Type of Evidence: DNA Evidence
Representative Objects and Materials Constituting DNA Evidence
Anything can have DNA evidence on it so long as it contains human cells. It’s easier to extract epithelial cells and human sex cells; to take them from urine samples, drops of saliva, buccal swabs, left-behind sperm and blood, or a mixed epithelial/sperm sample on a piece of fabric, as in an example pair of underwear, there are many ways and places to obtain it from. What it requires is cells from which to extract human cells, be they haploid sex cells or the diploid rest-of-them. Swab it from the lightswitch, then; swab it from a piece of evidence, so long as you sign the chain of custody.
Chemical and Physical Tools and Techniques used to Analyze DNA Evidence
At the scene, the investigator can and should identify first priority samples, second priority samples, and third priority samples, each defined by how likely they are to contain usable DNA and how well they will yield non-mixed DNA profiles. This requires the use of swabs and proper packaging techniques for items of evidence that can be collected.
First priority DNA samples are defined by their high DNA concentration from a single source, most often the suspect. Second priority DNA samples are defined by their moderate DNA concentration, usually left through aggressive touching, left most likely from a single source (or sources), hopefully the suspect or suspects. Third priority DNA is in low concentration, left by lots of touching from multiple possible sources and, as such, forms multiple DNA profiles. Swabbing areas get larger as priority tier increases and as DNA becomes less concentrated, as a result.
They should also take care to collect control samples (blank swabs or “blank swabs” to collect the DNA background noise of the area) and reference samples to serve as knowns and comparison tools.
At the scene, this type of trace evidence can be identified with both white and various colored light sources and contrasting barrier filters within the visible light spectrum. Colored alternate light sources are most useful for biological fluids like saliva, urine, semen, and blood, and for certain non-epithelial and non-gametic parts of the human body such as the bones that tend to fluorescence due to chemical compounds within them; using these light sources in tandem with the correct barrier filters will allow the investigator not only to identify the location of potential samples, but also to correctly document them. Just as any place where a weapon is found is a place where blood can be, sperm can be found pretty much anywhere.
When it comes to documenting blood samples, something similar is true, but there is more to it. Blood tends not to fluoresce in the presence of ultraviolet light, but is photographable with a combination of infrared photography techniques and ultraviolet light; these may be utilized to find it. If luminol is applied to blood, causing chemiluminescence, it can be seen much more easily; but this should be used carefully and with discretion.
The general process concerning DNA concerns the collection of the unknown and a series of knowns, then the extraction, quantification, amplification, separation, analysis, interpretation, and quality assurance of each.
Extraction can be done very simply, using a series of proteinases, detergents, and other assorted reagents, then precipitated with ethanol.
The extracted DNA must then be amplified using a thermal cycler. The polymerase chain reaction conducted here requires a buffer, MgCl2 (a DNA polymerase cofactor), dNTPs, an optimized primer, and, obviously, the target DNA.
Separation follows amplification. Geneticists have done this for years, with many different techniques that have been widely accepted by the scientific community (and are therefore acceptable). In the future, tools like Rapid DNA could be used widely in the field following correct validation and the acceptance of its scientific principles in the field; but, as of yet, it is only used in the state of Connecticut for the sake of generating leads and nothing else. The current standards for Rapid DNA are outlined under the “Rapid DNA Act of 2017,” and the tool itself is not to be used beyond the scope outlined by the working group established in congruence with this.
Historically, alternate forms of electrophoresis were used, including the combination of slab gel electrophoresis (using specifically polyacrylamide gel) and silver staining. Currently, however, the most commonly-used techniques for the purposes of amplicon separation are slab gel electrophoresis and capillary electrophoresis.
Following separation, some sort of interpretation or analytical tool is needed. Tools associated with this process are FMB10 Fluorescence Imaging Scanners and ABI Prism 373 or 377 DNA Sequencers. The use of each requires knowledge of the necessary software and data interpretation techniques, as well as underlying scientific principles of fluorescence, fluorescence tagging, and electrokinesis. This can assign a genotype to each fragment found, after which analysis can be performed.
