DNA And DNA Evidence, part2

21-09-2010
Forensic Dentistry

 

part 1  

 

1.7 DNA Databases
Over the past two decades, forensic odontologists have witnessed a series of computer software programs that provide the ability to store highly detailed antemortem and postmortem records. The Computer-Assisted Postmortem Identification System (CAPMI) and Wind-ID are memorable examples, as is the 2004 version of the National Crime Information Center (NCIC) that accommodates more than just dental fields for missing and unidentified remains.

The International Criminal Police Organization (INTERPOL) contracts with PlassData in Denmark to administer DVI System International, which is a similar database application that stores dental and other identification data, including DNA profiles, for use in disaster victim identification responses worldwide. These programs include elaborate search algorithms that enable the investigator to scan hundreds or thousands of records quickly in search of a match between the questioned and known sets of records.
Inevitably, following the generation of these best-possible matches, the associated records are retrieved in original hard copy or high-quality digital form and examined by a qualified forensic odontologist to determine if the threshold for a dental identification has been achieved.
Other forensic specialties have developed similar capabilities. The fingerprint community queries the Integrated Automated Fingerprint Identification System (IAFIS) located at the FBI’s Criminal Justice Information System in West Virginia that contains over 55 million subjects.
Firearms examiners use the Integrated Ballistics Identification System (IBIS) maintained by the Bureau of Alcohol, Tobacco, Firearms, and Explosives at the National Integrated Ballistic Information Network.
Less familiar forensic databases belie their purposes with names like Ident-A-Drug, ChemFinder, National Automotive Paint File, and Forensic Information System for Handwriting.

Each provides a set of references of class or individual characteristics that hopefully lead the investigator toward individualization of the evidence on his or her own case. Among all the law enforcement databases in the United States, none has had a more dramatic impact on American culture and crime-fighting success than the national DNA database, the Combined DNA Index System (CODIS).

 

1.7.1 CODI S
CODIS has generated such intense media attention and concerns regarding genetic privacy that some may fail to understand how the program really works. The FBI laboratory began development of CODIS software in 1990 as a pilot project. Initially, the program included only fourteen state and local laboratories. However, when the DNA Identification Act of 1994 was passed, it affirmed the FBI’s authority to establish an all-states laboratory network.
Now, every CODIS-approved laboratory is required to maintain certain quality control standards in order to contribute to and query the National DNA Index System. The database uses two distinct indexes.

 

1.7.1.1 Convicted Offender Index
One data set is called the Convicted Offender Index that contains the known DNA profiles of individuals submitted by state and federal law enforcement officials. Each sample is obtained in accordance with that state’s respective DNA collection statutes. The laws governing sample collection and whether an individual must be convicted of a violent crime or simply arrested before uploading the profile varies from state to state. Matches made between an evidentiary DNA profile and the Convicted Offender Index provide investigators with the real-time identity of a potential perpetrator.

 

1.7.1.2 Forensic Index
The second data set is the Forensic Index. It contains unnamed DNA profiles recovered as crime scene or sexual assault evidence. A match made within the Forensic Index may not lead immediately to the perpetrator’s name, but it can link crime scenes together and detect serial offenders whose activities span several jurisdictions. Although local law enforcement agencies are expected to maintain their own freestanding data, the true value of the NDIS program derives from multiple laboratories uploading quality data from thousands of offenders in addition to crime scene evidence, so it can be searched by law enforcement agencies nationwide. In this way, police from all over the country can coordinate their independent investigations and share whatever leads they may have developed in an attempt to defeat criminal activity. Currently, over 170 public law enforcement laboratories at the federal, state, and local levels perform electronic data comparisons and exchanges through NDIS by using CODIS software. CODIS software has also been loaned to over forty international law enforcement laboratories in over twenty-five countries for their own database programs.

The recent passage of the DNA Fingerprint Act of 2005 will expand the reach of CODIS submissions to include non-U.S. citizens detained or arrested by other federal agencies during border protection and homeland security activities.
The successes of the CODIS software and the NDIS program has logically led to an expansion of both in other areas too. In 2000, the FBI laboratory began to plan for another database to be called the National Missing Persons DNA Database (NMPDD) to aid in finding over one hundred thousand persons listed as missing by the Bureau of Justice Statistics and identifying the over forty thousand unnamed remains that are held in various jurisdictions across the United States. The NMPDD will comprise three indices, to include the STR profiles and mtDNA sequences of persons known to be missing whenever a source of DNA is available, close family members of the missing person, and any unidentified human remains.

