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<p><img src="dekkercover.jpg" height="120"><br>
<font size="-1">Source: 
Benecke M (2005) <br><i>Collection and handling of forensic DNA samples</i><br> In: Fuchs J, Podda M (eds.)<br> Encyclopedia of Diagnostic Genomics and Proteomics (EDGP)<br> Dekker, New York (DOI: 10.1081/E-EDGP-120020544; <br>ISBN 0824755022 (print) &amp; 0824755014 (web), Vol. 1, p. 500--504)<br>
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<HEAD><H1>Collection and handling of forensic DNA samples</H1></head>
<b><P align="justify"><a href="index.html">Mark Benecke</a>, Forensic Biologist </P></b>

<p align="justify"><a href="dnacollection.pdf"><img src="dekkercover2.jpg" alt="DNA Collection" height="400" ALIGN="RIGHT" HSPACE=5 VSPACE=2></a>

<b>Introduction</b>

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Under dry and cool conditions, DNA is a relatively stable molecule. From a technical and criminalistic point of view, DNA can be collected and stored like most visible biological stains. Crucial steps are examination of evidence including photographic documentation, and storage under dry and cool conditions. Special aids such as sexual assault kits, swabs, drying devices, and filter paper treated with denaturants are available and should be used. However, DNA collection in forensic environments is not a merely technical but also a criminalistic task. Two questions are of special importance: 1) whether a stain is of relevance for the actual crime, e.g., if it could have been left at the scene some time ago by persons who are not related to the crime, and 2) if a stain should be used for extraction straight away, or stored as long as possible for morphological measurements and crime (scene) reconstruction, e.g., the form of blood stains on wallpaper, the exact location of sperm stains on clothing, or the exact location of skin cells found on furniture.
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<b>Evidence Examination</b>

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Irrespective of possible chain of custody rules, examination of evidence starts with photographic and/or drawn descriptions of the items received by the forensic biologist. In every photograph, an absolute scale must be visible (millimeters/centimeters; no pennies, no matches). Resolution should be 4 MPixel to allow blowing up of the pictures. Flash light should be avoided because brighter parts of the objects will flash out (become white; a digital artefact).

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Biological stains that were detected either by their surface properties (detection by touch: e.g., sperm stains on dark clothing), monochromatic light (e.g., saliva), or regular bright light (e.g., hair or small blood stains) are circled and numbered by use of a water-resistant pen or neon color.


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<b>Collection of Biological Stains

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Swabs</b>

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Practically all stains can be collected by rubbing them off with a cotton swab. Stains on fabric should be cut out first. Swabs are soaked with one drop of fresh distilled sterile water. After transfer of the stain to the swabs, they must be dried immediately. Then, they are put in paper containers. The most convenient way to dry swabs is to put them straight into a closed cardboard box at room temperature (Fig. 1). There, they can neither touch neighboring objects nor develop mold. (1)

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<img src="dekkerfig1a.gif" ALIGN="LEFT" HSPACE=5 VSPACE=2>
<i>Fig. 1. Cardboard box allows simple and safe drying and storage of swabs. [Swissforensix, Switzerland (patented).] </i> 


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In professional forensic environments, contamination caused by airflow during the drying process has not been reported to be a problem. Some laboratory manuals ask for drying in closed cupboards (Fig. 2) or under sterile laminar airflow. Cupboards must never tightly close to avoid building up of humidity and mold.

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<img src="dekkerfig2a.gif" ALIGN="right" HSPACE=5 VSPACE=2>
<i>Fig. 2. Some laboratory standard procedures request cupboards to be used for drying of swabs. Sterile conditions are not necessary; contamination is not a problem as long as the swabs do not touch each other or the wall. Note that the cupboard must not be tightly closed to avoid building up of humidity. </i>


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Swabbing is performed by intense, multiple rubbing of the stained surface to collect a maximal amount of DNA. Inside of oral cavities, the cotton swab is rubbed against the mucous membrane; saliva alone may not contain enough cells.


