues reported several technical improvements to the HAADF method, including a combination of aberration corrected electron optics and low accelerating voltage. The latter is crucial for imaging biological objects, as it allows to reduce damage by the beam and increase the image contrast for light atoms. As a result, single atom substitutions in a boron nitride monolayer could be imaged. Halcyon Molecular is developing its single-molecule sequencing platform based on the technology utilized in this paper.
September 2010: The Toste research group at University of California, Berkeley, received an Advanced Sequencing Technology Award from the National Human Genome Research Institute to continue research into single-molecule sequencing by transmission electron microscopy, in collaboration with Halcyon Molecular.
Principle
The electron microscope has the capacity to obtain a resolution of up to 100 pm, whereby microscopic biomolecules and structures such as viruses, ribosomes, proteins, lipids, small molecules and even single atoms can be observed.
Although DNA is visible when observed with the electron microscope, the resolution of the image obtained is not high enough to allow for deciphering the sequence of the individual bases, i.e., DNA sequencing. However, upon differential labeling of the DNA bases with heavy atoms or metals, it is possible to both visualize and distinguish between the individual bases. Therefore, electron microscopy in conjunction with differential heavy atom DNA labeling could be used to directly image the DNA in order to determine its sequence.
Procedure of transmission electron microscopy DNA sequencing:
Step 1 – DNA denaturation
As in a standard polymerase chain reaction (PCR), the double stranded DNA molecules to be sequenced must be denatured before the second strand can be synthesized with labeled nucleotides.
Step 2 – Heavy atom labeling
The elements that make up biological molecules (C, H, N, O, P, S) are too light (low atomic number, Z) to be clearly visualized as individual atoms by transmission electron microscopy. To circumvent this problem, the DNA bases can be labeled with heavier atoms (higher Z). Each nucleotide is tagged with a characteristic heavy label, so that they can be distinguished in the transmission electron micrograph.
ZS Genetics proposes using three heavy labels: bromine (Z=35), iodine (Z=53), and trichloromethane (total Z=63). These would appear as differential dark and light spots on the micrograph, and the fourth DNA base would remain unlabeled.
Halcyon Molecular, in collaboration with the Toste group, proposes that purine and pyrimidine bases can be functionalized with platinum diamine or osmium tetraoxide bipyridine, respectively. Heavy metal atoms such as osmium (Z=76), iridium (Z=77), gold (Z=79), or uranium (Z=92) can then form metal-metal bonds with these functional groups to label the individual bases.
Step 3 – DNA alignment on substrate
The DNA molecules must be stretched out on a thin, solid substrate so that order of the labeled bases will be clearly visible on the electron micrograph. Molecular combing is a technique that utilizes the force of a receding air-water interface to extend DNA molecules, leaving them irreversibly bound to a silane layer once dry. This is one means by which alignment of the DNA on a solid substrate may be achieved.
Step 4 – TEM imaging
Electron microscopy image of DNA: ribosomal transcription units of Chrironumus pallidivitatus. The image was recorded with the relatively old technology (ca. 2005).
Transmission electron microscopy (TEM) produces high magnification, high resolution images by passing a beam of electrons through a very thin sample. Whereas atomic resolution has been demonstrated with conventional TEM, further improvement in spatial resolution requires correcting the spherical and chromatic aberrations of the microscope lenses. This has only been possible in scanning transmission electron microscopy where the image is obtained by scanning the object with a finely focused electron beam, in a way similar to a cathode ray tube. However, the achieved improvement in resolution comes together with irradiation of the studied object by much higher beam intensities, the concomitant sample damage and the associated imaging artefacts. Different imaging techniques are applied depending on whether the sample contains heavy or light atoms:
Annular dark-field imaging measures the scattering of electrons as they deflect off the nuclei of the atoms in the transmission electron microscopy sample. This is best suited to samples containing heavy atoms, as they cause more scattering of electrons. The technique has been used to image atoms as light as boron, nitrogen, and carbon; however, the signal is very weak for such light atoms. If annular dark-field microscopy is put to use for transmission electron microscopy DNA sequencing, it will certainly be necessary to label the DNA bases with heavy atoms so that a strong signal can be detected.
Annular bright-field imaging detects electrons transmitted directly through the sample, and measures the wave interference produced by their interactions with the atomic nuclei. This technique can detect light atoms with greater sensitivity than annular dark-field imaging methods. In fact, oxygen, nitrogen, lithium, and hydrogen in crystalline solids have been imaged using annular bright-field electron microscopy. Thus, it is theoretically possible to obtain direct images of the atoms in the DNA chain; however, the structure of DNA is much less geometric than crystalline solids, so direct imaging without prior labeling may not be achievable.
Step 5 – Data analysis
Dark and bright spots on the electron micrograph, corresponding to the differentially labeled DNA bases, are analyzed by computer software.
Applications
Transmission electron microscopy DNA sequencing is not yet commercially available, but the long read lengths that this technology may one day provide will make it useful in a variety of contexts.