Because both the unknown and known can be compared to a central database, these, too, must be known resources to the examiner. CODIS is the Combined DNA Index System; NDIS is the National DNA Index System. Together, they contain standards of genotypic information to which the examined unknown can be compared.
Forensically Valuable Properties of DNA Evidence and the Chemical and Physical Processes That Create Them
DNA is fairly individual. It can tell you the allelic makeup of who left a certain cell behind; but it is not infallible and, when a sample is mixed, it can be hard to extricate, barring mixed epithelial-gametic samples. Additionally, the sample can be contaminated at any point by any examiner, making the situation more complicated. For this reason, understanding the biology behind DNA and the human cell is incredibly important.
DNA, or deoxyribonucleic acid, is a polymer formed along a phosphate backbone that is stored within the nucleus of the cell— specifically within nucleotides. (Mitochondrial DNA also exists, but that’s not the kind we are talking about and not the kind we extract using these processes.) These helices are bound when outside the nucleus, tightly wound into chromosomes for easy crossing-over and recombination at a point called the centromere. We typically imagine the actual polymer in a double-helix form beyond the general X-shape of the chromosome, based on the research of Watson, Crick, and Franklin. Whether it’s in its single- or double-stranded form, the polymer is composed of that phosphate backbone and carbon, oxygen, nitrogen, and hydrogen (CONH) compounds bonded to it. Those major molecules make up each of the four common bases within DNA: cytosine, guanine, adenine, and thymine (and uracil, in RNA).
Cytosine and guanine hydrogen-bond to each other, and adenine and thymine do the same. Hydrogen bonds are, essentially, bonds formed by a hydrogen atom in a given molecule being attracted to an electronegative atom in another molecule. In this case, it would be the nitrogen in a compatible structure along the polymer. Adenine and guanine, both two-ring structures, can share two hydrogen bonds with each other at their respective available nitrogens; cytosine and guanine, one-ringed, can share three bonds at their similar nitrogens. Because of this double helix form, a series of major and minor grooves the form allows, and the spacing of the molecules, there is a fairly consistent series of negative charges along the polymer itself that make the process of electrophoresis so possible, when it occurs.
That being said, this polymer chain contains the genetic information of human beings created through any necessary recombination and crossing-over of parental traits. Large sections of it have no currently-known function, but those that do are incredibly valuable, both for medical purposes and for our forensic identification purposes. Because they are so specific, and there is a specific, calculable statistical likelihood of a given profile occurring in a given population, genetic information derived from this evidence is so valuable for comparative purposes.
Additionally, DNA is found in any cell with a nucleus. Though it is not found in reticulocytes (specific mammalian red blood cells that lack a nucleus because they eject it in favor of more hemoglobin), it is found white blood cells, making blood a useful source of DNA evidence so long as it has not separated into parts. Even then, it can be extracted from the white blood cells, meaning that the remaining plasma can be swabbed. Generally, however, DNA is found in any sample taken from the human body; in any body fluid that can be a carrier for epithelial cells, from tears to vomit to earwax; in hair, bone, and skin cells itself; and, naturally, in blood, semen, and vaginal secretions. The question, then, is how useful those collected samples can be. After all, some samples can be harder to extract and interpret than others, especially in the case of hair and especially in the case of mixed profiles.
All of the important parts of the DNA we’re looking at are found on the structure itself, in what are called short-tandem repeats. Standard forensic DNA testing has fairly little to do with proteins formed by DNA and everything to do with short-tandem repeats. These are sets of four base pairs that repeat over and over again in a certain identifiable pattern.
Historically, and currently, multiple locales on the human genome are looked at; CODIS currently limits it to twenty, an increase from the previous thirteen. For example, D1S80, which is more commonly singled-out in silver staining (though still used in modern fluorescence tagging), is on chromosome 1 in humans; its use in silver staining was phased out in favor of the current preference STRs. The delineation of what we are looking at, then, proceeds from the chromosome to the locus; from the locus to the STR; then, following the processes undergone, from the STR to the allele; and the allele to the DNA profile.