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After complex shooting situations, used bullets can be matched to the victims by swabbing off traces of tissue that remain on the bullet once it enters the body. (2)

<br><br><b>

Early Swabbing
</b>

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Swabbing of clothing items, especially of skin, should be performed as soon as possible in forensic and police investigations. For example, DNA typing was possible in the following cases where swabs had been collected early at the scene of the crime. Before swabbing, intelligent criminalistic assumptions concerning the location of the invisible yet possible stains had been made.

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Epithelial cells of an unknown suspect were swabbed off the front side of a collar of a polo-neck pullover. The victim had been stabbed; the stains had not been visible on the collar.

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DNA contained in epithelial cells that had been transferred by saliva of an offender was swabbed off the skin of an experimental victim that had showered. Amplification of the offender's STRs was successful up to several hours after transfer of his saliva to the skin of the victim.

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In contrast to common belief, corneocytes contain DNA. Therefore, all surfaces that may have been touched by an offender (through grabbing of ropes, wearing of baseball caps, hitting a person, inside of gloves) may be swabbed successfully. (3-5)




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Early swabbing is also necessary whenever cells from the top edge of bottles, beer cans, etc. are collected. Collection of the complete bottle or can frequently leads to spilling of its contents and dilution or washing off the cells. If early swabbing is not possible, the liquid must be drained out of the container by drilling a hole in its bottom.


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Filter Paper<br><br>
</b>

Liquid blood can be stored on filter paper that is then dried in the same way as cotton swabs. Filter paper that contains denaturants, buffer, and a free radical trap (e.g., FTA paper (6)) will lyse the blood cells and immediately deactivate bloodborne pathogens such as herpes, cytomegalovirus, and HIV.

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Filter paper can also be used to store saliva and liquids from decomposed bodies. In automated laboratories, standard-sized filter paper is the preferred option. Pieces can easily be punched out of it by a machine and subsequently processed by a DNA extraction and PCR robot. The advantage of FTA paper over regular filter paper is that it can be used for multiple PCR reactions. Template DNA will stick to FTA paper after washing off the PCR products and can then be reused.

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Urine and Feces</b>

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Because feces is found especially at scenes of (serial) burglaries, it should be collected irrespective of its repulsive nature. Fresh feces as well as liquid urine should be frozen below - 20&#176;C to avoid bacterial activity. DNA typing of urine is successful especially if it was excreted in the morning (when the highest number of epithelial cells are found compared to the rest of the day), and from feces after PCR inhibitors are removed. To recover the cells, urine needs to be centrifuged (cells are located in the sediment), whereas stool samples can be extracted straightaway or from swabbings with mini spin columns. The estimated number of up to 6 × 105 pg human DNA/mg stool is never reached in practice because of bacterial and digestive action; nevertheless, up to 170 pg DNA/mg stool were successfully extracted and amplified under case work conditions. (7&amp;amp;8)

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<b>
Sexual Assault Kits
</b>

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After sexual assaults, biological stains are often collected in a hospital environment, at home, at a general practitioner's office, or at a police station. To avoid contamination of the samples and to allow full collection following a checklist, sexual assault kits are available. Their use is generally and strongly recommended to guarantee collection of all stains in the best possible way even under highly stressful conditions or in cases where lay personnel has to collect the evidence. The kits consist of prepacked envelopes in a cardboard box, which can be stored and stacked at room temperature (e.g., Sexual Assault Care Kit, University of Bern/Swissforensix, Fig. 3). The envelopes contain swabs, combs for hair (head and pubic), filter paper, sterile distilled water ampoules, large paper bags, and standardized protocol sheets. (1)

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<img src="dekkerfig3a.gif" ALIGN="LEFT" HSPACE=5 VSPACE=2>Fig. 3. Sexual assault kit (University of Bern/Swissforensix, Switzerland): Standardized descriptions and checklists are printed on the actual envelopes that contain the collection materials. </i>

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<b>Skin Line Prints ("Fingerprints")</b>


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DNA is resistant against many histological stains, including substances used to develop fingerprints (or other skin lines). DNA typing was successful from developed skin line prints after cyanoacrylate (super glue fume) or color reagents such as amido black, leucomalachite green, Hungarian Red, DFO, or luminol had been applied. (9&amp;10)


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Developed skin line prints should first be documented with a high-resolution camera (5--12 megapixels) or scanner (1200 dpi minimum). The original skin line prints can then be submitted to DNA storage and extraction like any other biological stain. The stronger the initial fingerprint, the more likely a DNA profile may be obtained.


