Once the evidence collected during the inspection has arrived in the laboratory, it is subjected to a series of tests according to a standardized protocol in forensic laboratories, which can be summarized in the illustrative passages which follow.
In order to establish the presumed biological nature of samples taken during an inspection or present on items under examination, presumptive tests are carried out, which allow a generic identification to be made of the presumed biological material (e.g. blood, seminal fluid, saliva, etc). However, these tests must be confirmed by other methods which are able to establish the nature of the material under examination. The presumptive reactions for the generic identification of blood are based on the properties some substances have, when reduced to a colourless state (leuco base ) to turn a particular colour when in the presence of a peroxide (e.g. H²O²) and a peroxidase (e.g. haemoglobin) due to oxidation. In current use is Adler’s reaction, which consists of testing the item under examination with a saturated solution of benzidine in acetic acid, subsequently adding drops of H²O². If blood is present, the solution rapidly turns blue. Recently, reagent strips (Combur Test) were introduced into laboratory practice: [the strips are] impregnated with organic hydroperoxide and tetramethylbenzidine as a colourmetric indicator; if haemoglobin is present, causing oxidation, [the strips] turn from yellow to green-blue. The test is quick and easy to carry out, and the sensitivity varies, according to the Authors, from 1:300,000 to 1:500,000. False positives are known due to the presence of oxidants (e.g. metals like copper and iron), vegetable or animal peroxidases etc, while false negatives [can occur] due to strong reductive substances (e.g. cyanide) inhibiting the action of the haemoglobin.
If the traces are not visible to the naked eye (e.g. if present on a dark substrate) the Luminol test can be used (composed of an alkaline solution of luminol, sodium carbonate and sodium perborate). The solution is sprayed onto the substrate and, on reacting with haemoglobin, produces a chemiluminescence which is visible for a few seconds. There may also be false positive results with this test if non-haematic peroxidases are present.
In order to confirm that the material under examination is human blood (species identification) specific tests are employed based on immunochromatographic reactions which use monoclonal anti-human haemoglobin antibodies combined with a chromogen substance. The extracted material is placed on a reagent strip on which anti-human haemoglobin antibodies are immobilized: if human blood is present, the haemoglobin-antibody complex will concentrate the chromogen particles in a blue line. A positive control allows the accuracy of the reaction to be verified.
For the identification of seminal fluid, light sources can be used which cause the appearance of a white-blue light (known as ‘lunar’) if seminal fluid is present.
Among the most widely used generic and species-specific tests for seminal fluid are the immunochromatographic methods. These are quick and easy to perform, and allow the detection of two of the major components of human sperm: the Prostate Specific Antigen (PSA) and Semenogelin (Sg) . In addition to this, of course, microscopic identification of the spermatozoa (if present) is carried out, after they have been suitably stained [for viewing].
For the identification of saliva, immunochromatographic tests with monoclonal antibodies [specific for] human alpha-amylase are used, an enzyme present in large quantities in human saliva.
Once it has been confirmed that the collected material is a biological substance of human origin, laboratory investigations proceed with the aim of arriving at an individual identification.
For this purpose, [the direction of] the testing turns to the search for a genetic profile through DNA analysis.
Individual identification by DNA analysis: The cells of the human body have a structure constituted of a membrane, cytoplasm and the organelles associated with it, and the nucleus. The genetic material of the cell (DNA) is located inside the nucleus: it forms a complex with proteins, and is organized in linear structures called chromosomes. The human genome is constituted of two types of genetic material: the nuclear DNA (consisting of 23 pairs of chromosomes, 22 of these being autosomal pairs, and 1 pair of sexual chromosomes) and mitochondrial DNA. The nuclear DNA (deoxyribonucleic acid) contains all the information necessary to construct an organism, to make it function and to maintain it, as well as transmitting life from one generation to the next. It is a macromolecule made up of sub units called nucleotides, each of which is composed of a five-carbon sugar (deoxyribose) attached to a nitrous base (adenine, guanine, thymine and cytosine) and a phosphate group. The characteristics of an individual transmitted from one generation to the next are controlled by features of DNA called genes. The genetic constitution of an organism is called its genotype, while the phenotype is the physical manifestation of these genetic characteristics. The location in the chromosome of a particular gene is called its locus. Genes can exist in different forms, called alleles, which may give rise to the expression of different characteristics. An organism which inherits two identical alleles from its parents (for each specific locus, one allele is of paternal origin and one of maternal origin) is defined as homozygote, while those which possess two alleles that differ from one another are termed heterozygote. It is believed that the human genome contains only 20,000-25,000 genes, and only 1-1.5% of the genome is directly involved in the codification of proteins. About 75% of the genome is termed extragenic and more than 50% of this is composed of DNA repeats; 45% of these are interspersed repeated sequences, and the rest are sequences of DNA in tandem repeats (Lander et al., 2001; Li, 2001). It is these latter, made up of satellites, minisatellites and microsatellites, which are the regions of the genome most used for individual identification. At the end of the ‘80s, in the same period in which minisatellites were discovered, microsatellites were identified [descritti] – better known as Short Tandem Repeats or STRs. These have a repeated sequence from 2-7 bp, and to this day represent the most commonly used technique in the field of forensics. Tetrameric repetitions (a unit repeated in four bases) were combined in the so-called multiplex [technique] in order to obtain 16 loci amplified simultaneously in a single PCR reaction. The STR regions are highly polymorphic , and are therefore able to provide a very high power of discrimination (Butler, 2005).
