D1
Establishing Parentage in Forensic Cases
Amanda
C. Sozer*, Ph.D.1
and George R. Riley, Ph.D.2 1DNA
Technology Consulting Services, 2 Fairfax Identity Laboratories
Molecular genetic techniques are powerful
tools used in performing human identity testing and parentage analysis.
Through parentage analysis, DNA typing can be used to identify the origin
of a biological sample when the alleged contributor is not available but samples
may be obtained from relatives. In
addition, parentage analysis can be used to determine the perpetrator of
unlawful sexual contact resulting in pregnancy.
Historically, many forensic DNA laboratories
have avoided performing parentage analysis because the statistical
interpretation of the data differed from the statistical interpretation
typically used in identity testing. In
addition, genetic mutations are encountered in parentage testing and must be
taken into consideration when forming a conclusion.
We will present several forensic cases where
molecular genetic techniques were used to establish parentage.
In each case the mathematical formula used to analyze the data will be
explained. Unusual DNA profiles may
complicate parentage interpretation. Several
case examples will be given to illustrate the importance of careful
interpretation of the data so as to not falsely exclude a parentage
relationship.
Due to the low but not insignificant
mutation rate of repetitive sequences, an exclusion from parentage cannot be
based on an exclusion in a single DNA system (with the exception of cases
involving closely related potential parents).
We will present the mutation rates for commonly used RFLP and STR systems
as well as mathematical equations used to incorporate the mutational events into
the statistical analysis of the data.
Please Note:
After attending this presentation the
participants will understand the basic equations used in parentage analysis.
In addition participants should be able to recognize potential mutations
in parentage data and correctly incorporate the data into the analysis of the
forensic case.
D2 On
the Cutting Edge, New Chalanges at the Armed Forces DNA Laboratory
Amanda Blanchard*, M.S., Suzanna M. Barritt, M.S. Demris A. Lee, M.S.F.S.,
Col Brion C. Smith, D.D.S., Armed Forces DNA Identification Laboratory,
Rockville MD
New Technologies, increasing case loads and
training new employees can be an enormous challenge for any forensic laboratory.
Backlogs and increased demands for DNA identification cases have led AFDIL to
plan and research modifications in case processing methods, bioinformatics and
overall laboratory structure. This presentation will be a brief overview of some
of our recent accomplishments and objectives for the upcoming year. Strategies
and goals will be discussed for staff expansion, implementing a laboratory
information management system, and executing a high throughput robotics system
for processing blood references. The upcoming challenges of identifying remains
from the Korean War including unilateral turnovers as well as remains exhumed
from the punchbowl will also be discussed.
D3
Discrimination for Common
Haplotypes: Targeting of Additional Information Outside the D-loop
Hypervariable Regions
Michael
D. Coble*, MFS; Christine Harvie, MFS; Ilona Letmanyi, BS; and Thomas J.
Parsons, Ph.D. The Armed Forces DNA Identification Laboratory. 1413 Research
Blvd. Building 101, 2nd floor. Rockville,
MD 20850.
Several characteristics of mitochondrial DNA
(mtDNA) make it useful for the forensic identification of individuals.
These include the high copy number, maternal inheritance and lack of
paternal recombination in humans. The
use of mtDNA testing has found a vital niche in the forensic testing of degraded
samples and shed hairs. The
greatest variability of sequence differences is found in the non-coding D-loop
(or control region) of mtDNA. Since
this region of the genome is not under the same evolutionary constraints as
coding regions, a higher number of mutational differences can accumulate.
In fact, the evolutionary mutation rate in the control region is about
ten times the rate of the gene-coding region.
Currently, most forensic laboratories using
mtDNA analyses focus on sequence information from two hypervariable regions (HV1
and HV2) of about 600+ bases within the control region.
Due to the relatively small size of HV1 and HV2, mtDNA cannot be used to
uniquely identify individuals. Therefore,
the significance of a mtDNA match is determined in relation to the frequency of
that type in a relevant population. The
distribution of mtDNA types is highly skewed toward rare types, making the
significance of a match for a mtDNA type previously unseen in a database quite
substantial.
Interestingly, there are a small number of
common mtDNA types in all populations studied to date.