Because DNA is found within the nuclei of human cells, and we are typically looking for epithelial or gametic cells, there must be a way to remove it from them. This is where the specific reagents used in extraction come in. If you think of how dish soap and cold rubbing alcohol are utilized in the classic strawberry DNA extraction experiment, you can see how fairly one-to-one this can be. The difference here is, strawberries exhibit polyploidy. Classically, they’re octoploids, meaning that they have eight copies of their parental genomes. Humans, in contrast, are diploids. What this means is that human DNA is a little harder to access than a strawberry’s would be— or, rather, that a strawberry’s DNA is a little easier to access. I like to say that it’s fragile, but that’s not necessarily the case. Because strawberries are octoploids and humans are diploids, the structure of their cell membranes differs, and so too does their DNA. This makes strawberry DNA suitable for an introductory exercise when getting a handle on the subject (and incredibly fascinating when studying polyploidy), and thus a suitable comparative base for the extraction of human DNA from human cells. They are not, however, the same, even if we do compare the process.
With that one-to-one idea in mind, it’s very (no fruit puns, please) easy to think of what one might need in order to access the DNA within a cell; and, from there, what biological and chemical processes might be at play. Just as salt and dish soap are combined to create an “extraction” solution for a strawberry cell, human DNA requires a series of reagents for a proper extraction— to lyse the cell membrane and extract the DNA itself. During the process of biological stain extraction, the following are used:
EDTA is added to the stain during resolubilization to assist in the permeation of the outer cell membrane through its interactions with lipopolysaccharides alongside it. Tris already accomplishes this, but EDTA enhances the effect.
Cells are lysed using a detergent like SDS or sarkosyl, specifically to: lyse the cell membrane denature histone proteins; and destroy the secondary and tertiary structures of proteins, which serves to decrease their solubility in aqueous solution. This allows the DNA molecules to essentially “break free” of their cell membrane. Think again of the way that the combination of dish soap and salt allows the DNA of an octoploid strawberry cell to break free of its cells bonds. Here, similar processes are occurring. SDS is typically used, except in colder circumstances, where it would precipitate out of the solution. In a lysis procedure conducted under refrigerated conditions, you can use sarkosyl, which doesn’t have the same issue.
Proteinase K is an endolytic protease that cleaves peptide bonds at the carboxylic sides of amino acids; it serves a similar function to detergents, in that it hydrolyzes the histones, but is notably active in the presence of SDS and is not affected by EDTA. This makes it an incredibly useful reagent here, and especially in the lysis of epithelial and white blood cells.
Dithiothreitol (DTT) reduces disulfides to dithiols. This is the reagent used for gametic samples to break disulfide bonds, allowing the release of DNA from those protective proteins and for further degradation of the protein by proteinase K.
Following this, the DNA must actually be removed from the solution, like using a cold rubbing alcohol to remove strawberry DNA from a test tube. Here, the denatured proteins can be removed with chloroform. Water will rest atop the chloroform and phenol chloroform isoamyl alcohol (PCI) will be added to it. The resulting product can be purified further with cold ethanol. Remember, DNA “doesn’t like” cold ethanol— recall the way that strawberry DNA reacts in the same conditions. It precipitates out of the solution by “clumping up” in its presence (i.e., precipitating). This is because ethanol has a lower dielectric constant than the salt neutralizing the charges on the backbone of DNA, causing the DNA to effectively become less hydrophilic than normal and drop out of the solution. If conducting a two-step process to remove an epithelial sample from a sperm sample (or, otherwise, purification with ethanol is not desired), use of a centrifugal filter unit is also possible.
Pertaining to the above: while it is true that human DNA is diploid, the ingenuity of the process is that sex cells (gametic cells) are haploid. By utilizing DTT in a second round of extraction, you can get two separate samples from a mixed epithelial-sperm sample because, without that stronger cleaving agent, those disulfide bonds won’t break. The sperm sample will sediment at the bottom when centrifuged instead, meaning that it can be subjected to extraction on its own after the epithelial sample has been decanted out; the only difference is that you have to use DTT this time.