In 1997, the Federal Bureau of Investigation (FBI) laboratory proposed the establishment of a group of STR loci to be used in a national DNA database known as CODIS (Combined DNA Index System) (Budowle, 1999). The thirteen CODIS loci are: CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51 and D21S11. A DNA profile obtained with the analysis of the 13 STR loci provides an average random match probability of about 1 in a trillion in randomly chosen individuals in the population (Butler, 2005). These loci are recognized in the United States as the standard for the purposes of human identification.
In the field of forensic DNA analysis within Europe, on the other hand, the Official Journal of the European Union, dated 5.12.2009, established that Member States of the European Union would be invited to use at least the markers indicated in the relevant Annex – which form the European Standard Set (ESS) – in order to facilitate the exchange of DNA analysis results between laboratories of the Member States. The European Standard Set comprises the following markers: D3S1358, VWA, D8S1179, D21S11, D18S51, HUMTH01, FGA, D1S1656, D2S441, D10S1248, D12S391 and D22S1045.
DNA extraction: a biological sample taken from a crime scene (e.g. blood, saliva, seminal fluid etc), as with a blood or saliva sample taken from a suspect or for a paternity case, contains many substances as well as DNA. The DNA must therefore be separated from the remaining cellular material before it can be examined. The cellular proteins which encircle and protect DNA in the cell can, in fact, inhibit DNA analysis. Many extraction techniques were developed in order to purify DNA molecules of proteins and other cellular material, but the fundamental principles of this technique can be summarized in three main phases: 1) fragmentation and break-down of cell membranes to allow the release of nucleic acids; 2) denaturation of proteins; 3) separation of DNA from the proteins and removal of contaminants.
The specific method of extraction applied in each individual case depends on the type of biological sample to be examined: for instance, whole blood must be treated differently to a haematic trace or a bone fragment. Organic extraction in phenol-chloroform was the extraction method commonly used in the past, and it is characterized by the use of different chemical substances through successive passages. If it is true that this method functions well for DNA to a high molecular weight, it also takes a long time, requires the use of toxic chemical substances, and requires transferring the sample between different test-tubes, therefore increasing the risk of error and contamination.
Extraction with Chelex 100 resin is characteristically much quicker than the organic technique, taking only a few passages inside a single test-tube, and therefore offers minor possibilities of contamination inside the laboratory. Introduced into the forensic DNA community in 1991, Chelex 100 (Bio-Rad Laboratories, Hercules, CA) is an ion exchange resin, which is made into a suspension [with distilled water] (usually at 5%), and added to the samples to be examined (Walsh et al, 1991). The samples are then subject to boiling for a few minutes in order to break down the cells and release DNA. The Chelex denatures the double-stranded DNA, converting it to single-stranded DNA.
Solid phase DNA extraction methods were developed in recent years in order to allow high yield DNA extraction. One very popular technique uses spin-columns, in which the nucleic acids selectively bind to a silica surface (e.g. in the form of small beads) in the presence of high concentrations of chaotropic salts; these disrupt the hydrogen bonds in water, denaturing the proteins. In a pH solution of less than 7.5, the adsorption of DNA to the silica is around 95%, and the impurities can then be removed by washing. Subsequently, the DNA can be easily eluted from the silica material using a low salt alkaline solution. This method can be carried out with the aid of centrifugation or vacuum aspiration.
A further solid phase extraction approach uses a silica-coated paramagnetic resin (Tereba et al., 2004); [with this method] isolation of the DNA can happen in a single test-tube, simply by adding and removing solutions. The DNA molecules bind reversibly to the magnetic particles, and a magnet is used to isolate these particles on one side of the test-tube, thus holding the impurities (proteins, cellular debris) in solution, to be removed through subsequent clean-up. Finally, the DNA is released from the magnetic particles by a phase of heating for a few minutes.