For example, approximately 7.5% of Caucasians in an international
database share the same mtDNA haplotype in HV1 and HV2 compared to the Cambridge
Reference Sequence (ignoring the 309.1 C-stretch in HV2).
There are also thirteen additional Caucasian HV1/HV2 types that occur at
levels of 0.5% or greater in the database.
Other populations, such as African Americans and U.S. Hispanics, also
show a small number of common types and a large number of rare types.
However, the common type seen in one population is not the same in other
groups. This is likely due to
different evolutionary histories of different ethnic groups. Thus, the greatest limitation for mtDNA testing lies within
the small number of common types for which the power of discrimination is low.
Current mtDNA testing of HV1 and
HV2 identifies polymorphic sites concentrated within the control region.
Given that the coding region is fifteen times larger than the control
region, the greatest portion of total mtDNA variability is dispersed throughout
the coding region. Using RFLP
sites, evolutionary biologists have discovered an enormous amount of sequence
variability throughout the entire mtDNA genome.
These sites have been used to classify 99% of all European mtDNAs into
ten mtDNA haplogroups (e.g. H, I, J, K, M, T, U, V, W, and X).
The most common Caucasian HV1/HV2 type belongs to haplogroup H.
The most frequently used restriction enzymes in RFLP analyses examine
only about 20% of the mtDNA genome. This
leaves the possibility that a number of informative polymorphisms may remain
undetected.
We will present an automated,
high-throughput strategy for sequencing the entire mtDNA genome to identify
forensically informative coding region sites for common mtDNA types.
We have determined the sequence of 70 mtDNA genomes corresponding to
common HV1/HV2 types (e.g. haplogroups H, J, and T).
The discovery of novel, neutral polymorphisms in the coding region have
resolved common HV1/HV2 types in nearly all cases examined.
The utility of this approach can be gained by assaying a small number of
informative single nucleotide polymorphisms (SNPs) rather than sequencing the
entire mtDNA genome. We have
discovered that the analysis of a small number of shared, yet forensically
informative, sites can segregate a large number of individuals having an
identical HV1/HV2 background (rather than assaying one unique polymorphism for
each individual).
The Armed Forces DNA Identification
Laboratory (AFDIL) has a major effort underway to develop fluorogenic,
homogeneous assays for informative SNPs within the mtDNA coding region.
These assays are based on the Applied Biosystems 7700 platform (TaqMan)
and related chemistries. The
results of our mtDNA genome survey and assay development efforts will be
presented and discussed.
We have developed an automated,
high-throughput strategy for sequencing the entire mitochondrial DNA (mtDNA)
genome. Polymorphic sites in the
mitochondrial coding region reveal informative sites for the forensic identity
of common haplotypes. After attending this presentation, the audience will
understand that additional sequence information outside of the control region
can be used to increase the forensic discrimination of common mtDNA haplotypes.
D4
Calculation of Peak Height Ratios from Known
Mixtures and a Comparison of Related Statistical Calculations (DNA)
Elizabeth Ballard*,MAAFS scholarship award recipient , Virginia Commonwealth
University, Richmond VA
On a routine basis the Forensic DNA examiner
encounters evidence samples containing mixtures of two or more individuals.
Therefore, it is important that the DNA examiner be able to identify the
foreign DNA profile in a mixture when the victim and suspect may share alleles.
In order to aid the DNA examiner in the evaluation of this data, a study
will be conducted to determine optimum peak height ratios that could be used to
separate and identify the different profiles seen in mixtures. In addition, it
is imperative that the Forensic DNA examiner assigns statistical values to his
analysis. Two commonly employed methods are the likelihood ratio and
combined probability of exclusion calculations. A comparison will be made of these two methods. Several sets
of mixtures will be prepared in the following percent ratios using two known DNA
samples: 100/0, 90/10, 80/20, 70/30, 60/40, 50/50, 40/60, 30/70, 20/80, 10/90,
0/100. One of the sets of mixtures
will be prepared using K562 and GM9947A, while the other sets will be prepared
using samples from several employees of the Virginia Division of Forensic
Science. Casework-like samples used will include mixtures containing DNA from
numerous contributors and mixtures in which the major and minor contributors are
difficult to discern. The known mixtures and casework-like samples will be
amplified and typed. Peak height
ratios from the known mixtures will be calculated as well as the heterozygousity
ratios for the individual profiles. The data obtained will be used to determine
if foreign profiles in the casework-like samples can be identified, and if so,
what the optimum cut-off values for those ratios may be. Secondly, both the
likelihood ratio and the combined probability of exclusion will be calculated
using data from the above mentioned known mixtures and casework-like samples.