To facilitate potential matching of unidentified remains with their relatives’ DNA profiles, CODIS software will be upgraded to perform kinship analyses or familial searching.
This software will be similar to those previously used by the Armed Forces DNA Identification Laboratory (AFDIL) to identify American war dead and by the New York City Office of the Medical Examiner and AFDIL to identify victims from the terrorist attacks on September 11, 2001.

 

Glossary
Like most technical specialists in the forensic community, DNA scientists use terms that can make an already complex subject even more confusing.
Most odontologists will be familiar with the fundamentals of biology and biochemistry because of their own educational background. However, the words and phrases used in this chapter and explained below will provide the odontologist with the additional vocabulary necessary to understand basic forensic DNA oral and written presentations.

Allele: The variant in a DNA fragment size or in a DNA sequence at a particular locus is called an allele. The more alleles that naturally occur at a given locus in a given population increase the discriminating power of that locus. When paired (homologous) chromosomes each have the same allele at the same locus they are called homozygous.
When the two alleles are different between the paired chromosomes, they are said to be heterozygous.

Amplicon: The amplified segment of DNA that is the product of PCR is called the amplicon. The two boundaries of this target DNA are usual marked by forward and reverse primers before the PCR process begins.
Amplification: The process of increasing the quantity of original DNA template by using the polymerase chain reaction (PCR) is referred to as DNA amplification.
Analysis: The third step in laboratory processing is called analysis. Once a DNA sample has undergone extraction and amplification and is loaded into a genetic analyzer, data are generated that represent either a genetic profile based on the size of the alleles at each locus tested (autoDNA, yDNA) or the actual sequence of the targeted DNA (mtDNA). The raw data are then reviewed by the analyst, who uses his education, training, and experience to confirm the result and, when appropriate, compare sets of data, draw conclusions, and calculate statistical values. Some laboratories call the instrumentation portion of this process detection and reserve the word analysis for the final data review step by the analyst only.
Bases: DNA is composed of nucleotides strung together in a twisted double strand. Nucleotides are ring-shaped molecules with various combinations of carbon, oxygen, hydrogen, and nitrogen with a phosphate group attached. The nitrogen-containing portion of the nucleotide is called a base. Differences in base design result in four variations of bases in DNA: adenine (A), guanine (G), thymine (T), and cytosine (C). The distinctive sequence in which these four bases occur within specific locations along the DNA strand provides the genetic code for protein production, as well as the power of individualization used by the forensic scientist.
CODIS and NDIS: The Combined DNA Index System (CODIS) is a software application that was developed and distributed by the Federal Bureau of Investigation. The software allows the storage and searching of large amounts of DNA data. Crime laboratories that qualify for
CODIS access at the local, state, and national levels can search their own profiles and, more significantly, also search the DNA profiles posted by other laboratories through the shared data in the National DNA Index System (NDIS).
Complementary: In its natural configuration, DNA is double stranded. In the successful pairing of two single strands, the opposing sequences must be complementary. This means that adenine will only align opposite thymine, and guanine will align exclusively with cytosine.
If the sequences within the opposing segments of DNA do not meet these exacting requirements, regardless of their length, the single strands are not complementary and will not pair together.

Short segments of complementary DNA are used as oligonucleotide primers to mark the starting point of target DNA during PCR.
Denature: During the laboratory process of amplification (PCR) and again during the analysis step, the double helical strand of DNA must be unwound into separate single strands. The separation of strands, or denaturation, can be accomplished by the addition of certain chemicals or by elevating the temperature to approximately 98°C. The latter approach occurs during PCR. Denaturation is sometimes referred to as melting. The opposite of denaturing is annealing, which describes two complementary strands binding together.
DNA: Deoxyribonucleic acid is one of the body’s macromolecules and codes for all proteins. Forensic scientists currently focus on three major types of human DNA. All are identical at the molecular level, but each varies in its protein-coding responsibilities and its location within the cell. Y-DNA and X-DNA are the male and female sex chromo somes, respectively. Autosomal DNA (autoDNA) refers to any of the remaining twenty-two pairs of nonsex chromosomes. Sex and autosomal chromosomes are all located within the nucleus of the cell and contain the popular forensic targets called short tandem repeats (STRs) and single nucleotide polymorphisms. Mitochondrial DNA (mtDNA), on the other hand, is found outside the cell nucleus inside organelles called mitochondria, which reside in the cellular cytoplasm. Forensic scientists usually target the regions of the mtDNA genome with known hypervariable sequences or single nucleotide polymorphisms.
Electrophoresis: In the appropriate media, the negatively charged fragments of DNA migrate in an electrical field according to their molecular weight. Generally speaking, larger DNA fragments will move through the medium slower than smaller fragments of DNA. In this way, the analyst uses electrophoresis to separate DNA fragments of different sizes to visualize the results of PCR analysis. The most informative electrophoresis process takes place on a genetic analyzer.

