Whatman
200 Park Avenue
Florham Park, NJ, 07932
Website: http://www.whatman.com
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Application Of Dry Storage Matrices For DNA Sample Collection And Preparation For Forensic Analysis
Figure 1. Indicating FTA Micro Card.
Colored matrix turns white upon sample application to locate the area from
which to sample DNA. |
DNA analysis in forensics is commonplace today. The representations of crime scene
analyses on various popular television shows has raised public awareness to the
point that jurors are taking it upon themselves to request DNA information in
cases they are reviewing.
DNA analysis is a very powerful technique that can be used to both apprehend the
guilty and to clear the innocent. Unless a person has an identical twin, DNA doesn't
lie. It is the ultimate barcode, and it identifies each and every one of us. The
technologies used in DNA analysis have evolved to the point that less and less
DNA is required to gain a full profile to identify an individual. Full profiles
can be gotten from chewing gum, cigarette butts and the drinking surface of a
glass or beverage can. The use of Low Copy Number (LCN) DNA analysis means that
DNA can also be obtained from fingerprints. This has opened up a whole new area
for DNA profiling in the investigation of crimes such as burglary and breaking
and entering. It is widely accepted that repeat criminal offenders exhibit recidivism,
and that they generally progress to more serious and more violent crimes. The
use of DNA indexing systems or databases on the local (LDIS), state (SDIS) and
national (NDIS) levels, which is then subsequently combined in the FBI's CODIS
database, has been instrumental in matching DNA found at crime scenes to DNA profiles
of criminals in the database systems. This has resulted in the solving of a number
of "cold cases".
Figure 2. Indicating FTA Micro
Card (A) with nine sample punches removed corresponding to zone template.
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(B) Three major areas identified
as periphery (zones 1-4), middle (zones 5-8), and center (zone 9).
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DNA sample collection
Trends in the collection of DNA samples have resulted in a three-phase approach.
First, DNA was collected from all persons convicted of violent or sexual offenses.
DNA sample collection in most states then progressed to include all felons. Second,
legislation passed that required the results of DNA testing to be shared with
the accused. This legislation also increased funding to be used to reduce the
backlog of DNA samples for analysis in post-conviction claims of innocence. Lastly,
and somewhat controversially, is the taking of samples from all arrestees, thus
making DNA collection as common as fingerprinting. Several states have enacted
laws for DNA collection of all felony arrestees including California, Kansas and
Virginia.
With these legislative changes there will be a large increase in the number of samples taken from criminals. Even those states that have not enacted the encompassing legislation project increases in the number of DNA samples collected annually. The federal government also projects an increase in DNA sampling based on the DNA Fingerprinting Act (S.1601) which will collect DNA samples from illegal aliens and those arrested for federal offenses. There is clearly a need for a faster and easier way to collect DNA samples and to process the increased number expected in the near future.
Buccal cell samples
There is a growing trend in local and state police facilities to move away from the use of blood samples, which require the use of a trained phlebotomist for collection, to taking non-invasive buccal cell samples. Buccal cell samples are easily obtained by scraping the inside of the cheeks to collect epithelial cells. In order to collect high quality DNA samples, 38 state police laboratories, several federal laboratories and over 10 countries globally apply buccal samples to FTA Cards (Whatman, Inc., Florham Park, NJ.
Figure 3. FTA Elute card.
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FTA Cards are chemically treated devices for the collection, stabilization, long-term
room temperature storage and purification of DNA for purposes of database storage.
DNA is entrapped in the matrix of the FTA Card, is protected against degradation
from environmental factors, and is analyzed in situ by amplifying the DNA directly
from a small portion (1.2 mm disk) of the FTA Card. The FTA Cards are easy to
use, do not require professionally skilled individuals for sample collection,
and can be adapted for high throughput processing of many samples. For buccal
cell collection, Indicating FTA Cards are used. These cards contain an inert indicating
dye that changes color upon sample application so that the buccal sample is easily
located (Figure 1).
In work done by Xavier Aranda in the lab of Dr. Arthur Eisenberg at the University of North Texas Health Science Center,(1) the distribution of cells was examined in a sample area after the buccal cells were applied. In this experiment, the buccal cells were collected with a foam tipped swab then transferred to the FTA Card. Disks of FTA were sampled from around the application area and analyzed for 16 Short Tandem Repeats (STRs) in a multiplex PCR amplification. The distribution of the cells was determined by the success of amplifying all 16 of the STRs in the 9 sample areas examined (Figure 2 and Table 1). In the sample areas examined, full robust profiles were achieved 95% of the time. Total complete profiles were achieved over 97% of the time.
Sample quality and quantity
Figure 4. Real-time PCR analysis of
blood samples applied to FTA Elute Cards. Blood from 10 individuals was
applied to FTA Elute, extracted by the heated water method and analyzed
by qPCR. The average Ct across the samples analyzed is 26.6.
Click
to enlarge. |
A recent trend in forensics is to analyze the DNA not only for quality but for
quantity. In scene-of-crime and case-work analysis the total amount of DNA recovered
is critical. Knowing the quantity of DNA input into STR amplifications ensures
reproducible DNA profiles. The use of real-time or quantitative PCR (qPCR) is
becoming more commonplace. Several companies have kits available for accurately
measuring the amount of DNA purified from blood and buccal samples. For qPCR,
having DNA in solution is highly desirable, since insertion of the FTA disk can
interfere with the fluorescent signal of the amplified DNA. FTA Elute eliminates
this concern in those instances where qPCR is required.
FTA Elute (Figure 3) is also a chemically treated matrix device for the collection of biological samples. FTA Elute differs from FTA in that the DNA inhibitors are irreversibly bound to the matrix and the DNA is eluted in water in a simple heating step. The DNA recovered from a 3 mm disk of FTA Elute is ample enough for many PCR analyses, and for both real-time and multiplex amplifications.
The amplification of DNA purified from FTA Elute is highly reproducible, as can be seen in Figure 4. Blood samples from 10 individuals were applied to FTA Elute and the DNA recovered in water by the rapid (30 minute) heat elution step. The DNA was then analyzed by real-time PCR. The 10 curves are very reproducible, and have an average Ct of 26.6. These data show that FTA Elute is adaptable to real-time PCR applications.
Summary
Rapid advances in forensic DNA analysis are challenging the processes of how DNA
is purified. Having a system that will archive and preserve DNA in a stable and
space saving format is desirable because, as analytical techniques develop, there
may be need to return to archived samples for further testing.
| Table 1. Assessment Of Nine Different Zones For The Transfer Of Buccal
Epithelial Cells Onto Indicating FTA Micro Card. |
FTA Sample Zone |
Complete Profile (++ Robust)
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Complete Profile ( + Weak) |
Incomplete Profile (- Alleles Absent |
1 |
15 |
0 |
1 |
2 |
15 |
1 |
0 |
3 |
14 |
0 |
2 |
4 |
15 |
1 |
0 |
5 |
16 |
0 |
0 |
6 |
15 |
0 |
1 |
7 |
16 |
0 |
0 |
8 |
15 |
1 |
0 |
9 |
16 |
0 |
0 |
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Total Percentage 95.1% |
Total Percentage 2.1% |
Total Percentage 2.8% |
Total Punches |
137 |
3 |
4 |
About the Author
Betsy Moran, Ph.D. Technical Marketing Manager for the FTA Products BioScience
Business Development Group at Whatman, Inc 200 Park Avenue, Suite 210 Florham
Park, NJ 07932
P : 973 245-8345
F : 973 245-8329
betsy.moran@whatman.com
References
1. Poster presented in 2005 at the Promega International Symposium on Human Identification.
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