The results of these calculations will be compared.
D5 The
Comparison and Validation of FTA®, Chelex, and Qiagen Extraction
Methods on Blood Spotted FTA® Cards for the Analysis of the 13 Core
STR Loci
Linda B. Jankowski*, M.S., DNA Technical
Director, Maureen
M. Low-Beer, M.S.,Senior Forensic Scientist, Edward J. LaRue, B.A., DNA Unit Supervisor,New Jersey State Police
Blood spotted FTA® cards are
frequently the method of choice for preservation and storage of reference
samples from suspects, victims, and convicted offenders. The advantages of the
cards are well known, and include the inactivation of blood borne pathogens, the
binding of DNA to the matrix of the card, and their recommended storage at room
temperature. This paper describes the comparison of four DNA extraction methods
on FTA® cards. Traditionally, a small punch from the FTA®
card is washed according to the manufacturer’s protocol, and the punch is
amplified directly without quantitation of the DNA. Our laboratory validated
this procedure, which turned out to be problematic in casework. The quantity of
DNA on the 1.2mm punch is quite variable. The absence of a quantitation
mechanism often results in electropherograms with off-scale data, even though
the number of cycles was reduced to 25. Furthermore, presumably due to
inhibitory factors present in the card, preferential amplification of the
smaller alleles sometimes results in partial and/or unbalanced profiles seen
both among and within loci. This is evident by a downward slope of peaks from
the smaller to the larger loci, and unbalanced heterozygote peak ratios.
Although DNA is supposedly permanently bound to the FTA® Card, a
standard 200 µl Chelex Extraction results in high quantities of DNA in the
extract. This allows the DNA to be quantitated and a controlled amount to be
amplified. Like others, however, we found that in a significant number of cases,
inhibitors prevented amplification of the standard 1ng of DNA. To overcome this,
we validated a 400 µl Chelex Extraction, which sometimes alleviates the
problem. However, there were still a number of samples for which we got no
profile or an incomplete profile. The Qiagen extraction of FTA®
cards has proved to be the best method. Full profiles were obtained from
specimens which gave no results with the previously described methods.
Additionally, the balance among the 13 loci is greatly improved, as is the
heterozygote peak height ratio.
D6
Extraction and Analysis of Mitochondrial
DNA from Processed Fingerprints
Robert
Bever*, Nicole Gross and Susan Currence. The Bode Technology Group, Inc.
Springfield Va. 22150
Fingerprints are routinely used in
investigations to characterize individuals associated with forensic evidence.
However, sometimes fingerprints are smeared or incomplete and cannot be
interpreted. The use of mtDNA for
the characterization of these fingerprints would be valuable in forensic
investigations. Three extraction methods were compared for their ability to
purify amplifiable mtDNA from processed fingerprints. Fingerprints collected on
porous and nonporous substrates were treated with ninhydrin solution, physical
developer, and magnetic powder. Total
DNA was extracted from these fingerprints using the Qiagen QiaAmp, Promega
Wizard kit, and InstaGene Matrix. Due
to the sub-nanogram quantity of starting template, DNA recovered in these
extractions could not be quantified prior to amplification. Employing the three extraction methods, a range of 2ng to 40
ng of amplified mtDNA product could be detected.
Direct sequencing of the amplified products produced a clean
interpretable DNA sequence. A discussion comparing the strengths and weaknesses
of each extraction method will be presented.
D7
Validation of Reduced Volume PCR Amplification Reactions using AmpFlSTR
Kits
Michelle
L. Gaines*, North Louisiana Criminalistics Laboratory, Shreveport, LA
Current forensic
DNA typing methods use genetic typing systems based on the polymerase chain
reaction (PCR). The PCR-based
systems require very little sample and offer the advantages of high sensitivity,
complex multiplexing, and enhanced interpretation of forensic samples.
