DNA sample Collection procedures.

(a) DNA samples will be collected, handled, preserved, and

submitted to the FBI in accordance with FBI guidelines.

(b) CSOSA has the authority to use such means as are reasonably

necessary to collect a sample from an individual who refuses to

cooperate in the collection of the sample. Unless CSOSA determines that

there are mitigating circumstances, CSOSA will consider that an

individual is refusing to cooperate if:

(1) The individual is being ordered or transferred to CSOSA's

supervision, but fails to report to CSOSA for collection of the sample

within 15 business days of being sentenced to probation or being

discharged from a correctional institution; or

(2) The individual is already under CSOSA supervision and has been

notified by his or her Community Supervision Officer of the time to

report for collection of the sample, but fails to report for collection

of the sample; or

(3) The individual has reported to CSOSA for collection of the

sample, but fails to provide the sample after being given a minimum of

one hour to do so; or

(4) The individual specifically states that he or she will not

cooperate.

(c) When an individual has refused to cooperate in the collection

of the sample, CSOSA deems the following to be reasonably necessary

means for obtaining the sample:

(1) Impose administrative sanctions;

(2) Request a revocation hearing by the releasing authority; and/or

(3) Refer the individual who refuses to cooperate for criminal

prosecution for a class A misdemeanor pursuant to section 4(a)(5) of

the DNA Analysis Backlog Elimination Act of 2000 (42 U.S.C.

14135b(a)(5)).

References:

STATE CRIME LABORATORY Chapter 10-17-01 DNA Analysis.

DNA identification information: collection.

DNA Identification in Mass Fatality Incidents

Genetic Privacy Act: File 5

DNA identification information: collection from certain offenders.

Application of dry storage matrices for DNA sample collection and preparation for forensic analysis.

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.

Sample protocol for material to be used in DNA barcoding

Effective DNA barcoding depends on the quality of the biological material. Following this simple sampling protocol will ensure proper preservation of biological samples for DNA studies.

For mammals, fish, birds and large invertebrates

1. Freeze whole individual specimens in plastic bags; use a write-on label to record vessel/expedition name/code, locality or station number, latitude and longitude, date, species name and collectors name. Store labelled specimens in freezer.

2. For large specimens that are impractical to freeze and return to the lab, take a small piece of muscle tissue from any location on the body (a half thumb size piece of muscle tissue) and freeze in a labelled clip-top mini-grip bag or a cryo-vial. Label the bag/vial with vessel name/code, locality or station number, date, species name, and name of scientist making the identification. Photograph the whole specimen before discarding, and cross reference the digital photo to the tissue sample code. It is essential that species-diagnostic characters can be seen on the photograph.

3. Avoid formalin work areas for handling specimens.

For small fish and invertebrates

1. Use 96% pure ethanol (~80-85% for fragile arthropods) for fixation and preservation. Do not use denaturised alcohol.

2. Label all samples with locality, coordinates, date and collector.

3. If sample contain considerable amount of water (e.g. kick samples etc.), exchange the sample liquid with fresh ethanol after a day or two if possible.

4. Always fill sample containers full with ethanol to avoid damage to material during transport. Record specimen collection data on a waxy paper label, use pencil. Add label to ethanol filled jar. Record the vessel/expedition name/code, locality or station number, date, species name, and name of scientist making the identification.

5. Keep samples cool and dark (to avoid DNA degradation).

If ethanol is impractical or unavailable in large amounts, the samples (or specimens) can be subsampled in ethanol (as above). Cross reference labelling with unique identifiers is important to link subsamples or tissue samples with primary samples.

Formaldehyde solutions degrade DNA. If samples must be fixed in formaldehyde, make sure that they are kept cool and transferred to ethanol as soon as possible (at the latest within 14 days). Also make sure that the ethanol is exchanged with fresh ethanol after a few days. Note the formalin fixation on the sample record

Specimens that must be kept dry for morphological studies (such as butterflies) should be kept frozen (at -20°C or lower temperatures) or quickly dried in an oven or incubator.