Amplification functions by a polymerase chain reaction (PCR) similar to the process of DNA replication within the cell, using two oligonucleotide primers designed to mark the segment containing the desired STR. Once marked, amplification is achieved by twenty-five to thirty cycles in a thermal cycler, which allows the sample to undergo three crucial steps: first, by the mechanism of heat, denaturation (which separates the DNA from a double helix structure into two separate strands); second, cooling to let the primer anneal to the separated segments; and, third, also by heat, the production of copied DNA through DNA polymerase’s addition of nucleotides (those A, T, C, and G groups). This forms the necessary amplicons, which grow exponentially in number— to about a billion by cycle thirty. In short, the process is denaturation, annealing, and extension, in those several cycles, until you have reached the desired number of replicated amplicons.
Multiplexing, the process that allows for the simultaneous amplification of multiple loci by adding more than one primer and optimizing the number of thermal cycles used, then takes into consideration the competing demands of locus size, the number of alleles, and the necessary balance across primers. This can cut down on the amount of time used per amplification process, if that is a necessary consideration.
The primer itself is meant to be optimized based on the values of free energy (ΔG.) as measured and calculated five base pairs away from the 3’-binding site and amplicon length (generally 18-30 base pairs in length; sequences outside those bounds exist, but are rarer on a statistical level. The amplicon length is calculated by taking the position of the antisense primer and subtracting the position of the sense primer from it, then adding one to represent a specific addition by Taq polymerase). If the primer is not optimized, this can lead to certain issues in amplification, such as the plateau effect— a loss of product compared to the theoretical yield. If the theoretical yield is linear, the plateau effect is a sort of “drop off” after a certain point in time that exists in contrast to it. While it can be affected by incomplete denaturation early on in thermal cycling, it’s almost likely affected by primer design. Nonspecific primer products can result in mispriming. Either way, it can be counteracted by optimizing the number of PCR cycles or by optimizing the temperature. (The optimal annealing temperature can be calculated by the sum of the melting point of the primer multiplied by 0.3; and 0.7 multiplied by the melting point of the product. Subtract twenty-five from this; the temperature should be measured in degrees Celsius.)
Inhibitors are often present in PCR, however, not just through ineffectual primer design or incomplete denaturation by heat; the process itself can be affected by compounds interfering with the interaction between DNA and Taq polymerase, and so these inhibiting compounds must be removed during the extraction process. Some of these are internal, coming from bodily fluids; others come from the substrate; and still others come from reagents and analysis materials. These can be counteracted with ammonium ions and dimethyl sulfoxide.
After extraction and amplification, DNA must be separated. This process depends on two primary factors: charge and mass. The general principle delineates as such: the smaller a fragment of DNA is, the quicker it will travel from the cathode to the anode. The reasons for this are more complicated than the principle (though, admittedly, simple). DNA is formed along a phosphate backbone. Because it takes the general form of CONH along this backbone (which allows for easy hydrogen-bonding and separation during the cell’s very common reproductive cycle), it has an evenly-spaced negative charge. This allows the anode to attract the molecule while the cathode, negatively-charged, repels it. At the same time, the slab itself acts as a sort of sieve or colander through which the fragments must trudge and filter, meaning that the smaller ones filter through quicker. This sorts them by size based on mass and propels them by charge at the same time. The consistent negative charge along the double helix structure of DNA allows it to, in essence, move along the medium of the gel. Because like charges repel each other, the cathode repels DNA in the same way that the anode attracts it. Any difference from this within the observed mechanism of slab gel electrophoresis is an indication that something has gone wrong within the process itself, causing a shift with the electric field or with the polymer such that it is not migrating or filtering through the gel correctly. These electric effects are based on Ohm’s Law, V=IR, where I is the current, R is resistance, and V is voltage. Here, if we keep resistance constant, an increase in current increases voltage, and vice versa. They are proportional to each other. This also means that, because electricity is also linked to the amount of heat generated by a system (in effect, energy), you must keep an eye on that, as well.