The most frequently used PCR-based systems are based on the short tandem
repeat (STR) loci. PCR-based STR
multiplex typing kits are now produced commercially, making DNA profiling more
efficient yet costly. The
commercial PCR amplification kits were developed and validated for a fixed
volume reaction. PCR amplification
kits produced by Applied Biosystems (ABI) were optimized for a 50 mL
reaction. The kits from Promega
were optimized for a 25 mL
reaction.
Our laboratory has
explored the option of reducing the volume of the PCR amplification reaction.
The potential advantages are to decrease the amount of sample consumed
and increase the sensitivity of the reaction.
Our concern was that reducing the reaction volume could significantly
alter the amplification kinetics, affecting the quality and reliability of the
resulting DNA profiles. To
determine how a reduction in reaction volume would affect kinetics, sensitivity,
and interpretation, we conducted the following experiments:
1.
The quantity of template DNA was reduced
proportionally with the reaction volume.
2.
The reaction volume was reduced while
maintaining a constant quantity of template
DNA.
3.
The efficiency of the PCR reaction was
evaluated at reduced reaction volumes.
4.
The lower limit of detection of the PCR
amplification was evaluated at reduced
reaction volumes.
5.
The ability to interpret mixtures was
assessed by preparing and amplifying serial
dilutions of known mixtures at reduced
reaction volumes.
When the quantity of template DNA was
reduced proportionally with the reaction volume there was no significant change
in the intensity of the signal in the resulting electropherograms.
At 0.4 ng and lower of template DNA we observed an increase in peak ratio
variance resulting from stoichastic effects.
When the quantity of template DNA was maintained at 2.0 ng, the total
amount of PCR product was approximately equal for reaction volumes of
50 mL,
25 mL,
and 15 mL.
There was very little variance in the peak ratios at the heterozygous
loci. The kinetics of the PCR
reaction appeared to be unaffected as the volume of the PCR reaction was
decreased. However, the same
concentration of PCR product was achieved with fewer cycles in the lesser volume
reactions. The lower limit of
detection of the amplification reaction decreased as the volume of the reaction
was reduced. The increase in
sensitivity was observed for both single source DNA samples and for the major
and minor components of the known mixture reactions.
The reactions with lower quantities of template DNA were observed to have
an increased incidence of stoichastic variance in the peak heights.
This series of experiments verified that it
was feasible to decrease the volume of the PCR amplification reaction and still
produce reliable DNA typing results. However,
laboratories considering this as a cost effective option alone, should be aware
of the limitations of this method and at what point a reduction in volume will
begin to affect the quality of the work product of the laboratory.
D8
Identification of Plant Trace Evidence Using Molecular Techniques
Matthew
Cimino, Lauren Brinkac, Emily
Hopkins, Nicole Gross, and Robert Bever*, The Bode Technology Group,
Inc., Springfield, VA
The
identification of plant materials associated with trace evidence can provide
useful leads during criminal investigations.
Historically, morphological and histological characteristics have been
used to analyze botanical trace evidence. However,
based on their morphological features, many plant particles found as trace
evidence cannot be identified. An
alternative method of botanical evidence identification employs DNA molecular
markers. The rbcL
gene of chloroplast DNA and the Internal Transcribed Spacer (ITS) region of
nuclear ribosomal DNA are moderately conserved sequences of DNA that can be used
for the identification of plants. We
have used these gene sequences to identify plant particles associated with
environmental trace evidence.
The
molecular methods developed to analyze trace evidence involve the following
steps:
·
Isolation and purification of DNA from trace evidence.
·
PCR amplification of target loci..
·
Cloning of mixed amplified products.
·
Prioritization based on restriction fragment length polymorphisms.
·
Sequencing of the cloned PCR products of interest.
·
Identification using NCBI BLAST database and PAUP phylogenetic software.
Molecular
markers are useful for investigating botanical samples of various composition
and magnitude. Plants have been
identified from as little as a 1-mm punch of dried plant tissue, 5 grains of
fresh pine pollen, and 10 picograms of DNA.
Additionally, methods have been developed to identify plant materials in
heterogeneous mixtures of soil or dust collected from various environments and
substrates. These include clothing,
roadside debris, and packaging material. It
is envisioned that molecular analysis of botanical trace evidence will be useful
in providing investigative leads for solving crimes such as kidnapping,
distribution of drugs, murder, and acts of terrorism.
