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DNA Analysis in Criminalistics

DNA Analysis in Criminalistics

Criminalistics applies scientific techniques to collect and analyze physical evidence in criminal cases. Criminalists primarily focus on identifying, documenting, collecting, preserving, and analyzing physical evidence. Physical evidence often analyzed, compared, or identified includes firearms (ballistic evidence), drugs, fingerprints, fibers, hairs, and blood. The goal of a criminalist is to identify the source of physical evidence to provide the court with a connection to a certain crime scene. Examining and analyzing any biological materials present at the crime scene is one of the most effective methods to achieve this (Elkins, 2012). Subsequently, criminalists use scientific methods to identify the origin of biological crime scene evidence. These methods involve identification, analysis of DNA collected from the crime scene, and interpretation of results.

DNA Identification

Crime scene investigators responsible for recovering evidence from crime scenes are needed to identify, collect, handle, package, and label evidence materials from the crime scene correctly. They must also carefully document the location or position of the evidence at the crime scene, for instance, through photographs. Deoxyribonucleic acid (DNA) can be extracted from almost every biological material, such as blood (except red blood cells) and hair cells. DNA extraction separates DNA from the other cellular material (membranes and proteins). Biological items should be handled with care to avoid contamination.

DNA extraction involves three steps: lysis, where cells are broken and opened; separation of DNA from the other components of the cell; and isolation of the DNA. Heat (which increases fluidity) and a detergent, like sodium dodecyl sulfate (SDS) or dithiothreitol (DTT) that reduces disulfide bonds, are some of the methods used to disrupt the membrane. Proteins can be inactivated by heat denaturation or by digestive enzymes, such as proteinase K, to cut them. Additionally, the temperature should be kept below 60°C for 15 to 20 minutes. The magnesium needed for nuclease activity or DNA is immobilized on a solid phase and eluted by buffer or salt.

Suppose the DNA remains in the aqueous state; it is separated from the other cellular materials by centrifuging the proteins and lipids to the bottom of the tube or separating them in organic solvents. The commonly used procedure for DNA extraction is the organic method. In organic extraction, cells are lysed by gently disrupting the membranes with heat alongside SDS and DTT. Enzyme proteinase K is then added to digest the proteins and nucleases. The phenol-chloroform-isoamyl alcohol (PCIA) reagent is used to separate DNA from the other cellular components.

The addition of the PCIA results in the formation of two phases: an organic phase at the bottom and an aqueous phase containing DNA at the top. The DNA portion is obtained by pipetting the top aqueous layer. Since DNA is more soluble in its aqueous state, removing all the organic components is essential; otherwise, the phenol will degrade DNA. Finally, the DNA can be filtered and concentrated by centrifugation such that DNA is captured on the membrane. At the same time, all other aqueous components, like the protein fragments, pass through the membrane. The filter is inverted to obtain the DNA extract. The filter accumulates DNA into small volumes for subsequent amplification procedures. (Elkins, 2012).

DNA Analysis

This involves determining the quality and quantity of obtained DNA extract. DNA quality and quantity determination techniques include polymerase chain reaction (PCR) and Agarose gel electrophoresis techniques. Gel electrophoresis is the polarity-based separation of molecules. To elaborate, when identically charged ions are placed between two oppositely charged electrodes in a solution, the small ions migrate faster towards the electrode of opposite charge than the large ions. Similarly, the more highly charged ions move faster than the lowly charged ions when differently charged ions are placed in a solution. Subsequently, since DNA is negatively charged, it migrates through the gel toward the positive electrode (cathode).

Agarose gel electrophoresis is relevant in evaluating genomic DNA obtained from samples. For instance, tight bands illustrate intact DNA, while a smear signifies degraded DNA. This technique also determines the quality of DNA extract. For example, intense bands show high-quantity DNA, while faint ones show low-quantity DNA. Dyes are used to visualize the distance covered by the DNA extract. The distance traveled by the DNA extract is related to its length and structure.

