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Jul 1, 2011

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 Des Moines. PCR is a sensitive, fast, and highly discriminatory method of analysis. One of the most essential aspects of DNA evidence analysis at the lab is that a basis knowledge of evidence collection principles is necessary at the initial stage. PCR allows the criminalist to examine evidence which has been properly collected and preserved with expectations that accurate results will be found as result of the analysis.

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 Des Moines be made with cotton tipped swabs (ie: Q-tiptm). The process is simple, and the following outlines the procedure:

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.

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.

Prepared solutions

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
authorM 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

authorP 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

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 Cambridge Health-tech Institute (CHI) Sequencing Conference in San Diego, held from 23–24 April 2008.

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.

Yeast DNA and RNA Methods

Yeast DNA and RNA Methods

Beta-gal Assay

Bgal filter assay

Bgal liquid assay

Bgal plate overlay assay

Determination of replication Timing of yeast by Comparative Hybridization

Determination of replication Timing of yeast by Density Transfer

Determination of replication Timing of yeast by Transient Hemimethylation

DNA Replication Techniques

Isolation of yeast DNA by smash and grab method

Liquid Sporulation

Plasmid Rescue from Yeast from Liquid Culture and Clony Rescue

Preparation of Yeast DNA Embedded in Agarose Plugs

Quantitative Mating Assay

Rather Rapid Genomic Prep

RNA Prep

ß-GAL Filter Assay

ß-gal Lift method

Yeast Chromosomal DNA Preparation

Yeast DNA Miniprep Protocol (Haber Lab)

Yeast DNA Preparation by rapid glass bead method

Yeast Genomic DNA Extraction 2-D Gels

Yeast Genomic DNA Isoaltion

Yeast Genomic DNA Miniprepartion

Yeast protocol (for microarray)

Yeast quick plasmid DNA Preparation

Yeast RNA Minipreparation

Databases of Yeast Genetics

Yeast Genetics

EMS Mutagenesis

EMS mutagenesis of yeast

Generating a Temperature Sensitive Allele

Genome-wide Gene Expression Analysis

Pheromone halo assay (Dohlman Lab)
UV mutagenesis (Corbett Lab)
Replication timing by density transfer (Fangman / Brewer Lab)
beta-Gal reporter gene assay (Dohlman Lab)
beta-Gal filter assay (Dohlman Lab)
beta-Gal filter assay (Breeden Lab)
b-Gal filter assays (Herskowitz Lab)
EMS mutagenesis of yeast (Hahn Lab)
Replication timing by comparative hybridization (Fangman / Brewer Lab)
Replication timing using transient hemimethylation (Fangman / Brewer Lab)
X-Gal agarose overlay assay (Herskowitz Lab)
Procedure for generating ts alleles of NUT2 (Herskowitz Lab)
Yeast Genetics Protocols (Herskowitz Lab)

Yale Genome Analysis Center

Two-Hybrid System

Brent Lab Two-Hybrid Information

Development of an optimized interaction-mating protocol for large-scale two-hybrid analyses.

GST fusion protein purification from Yeast (Dohlman Lab)

Handling and using insertion libraries

Nature Protocols: Smart-pool-array protocol for a yeast two-hybrid assay.

Performing a hunt by interaction mating (Finely Lab)
The yeast two-hybrid system for beginners (Newman / Collinge Lab)
Two-hybrid system (Millner Lab)
Two-hybrid analysis of genetic regulatory networks (Finely Lab)
Two-hybrid system (Finely Lab)
Two-hybrid system (Gietz Lab)

Two Hybrid System Transformation

Yeast Two-Hybrid : State of the Art (Van Criekinge, Beyaert)

Dec 4, 2010

US Scientists Reverse Signs of Aging in Mice

Elderly mice restored to middle age

Harvard scientists say they were able to reverse signs of aging in mice by tweaking a gene which protects cells from the harmful, cumulative effects associated with growing old.

Harvard scientists say they were able to reverse signs of aging in mice by tweaking a gene which protects cells from the harmful, cumulative effects associated with growing old.

Scientists say they have reversed age-related degeneration in mice, resulting in an improvement in the rodents' fertility and the growth of new brain tissue. But it could be some time before the technique might be used in humans.

Fountain of youth

Scientists at Harvard University's Dana-Farber Cancer Institute in Boston report they were able to reverse signs of aging in mice by tweaking a gene that protects cells from the harmful, cumulative effects associated with growing old.

The gene is involved in the production of structures at the tips of DNA chromosomes called telomeres.

Telomeres are like the plastic caps on the ends of shoe laces that keep them from becoming frayed. In the case of chromosomes, the telomeres protect the strands of DNA from environmental assaults such as chemical and radiation exposure.

But every time a cell divides, its telemeres shorten, eventually leading to DNA damage and aging.

In studies with mice, researchers switched off the telomerase gene and watched the rodents rapidly develop age-related impairments.

Eternally young?

However, when they turned the genes back on on, the animals' declines reversed.

"Their fertility was restored. We also saw a big effect on the lining of the intestines and as well as in the brain, which was a little bit unexpected," says lead researcher Mariela Jaskelioff. "We actually saw a decrease in the size of the brains of these mice with premature aging. And we could reverse these by reactivating telomerase."

The mice in the study were at an age equivalent of an 80- or 90-year-old human. Researchers restored them to middle age by turning on the telomerase gene.

Despite the encouraging results, the genetic manipulation is not the secret to eternal youth for humans. Jaskelioff says the telomerase gene is involved in the growth of both normal and cancerous cells.

"The fear is that in humans, adult humans, we accumulate mutations all through our lifetimes," she says. "And if we were to reactivate telomerase in cells that have malignant mutations, then the propensity to develop cancer would probably be exacerbated."

However, according to Jaskelioff, it might be possible to stimulate the telomerase gene for short periods of time in people with a rare disorder which causes premature aging.

Scientists describe how they reversed aging in mice in an article published in the journal Nature Medicine.