D9
Short Tandem Repeat Analysis in the FBI Laboratory: Some Interesting Casework
Examples
Alice L. Brown*, Deborah Hobson,
Frederick Keller, and Jenifer Smith DNA
Analysis Unit I, Federal Bureau of Investigation Laboratory, Washington, DC
The
DNA Analysis Unit of the FBI Laboratory began using STR (Short Tandem Repeat)
analysis on forensic casework samples in January of 1999.
The FBI began casework analysis with the Profiler Plus Kit and added the
COfiler Kit four months later. It has been demonstrated that with the
sensitivity of PCR, multiple alleles/locus and quantitative data, STR analysis
has increased the FBI’s ability to interpret DNA mixtures.
In many cases, meaningful results would not otherwise have been obtained
with pre-STR techniques. One FBI
examiner/technician team has worked over 240 submissions using STR analysis.
Some of these cases have involved recovering and typing DNA from items
not initially thought to provide probative DNA profiles.
Most of the DNA recovered was sufficient for analysis at all 13 CODIS
loci. Some of the more interesting
items in these cases were hair clips, fingernails, a paper bag and a toilet
seat. Interesting cases and the STR
results will be presented.
D10
An Interesting DNA Data Bank Hit
from a Mixture STR Profile
Wendy
M. Cohn * M. S., George C. Li M. S. , Virginia Division of Forensic Science, Central
Regional Laboratory, Richmond VA
Evidence from an alleged drive-by shooting
was submitted by a local police jurisdiction to the Virginia Division of
Forensic Science for forensic biology analysis. Eye witness account indicated that 3 males were in the
vehicle from which shots were fired, resulting in serious injuries to several people.
Through police investigations, three suspects (A, B, and C) were arrested
and charged with the crime. The
police also recovered a car that they
think was the vehicle involved , which turned out to be a rental car
The laboratory was requested to help
establish any link between the suspects and the vehicle.
The evidence from the inside the car included a glass bottle, a plastic
water bottle and a cigar holder. The blood samples from all three suspect were
also submitted for comparison.
DNA STR analysis was conducted on the
evidence using the Promega PowerPlex 1.1 typing kit, and useable results were
obtained from all the above evidence.
The DNA profile from the mouth of the
glass bottle was found to be consistent with suspect A.
The profile from the mouth of the plastic water bottle turned out to be a
mixture of suspect A and B. The DNA
profile from the cigar holder was also a mixture profile, but it did not match
any of the three suspects.
A search was made with this unknown mixture
profile from the cigar holder against the Virginia DNA Data Bank, which
contained more than 120,000 STR profiles at the time.
The search resulted in 22 moderate stringency hits,
all but one of which were eliminated due to the presence of a homozygous
locus in the mixture profile. The
remaining one matching profile belonged to
a female convicted felon. The
inmate information on this individual was provided to the police for further
investigation, which subsequently revealed that the female turned out to be the
mother of suspect C.
D11
Potential Sources of Low Level DNA Profiles Detected in Forensic Specimens
Julie
Ann C. Kidd*, Stacy Stouder, Deborah Hobson, and Jenifer A. L. Smith DNA Analysis Unit I,
FBI Laboratory, Washington, DC
Evidentiary items submitted in forensic
casework often contain DNA from multiple contributors.
PCR-based short tandem repeat (STR) analysis can allow for detection and
evaluation of low-level contributions from various biological sources in these
mixtures. The DNA Analysis Unit I
of the FBI Laboratory is currently studying the effects of potential low-level
biological transfers to commonly submitted evidentiary items using the 310
Genetic Analyzer from Applied Biosystems. The data will focus on the transfer of
low-levels of DNA to machine washed and dried garments as well as the effects to
co-mingled clothing.
D12
The Effects of the 310 Genetic Analyzer’s Optics on Sensitivity
Maribeth
A. Donovan*, Jill B. Smerick, Deborah L. Hobson, and Jenifer A.L. Smith DNA
Analysis Unit 1, FBI Laboratory, Washington DC
The 310 Genetic Analyzer from Applied
Biosystems uses laser induced fluorescence to detect labeled short tandem repeat
(STR) products of the polymerase chain reaction (PCR) that have been separated
by capillary electrophoresis. The
optical system on the 310 Genetic Analyzer includes an argon laser, a series of
lenses including a prism, and a Charge-Coupled Device (CCD) camera. The wavelengths of light emitted by the laser excite the
fluorescent dye labels on the STR products as they migrate past the detection
window of the capillary. The
fluorescent emissions from the STR products are directed and focused through a
series of lenses and the resulting light spectra is captured by the CCD camera.
