Category: DNA

Molecular Diagnostics: Forensics DNA Profiling

Abstract

DNA analysis is very important in forensics as it is a method to discover a victim or perpetuator of a crime. The study done was to extract DNA using a buccal swab and analyse it using a capillary gel electrophoresis which was then compared to determine the perpetuator of a crime. The DNA was extracted, quantity of DNA determined using a nanodrop and then a capillary gel electrophoresis was done. The DNA collected was of low quantity being 0.0686 ug/ul. The capillary gel electrophoresis results presented as an electrophoretogram showed that the sample collected from the coffee cup matched the DNA of the individual tested.

Introduction

DNA profiling also called DNA fingerprinting is a process of determining an individual’s DNA characteristics. DNA profiling is a technique used in forensics to investigate victims or perpetrators of a crime by comparing their DNA to the DNA evidence found in a crime scene (Murphy, 2018). A buccal swab is a method of retrieving DNA samples from an individual in a cheap, reliable and non-invasive way (McMichael et al., 2009). It collects a sample of DNA in the form of saliva from the inside of the cheek in order to form a basis of comparison. Before the obtained sample can be used it needs to be purified and DNA needs to be extracted from the cells. It can be easily done using a Buccalyse DNA release kit also known as Isohelix BEK 50 kit, also prepares the sample for PCR (Scientific, 2019). The DNA sample is then analysed for sequences called short tandem repeats (STR) which are unique for every individual. They are repeated units of bases and has multiple alleles defined by the number of repeat units called flanking regions (HMM202 practical manual, 2019). It is done using a capillary gel electrophoresis which provides a rapid, high resolution separation of the amplified DNA (Mansfield et al., 1998). In this practical a sample of DNA was collected using a the Isohelix kit mentioned and prepared for STR analysis using capillary gel electrophoresis. Due to time constraints an already prepared electrophoretogram was given to analyse. The purpose of this experiment is to be able to extract DNA and analyse it as potential suspects of a crime.

Method

The first step was to obtain a buccal swab. Drinking caffeine had to be avoided prior to the experiment and mouth had to be rinsed to avoid any contamination. A tube was labelled with individuals initials and 200ul of Buccalyse was added. The swab was rubbed on the inner cheeks around 10 times to ensure a good sample, and then was placed in the tube guaranteeing most of the liquid was captured. The tube was then incubated at 70 degrees for 15 minutes and 95 degrees for 2 minutes. The tube was vortexed in between each incubation. The next step involved the nanodrop. From task 1, 2ul of the isolated genomic DNA was obtained and the concentration detected.

This then followed an agarose gel electrophoresis at 120V for 30 minutes. A sample of 10ul of DNA was obtained and aliquot into wells. The final step was DNA profiling done by a capillary gel electrophoresis with the results given by the staff (HMM202 practical manual, 2019).

Discussion

The results in figure A mean that the DNA sample collected from the buccal swab was successful as there was a black band present behind the well. The DNA was condensed, and the weight of the DNA was too heavy for it to migrate through the matrix thus producing that black colour behind the wells (Chauhan, 2018). The smearing present represents that the DNA is contaminated with RNA (Chauhan, 2018). Compared to other samples in the gel electrophoresis there were high quality DNA samples present like in lane 6, 7, 11 and 12. This indicates that the sample collected in lane 14 was of low quality which could be due to insufficient swabbing of the inner mouth, which in turn did not collect enough DNA sample. The nanodrop results obtained also showed that the sample of DNA collected was contaminated with protein, phenol or other contaminants, thus yielding a low quantity of DNA (Scientific, 2019). The highest ratio for A260/280 obtained by the nanodrop was 1.03 however still lower than the preferred range. To ensure less contamination enzymes can be used like proteinase K or RNase and then incubated overnight (Bradburn, 2018). It is important to reduce contamination as it interferes with the results and could cause an individual to become a suspect that has no relation to a case.

