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Progress in the field of forensic DNA profiling

Collecting biological fluid samples on a solid matrix is a standard method in both newborn screening and forensics. Blood spot collection on 903 filter paper for newborn screening was first described by Dr. Robert Guthrie in 1963.1 Heel-stick blood samples were blotted onto 903 filter paper and signature metabolites tested for diagnosing inborn errors of metabolism. Later McCabe et al. (1987) discovered that DNA could be extracted from these dried blood spots (DBS).2 As a result, DBS technology quickly became adopted as a simple means of obtaining DNA samples for human genetics and forensic DNA identification.

While metabolites in dried blood samples, such as those measured in newborn screening tests, are often stable, DNA undergoes degradation over time. In addition, if blood samples are not dried and stored properly at low temperature, they can become contaminated with microorganisms that make DNA analysis impossible. These observations led researchers at Flinders University in Australia to develop a unique chemical treatment of solid matrices that can stabilize DNA at room temperature. The chemically coated matrix, FTA, also circumvented the problem of DNA contamination by inhibiting growth of both bacteria and mold, which was a common problem when samples were kept under conditions of high heat and humidity or were improperly collected. The benefits of DNA stabilization and long term room temperature storage for genetic identification were immediately apparent and led to the FTA matrix being quickly adopted by the forensic community for collecting blood samples for DNA analysis.

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Benefits of FTA

1. FTA preserves and stabilizes DNA, preventing environmentally induced degradation.
In a series of experiments conducted by the Midwest Research Institute (MRI), DNA on an FTA treated and an untreated matrix was challenged with a UV source to induce the sample aging process; DNA degradation was measured using real-time PCR After UV treatment there was a 2-fold loss of DNA on the FTA treated matrix whereas there was a greater than 850 fold loss of DNA on the untreated matrix.

2. FTA allows for the room-temperature storage of biological samples for DNA analysis.
Blood samples stored on FTA matrices at room temperature for over 17 years were tested at the University of North Texas Health Science Center, DNA Identification Lab for the ability to undergo PCR amplification for Short Tandem Repeat (STR) analysis and were found to be fully profiled without stutter, peak drop-ins/outs, or peak imbalance.

3. FTA inhibits microbial growth that can damage DNA.
The Health Protection Agency in the UK tested over 400 strains of bacteria and 72 strains of mold and fungus and found that FTA inhibited the growth of these organisms.

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Adoption of FTA
FTA matrices were developed for the field of forensics as a means for collecting and storing blood samples when DNA preparation was required. With the growth in number of forensic DNA database samples, and the development of the Combined DNA Indexing System (CODIS) program, FTA provided a quick and easy mechanism for collecting, shipping, and storing DNA samples. FTA is used by the majority of state crime labs in America. Several states including Michigan, Florida, Alabama, New York, New Jersey, and Washington have been using FTA in their CODIS databasing programs for more than a decade. FTA is accepted internationally by many police forces in Europe (e.g. France and Sweden) and further a field (e.g. Australia and India).

FTA has evolved into a universal means of preparing DNA from a wide variety of sources for analysis. Outside criminal forensics, the technology has been cited in over 325 papers for isolation of genomic DNA from diverse sample types such as plant, animal, avian, marine, microbial, and viral samples.

Buccal Cell Collection
DNA preparation and handling in the field of forensics is constantly evolving to meet new challenges. Expanding numbers of samples and pressure on crime labs for increased sample throughput have changed the way forensic molecular biology is conducted. One significant change has been the shift from collecting blood samples to buccal samples for CODIS databasing programs. Buccal cell samples are easier to obtain from individuals than blood samples, as law enforcement personnel require very little training before being able to collect samples in this way. One potential challenge with buccal samples, however, is obtaining a consistent sample. This issue has been addressed by collection systems which serve as all in one cell collection and transfer devices that load cells onto the FTA matrix for DNA capture. Whatman’s EasiCollect collection and transfer kits have been adopted by the U.S. Army Crime Lab and in Europe by La Guardia Civil.

Recovery of DNA from FTA Cards
Many laboratories wish to not only amplify DNA directly from the FTA matrix but also to extract DNA for quantitative analysis. Whie several methods have been developed to obtain DNA in solution from FTA punches, commercially available kits are usually preferred over the classic time consuming solvent extraction method.

High Throughput
There has been a rapid increase in the number of DNA databasing samples passing through crime labs due to changes in state and federal legislation such as sampling from all arrestees, which has put pressure on labs to increase sample throughput. Because of this, many labs have adopted automated or semi-automated punching devices and liquid handling robots to process samples and set up amplification reactions. FTA cards are compatible with automation of both disc punching and washing; discs can be punched with devices such as those offered by Perkin Elmer or BSD Robotics. Once FTA cards have been punched and the discs dropped into 96 well PCR plates, further processing can be done by liquid handling robots, using standard protocols.

Recent improvements in analysis of samples collected on FTA cards have centered on development of STR multiplex kits that allow the direct amplification, thereby eliminating the punch washing step or need for quantifying DNA. The Amp FlSTR Identifiler Direct PCR Amplification Kit (Applied Biosystems Incorporated) and the PowerPlex 16HS Kit (Promega Corporation) can directly amplify DNA on an FTA card punch in this way.

Summary
Consistent and reliable DNA preservation, room-temperature storage, ease of use and sample collection, and simple processing of punches for PCR have made FTA the most widely used matrix in forensics. Recent developments in automation and chemistry have made FTA cards easier to use than ever before. FTA technology has grown with the demands and challenges of today’s forensic labs, and has been adopted worldwide as the gold standard for DNA sample collection.

References:

  1. Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 32:338–43. 1963.
  2. McCabe ERB, Huang S-Z, Seltzer WK, Law ML. DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening. Hum. Genet. 75:213–16. 1987.

 

Betsy Moran, Ph.D. is a Field Applications Scientist with Whatman GE Healthcare. She has worked in the field of Forensic DNA applications for six years.

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