Quantification of DNA From Forensic Exhibits

Quantification of DNA From Forensic Exhibits

Deoxyribose nucleic acid(DNA) as we know holds the blueprint of every individual’s character and traits. When it comes to forensic science, DNA is worth a million gold for the scientists who analyze the sample. Multiple criminal and civil cases across the globe were solved due to this miraculous invention. 

Edmond Locard gave Locard’s principle of exchange in forensics stating every contact leaves a trace. When culprits leave the crime scene, they tend to shed evidence without their knowledge. Now, this could include their bodily cells or any kind of body fluids.

These samples hold a huge amount of DNA traces. This biological material can also end up being contaminated if not properly collected and packed from the crime scene.

Sources of DNA

DNA can be extracted mostly from biological evidence. However, few physical evidence like fabrics or any kind of surface could hold this biological evidence or even direct touch DNA.

When a person comes in contact with any surface, even a minute touch of their body part to the surface could lead to shedding of their body cells which can contain DNA and this is referred to as touch DNA. Only a very small amount of touch DNA is required for analysis.

Other biological evidence that holds DNA could be blood, semen, hair, saliva, sweat, and vaginal swabs. Though DNA extraction and amplification from saliva and sweat are not preferred usually due to lack of sufficient sample quantity.

DNA is a powerful investigative tool because, except for identical twins, no two people have the same DNA. Therefore, DNA evidence collected from a crime scene can be linked to a suspect or can eliminate a suspect from suspicion. 

The effective use of DNA as evidence may also require the collection and analysis of elimination samples to determine the exact source of the DNA. Elimination samples may be taken from anyone who had lawful access to the crime scene and may have left biological material.

Collection of Samples For Analysis

DNA samples are prone to contamination, hence a thorough and safe collection of the samples is necessary. When body fluids are deposited on cloth materials or any movable surface as such, it is air-dried and is packed in a breathable format inside a paper.

If the surface is not movable as a whole, the portion containing the potential evidence is cut out or if the fluid is still wet and is present on an immovable surface, swabs are done. Once these swabs are air-dried they will be packed.

In cases, like sexual assaults which are extremely sensitive, the sample collection differs. DNA is the most abundant form of evidence in sexual assaults, hence the collection is mostly done from the victim’s body. If the victim is alive post the assault, it is of utmost necessity that evidence collection needs to be conducted considering the mental trauma they would have faced and the situation they would be in.

To assist in the collection, victims of sexual assault should not change clothes, shower, or wash any part of their body after the assault. Such evidence as semen, saliva, and skin cells may be found on clothing or bedding, under fingernails, or in the vaginal, anal, or mouth region.

Evidence on or inside a victim’s body should be collected by a physician or a medical examiner. A medical examination should be conducted immediately after the assault to treat any injuries, test for sexually transmitted diseases, and collect evidence, such as fingernail scrapings and hair. Typically, the vaginal cavity, mouth, anus, or other parts of the body that may have come into contact with the assailant are examined. The examiner should also take a reference sample of blood or saliva from the victim to serve as a control standard.

Reference samples of the victim’s head and pubic hair may also be collected if hair analysis is required. A control standard is used to compare known DNA from the victim with that of other DNA evidence found at the crime scene to determine possible

Avoiding Contamnation & Preserving the Samples

DNA evidence can be contaminated if DNA from other sources gets mixed and ruins the relevance of the case. Right from the officers who collect evidence from the crime scene to the scientists who analyze the samples at the lab are advised to wear disposable gloves, use clean instruments and avoid touching other objects including their own body while handling the evidence.

The degradation of DNA samples is not only affected by cross-contamination but also by environmental factors such as heat and humidity. If the sample is wet and is packed tightly in a plastic bag it can contribute to the growth of additional bacterial cells within the existing evidence.

Or if the heat is too much the DNA itself can get scorched out or degraded. Hence, with the proper collection procedure and handling of evidence, DNA can be stored for years without risk of extensive degradation even at room temperature.

Extraction of DNA From Forensic Exhibits

The most common evidence found at the crime scenes in serious assaults includes murder, rape, attempt to murder, etc. are blood, semen, hair, and traces of saliva. These four kinds of evidence can be used for extracting the DNA. 

DNA extraction is the isolation of total cellular DNA from the desired evidence matrix for analysis. The method commonly used for the extraction is phenol-chloroform in terms of organic extraction. For lysing the cellular components of the sample obtained, Lysis buffers I, II, III, and IV can be used depending upon the nature of the sample. Protein walls of the cells are digested using proteolytic enzymes such as proteinase K before the extraction starts.

The Phenol-Chloroform Method

This method is preferred because it helps in removing all other organic substances that might interfere in DNA identification and it is easy to perform. Chloroform is used along with phenol due to its higher density which helps in forming an aqueous solution of phenol and chloroform. To reduce the forming that might be caused when phenol and chloroform are mixed, isoamyl alcohol is used framing a sharp interface between the two.

Requirements for phenol-chloroform extraction include EDTA tubes, lysis buffer I, Lysis buffer II, sodium lauryl sulphate, proteinase K, phenol, chloroform, iso methyl alcohol, sodium acetate, ethanol or absolute alcohol, Tris EDTA buffer, micropipette, filter paper, incubating chamber, centrifuge, and water bath. The role of TE buffer in extraction is to adjust the pH to approximately 7.5 to 8 making the mixture polar and easy to dissolve DNA.