In this study, the effects of the components of the optical system on
instrument sensitivity were monitored for eight (8) 310 Genetic Analyzers.
The effects of the laser, CCD camera and preventive maintenance
procedures on instrument sensitivity will be presented.
D13
PowerPlex
16 – Validation Studies on the ABI 310 and
ABI 377 Genetic
Analyzers
J.
Zachetti* , C. Tomsey, F. Kist, Pennsylvania State Police DNA Laboratory
Greensburg, PA
STR
(Short Tandem Repeat) loci consist of short, repetitive sequence elements 3 to 7
base pairs in length. These repeats
are well distributed throughout the human genome and are a rich source of highly
polymorphic markers that can be used to discriminate from one individual to
another. The Geneprint PowerPlex 16
System allows the coamplification and color detection of sixteen loci (15 STR
loci and Amelogenin) that satisfy the needs of several major law enforcement
agencies throughout the world. The
Pennsylvania State Police will utilize this rapid and robust system in the
future to expedite its DNA casework as quickly and accurately as possible.
This presentation is a compilation of the validation efforts performed by
the Pennsylvania State Police of PowerPlex 16 on the ABI 310 & 377
instruments.
LEARNING OBJECTIVES: After attending this
presentation the participant will understand: 1) the significance of the
post-recovery handling of physical evidence, and 2) the value of designing a
facility and identifying procedures that will minimize the possibility of the
contamination or cross-contamination of physical evidence.
D
Mixture Ratios
Deborah
Hobson*, Jill Smerick, Debra Defenbaugh, James Corcoran,Frederick Keller, and
Jenifer Smith,
DNA
Unit I, FBI Laboratory,Washington, DC
Short
tandem repeat (STR) analysis is rapidly becoming commonplace in forensic DNA
laboratories. Using current
methods, STR analysis allows for quantitative evaluation of data resulting in
interpretation of complex mixtures. Currently, the FBI Laboratory uses
guidelines for stutter, non-template nucleotide addition, and peak ratios in the
interpretation of mixed specimens. The
DNA Analysis Unit I is currently investigating the utility of mixture ratios for
mixture analysis. The data from
completed and on-going studies focusing on mixture ratios will be presented.
D15
Low level
DNA profiles using the 310 Genetic Analyzer
Jill Smerick*, Deborah
Hobson and Jenifer Smith , DNA Unit 1 , Federal
Bureau of Investigation Laboratory, Washington DC
Laboratories participating in th e Combined
DNA Index System (CODIS) have the opportunity to type the 13 core Short Tandem
Repeat (STR) loci using various STR kits and platforms.
The DNA I of the FBI Laboratory currently analyzes these loci with the
Profiler Plus and Cofiler kits by capillary electrophoresis using the 310
Genetic Analyzer. PCR product preparation including formamide, concentration and
filtration, and injection time were studied to identify the effects of
sensitivity on data quality. This
presentation discusses the sensitivity of these instruments and the necessity to
implement match thresholds to insure reliable results.
D16
An Investigation of Forensically Important Blow
Flies
(Diptera:Calliphoridae)
Occurring in Brazos and Burleson Counties of Central Texas
F.
M. Tenorio*, Jimmy K. Olson, PhD, Craig Coates, PhD,
Department of Entomology, Texas
A&M University, College Station
Texas
Blow flies are the insects of most
importance to forensic entomology as they are the first to colonize a corpse or
carcass, often within minutes of exposure. There are a number of taxonomic keys
available for the identification of adult blow fly specimens. However,
differences between some closely related species are subtle and often difficult
to observe.
Larvae can also be identified using
morphological characteristics, but there are few keys available for the
identification of larvae younger than the third instar.
Exposed pig carcasses were used to survey
the species of blow flies present in Brazos and Burleson counties. Cynomyopsis
(=Cynomya) cadaverina (Townsend), Calliphora
vicina (Robineau-Desvoidy), and Calliphora
livida (Hall) have proven to be the three most important species colonizing
a carcasss during the winter and early spring. These are all blue bottle flies
and are morphologically very much alike. The larvae are so similar that
taxonomic keys cannot distinguish between C.
livida and C. vicina.
