Forensic medicine
DNA identification test
Principle of the genetic identification test
Each cell in the human body contains two strands of nuclear DNA divided among 22 chromosome pairs (the autosomes) and two gender chromosomes (two X chromosomes in women and one X and one Y chromosome in men). Half of these chromosome pairs are inherited from the mother via the ovum and the other half from the father via the sperm cell. These sperm cells contain either an X chromosome or a Y chromosome, which means that they will determine the sex of the child. In addition to the nuclear DNA, each cell also contains between 1,000 and 5,000 strands of mitochondrial DNA found in separate compartments, the mitochondria or "power sources" of the cell. In contrast to the nuclear DNA, the mitochondrial DNA is only inherited from the mother. Both types of DNA can be used for the genetic identification of people or biological traces, whereas only the nuclear DNA can be used to determine paternity. The purpose of a genetic identification test is to analyse a whole series of DNA loci or DNA markers in each of the people and/or biological samples (blood, sperm, saliva, urine, muscle, etc.) tested. Each of these DNA markers displays great variation in the population, either in length (number of nucleotides) or in composition (sequence of nucleotides). This variation is strictly hereditary and is present in each cell of the human body, which means that these DNA loci can be used as a hereditary marker. The hereditary variation of the nuclear DNA markers in the population is so great that the chance that the same combination of alleles may occur in two non-related individuals is less than 1/10,000,000,000. With the exception of identical twins, who have the same DNA, each person can be genetically identified on the basis of his or her DNA profile. The DNA test enables us to compare the combination of genetic markers or the DNA profile of people (victim, suspect) with the combination found in biological traces such as blood stains, sperm, hair, saliva, fingerprints, etc.
Technical notes
A number of steps can be distinguished in the process of genetic identification testing:
- examining the evidence with description and identification of the biological material and traces;
- isolating the DNA from the cell material in the traces;
- determining up a DNA profile;
- comparing the DNA profiles obtained;
- reporting on the results.
Each cell, be it blood, sperm or saliva, contains the same DNA if it comes from the same donor. However, the DNA profile of these cells does not “tell” whether the DNA comes from blood, sperm or saliva. Consequently, before starting the DNA test it is important to carry out an identification of the biological trace that makes it possible to prove that it is blood or sperm, for instance. A second aspect of this test involves demonstrating that the biological trace is of human origin. Although the forensic DNA test focuses on human DNA, a negative result (no DNA profile) may be due either to the fact that the DNA is of animal origin (e.g. blood from a pet) or to the presence of an inhibiting substance in the support on which the biological trace was found. The technique of DNA amplification or the "Polymerase Chain Reaction" (PCR), which is based on an enzymatic process, is used to analyse the DNA markers. Some chemical substances can prevent this reaction, in which case no result is obtained. This problem can be identified by DNA quantification whereby the presence of human DNA is proved.
The PCR technology makes it possible to copy specific DNA segments in a test tube, which means that millions of copies of DNA are available for further analysis. Various genetic markers from the nuclear DNA and known as STRs (Short Tandem Repeats) are analysed simultaneously in one test tube, which means that only a small quantity of DNA is needed for the test. Usually 1 ng of DNA is used (approximately 300 copies of nuclear DNA), but it is also possible to determine a DNA profile with less than 100 pg DNA. This is referred to as "Low Copy Number" DNA or LCN-DNA. This situation occurs when analysing micro traces or cell material in fingerprints.
After amplification, the various DNA fragments are made directly visible once they have been separated according to length (electrophoresis) and automatic laser detection. After amplification, the mitochondrial DNA is characterised by means of an enzymatic process whereby the sequence of the four building blocks or nucleotides (A, T, C and G) are made visible. Two significantly variable segments (HV1 and HV2) each of 400 nucleotides are analysed here.
Sequence of a segment of the mitochondrial DNA.
The result of the DNA test is reported in the form of a table in which the various traces examined and the reference samples from the victim and/or the suspects are collected together with the DNA profiles or the mitochondrial DNA sequences obtained. The profiles consist of a series of figures for each DNA marker used in accordance with an internationally recognised nomenclature so as to enable comparison with tests conducted in other countries or with foreign databanks. The DNA profile also indicates the sex (XY for a man and X for a woman) of the donor of the profile on the basis of a DNA analysis of the amelogenin gene (AMEL) that is located on the X and the Y chromosome. The mitochondrial DNA profiles show the differences with a reference sequence used at international level ("Anderson" or "Cambridge Reference Sequence"). An indication is also given of the positions between which the sequence is determined.