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<b>


Storage and Extraction
</b>
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Dried biological samples should be stored in standardized paper bags (envelopes, brown paper bags) in a dry and cool environment. This will preserve the DNA over months to years. If dry samples need to be stored for more than 2 years, freezing below - 20&amp;deg;C is recommended. To avoid paper layers sticking to each other in the freezer, the envelopes should be put into plastic bags. Never write on plastic surfaces that become frozen because any type of ink will easily come off. Use paper labels instead.
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In temperate parts of the world, DNA was successfully extracted out of clothing and smears on slides that had been stored more than 10 years in dark environments at room temperature. In tropical countries, freezing is always necessary because of the high humidity, which allows bacteria and mold to build up.


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Biological stains on glass slides, either embedded (histological tissue samples) or just regular smears (vaginal smears or blood), generally lead to good extraction results. The slides should be stored in standard cases for microscopic slides. Alternatively, they can be fixed with sticky tape inside of a paper envelope. Traces of dust generally do not affect the quality of dry stains but should obviously be avoided.
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Insects collected at crime scenes or from corpses should not be dried because museum beetles will frequently destroy the samples within months. The insects should be preserved in 90% EtOH. At room temperature, DNA extraction of such material will then be possible up to several weeks after storage; at temperatures below - 20&#176;C, extraction will be successful for several years. (10-12)  Never use formalin to preserve samples; it will degrade the DNA.


Cigarette butts, envelopes with stamps, fingernail clippings, and dried nasal secretions should be stored dry in paper bags, envelopes, or cardboard boxes. Fingernails can be thoroughly swabbed if clipping is not an option. (13-15)


Because telogenic hair and broken-off hair shafts have been successfully used for DNA extraction, hair should be carefully stored, e.g., by attaching one end of every hair with sticky tape to the inside of an envelope or between two layers of filter paper. If hair is collected by the police using sticky tape for fiber collection, all material (fibers, lint, and hair) should remain on the tape until extraction becomes necessary. (16&amp;17)

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<b>Extracted DNA Stored in Buffers</b>

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Depending on the applied extraction method, DNA stored in TE [10 mM Tris--HCl (pH 7.5), 1 mM EDTA] or similar buffers may be stable for weeks (after Chelex extraction) or months (phenol/chloroform extraction or use of spin columns) in the refrigerator at + 4&#176;C to + 12&#176;C. Freezing of extracted DNA in TE buffer below - 20&#176;C will preserve the sample for years. Before freezing, it is strongly recommended to distribute the DNA in small aliquots (e.g., 10 &micro;L each) to avoid repetitive thawing and freezing of single samples.


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 <b>Emergency Buffers</b><br><br>

The standard storage buffer for extracted DNA is TE buffer. Before its use, the buffer is autoclaved or cleaned with a sterile filter. It can then be stored at room temperature. Under extreme conditions in the field, if drying of the samples is impossible (because of dust, humidity, chaotic mass disaster environments), TE can be used to collect samples by aliquoting 1 mL TE into sterile 1.5-mL plastic tubes. The collected biological stains can be put inside these emergency containers. DNA must still be extracted as soon as possible, and the samples should be stored as cool as the situation allows.
<br><br><b>
Main Destructive Influences on DNA
</b>
<br><br>
Under the influence of UV light (including sunlight) and acids, DNA contained in biological stains as well as extracted DNA breaks into pieces (degrades). Depending on the intensity of fragmentation, PCR might still be possible. Humidity does not directly affect DNA but will allow mold and bacteria to destroy the sample including the DNA within days. Frequent freezing and unfreezing of stains or extracted DNA will also lead to degradation. Household use of detergents and cleaners does not necessarily destroy DNA. (19)  Sperm heads on fabric can survive machine washing at 30--40&#176;C if no bleach was used.