On the other hand, PCR is used to make copies of the DNA extract. It rapidly amplifies and simultaneously quantifies the targeted DNA molecule in a specific region. Materials for a PCR procedure include a DNA template, two primers (5′ and 3′), magnesium ions, a buffer, and bovine serum albumin.

Factors That Make DNA Evidence Inadmissible

Interpretation of the results can be challenging when forensic samples are of poor quality and quantity or contain a mixture of DNA from several persons. It is uncertain where testing problems associated with low-quality or low-quantity samples may arise in a single case. An analyst may not realize that the results are unreliable and may even make wrong interpretative decisions that the court can consider inadmissible. For instance, for samples containing biological material from different people, tests cannot precisely reveal how much each person contributed to the sample, whether they contributed to the alleged crime at the same time, at different times, or when the biological material was deposited (Murphy, 2018).

The other challenge revolves around the issue of contamination. Factors contributing to contamination include improper laboratory environment, poor laboratory protocols, and poor quality control measurements. DNA is sensitive to environmental conditions. Therefore, exposure to extreme temperatures, water, sweat, oxygen, or sweat can lead to DNA degradation before and at the laboratory. Similarly, DNA testing errors may result from mislabeled samples (before and at the laboratory), cross-contamination of samples of the same case, or other cases. All these errors yield inaccurate result interpretations that can be considered inadmissible in court.

Tissue Matching

Other than protein-based assays, an alternative approach to examine biological material is tissue identification through the analysis of species of messenger RNA (ribonucleic acid) (mRNA) and microRNA (miRNA) (Frumkin et al., 2011). Tissue identification often applies DNA methylation-based procedures. Methylation occurs at the C5 position of cytosine in some CpG dinucleotides in mammalian DNA. 70 to 80% of all CpGs in the human genome are methylated, while unmethylated CpGs are grouped in ‘CpG islands’ at the 5′ ends. Consistently methylated loci form patterns; therefore, unmethylated loci can be easily identified within natural DNA from the collected forensic samples.

The first step of DNA methylation-based tissue identification is collecting biological material containing tissues. These can be saliva, blood, urine, semen, and vaginal secretion. The next step is DNA extraction and quantification. DNA can be extracted through organic extraction or any common extraction methods and quantified using PCR analysis. Genetic loci and primer design are then selected. The available software programs can then be used to select suitable genetic loci and primer designs. Digestion of the endonuclease, PCR, and capillary electrophoresis then follows. The endonuclease is digested by HhaI and amplified through PCR reactions. Data analysis for the tissue identification assay is carried out using software that can be easily interpreted (Frumkin et al., 2011).

Importance of Utilizing Different Analysis Methods

It is essential to utilize different DNA testing methods as different methods aim to achieve different goals. This enables the analyst to gather additional relevant information in a certain case. Moreover, different additional testing techniques are used as confirmatory tests. For instance, additional PCR tests can be carried out to confirm the positivity of DNA quantification results obtained from Agarose gel electrophoresis.

Similarly, some methods may be preferred over others. Factors that determine a method’s preference over another depend on the accuracy and complexity of the method, the time and resources required for the technique, and the stability of DNA. Analysis in some assays requires a human decision, like in most protein-based kits, while others, like the DNA methylation-based identification assays, are user-independent, thus increasing their admissibility and reliability in legal proceedings. Further, DNA methylation-based identification assay offers a complexity advantage as it can be combined with DNA profiling. It is also automated, reducing the risk of contamination and labor and saving time. Further, DNA is more stable than RNA; therefore, DNA-based assays are preferable.