Generally, the gels utilized in amplicon separation through slab gel electrophoresis are created from one of two compounds: agarose or polyacrylamide. Agarose, a non-toxic gel derived from seaweed, is made of long chains of polysaccharides and, as such, has large pores and a high gel strength. This makes it suitable for separating large DNA molecules. Polyacrylamide, on the other hand, is used to sort smaller DNA molecules. It is formed through the polymerization of monoacrylamide and bis-acrylamide in the presence of free radical catalysts and accelerators. The covalent bonding of the two (monoacrylamide, which is a neurotoxin and must be handled with care, and bis-acrylamide) produces the gel itself, which is also toxic and transparent. Otherwise, the process of slab gel electrophoresis requires a buffer system and the use of a stain for banding. Of the two possibilities, agarose is preferred for larger DNA molecule separation; polyacrylamide is preferred for smaller.
Capillary electrophoresis, on the other hand, requires no such gel. In contrast, it requires a different process predicated on the same electric principles. The capillary itself is a hollow, fused silica tube with a polyamide coating. The coating is burned away to create a detection cell window through which to see the flagged bits of DNA. Similar to gel electrophoresis, the amplicons are inserted at the cathode and migrate to the anode in accordance with their own negative charge.
Contrastingly, however, the process runs differently. For one, the preferred process of insertion is electrokinetic injection (though siphoning and hydrodynamic pressure injection are also methods of sample introduction you could use when introducing your sample). Electrokinetic injection, as with gel electrophoresis, relies on electromotive force moving the sample from the cathode to the anode. DNA introduced at the cathode sees no significant loss of sample; rather, its transfer is influenced by ionic strength. The capillary ends themselves must be deionized, then, and stored in water or a buffer to ensure they don’t dry out.
Capillary electrophoresis uses spectroscopic detection based on laser excitation visible through the window. Each fluorescence tag associated with each bit of DNA going through it emits a different wavelength. This is measured by a charge-coupled device camera that outputs the data represented in peaks in an electropherogram with an x-axis representing the scan data points and a y-axis representing the relative fluorescence units, or the quantity of DNA based on its response from the detector.
This electropherogram data is analyzed through fragment sizing, where peaks or areas are assigned a height or band density, then given a genotype assignment. After that, the data is reviewed by an analyst and their results are confirmed by a second analyst in congruence with the ACE-V process.
Interpretation of genotypes is based on a pattern of peaks or bands, which are visual representations of DNA fragments. These are typically built on a bedrock of interpretation parameters including sensitivity, reproducibility, sensitivity, precision, and heterozygosity— like any good genetics-based science (or any good science, barring the bit about heterozygosity).
The Probative Value of DNA Evidence and the Range of Conclusions
When interpreting, we call these examined alleles “heterozygous” or “homozygous.” These are classic terms in the vein of Mendelian genetics (even Davenport used them, as eugenics-minded as he was), where homozygous refers to two similar alleles and heterozygous to two different alleles.
When looking at these alleles, we aren’t necessarily considering factors such as epistatic pathways, sex-inherited traits, and intersex conditions, but they are things we must be aware of in our interpretation of any information as they pertain to a human population and their relevant genetic statistics. If, for example, your non-mixed epithelial sample has a peak indicating no Y chromosome and one X chromosome, it would serve the examiner well to know about certain forms of trisomy, including XO mosaicism. While mixed samples are always a possibility (and would then contribute to an “inconclusive” conclusion), so too are conditions of aneuploidy; and both must be taken into account in any sort of analysis.
Because the goal of interpretation is to form a DNA profile and assess its statistical likelihood in a given population; and, ostensibly, to compare it to a known collected from someone associated with the scene or a known from CODIS/NDIS. There is so much science to be done within DNA (and currently being done), and humans are constantly shedding DNA everywhere they go. With that in mind, there still must be a conclusion on the part of the examiner. The American Academy of Forensic Sciences recommends the language “inclusion” or “inclusionary conclusion” (meaning that an individual is a potential contributor to a given profile, without confirming they are its sole contributor); “inconclusive” (i.e., lack of support for inclusion or exclusion); and “exclusionary conclusion” or “exclusion,” with the same logic as the definition for inclusion. A conclusion must be written for each sample tested.
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