USFWS
Genetic Monitoring for Managers
Alaska

 

DNA Collection and Archiving

Proper techniques for collection

After identifying the appropriate type of sample to be collected, the first step to ensuring reliable genetic analysis is to collect each sample in such a manner as to minimize contamination and degradation. This may mean the sample is temporarily placed in a different medium than what will be used for long-term storage.

Collection methods are extremely varied, ranging from truly noninvasive methods such as scat or shed feathers, to considerably more invasive approaches including biopsy darts and blood samples taken from live-captured animals. Typically, more invasive collection methods will yield higher quality/quantity DNA samples and have less opportunity for contamination or degradation.

Conversely, noninvasive collection methods such as hair or scat begin to degrade immediately after being deposited by the animal. Depending on the field conditions, DNA degradation can be rapid. For example, scat samples (or at least the DNA within them) may not persist for more than a couple of days under tropical conditions or in places where scavengers will utilize the scat (Murphy et al. 2007, Sanchez et al. 2004). Therefore, in addition to collection methods, sampling design itself must be influenced by collection conditions.

Noninvasive methods often also carry a risk of contamination by multiple individuals. One of the most common applications of noninvasive sampling is hair left on barbed-wire "hair traps." Although relatively rare, multiple, sometimes closely related individuals can leave hair on the same barb. This usually prevents indentification of individuals due to the presence of >2 alleles at >1 locus. As with issues of degradation in the field, contamination can be reduced with frequent visits to sampling stations. However, the frequency of visits must be balanced with other concerns such as budgetary limitations, logistical realities, and allowing adequate time for animals to encounter stations and deposit samples.

When determining the nature of the samples to be collected and how to store them (both short and long term) keep in mind other potential applications. Beyond genetic analysis, many types of tissue can yield other valuable information about the sampled individuals: stress and reproductive hormone levels, stable isotopes (diet), and elemental analysis (e.g., contaminant load).

Tenotyping success rates deciline sa DNA deteriorates and breaks into smaller pieces.
Common threats to sample integrity include:

Moisture: plastic holds moisture and causes o f DNA degradation while, paper breathes and allows dessiccation at lease in dry climates
UV radiation: keep samples out of the sun
Freeze / thaw cycles: If the sample becomes frozen, keep it frozen!
Age: use samples that are as fresh as possible
Composition of sample: composition may affect sample analysis, e.g. animal diet can affect genetic analysis of scat (Murphy et al. 2003)
Mixture: separate container for each sample, sterilize tools between each sample, use sample containers that don’t leak

Additional media: additives to prepare/preserve the sample, e.g. blood anticoagulant, lysis buffer, preservative

The following lists collection media and short-term storage methods recommended for various DNA-sources:

Sample Type
Collection Media Short-term Storage
Blood
Blood Buffer Room Temperature
Blood
Filter Paper in Envelopes
Dry, Room Temperature

Blood quills

Tissue Preservation Buffer
Room Temperature
Bone Envelopes or Dry Microtubes
Dry, Room Temperature
Egg shell membranes Envelopes or Dry Microtubes
Dry, Room Temperature
Feathers from museum skins Envelopes Dry, Room Temperature
Feathers from nests Tissue Preservation Buffer Room Temperature
Feathers from nests Envelopes Dry, Room Temperature
Hair Envelopes or Dry Microtubes Dry, Room Temperature
Muscle Tissue Preservation Buffer Room Temperature
Scat Ethanol Room Temperature
Scat Silica Beeds/Gel Room Temperature
Teeth Envelopes or Dry Microtubes Dry, Room Temperature

*table adapted from http://www.absc.usgs.gov/research/genetics/dna_sampling_protocol.htm

Sample Collection
Almost all wildlife genetic studies require complex and expensive field operations to obtain samples. In fact, once field personnel, transportation, equipment, housing, communications, and other field costs are accounted for, laboratory costs often pale in comparison. In some instances, field data are meticulously collected, yet samples are treated improperly or are inadvertently contaminated. Below we discuss the handling and treatment of field samples.

Contamination
Contamination can occur in the field or laboratory and can be a major concern for noninvasive sampling studies (depending on the objective). In the field, for example, contamination can be caused by baits, lures, previously handled animals, accompanying pets, or field personnel. Considering that the target sample may comprise only a few cells at the end of a hair, it is important to limit contact with material that can mask the target sample. We recommend the use of new latex gloves and sterile mechanical devices (e.g., tweezers, wooden picks) for handling all samples in the field. Gloves should be changed between the handling of different samples, and mechanical devices can be sterilized with ethanol and a lighter, washed in a weak bleach solution, or replaced between samples. Some of these safeguards can be diminished depending on the research question at hand. For instance, if microsatellites are to be used to determine the individual identification of nonprimates, contamination from field personnel is less of a concern-although cross-sample contamination must still be guarded against. But contamination can be an issue for studies that use universal genetic tools (e.g., mitochondrial DNA to identify species for estimating occupancy, or gender-specific markers to identify sex). Given that the goals of many studies change over time, we recommend implementing protocols that minimize all types of contamination.

Every laboratory has protocols in place for detecting contamination (see Selecting a lab/geneticist for what to consider when choosing a laboratory), and laboratories that routinely process noninvasive genetic samples have separate facilities for receiving and extracting DNA. In addition, the bleaching or UV irradiation of laboratory surfaces is routine. Furthermore, laboratories specializing in noninvasive genetic sampling may limit access to areas where noninvasive samples are analyzed and discourage technicians from entering a main laboratory before processing samples in the satellite facility. Finally, all laboratories will routinely run both positive (known genotypes) and negative controls (e.g., samples comprising simply distilled water) to detect laboratory contamination. While such measures minimize contamination from other samples or PCR products found within the laboratory, even the most stringent lab will unlikely be able to discern field contamination (e.g., the cross-contamination of samples between hair snares as a result of improper handling).

Labeling, tracking, and shipping genetic samples  
It is critical to have strict, well thought out protocols for label and track samples in the project office , in the field and at the genetics lab, especially for larger studies. Each sample requires a unique and obvious identifier. While this may seem trivial, it is not uncommon to end up with envelopes or vials having similar labels if multiple field crews are working concurrently (e.g., “sample collected 9/27/04, hair #1”). If samples arrive at the lab without clear and accurate documentation, they may become useless due when study objectives require error-free collection location and dates. Thus, we recommend that a designated field coordinator be assigned to organize all samples, ensure that each sample is assigned a unique identifier, compile a master list, and send all this information to the lab. Our second recommendation is to use a barcode system; a number of labs that process many samples have purchased bar code readers in recent years (see figure 6.16 for a description of barcode labeling). From the perspective of the lab, the following information is helpful to include in a shipment of samples: 1 .Sample list. Many researchers include an electronic and hard copy sample list with their samples. Often, this list includes the field data associated with a sample (e.g., location, collector, comments). After genotyping a sample, genetic data is entered into the initial electronic sample list. This reduces transcription errors and errors associated with manipulating spreadsheet files at the lab. 2. Copies of the necessary permits. Many samples are collected under state, national, and international permits. Most labs  maintain a file of these permits. Additional permits (e.g., CITES permits) may be necessary if samples are from species of concern and are sent to a lab outside the country of collection. 3. Chain of custody form. If samples are potentially contentious, a chain of custody form should be completed to track access to each sample.