DNA sequencing and crime scenes
DNA sequencing is a scientific process that determines the order of nitrogenous bases in a DNA molecule, which is crucial for various applications, including forensic science. In crime scenes, biological samples can often be degraded or contain insufficient nuclear DNA for standard analysis. In such cases, DNA sequencing serves as an alternative and more detailed method for analyzing these samples. Although DNA sequencing is more time-consuming than traditional nuclear DNA typing, it can yield valuable information when other methods fail.
The process begins by selecting a specific portion of the genome for sequencing, followed by amplifying that DNA segment. This amplified DNA is then subjected to a sequencing reaction that employs fluorescently labeled modified nucleotides. The resulting products are subsequently analyzed using high-throughput genetic analyzers, allowing for a precise determination of the DNA sequence based on the color emissions from the labeled nucleotides. Overall, DNA sequencing enhances the ability to analyze compromised samples from crime scenes, providing crucial evidence for investigations.
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DNA sequencing and crime scenes
DEFINITION: Process in which the nitrogenous bases that make up a DNA molecule (adenine, guanine, cytosine, and thymine) are “read” to determine their order and sequence in a given genome.
SIGNIFICANCE: Biological specimens obtained at scenes of crimes are not always intact; some have been exposed to harsh environmental conditions and others do not possess the necessary amount of nuclear DNA for standard analysis. When analysts encounter these types of specimens, they can use the alternative method of DNA sequencing to discriminate between samples.
In some instances, biological samples are not suitable for performing the relatively fast and standard nuclear analysis and typing of (deoxyribonucleic acid). On occasion, the copy number of nucleated cells is too low, and the analyst cannot obtain enough nuclear DNA to acquire a strong enough signal to interpret after typing the DNA. DNA sequencing, although more time-consuming and complicated than the standard analysis, may provide the necessary clues when other methods to obtain a good profile using conventional typing methods are exhausted.
The first step in sequencing a DNA sample is to determine what fraction of the genome is to be read (usually between three hundred and one thousand base pairs) and perform an amplification reaction using unlabeled primers that target the region of choice. After the DNA is amplified, the Polymerase chain reaction (PCR) product is cleaned to remove any unbound nucleotides and residual, nonincorporated primers. This purified product is subsequently quantified and diluted to a predetermined concentration that is dependent on the length of the fragment of DNA being sequenced. The sequencing template is amplified with a single primer (forward or reverse) so that the end products are single-stranded DNA molecules. Unlike in the more common PCR reactions, a pair of primers cannot be used because the end product would result in a double-stranded molecule that could not be interpreted. This would be analogous to attempting to read two lines of text when one is superimposed on the other, a task that is difficult to perform.
In the sequencing reaction, fluorescently tagged modified nucleotides called dideoxyribonucleic acids (ddNTPs) are present. Each is labeled with a different color fluor and is randomly incorporated into the newly synthesized DNA strand. Because ddNTPs lack the hydroxyl group normally involved in the elongation step, the synthesis of the new strand terminates with the labeled ddNTP. The cycle-sequencing product is then cleaned to remove any unbound nucleotides and dried.
The samples are now ready to be “read,” or separated. The dried samples are resuspended in formamide, a denaturant, before they are loaded onto high-throughput genetic analyzers. The sample is electrokinetically injected into a capillary filled with a gel polymer that is able to provide the one-base-pair resolution needed to determine the order of nucleotides in a particular DNA fragment. Because each nucleotide is labeled with fluors that will emit at different wavelengths, color distinction can be used to determine the sequence of the sample. The fluorescence is captured by a camera and transferred to the computer’s software, which makes the data available to the analyst for further interpretation.
Bibliography
Bukyya, Jaya Lakshmi, et al. "DNA Profiling in Forensic Science: A Review." National Library of Medicine, 31 May 2021, www.ncbi.nlm.nih.gov/pmc/articles/PMC8635824/. Accessed 14 Aug. 2024.
Kieleczawa, Jan. DNA Sequencing: Optimizing the Process and Analysis. Sudbury, Mass.: Jones & Bartlett, 2005.
Nunnally, Brian K. Analytical Techniques in DNA Sequencing. New York: Taylor & Francis, 2005.
Sharman, Sarah. "Forensics and DNA: How Genetics Can Help Solve Crimes." Hudson Alpha, 11 Nov. 2021, www.hudsonalpha.org/forensics-and-dna-how-genetics-can-help-solve-crimes/. Accessed 14 Aug. 2024.