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If more than a few hours are expected to pass before freezing or drying is possible, any solid biological sample in the field should be stored in centrifuge tubes containing aliquots of 95% EtOH. At room temperature, this will preserve the sample's DNA for weeks. (18)

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<b>Contamination</b>

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Under conditions of normal case work, contamination is only observed after careless manipulation or purposeful spraying of high (nanogram) amounts of DNA near or directly into open tubes before PCR. Secondary transfer via door handles, etc. is only a problem under extremely careless, unprofessional conditions. (20&amp;21)

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Obviously, mixtures of DNA might be present in the samples themselves. Mixtures of epithelial cells with sperm can be separated by differential lysis (separation of sperm from epithelial cells (22)). Other mixtures may show distinctively different peak heights after electrophoretic separation of the PCR products. For example, an object at a scene of crime may have been touched by Person A days before a biological stain (such as blood) of Person B is deposited on the same surface. In that case, a DNA mixture might be present later. It can often be detected by the different peak heights of the STR alleles.
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Irrespective of the possible presence of mixtures, swabbing is always recommended if the items cannot be moved, are bulky, or if the stain is located on a person. Subsequent procedures like differential lysis should not be performed before DNA extraction becomes necessary. Generally, once evidence examination is completed, all biological samples should simply be stored cool and dry, and left intact as long as possible.
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<b>
Withdrawal of Samples out of Storage

</b>
<br><br>
If parts of a stored biological sample need to be withdrawn for DNA extraction, forceps and scissors must be wiped with paper towels and 70% EtOH (or methylated spirits) every time they are used. In routine use, cross-contamination caused by wiped, smooth-surface forceps has not been observed. An exception is forceps with grooves. They must be autoclaved before every use because the groves quickly fill up with contaminants.

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Still, especially during evidence examination and withdrawal, it is essential to take care of cross-contamination caused by contaminated distilled water, touching the swabs with used gloves, etc. Standard bacteriological procedures are an optimal guide.

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Sample Retainment</b>
<br><br>

It is recommended to always retain at least half of a stain in storage. One reason is that extracted DNA in liquid buffers is less durable than the original, dried stain. In addition, the defense should have a chance to reexamine the stain beginning with the original sample, not the extracted DNA. Only if DNA extraction and PCR seem to fail because of low amounts of DNA may stored samples be used up completely. This needs the consent of the prosecutor's (D.A.'s) office. Even in these cases, at least a minute amount of the original material should be stored so that future DNA technologies may be applied later on.
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Conclusion</b>
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Collection of biological stains should be documented by photographs and drawings. Dry and cool storage will allow biological samples to be stored over years.
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Extraction of DNA should be performed only if necessary for a current investigation; the original stains should never be extracted completely. Contamination in the laboratory does not occur if the sampling is performed by trained personnel. Because many surfaces and even stains like fingerprints (skin lines), corneocytes on ropes, telogenic hair, the surface of skin after showering, etc. may contain material that is suitable for DNA typing, intelligent criminalistic decisions have to be made before collecting the evidence.
<br><br>

Intense swabbing and the use of sexual assault kits are simple yet very important procedures that guarantee maximum yield of DNA and collection of biological material even if it is not visible at the moment of collection. Even difficult stains such as feces can be extracted and should be stored frozen whenever possible. Under extreme field conditions, 90% EtOH may be used as a collection and storage liquid.
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References</b>
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1   Hochmeister M., Eisenberg A., Budowle B., Binda S., Serra A., Whelan M., Gaugno D., Fitz J., Dirnhofer R., The Development of a New Sexual Assault Kit for the Optimization of Collection in Handling and Storage of Physical and Biological Evidence, Proceedings of the First European Symposium on Human Identification, Toulouse, France, 1996, Promega, Madison, WI, 1996.