The goal of this research is to develop a
DNA identification procedure to be used in conjunction with morphological
differences, to enable a faster and more accurate identification of egg, larval,
and pupal samples. Mitochondrial DNA (mtDNA) is particularly well suited as a
template for identification purposes as there are hundreds of copies in each
cell, and it is relatively resistant to degradation. Most importantly, the
mutation rate of mtDNA is high enough to provide numerous sequence differences
between closely related species.
The results of field collections relating to
blow fly species in the area, and preliminary studies to develop a DNA- based
identification protocol for the three species of forensic importance in central
Texas will be presented.
D17
Technological Advances for
Forensic DNA Testing
Y.
Daoudi*, P. Collins, C. Leibelt, L. Hennessy, F. Shadravan, M. Bozzini, and R.
Roby, Human Identification Group, Applied Biosystems,
Foster City, CA
Forensic DNA
testing has progressed rapidly in the last decade due to advances in DNA
analysis technology and automation. The
development of fluorescent short tandem repeat (STR) analysis provided a highly
automated and informative tool for a variety of genetic assays, including human
identification applications. An
advantage of fluorescent dye detection systems is that DNA fragments overlapping
in size range can be labeled with different dyes and thus be simultaneously
detected in a single lane or injection on analysis instrumentation.
Applied Biosystems’ 4-dye technology, using 3 dyes to label DNA
fragments and a fourth dye for the internal size standard is a well established,
reliable technique for a number of DNA fragment analysis, including STR
analysis. This 4-dye technology provided a significant increase in
throughput over the more traditional methods (e.g. radioactivity,
chemiluminescence). However,
increasing demand for genotypic information has spurred the need for even
higher-throughput solutions. To address this issue, Applied Biosystems developed
two multicapillary instruments (the ABI Prism® 3700 DNA Analyzer and the ABI Prism® 3100 Genetic Analyzer) and a new 5-dye system for
automated fragment analysis, permitted by the development of two new fluorescent
dyes. The development of a 5-dye system further enhances throughput capabilities
on ABI Prism® instrument
platforms. In this talk we will
present information on the 16 capillary ABI Prism®
3100 Genetic Analyzer, the 96 capillary ABI Prism® 3700 DNA Analyzer, and the AmpFlSTR®
Identifiler™ PCR Amplification Kit. This
kit incorporates the new 5-dye technology and will amplify 15 tetranucleotide
loci and the gender identification locus Amelogenin in a single PCR reaction
which provides higher throughput capabilities.
D18
P30 Antigen Detection Using the
OneStep ABAcard System for the Identification of Semen: The
FBI Experience
Rhonda
L. Craig*, Anthony J. Onorato , Jennifer C. Luttman and Jenifer L. Smith, FBI Laboratory, DNA Analysis Unit I, Washington, D.C.
Since its characterization in the 1970’s, the use of Prostate
Specific Antigen (PSA), or P30, for the chemical identification of semen has
become well established. In the
1980’s, the first immunometric assays were developed and employed for the
detection of P30 in semen stains of potential forensic value in alleged cases of
sexual assault. Since that time,
efforts have been made to develop a simple, reliable, and cost effective
chemical method for the identification of semen in forensic samples that also
demonstrates both the sensitivity and specificity required of confirmatory
tests. In 1998, the FBI DNA
Analysis Unit I (DNAUI) adopted the
Abacus Diagnostic OneStep ABA P30 system as its routine confirmatory test for
the chemical identification of semen.
As with any testing methodology of potential forensic value, extensive
validation studies were performed on this card-based system to evaluate its
stated performance specifications, recommended
testing protocol, and to compare its performance to that of the solid-phase
double-antibody sandwich Enzyme-Linked Immunosorbent Assay (ELISA) method in use
by the Laboratory at that time. Also
during this evaluation period, quality assurance/quality control protocols were
developed to insure the uniform performance of each new lot of product received
by the Laboratory. Since
our implementation of this system, our QA/QC measures have identified
product lots that did not meet the performance standards established by our
laboratory during the validation process. These
substandard lots were subsequently exchanged and served as the beginning of a
series of productive exchanges between the FBI and Abacus Diagnostic, Inc. that
has helped us to resolve these performance issues.