For vascular plants

High-quality DNA is most easily obtained from plants when the tissue is dried rapidly. Botanists now routinely use silica gel for field preservation of leaf material for DNA analysis. Silica gel can be purchased from most biological supply companies.

1. The ratio of silica gel to leaf tissue should be approximately 5-10:1. For best results, tissue should be completely dry within 24 hours.

2. Choose green, healthy leaves from a single individual plant. In general, a minimum of 2-3 square cm is necessary for 1-2 DNA extractions, but some small individuals will have less leaf material. A good rule of thumb to follow when deciding how much leaf material to sample is: more is always better.

3. Keep track of the individual from which you sampled leaf material. Small tags work well and they do not interfere with regular pressing activities. It is critical that voucher specimens be preserved from individuals that have associated material collected for DNA studies. A DNA sample that does not have an associated voucher specimen has extremely limited value in biological research.

4. Place the leaf tissue (or other green tissue if there is not a lot of leaf tissue available) into a small sealable bag with the silica gel. Tear or cut the leaf material into smaller pieces before inserting into the bag – this increases the surface area of the leaf that is exposed to the silica, and the drying process occurs more rapidly.

5. Write the collection number of the individual on a small piece of paper, and insert this into the Ziploc bag. Also write this information on the outside of the bag with a permanent marker.

6. Store the silica gel packets in a sealed bag or container to keep moisture out, and ensure that they do not get re-hydrated over time. For long term storage, silica gel packets can be stored in a freezer.

Collection of Materials for DNA Analysis

Blood (Blood/1 Vial/Blood Buffer/Room Temperature)

For birds, blood is the preferred tissue type for nuclear DNA work, but less valuable than muscle for work that includes mtDNA analyses. We have provided 1.5 mL eppendorf tubes with a blood buffer storage solution (Longmire Buffer). The chemicals are not toxic, but this solution should not be ingested.

The buffer and any blood/buffer combination can be stored at room temperature until you return from the field, at which point they should be frozen. Only a few drops of blood are needed from each bird, enough to turn the buffer red (usually about 5 drops). Blood can be obtained by pricking the tarsus vein and transferring the drops to the tubes by way of capillary tubes (DO NOT USE heparinized tubes). Using a capillary bulb, blood can be blown from the capillary tube into the buffer after collection. If the blood clots inside the capillary tube, break off the tube inside the buffer vial and just leave it there. Blood can also be collected from the brachial or jugular vein with a sterile syringe. Use a new, sterile syringe for each individual.

Blood on filter paper (Blood/Whatman Filter Paper/Dry/Room Temperature)

5-6 drops of blood can be placed on filter paper (Whatman). The paper should be kept separate from other samples to avoid contamination. Allow the damp filter paper to dry and store separately in either: (1) a ziploc bag with silica gel, or (2) a separate envelope.

Blood Quills. (Blood Quills/1 Vial/Tissue Preservation Buffer/Room Temperature)

Blood quills are put into tubes containing tissue preservation buffer provided by our lab. Pull two or more blood quills from the bird. We recommend sampling wing coverts rather than emerging primaries and secondaries, which are more critical to flight. We extract DNA from the bloody “skin end” of the quill, so if the quills will not fit into the tube, trim off the feather tips, leaving the bloody ends (calamus) to put into the tube.

We strongly suggest that latex gloves be used when sampling, and that instruments be cleaned with 10% bleach between sampling. This prevents between-sample contamination and protects the collector from infectious diseases and any preservatives that may have been used in the skin’s preparation.

Tubes can be stored at ambient temperature for shipping.

Egg Shell Membranes (Egg shell membranes/1 Envelope/Dry/Room Temperature)

DNA yields from eggshell membranes are very good, provided there is vascularization on the membrane. The easiest field technique is to collect each membrane and place it in a separate plastic bag: placing all membranes in the same bag causes cross contamination of samples. We do not use the hard shell at all, so that portion can be left in the field. Do not store feathers and eggshells from the same nest in the same bag. Give the nest a number, and then label each feather or egg sample with that number (e.g., nest number 100 has feather sample number 100 and membrane sample numbers 100(1), 100(2), etc.).