Paternity Tests

Paternity tests mostly involve Y-DNA testing. In the human cell nucleus, the first 22 pairs of chromosomes are called autosomes, while the 23rd pair comprises the sex chromosomes: an XY pair in males and an XX pair in females. Y-DNA is paternally inherited from father to son. Therefore, Y-DNA tests can only be administered to male individuals. Moreover, Y-DNA does not undergo recombination and remains unchanged for many generations (Genetic Science Learning Center, 2016). Y-DNA testing is divided into two tests: Short Tandem Repeat (STR) and Single Nucleotides Polymorphism (SNP) tests that classify unique sequences along the Y chromosome DNA and identify variations in a single DNA component, respectively (Bettinger and Wayne, 2016). The first step for this test is the collection of DNA samples, for instance, from saliva swabs from the individuals of interest. The analyst then chooses a test kit of choice to analyze the DNA of individuals under comparison.

Third-party application tools like GEDmatch are used to analyze and compare the test-taker’s results. The one-to-one application tool, for example, gives details of DNA matches and identifies DNA segments and the total amount of DNA shared by the test-takers. The total amount of shared (obtained in centiMorgans) is relevant in determining the genetic relationship of individuals under comparison.

Significance of Paternity Tests

Paternity cases can be criminal or domestic. The most common paternity case involves a parent and a child who are evaluated to pursue an alleged parentage. Suppose such a case is presented in court. In that case, paternity tests become significant where reference samples are collected from the alleged parent, known parent, and the child for genetic relationship analysis and identification. In other cases, paternity tests are useful in criminal parentage, where individuals are evaluated as the parents of a person who is the source of biological evidence. Paternity testing is also applied in immigration and mass disaster cases where a paternal family member is missing.


DNA testing plays a significant role in forensics. Criminalists apply DNA analysis to identify biological crime scene evidence to either include or exclude an individual from a crime scene. Biological samples need to be handled with care to avoid contamination, leading to inaccurate interpretation of results and causing the evidence to be considered objectionable. DNA testing is also essential in paternity testing, which can be significant in court cases like criminal parentage cases.


Bettinger, B., and Wayne, D. (2016). Genetic genealogy in practice. National Genealogical Society.

Elkins, K. (2012). Forensic DNA Biology: A Laboratory Manual. Elsevier Science.

Frumkin, D., Wasserstrom, A., Budowle, B., & Davidson, A. (2011). DNA methylation-based forensic tissue identification. Forensic Science International: Genetics5(5), 517-524.

Genetic Science Learning Center. (2016). Introduction to Molecular Genealogy. Retrieved 16 January 2022, from

Murphy, E. (2018). Forensic DNA Typing. Annual Review of Criminology1(1), 497-515.


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DNA Analysis in Criminalistics

DNA Analysis in Criminalistics

As you study DNA, you find that every person’s DNA is different from another’s. Identical twins are the only people whose DNA is the same. Consider your parents and your siblings. Even though your brother and your sister have the same parents, it is unlikely that you look exactly like them unless you are identical twins. You may have common features, and you will all share common DNA among yourselves, but only identical twins will have the same DNA. DNA testing is used for many reasons, such as the following:
• Identify potential suspects whose DNA may match evidence left at crime scenes
• Exonerate persons wrongly accused
• Match organ donors
Assignment Guidelines
• Address the following in 4–5 pages: In your first case, you have been asked to list and explain the steps that you would use to identify and analyze DNA from a person who has been in prison for ten years. The results of your test may forgive the person.
 Once the physical evidence has been delivered to the forensics lab, what is the process of identifying DNA? Be specific and explain in detail.
 Consider that the evidence is over ten years old.
 Once the DNA evidence has been identified, what is the process of analyzing the DNA? Be specific and explain in detail.
 What challenges exist that can make your analysis inadmissible in court? Explain. How are tissue matches made? Explain. Why is it important to utilize different DNA testing methods? Explain.
 Why is it that some methods may be preferred over others? Explain.
Other than forensic uses, DNA testing is essential in paternity testing.
 What are the steps used to carry out a paternity test? Explain in detail.
 Why might the results of a paternity test be significant in a court case? Explain.

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