Our experiences with the Abacus Diagnostic OneStep ABA P30 system will be
expanded upon as a part of this presentation as will our QA/QC protocols and
interactions with Abacus Diagnostic, Inc. Collectively,
this will serve to reinforce the importance of laboratories designing and
performing their own QA/QC standardization procedures for this specific product
as well as any and all other critical testing formats in the laboratory.
D19
Forensic Analysis of Mitochondrial vs Nuclear DNA Degradation Rates
Kari S. Yoshida* and David R. Foran, Department of Forensic Sciences,
The
George Washington University, Washington, DC
In poor or highly degraded forensic
specimens it has been found empirically that there is a much higher likelihood
of recovering mitochondrial DNA (mtDNA) over nuclear DNA. Because of this, mtDNA
is being utilized increasingly in forensic settings, although it does not have
nearly the discriminatory power nuclear markers.
The exact reason for the increased utility
of mtDNA in degraded samples is unknown. There are hundreds or thousands of
copies of mtDNA per cell, versus two copies of nuclear loci, and so it is often
stated or implied in the literature that the higher copy number results in its
robustness. However, there is contradictory evidence for this, in that high copy
number nuclear genes do not behave or degrade as do mitochondrial loci. This
indicates that other attributes of mtDNA may play an important role in its
usefulness in a forensic setting, and that understanding these attributes might
lead to increased utility of mitochondrial, and perhaps nuclear, markers.
The purpose of this study was to
objectively examine three unique attributes of mtDNA, including copy number, its
circular structure, and its cellular location away from the nucleus, to
determine which, and to what extent, these influence DNA degradation. A model
system was utilized, and a variety of different organs, which had been allowed
to degrade for various lengths of time, were examined. Our results indicate that
factors beyond copy number play an important role in the stability and utility
of mtDNA, and that these factors should be taken into account when mitochondrial
and nuclear markers are assayed in a forensic setting.
D20
A Simplified
Fingernail DNA Extraction
Protocol that Reliably Separates Exogenous DNA
from the Fingernail
Rachel
E. Cline*, Nicole M. Laurent and David R. Foran, Department of Forensic
Sciences,The George Washington University,Washington, DC
Fingernails can be useful and informative sources of DNA in
forensic casework. Not only can DNA
from the fingernail provide important identification information, but in the
case of rape or other violent crimes in which the victim scratched the attacker,
exogenous DNA from cellular material underneath the fingernails may also be
obtained, leading to the identification of the perpetrator.
Current DNA extraction protocols tend to focus
only on isolation of DNA from the fingernail itself, without providing a
reliable method of purifying exogenous DNA.
In addition, these protocols can be complex, time-consuming, laborious,
and require the use of equipment that may not be available in all laboratories.
We have designed a simplified fingernail DNA
extraction protocol that minimizes the time and steps involved and reduces
needed equipment. It also deals
with any contaminates or debris that may be present on the nail, such as
fingernail polish. Both fresh and
aged fingernail samples were examined at both nuclear
and mitochondrial loci.
The design and success of
this protocol was directly assayed through a PCR-based system that readily
differentiated between exogenous and endogenous DNA. In this way, each individual step in the extraction procedure
could be assayed for the level of separation between the two sources of DNA.
This has not only allowed us to develop a straightforward and forensically
useful DNA extraction protocol for fingernails, but also one that can reliably
isolate exogenous DNA from the fingernail.
This may be critically important in determining both the victim and
perpetrator of a crime.
D21
Customizing GenoTyper7 Macros to Individual
Laboratory Specifications
Brendan F. Shea*, Jill Smerick, Deborah
Hobson, and Jenifer Smith,Federal Bureau
of Investigation, DNA Analysis
Unit I, Washington, DC
When analyzing
short tandem repeats (STRs) utilizing the ABI Prism 310,Genotyper7 is commonly used to
assist in allele calls following an electrophoretic run. The macros
which are provided along with the software can aid in this process. This
talk will discuss some of the basics of the macros supplied with Genotyper7, and go into some detail as to how the macros can
be altered to better serve the user
based on the stutter and -A specifications of each individual
laboratory.