Feathers from nests. (Feathers/1 Envelope/Dry/Room temperature)

We get the best results with contour or tail wing feathers (those with a substantial sheath or rachis) deposited in nests or shed by birds during molt. The DNA is actually in the calamus, so feathers without the calamus cannot be used to extract DNA. We are unable to obtain DNA from down feathers. Please collect as many contour feathers from each nest as possible (we use 5 feathers per DNA extraction from geese and at least that many for passerines, but like to have extra in case it does not work the first time around). Feathers can be removed after the nest has failed or hatched, or when first discovered if you don’t plan to revisit the nest. Keep feathers dry after collection, since moisture can cause decay of feathers and subsequently the DNA. Place feathers in paper envelopes or, if bone dry, in plastic bags. Store feathers from different nests in separate bags/envelopes. Feathers do not need to be frozen. Envelopes can be placed in a plastic bag with 2 Tbsp. silica gel to aid in maintaining a dry environment. {See Pearce et al. (1997) for additional considerations when sampling feathers or egg membranes. However, note that chelex extractions are not recommended unless it is anticipated there will be no long-term use of the extracted DNA.}

Feathers from museum skins. (Feathers/1 Vial/Tissue Preservation Buffer/Room Temperature)

Success in extracting DNA from museum skins is variable, depending upon the way the skin was prepared. We have developed protocols for extracting from samples prepared using a number of preservatives (and combinations), including gasoline, arsenic and borax. We generally get better extractions from feathers plucked from museum skins (along with skin at the base of the feather), rather than from snips of skin alone. This may be due to inhibitors from preservatives used on the skins.

To minimize damage to museum skins, try to collect from areas that are less noticeable. Sampling can often be more easily done along suture lines, such as in the area of the cloaca. Feathers from the wing area (such as the marginal coverts) usually yield good DNA (perhaps because preservatives were used less often on the wings), but sampling from this area is difficult to do without affecting the integrity of the skin. Please abide by the instructions of the curators. We like to have at least 5 feathers and associated skin if possible.

Pluck feathers and associated skin from a small area. It helps sometimes to use forceps or tweezers. Take care to support the skin with one hand while gently pulling the sample; this will help to keep the skin from ripping. Place each sample in a sampling envelope and record museum numbering system, and species, sex, age, date and collection location. Often the museums will have much of this information on databanks. Keep the envelope dry.

We strongly suggest that latex gloves be used when sampling, and that instruments be cleaned with 10% bleach between sampling. This prevents between-sample contamination and protects the collector from infectious diseases and any preservatives that may have been used in the skin’s preparation.

If we are preparing skins, we always collect tissue samples from the carcass. We collect heart, breast muscle, and blood as it pools around the heart, and store each in separate tissue preservation buffer vials (see protocols below, and separate multiple tissue sampling protocol).

See Mundy et al. (1997) for additional information about collecting from museum skins.

Fin Tissues. (Tissue/1 Vial/Ethanol/Room Temperature) or (Tissue/1 Container/Dry/Room Temperature)

Fifty fin samples for population genetic analysis and three or four reference samples (whole fish) for phylogenetic analysis need to be collected from each location. There are two methods for sample collection: dry or in vials with 100% ethanol (EtOH). The preferred method of collection is to store the tissue in a vial with 100% ethanol (EtOH). (If this is not possible, the following dry method can be used.)

Use clean scissors or a clean scalpel blade to cut a small piece of tissue from one of the fins of the live fish. Tissue size should be approximately 5 mm2 ( about the size of this block). A wedge from the upper or lower lobe of the tail fin works fine. Because adipose fins contain a lot of complex lipids, they are not an easy target for DNA extraction, although some DNA can be extracted from this tissue. Eroded fins from dead salmon carcasses are highly degraded, and DNA is usually not readily extracted from such tissue. A well-dried 5 cm2 piece of skin tissue works best under these conditions.