Procedure:

1. 500ml of blood is taken in EDTA tubes and an equal amount of lysis buffer I is added to it.
2. The tube is then incubated at -20 degrees celsius for around two hours and is again kept at 60 degrees celsius for 10-15 minutes for lysing the cells.
3. The samples are then centrifuged at 4600rpm for ten minutes. The supernatant is discarded and the pellets are mixed with an equal amount of lysis buffer II.
4. 100 microlitres of 20% Sodium dodecyl sulphate or sodium lauryl sulphate along with proteinase K is added to the mixture. The sample is further incubated at 37 degrees celsius overnight.
5. An equal amount of Phenol is added to the incubated sample by the inversion method.
6. The sample is then centrifuged again at 4600rpm for ten minutes and the supernatant is transferred to another tube, to which phenol and chloroform are added in the ratio 1:1 and is mixed by inversion and centrifugation at 4600 rpm for ten minutes.
7. The process is repeated and the pellets are further dissolved by TE buffer and are kept for 60 degrees celsius for ten to fifteen minutes in a water bath. Once the pellets are dissolved, the sample is ready for amplification and profiling.

The process is almost similar in most of the cases for all the biological samples. In the case of hair strands, mitochondrial DNA is obtained mostly than nuclear DNA until there is a forceful retaliation between the culprit and the victim. Hence mitochondrial DNA will provide the maternal lineage of hereditary linking.

Amplification of DNA Samples

The most common method for amplification of DNA samples obtained after extraction is Polymerase Chain Reaction – The PCR technique. Polymerase chain reaction results in increasing the number of DNA samples present to millions of copies to perform a correct analysis and matching. Its requirements include,

• Thermal Cycler: It is the PCR machine inside which the reactions take place with samples. It has a thermal block with holes for loading the sample tubes containing reaction mixtures. It has the space to load three hundred plus vials usually distinctively set in three thermal blocks, along with preloaded coding features and screen to control the operation of the machine such as time set and program set. 
• DNA Polymerase: Polymerases are used in stabilizing the reaction so that the DNA would be successfully made at the high temperature required for the reaction. Hence the most important feature of a DNA polymerase is that it should be heat stable. A few of them are PFU polymerase, obtained from Pyrococcus furiosus, and TTH polymerase obtained from Thermus thermophilus. However, the apt and common polymerase used is Taq DNA polymerase. It is obtained from Thermus Aquaticus, which possesses the capability of tolerating very high temperatures and is found in hot springs across the world. It was first invented in 1969 at the hot spring of Yellowstone National Park. It is also believed to be the most ancient form of bacteria. They are capable of making new proteins such as DNA in high temperatures without getting degenerated, due to which Taq Polymerase is made from this group of bacteria for PCR. 
• DNA Primers: Primers are short sequences of nucleotides that provide a starting point for DNA synthesis. They are attached at the end of DNA strands that are being multiplied which also helps in elongation. They are usually around 20 nucleotides in length. For a nucleotide to be used as a primer it should contain certain properties: 
• Deoxyribonucleic Triphosphates(dNTPs): Nucleosides bonded with three phosphate groups containing deoxyribose as the backbone are dNTPs. They are organic compounds used as the building blocks of DNA. They help in enhancing the production of new DNA strands.
• Tris Buffer: Also known as Trisaminomethane buffers, they are used in TAE and TBE variations of buffers in solutions of nucleic acids. It helps in maintaining the pH of the reaction and has a melting point of 175 degrees celsius.
• Magnesium Chloride: The salt form of magnesium enhances the activity of Taq DNA polymerase and acts as a co-factor of the reaction by increasing the rate of amplification of DNA.
• Potassium Chloride: It neutralizes the charge present on the backbone of DNA and reduces repulsion between negatively charged DNA strands, i.e., the primer and the template, and stabilizes the primer-template binding.
• Bovine Serum Albumin: BSA helps in yielding results from the most impure form of sample DNA. It removes the contamination and enhances production. It also helps in the stabilization of enzymes during the storage of the samples. Sometimes Gelatin is used as an alternative to BSA or along with BSA. PCR takes place in four different stages which include:

1. Initialization: It is done only in the presence of DNA polymerase that requires manual heat activation by Hot Start PCR.
2. Denaturation: The target DNA, the sample to be amplified is heated at a temperature range of 94-98 degrees celsius for one minute, leading to the separation of the double-stranded DNA into two single-stranded DNA due to the breakage of hydrogen bonds between the base pairs in the given high temperature.
3. Annealing: The new strands are slowly built from the existing two template strands by attaching the primers in complementary directions(3’-5’: 5’-3’) using Taq polymerase. This is done by lowering the temperature to 50-54 degrees celsius.
4. Elongation: The multiple strands are now forming with one old and one new strand of DNA and the cycle is repeated thirty to forty times for obtaining around billions of copies.

Post amplification procedure using detectors to analyze the graphical result obtained and can support the applications as necessary. The amplified DNA bands can be distinctly visualized through gel electrophoresis too.

Other than phenol-chloroform extraction, certain other forms of extraction include Chelex extraction and Solid-phase extraction. Chelex works on the principle where the resin form of chelex prevents DNA degradation from degradative enzymes (DNases) and from potential contaminants that might inhibit downstream analyses.

In general, the Chelex resin will trap such contaminants, leaving DNA in solution. Solid-phase extraction (SPE) techniques have reduced analysis times and organic solvent consumption in DNA purification by applying samples to a sorbent material that reversibly binds DNA. However, SPE methods require tedious centrifugation steps, lack reusability, and suffer from high cost per sample.