De novo genome assembly
When sequencing a genome, it must be broken down into pieces that are short enough to be sequenced in a single read. These reads must then be put back together like a jigsaw puzzle by aligning the regions that overlap between reads; this process is called de novo genome assembly. The longer the read length that a sequencing platform provides, the longer the overlapping regions, and the easier it is to assemble the genome. From a computational perspective, microfluidic Sanger sequencing is still the most effective way to sequence and assemble genomes for which no reference genome sequence exists. The relatively long read lengths provide substantial overlap between individual sequencing reads, which allows for greater statistical confidence in the assembly. In addition, long Sanger reads are able to span most regions of repetitive DNA sequence which otherwise confound sequence assembly by causing false alignments. However, de novo genome assembly by Sanger sequencing is extremely expensive and time consuming. Second generation sequencing technologies, while less expensive, are generally unfit for de novo genome assembly due to short read lengths. In general, third generation sequencing technologies, including transmission electron microscopy DNA sequencing, aim to improve read length while maintaining low sequencing cost. Thus, as third generation sequencing technologies improve, rapid and inexpensive de novo genome assembly will become a reality.
Full haplotypes
A
haplotype is a series of linked alleles that are inherited together on a single chromosome. DNA sequencing can be used to genotype all of the single nucleotide polymorphisms (SNPs) that constitute a haplotype. However, short DNA sequencing reads often cannot be phased; that is, heterozygous variants cannot be confidently assigned to the correct haplotype. In fact, haplotyping with short read DNA sequencing data requires very high coverage (average >50x coverage of each DNA base) to accurately identify SNPs, as well as additional sequence data from the parents so that Mendelian transmission can be used to estimate the haplotypes. Sequencing technologies that generate long reads, including transmission electron microscopy DNA sequencing, can capture entire haploblocks in a single read. That is, haplotypes are not broken up among multiple reads, and the genetically linked alleles remain together in the sequencing data. Therefore, long reads make haplotyping easier and more accurate, which is beneficial to the field of population genetics.Copy number variants
Genes are normally present in two copies in the diploid human genome; genes that deviate from this standard copy number are referred to as copy number variants (CNVs). Copy number variation can be benign (these are usually common variants, called copy number polymorphisms) or pathogenic. CNVs are detected by fluorescence in situ hybridization (FISH) or comparative genomic hybridization (CGH). To detect the specific breakpoints at which a deletion occurs, or to detect genomic lesions introduced by a duplication or amplification event, CGH can be performed using a tiling array (array CGH), or the variant region can be sequenced. Long sequencing reads are especially useful for analyzing duplications or amplifications, as it is possible to analyze the orientation of the amplified segments if they are captured in a single sequencing read.
Cancer
Cancer genomics, or oncogenomics, is an emerging field in which high-throughput, second generation DNA sequencing technology is being applied to sequence entire cancer genomes. Analyzing this short read sequencing data encompasses all of the problems associated with de novo genome assembly using short read data. Furthermore, cancer genomes are often aneuploid. These aberrations, which are essentially large scale copy number variants, can be analyzed by second-generation sequencing technologies using read frequency to estimate the copy number. Longer reads would, however, provide a more accurate picture of copy number, orientation of amplified regions, and SNPs present in cancer genomes.
Microbiome sequencing
The microbiome refers the total collection of microbes present in a microenvironment and their respective genomes. For example, an estimated 100 trillion microbial cells colonize the human body at any given time. The human microbiome is of particular interest, as these commensal bacteria are important for human health and immunity. Most of the Earth's bacterial genomes have not yet been sequenced; undertaking a microbiome sequencing project would require extensive de novo genome assembly, a prospect which is daunting with short read DNA sequencing technologies. Longer reads would greatly facilitate the assembly of new microbial genomes.
Advantages and disadvantages
Compared to other second- and third-generation DNA sequencing technologies, transmission electron microscopy DNA sequencing has a number of potential key strengths and weaknesses, which will ultimately determine its usefulness and prominence as a future DNA sequencing technology.
Advantages
Longer read lengths: ZS Genetics has estimated potential read lengths of transmission electron microscopy DNA sequencing to be 10,000 to 20,000 base pairs with a rate of 1.7 billion base pairs per day. Such long read lengths would allow easier de novo genome assembly and direct detection of haplotypes, among other applications.
Lower cost: Transmission electron microscopy DNA sequencing is estimated to cost just US$5,000-US$10,000 per human genome, compared to the more expensive second-generation DNA sequencing alternatives.
No dephasing: Dephasing of the DNA strands due to loss in synchronicity during synthesis is a major problem of second-generation sequencing technologies. For transmission electron microscopy DNA sequencing and several other third-generation sequencing technologies, sychronization of the reads is unnecessary as only one molecule is being read at a time.
Shorter turnaround time: The capacity to read native fragments of DNA renders complex template preparation an unnecessary step in the general workflow of whole genome sequencing. Consequently, shorter turnaround times are possible.
Disadvantages
High capital cost: A transmission electron microscope with sufficient resolution required for transmission electron microscopy DNA sequencing costs approximately US$1,000,000, therefore pursuing DNA sequencing by this method requires a substantial investment.
Technically challenging: Selective heavy atom labeling and attaching and straightening the labeled DNA to a substrate are a serious technical challenge. Further, the DNA sample should be stable to the high vacuum of electron microscope and irradiation by a focused beam of high-energy electrons.
Potential PCR bias and artefacts: Although PCR is only being utilized in transmission electron microscopy DNA sequencing as a means to label the DNA strand with heavy atoms or metals, there could be the possibility of introducing bias in template representation or errors during the single amplification.
Posted by
DNAworld
at
4:08:00 AM
0
comments