Enzyme: Proteins that facilitate a biochemical reaction are called enzymes.
In forensic DNA science, the most commonly used enzyme is DNA polymerase that, in the proper chemical environment, assists with the synthesis of new strands of DNA from an existing template during the polymerase chain reaction. Additionally, an enzyme called proteinase K is used to break down cellular components and expose DNA during the extraction process.
Extraction: The first of three laboratory processing steps in forensic DNA science is called DNA extraction. It involves isolating the DNA by preparing the gross sample, sometimes by grinding or macerating it as in the case of bones, teeth, or tissues. Then proteinases are used to disrupt the cellular barriers and allow the naked DNA to go into solution. Separation of the DNA from the cellular debris and solvents is achieved by alcohol precipitation, filtration, or use of silica or paramagnetic beads.
Gene: Any segment of DNA that is transcribed into ribonucleic acid and eventually into a functional or structural protein is called a gene.
Most segments of DNA targeted during forensic analysis are believed to have little or no structural or functional role.
Genome: The full genetic makeup of an organism is called its genome.
The human genome is composed of 3 billion base pairs and 20,000 to 25,000 genes.
Genotype versus phenotype: The sum of genetic information in an organism’s genome is its genotype. The physical manifestation of that genetic information is called the organism’s phenotype.
Locus: The location of a particular gene or segment of DNA is called the locus (plural: loci). A locus can be described by using the chromosome number, designating the short or long arm of the chromosome, and the band or subband on that arm.
Mitochondrial DNA: Mitochondria are organelles that exist outside the cell nucleus in the cytoplasm. Mitochondria possess their own genome that is separate and distinct from the twenty-three pairs of chromosomes inside the cell nucleus. The human mitochondrial genome is 16,569 base pairs in length, carries 13 coding regions, and is inherited along maternal lines. It contains known regions of diversity that help forensic analysts to distinguish individuals of different maternal lineages from one another.
Mutation: An alteration in DNA coding sequence that is usually erroneous and occurs during DNA replication. It may be caused by an environmental influence, viruses, or simply a copying error during cell division.
Nuclease: Enzymes that digest nucleic acids and nucleotides are called nucleases. These proteins occur naturally in many microbial contaminants, and although human-specific primers will not recognize and amplify the bacterial genome, the nucleases within the bacteria can degrade the human DNA sample.
Nucleic acid: A long chain of five-sided sugar rings, nitrogenous bases, and phosphate connectors is a nucleic acid. The structural unit of the molecule is called a nucleotide. Without the phosphate group, the molecule is called a nucleoside.
PCR: The polymerase chain reaction is a laboratory technique that targets a specific segment of original DNA template placed in the appropriate chemical environment along with primers, polymerase, and nucleotides. During a series of carefully orchestrated temperature changes, the template undergoes denaturization, annealing, and extension. At the end of each cycle the quantity of DNA template has been doubled.
Polymerase and Taq: Polymerase is the enzyme that adds nucleotides to the new strand of DNA during PCR. The step is call extension.
Since the high temperature in the denaturing step of PCR deactivates most polymerases, the isolation of a thermostable polymerase from the thermophilic bacterium Thermus aquaticus (Taq) allowed multiple cycles of denaturing and annealing to occur without loss
of enzyme activity.
Primer: A small segment of complementary DNA that establishes the starting point for DNA synthesis in PCR is called an oligonucleotide primer. It signals the point at which polymerase begins to add nucleotides.
Sequencing: Current forensic mtDNA sequencing is based on modifications of the Sanger chain termination method that allows the scientist to determine the order of the nucleotide bases-adenine (A), guanine (G), cytosine (C), thymine (T)-in a given segment of DNA.
Short tandem repeat (STR): These are defined as two or more nucleotides in a fixed sequence and repeated in a continuous series. For example, if the tet ranucleot ide (four-nucleot ide) sequence [A-G-T-A] i s repeated nine times on one chromosome at a specific locus and eleven times at the same locus on the homologous chromosome, the STR profile for that individual can be said to be 9,11 at that locus. Most forensic analytical procedures will target six to seventeen loci on autoDNA and Y-DNA for STR profiling.

 

Acknowledgments
The authors gratefully acknowledge the support of Dr. Kevin Torsky at the Joint POW-MIA Accounting Command Central Identification Laboratory (JPAC CIL) and LTC Louis N. Finelli of the Armed Forces DNA Identification Laboratory (AFDIL) for their assistance.

 

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Forensic dentistry
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