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2   Benecke M., Schmitt C., <a href="elpho2.html">Five cases of forensic short tandem repeat DNA typing</a>, Electrophoresis, 18  (1997) 690--694.

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3   Wiegand P., Heide S., Stiller D., Kleiber M., DNA typing from epithelial cells recovered from the pullover and neck of the victim, Rechtsmedizin, 8  (1998)  226--228.
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4   Hammer U., Bulnheim U., Karstädt G., Meissner N., Wegener R., DNA typing of epidermal cells transferred after physical violence, Rechtsmedizin, 7  (1997)  180--183.
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5   Banaschak S., Baasner A., Driever F., Madea B., Interpretation von DNA-Spuren auf einem K&ouml;rper, Rechtsmedizin, 11  (2001)  148.

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6   Burgoyne L., A. Solid Medium and Method for DNA Storage US Patent 5,496,562 (1996)

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7   Prinz M., Grellner W., Schmitt C., DNA typing of urine samples following several years of storage, Int. J. Leg. Med., 196  (1993)  75--79.
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8   Martin L., STR Typing of Nuclear DNA from Human Fecal Matter Using the Qiagen Quiamp Stool Mini Kit, Twelfth International Promega Symposium on Human Identification, Biloxi,MS, 2001, Promega, Madison, WI, 2001.


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9   Van Oorschot R. A.H., Jones M. J., DNA fingerprints from fingerprints, Nature, 387  (1997)  767.

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10   Stein C. S., Kyeck S. H., Hennsge C., DNA typing of fingerprint reagent treated biological stains, J. Forensic Sci., 41 (1996)  1012--1017.

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11   Butler J. M. Forensic DNA Typing: Biology &amp; Technology Behind STR Markers,  Academic Press, San Diego, 2001.
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12   Benecke M., Wells J. Molecular Techniques for Forensically Important Insects, Entomological Evidence: Utility of Arthropods in Legal Investigations, Byrd J. H., Castner J. L. CRC Press, Boca Raton, 2000, pp. 341--352.
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13    Progress in Forensic Genetics, Brinkmann B., Carracedo A. Excerpta Medica International Congress Series 1239, Elsevier, Amsterdam, 2003.Vol. 9
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14   De Stefano F., Bruni G., Casarino L., Costa M. G., Mannucci A., Multiplexed DNA markers from cigarette butts in a forensic casework, Adv. Forensic Haemog., 6  (1996)  252--254.

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15   Wiegand P., Bajanowski T., Brinkmann B., DNA typing of debris from fingernails, Int. J. Leg. Med., 196  (1993) 81--83.

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16   Hellmann A., Rohleder U., Schmitter H., Wittig M., STR typing of human telogenic hairs—A new approach, Int. J. Leg. Med., 114  (2001)  269--273.
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17   Higuchi R., von Beroldingen C. H., Sensabaugh G. F., Erlich H. A., DNA typing from single hair, Nature, 332  (1988) 543--546.
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18   Borowsky R., Field preservation of fish tissues for DNA fingerprint analysis, Fingerpr. News, 3  (2)  , (1991)  8--9.
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19   Van Oorschot R. A.H., Szepietowska I., Scott D. L., Weston R. K., Jones M. K. Retrieval of Genetic Profiles from Touched Objects, First International Conference on Forensic Human Identification in the Millennium,  Forensic Science Service, London, 1999.
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20   Scherczinger C. A., Ladd C., Bourke M. T., Adamowicz M. S., Johannes P. M., Scherczinger R., Beesley T., Lee H. C., Systematic analysis of PCR contamination, J. Forensic Sci., 44  (1999)  1042--1045.
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21   Ladd C., Adamowicz M. S., Bourke M. T., Scherczinger C. A., Lee H. C., A systematic analysis of secondary DNA transfer, J. Forensic Sci., 44  (1999)  1270--1272.
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22   Gill P., Jeffreys A. J., Werrett D., Forensic application of DNA fingerprints, Nature, 318  (1985)  577--579.
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