Example of a table with the DNA profile determined in the DNA from a blood trace.
Example of a table with the mitochondrial DNA profile for two segments of the mitochondrial DNA from a hair.
The results are discussed and a conclusion is formulated. If the profiles do not correspond, then these people can be entirely ruled out as possible donors of the biological traces. If they are identical, then an indication is given of the expected probability that another non-related person in the population may have the same DNA profile. This is based on the occurrence of the different variations in a given population. If more than one DNA profile is revealed in a biological trace (e.g. blood stain from two people, victim and suspect), a value is also given as to which hypothesis (victim or suspect, or two unknown persons) is the most probable to explain the results.
Example of a DNA mixed profile
Genetic identification test in the laboratory
The law of 22 March 1999 (link to pdf containing law: Appendix X1) on the identification procedure by means of DNA analysis in criminal cases governs the legal and practical aspects of the forensic DNA tests in criminal cases in Belgium. The law provides for the establishment of two national DNA databases (convicted and non-identified traces) to be managed by the National Institute for Criminalistics and Criminology, and the recognition of laboratories to carry out the DNA analyses. The Royal Decree of 4 February 2002 (link to pdf containing Royal Decree: Appendix X2) describes the conditions for the recognition of these laboratories and the establishment of the DNA databases. The Forensic Genetics and Molecular Archaeology Activities Centre was officially recognised for performing DNA analysis in criminal cases by the Belgian minister of Justice on 28 May 2004 after it has been accredited by BELAC (BELTEST until August 2006) in accordance with the international standard ISO 17025.
Since 1998 the Laboratory of Forensic Genetics and Molecular Archaeology Activities has used thirteen different STRs to determine a DNA profile. These STRs are part of the CODIS-DNA database (Convicted Offender DNA Information System) of the FBI in the US and are partially selected by Interpol (European Standard Set or ESS loci). The combination of these STRs gives a DNA profile that is unique for each individual. This series of DNA markers has been expanded over the years so that we are now able to analyse 21 autosomal STRs and 12 STRs (DYS390, DYS391, DYS392, DYS393, DYS389I/II, DYS19, DYS437, DYS438, DYS439 and DYS385a/b) on the Y chromosome. Standard practice is to use the STRs in Table 1 to determine a DNA profile of biological trace material. This set is expanded to include the STRs Penta D and Penta E for reference samples and the samples for the national DNA databank of "Convicted offenders". The other autosomal STRs are used in complex cases of relationship determination.
Examples of STRs (pdf, 30.53 KB)(pdf)
The STRs on the Y chromosome are applied in the analysis of mixed traces of male and female cell material, primarily in the context of DNA tests conducted as part of investigations into sexual offences. They can also contribute towards demonstrating the number of male donors of the DNA in the mixed trace. In relationship testing with male descendants the STRs on the Y chromosome can be used to confirm inclusion/exclusion or when the biological father is not available for the DNA test (e.g. he is missing or has died). In the context of a genealogical test, DNA analysis of STRs on the Y chromosome can also contribute towards determining the inclusion or exclusion of common paternal relationship.
Apart from the STRs, the mitochondrial DNA is also used for judicial identification. Specifically in the cases of shed hairs found in balaclavas, this is a breakthrough. These hairs no longer have any hair roots in which the cell material containing the nuclear DNA is present. However, the hair shaft does still contain enough mitochondrial DNA for analysis. The disadvantage of the mitochondrial DNA is that brothers and sister have inherited the same mitochondrial DNA from their common biological mother. However, this is an advantage in the case of the identification of skeletons by comparison with the mitochondrial DNA of a maternal related person.
Quality management in the laboratory
The laboratory strives to offer the highest possible quality in DNA analysis and reporting as part of the forensic DNA test.
This is achieved by:
- the implementation of a quality system in accordance with standard ISO 17025 for test laboratories, such as:
- measures to prevent contamination (including the layout of the rooms, separate analysis of reference and trace samples – see figure)
- permanent training for staff members
- participation in external quality control processes
- thorough internal monitoring of analysis results (including using multiplex PCR reactions with partially common DNA markers)
- accreditation of all our services activities (judicial DNA test and determination of relationship) by BELAC
- implementation of state-of-the art technology and methods in the forensic DNA examination
- active participation in research and development in forensic DNA testing.
- Independent analysis of reference samples and trace samples to precent contamination.
Independent analysis of reference samples and trace samples to prevent contamination.
Print
Send to friend