Tissue preparation Deparaffinization method Xylene removal To remove the residual xylene, the samples were washed five times with Ethanol as follows. Tissue lysis Phenol-cholorophorm method For the DNA extraction from cell lysate, phenol-chloroform was used as following. References
The microtubes were then left in a 40 ºC oven to dry the tissues.
Mar 5, 2012
A simple method for DNA extraction from formalin-fixed, paraffin embedded tissue blocks
DNA reveals Neanderthal extinction clues
Neanderthals were already on the verge of extinction in Europe by the time modern humans arrived on the scene, a study suggests. DNA analysis suggests most Neanderthals in western Europe died out as early as 50,000 years ago - thousands of years before our own species appeared. A small group of Neanderthals then recolonised parts of Europe, surviving for 10,000 years before vanishing. The work is published in the journal Molecular Biology and Evolution. An international team of researchers studied the variation, or diversity, in mitochondrial DNA extracted from the bones of 13 Neanderthals. This type of genetic information is passed down on the maternal line; because cells contain multiple copies of the mitochondrial genome, this DNA is easier to extract from ancient remains than the DNA found in the nuclei of cells. The fossil specimens came from Europe and Asia and span a time period ranging from 100,000 years ago to about 35,000 years ago. The scientists found that west European fossils with ages older than 48,000 years, along with Neanderthal specimens from Asia, showed considerable genetic variation. But specimens from western Europe younger than 48,000 years showed much less genetic diversity (variation in the older remains and the Asian Neanderthals was six-fold greater than in the western examples). In their scientific paper, the scientists propose that some event - possibly changes in the climate - caused Neanderthal populations in the West to crash around 50,000 years ago. But populations may have survived in warmer southern refuges, allowing the later re-expansion. Low genetic variation can make a species less resilient to changes in its environment, and place it at increased risk of extinction. "The fact that Neanderthals in Europe were nearly extinct, but then recovered, and that all this took place long before they came into contact with modern humans, came as a complete surprise," said lead author Love Dalen, from the Swedish Museum of Natural History in Stockholm. "This indicates that the Neanderthals may have been more sensitive to the dramatic climate changes that took place in the last Ice Age than was previously thought." Neanderthals were close evolutionary cousins of modern humans, and once inhabited Europe, the Middle East and Central Asia. The reasons behind their demise remain the subject of debate. The appearance of modern humans in Europe around the time of the Neanderthal extinction offers circumstantial evidence that Homo sapiens played a role. But changes in the climate and other factors may have been important contributors. "The amount of genetic variation in geologically older Neanderthals as well as in Asian Neandertals was just as great as in modern humans as a species," said co-author Anders Gotherstrom, from Uppsala University. "The variation among later European Neanderthals was not even as high as that of modern humans in Iceland." The researchers note that the loss of genetic diversity in west European Neanderthals coincided with a climatic episode known as Marine Isotope Stage Three, which was characterised by several brief periods of freezing temperatures. These cold periods are thought to have been caused by a disturbance of oceanic currents in the North Atlantic, and it is possible that they had a particularly strong impact on the environment in western Europe, note the researchers. Over the last few decades, research has shown that Neanderthals were undeserving of their brutish reputation. (BBC) Researchers recently announced that paintings of seals found in caves at Nerja, southern Spain, might date to 42,000 years - potentially making them the only known art created by Neanderthals. However, this interpretation remains controversial.
Neanderthals were close evolutionary cousins of our own species - Homo sapiens
The last Neanderthals might have held out in southern Europe until 24,000 years ago
Were Neanderthals responsible for these cave paintings of seals at Nerja in southern Spain?
'DNA robot' targets cancer cells
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
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)
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)
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 …
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.
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