The date of collection, fish species and stock, type of collection method, and fish length, sex, and age (YOY, juvenile, adult will suffice) should be collected with each fin where possible.

If samples are to be sent through the mail, ethanol should be drained from the samples immediately prior to mailing; the samples will be rehydrated upon receipt at the Molecular Ecology Laboratory.

Dry Sample Collection (Alternative).

For the dry method, whirl-pack bags, cryo-tubes, or scale envelopes lined with high quality filter paper work well.

Either in the field after collection, or in the office immediately upon return from the field, samples should by air-dried on filter paper or paper towels until all mucus and moisture in the fin has evaporated and the fin feels dry to the touch. Sun drying in the field works best and can be done quickly. Drying fins inside usually takes 18-24 hours at room temperature. Fungus and bacteria immediately invade the fins upon collection and these factors break down the cell walls of the tissue and the DNA exudes into the surrounding medium, making DNA extraction in the lab difficult, if not impossible. DNA from moist-stored fins are often OK for up to 6-8 hours (it depends on the original condition and size of fin clip), but samples are best when packed on ice if drying is to be delayed for over 4 hours.

Dried fin clips should be repackaged separately (make sure the baggy or envelope is dry as well) and attached to field notes for shipment. Dry samples can be sent surface mail without special packaging.

Hair and Hard Tissue (Hair, Bone, or Teeth/1 Envelope/Dry/Room Temperature)

Hairs and hard tissue samples, such as bone or teeth, should be kept as dry as possible. DNA is present only in hair follicles, so hairs without follicles are not useful for genetic analyses. DNA is extracted from tooth pulp, so the whole tooth is preferred. These can be stored in containers or envelopes.

Muscle (Tissue/1 Vial/Tissue Preservation Buffer/Room Temperature)

Muscle tissue samples are the preferred samples for work that includes mtDNA analyses along with nuclear DNA analyses, particularly for birds. Among muscle tissue samples, heart is the most preferred for birds, since the mtDNA yield is very high relative to nuclear yield. DNA can also be extracted from tongue, skin, hair, teeth and bone. Soft tissue samples can be stored at room temperature in the field in the tissue preservation buffer. Any muscle or skin tissue will work and can be stored in this buffer solution. Please make sure that the storage buffer completely covers the tissue sample. Also, make sure to clean instruments between sampling different birds to prevent cross-contamination, using a 10% bleach solution followed by a water rinse. A sample about the size of a pencil eraser is all that is needed, but make sure the sample is entirely submersed in the buffer.

Scat (Scat/1 Vial/EtOH/Room Temperature) or (Scat/1 Vial/Silica Beads/Room Temperature)

Host DNA is very difficult to obtain from scat samples. Because of the low amounts of host DNA compared to bacterial and diet sources of DNA, extreme caution must be used to prevent contamination of one scat sample with scat from another individual. Therefore, we provide gloves, and tongue depressors with each sampling vial to be used only for one vial then discarded. We provide two different types of preservative for scat samples: (1) Liquid ethanol in a 50 ml tube, or (2) Silica beads/gel in a 50 ml tube. Unpublished research from our laboratory and others suggest that ethanol is superior to silica beads/gel for preserving scat samples for DNA analysis.

Regardless of which preservative you have, use a new pair of gloves and a new tongue depressor for each sample handled. If you are directed to aliquot one scat sample between the two preservative types, you do not have to change gloves and tongue depressors. Try to place an amount of scat approximately the size of a golf ball into the collection tube. Do not fill the tube; it is important to leave enough space for the sample and preservative to mix (easier done with the liquid ethanol than the silica beads). Samples are okay left at ambient temperature, but should be kept away from heat and out of sunlight for a few days. It is best to return them to the lab and freeze them as soon as possible. Unpublished data from several laboratories, including ours, indicate DNA yields decline dramatically in samples over about a week old, regardless of collection method. Scat samples preserved within 24 hours of defecation yield the highest amount of host DNA.