The FTA card contains chemical substances able to protect DNA from degradation by nucleases, and from bacterial growth (Burgoyne, 1996): the DNA therefore remains stable at room temperature for a period of several years. Use of the FTA card simply entails adding a drop of blood onto the card and drying it. The cells are lysed in contact with the card, and the DNA contained in the white blood cells is trapped in the matrix of the card. A small fragment of card is punched out at the area of the stain, and placed inside a test-tube for subsequent clean-up with solvents able to remove the heme and other PCR inhibitors.
Special methods were developed for isolating male DNA (sperm heads) from female epithelial cells (Gill et al., 1985) in sexual violence cases, thus enabling interpretation of the data by removing most of the female contributor from the resulting profile. This procedure, known as differential extraction, can be carried out through the selective digestion of epithelial cells, followed by isolation of the undigested sperm heads through centrifugation. The spermatic fraction is then further digested [i.e. to release the DNA] and extracted using dithiothreitol (DTT).
Automation of the DNA extraction process: many different instruments were produced with different properties and capacities to process larger or smaller numbers of samples, but the fundamental mechanism, implementation procedures and simplicity of use are similar. The most popular automated method uses magnetic particles. This procedure allows the simultaneous extraction of DNA from a large number of samples (up to 96) with the guarantee of maximum reproducibility, quality and productivity.
DNA Quantification: determining the quantity of DNA present in a sample is important for the majority of PCR-based analyses, because an excessive quantity can cause the appearance of extra peaks, or peaks which are outside the limits of the measurement technique, while too scarce a template quantity can cause allele drop-out in which the PCR reaction is affected by stochastic phenomena. PCR amplification can also fail due to the presence of inhibitors extracted along with the DNA from the sample, DNA degradation, an insufficient quantity of DNA, or a combination of all these factors. Many methods were developed for DNA quantification, including the slot-blot procedure and the analysis of fluorescence using a microtiter plate.
A relatively recent innovative method in the field of forensic DNA typing is quantitative PCR (qPCR), also known as Real-Time PCR. This technique requires the use of a machine able to measure DNA concentration during the PCR phases while the template is amplified. Quantitative PCR includes the same phases as traditional PCR (denaturation, annealing and extension); however a fluorescent marker sensitive to the formation of dsDNA is introduced into the reaction, and so allows the amplification product to be measured as it accumulates. The principle advantages of qPCR quantification include a vast dynamic range, high yield capacity, high sensitivity and target-specific quantification (Butler, 2005).
Commercially available kits, today in widespread use in forensics, allow the simultaneous calculation of the total quantity of human DNA and of male [DNA], using a primer sequence targeted at a specific region of the Y chromosome. Cycle by cycle, Real Time PCR analyzes variations in fluorescence from the amplification of a target sequence during PCR. There are three different phases which define the PCR process: exponential (or geometric) amplification; 2) linear amplification; and the plateau phase. The best point to measure fluorescence (through a comparison with samples of known concentration) in terms of the number of cycles is the exponential PCR phase, in which the ratio between the quantity of product and the starting amount of DNA is more reliable. The qPCR instruments use the ‘cycle threshold’ (Ct) for the calculations. The Ct value is the point at which the fluorescence of the signal exceeds an arbitrary threshold level: the fewer cycles required to exceed this threshold, the greater the quantity of DNA template molecule placed in the reaction.
Another DNA quantification method, less sensitive than qPCR and not specific to humans, is the Qubit Fluorometer 2.0, which utilizes a fluorometric technology using Molecular Probes markers; these emit fluorescent signals only when they are bound to specific target molecules, even in the presence of free nucleotides or degraded nucleic acids. With this method, samples with concentrations as low as 10pg/ul of DNA can be quantified (the analysis range of the Qubit dsDNA HS Assay kit is 0.2-100 ng, corresponding to a starting sample concentration in the range of 10pg/µl-100ng/µl).
Dna Amplification: The development of the Polymerase Chain Reaction (PCR) in 1985 by Kary Mullis gave a big boost to scientific progress in the field of DNA analysis, particularly in the forensic community. This technique allows the production of multiple copies of a nucleic acid sequence in vitro (Mullis, 1990). During the PCR reaction, the double-stranded DNA (dsDNA) is subject to a three-phase process: denaturation, annealing and extension. The strands of each template molecule are denatured through heating (~95ºC) and subsequent cooling (~55ºC), allowing the primers (small oligonucleotide sequences) to bind to the single strands. The temperature is then raised again (~72ºC) and a DNA polymerase enzyme extends the primers, absorbing the dinucleotide triphosphates (dNTPs). In this way, the synthesis of target sequences of dsDNA happens in a rapid cyclical process. At each cycle, the PCR generates a 2n increase of the target DNA (in theory, the quantity of DNA doubles). The PCR requires a mix of reagents and amplification parameters which must be optimized to produce sufficient amplification product. The major components included in the “master mix”, which must be aliquoted into each PCR test-tube, are the following: buffer, dNTPs, polymerases, primers and magnesium. PCR has become an important analytical tool in the forensic field due to its sensitivity, specificity, speed of analysis and ease of automation. The PCR amplification technique allows the analysis of low quantity (>1ng) forensic samples of extracted DNA, as well as [being] a valid and reliable approach to the analysis of biological samples recovered from a crime scene.
Capillary Electrophoresis: the PCR kits commonly used in forensic practice allow the simultaneous amplification of numerous DNA fragments, which, as STRs, are constituted by different numbers of repeating units: therefore different alleles in the generated amplicons present different lengths. In order to be analyzed, they must be separated using an appropriate technique with a high resolution [i.e. separation] capacity, so that even those alleles which differ from each other by a single base can be analyzed, in a range from 100 to 500 bp. Moreover, the method used must be reproducible to allow the results from different laboratories to be compared. To separate the various molecules in the amplicon mixture produced by the PCR reaction, the negatively charged phosphate groups in the DNA backbone are used: in an electrical field, the ions are attracted by the pole of the opposite charge; therefore in the case of nucleic acids, by the positive pole. This process is called electrophoresis, and it refers to the migration of electrical charges in a separation medium, to each end of which a potential difference is applied. The capillary electrophoresis technique (CE) was introduced in the early ’80s, and with subsequent development of the instrumentation, quickly gained in popularity in the fields of molecular biology and forensics. This instrumentation is completely automated, and allows the examination of multiple wavelengths simultaneously and therefore a high number of loci which overlap in length, with minimum sample consumption subject to the run. The signal emitted by fluorochromes, excited by a laser in proximity to the anodic extreme, is recorded by a detector consisting of a Charged-Coupled Device (CCD): the greater the number of photons hitting the surface of the silica matrix, the greater the accumulation of electrons and consequently the height of the digital signal into which it is converted. The data is finally sent to a computer which, correlating the fluorescence peak with the migration time, translates the fluorescence signal into a length measurement expressed in bp or in nucleotide sequence. The sizes of the PCR products of each sample are compared with the fragments contained in the allelic ladder. The ladder is constituted of a mixture of alleles of known length (these are the most common alleles of a particular STR present in the population, and therefore representative of the variability of a given STR) and is used to correlate the size of the amplification product with the number of replicates by which it is formed; in this way, the genotype of the sample can be established. Each peak of the sample must not differ in length by more than 0.5 bp from the corresponding ladder peak, or the allele is not assigned and the peak is defined off-ladder (OL). At the end of the electrophoretic run of each sample, a specific software produces a file called raw data: a graph which correlates the Relative Unit of Fluorescence (RFU) on the Y axis with the number of data points on the X axis.
Interpretation of the Results: the conversion of an electropherogram into a genetic profile is carried out through software, but the profiles generated from the samples must be interpreted by expert staff. Guidelines were developed for the interpretation of genetic profiles to ensure that the results obtained are reliable: this aspect is of crucial importance, especially when the samples to be analyzed contain very low quantities of DNA, degraded DNA, or mixed profiles. Each laboratory must develop a proper interpretative strategy based on internal validation studies, and on the results reported in the literature (Scientific Working Group on DNA Analysis Methods, SWGDAM, 2010). Some of the most important guidelines for the correct interpretation of electropherograms are the following:
- it is necessary to establish a minimum value for the heights of peaks to be considered alleles; all peaks below this value are considered background noise;
- the electropherogram must show balanced peaks – that is, peaks of a comparable height. In particular, individual heterozygous loci must have peaks of about the same height. To evaluate the peak height balance of a locus, the ratio between the height of the smallest allele and that of the largest allele is calculated: usually this ratio is always greater than 90% (>0.90) but the threshold value is placed at >0.60.
- it is necessary to consider the maximum percentage of stutter produced at each locus. Stutter are aspecific peaks due to [strand] slippage and to the erroneous pairing of the repeating regions of the two DNA strands during the PCR reaction. The peaks are usually shorter by one repetition than the nearest allelic peak. The presence of stutter is of great importance in the interpretation of mixed traces, in that it can be difficult to establish if a peak is stutter or a true allele. It should be recalled that the maximum stutter percentage observed in any individual locus is stated in the user manual of the specific amplification kit. Stutter is identified by the ratio between the height or area of the minor peak, and the height or area of the major peak: this ratio is generally below 15%.
 ‘Leuco base’: “a colourless compound formed by reducing a dye so that the original dye can be regenerated by oxidation”.
 A commercial example of such a test.
 Polymorphism: “Natural variations in a gene, DNA sequence, protein, or chromosome that have no adverse effect on the individual and occur with fairly high frequency in the general population”.