Every individual will have differing numbers of bases and repeated sequences in an STR region thus the graphs formed in an electropherogram based on those alleles would be unique to every individual (Encyclopedia Britannica, 2018). The aim of the experiment was achieved as DNA was successfully extracted however was highly contaminated. In future if the experiment was to be conducted the DNA sample should be purified to eliminate any contaminants. Not only is capillary gel electrophoresis used for DNA profiling it also helps in clinical and health studies. It is used to determine endogenous and exogenous compounds in body fluids and tissue extracts (Thormann et al., 1994). It also used in open-tubular column systems, identify separation patterns of serum proteins and even used to separate haemoglobin variants (Chen, Liu and Sternberg, 1991).

RefePrences

1. Dr Liza Raymond and Dr Richard Williams (2019), Molecular Diagnostics HMM202 Practical Manual, pp.23-27.
2. Mansfield, E., Robertson, J., Vainer, M., Isenberg, A., Frazier, R., Ferguson, K., Chow, S., Harris, D., Barker, D., Gill, P., Budowle, B. and McCord, B. (1998). Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis. Electrophoresis, 19(1), pp.101-107
3. McMichael, G., Gibson, C., O’Callanghan, M., Goldwater, P., Dekker, G., Haan, E. and MacLennan,, A. (2009). DNA from Buccal Swabs Suitable for High-Throughput SNP Multiplex Analysis, 20(5), pp.232–235.
4. Murphy, E. (2018). Forensic DNA Typing. Annual Review of Criminology, [online] 1(1), pp.497-515. Available at: https://www.annualreviews.org/doi/10.1146/annurev-criminol-032317-092127[Accessed 11 Sep. 2019]
5. Scientific, B. (2019). Buccalyse Rapid Buccal Cell DNA Isolation Kit 25 extractions. [online] Bocascientific.com. Available at: https://www.bocascientific.com/simhelix-buccalyse-rapid-buccal-cell-dna-isolation-kit-50-extractions-with-sk1-swabs-p-4452.html [Accessed 11 Sep.2019]
6. Scientific, T. (2019). Assessment of Nucleic Acid Purity. [ebook] Available at: https://s3-us-west-2.amazonaws.com/oww-files-public/d/d7/T042- NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf [Accessed 14 Sep. 2019]
7. Chauhan, T. (2018). A complete guide for analysing and interpreting gel electrophoresis results. [online] Genetic Education. Available at: http://geneticeducation.co.in/a-complete-guide-for-analysing-and-interpreting-gel-electrophoresis-results/ [Accessed 14 Sep. 2019].
8. Bradburn, S. (2018). The Nanodrop Results Explained – Top Tip Bio. [online] Top Tip Bio. Available at: https://toptipbio.com/the-nanodrop-results-explained/ [Accessed 16 Sep. 2019].
9. Encyclopedia Britannica. (2018). DNA fingerprinting | Definition, Examples, & Facts. [online] Available at: https://www.britannica.com/science/DNA- fingerprinting [Accessed 16 Sep. 2019]
10. Thormann, W., Molteni, S., Caslavska, J. and Schmutz, A. (1994). Clinical and forensic applications of capillary electrophoresis. Electrophoresis, 15(1), pp.3-12.
11. Chen, F., Liu, C. and Sternberg, J. (1991). Capillary electrophoresis–a new clinical tool. The American Association for Clinical Chemistry, 37(1), pp.1-2.

The Role Of DNA In Forensic Science

The origin of DNA fingerprinting was discovered in 1984 by Dr Alec Jeffreys (Jackson and Jackson, 2011, p. 158). Over the decades, with technical developments in genetics, the original DNA fingerprinting procedure has undertaken a variety of modifications and refinements. DNA profiling has become so precise and sensitive that in the United Kingdom it is no longer allowed to be used as a sole piece of evidence in a criminal investigation, it has to be used alongside other procedures. Nevertheless, when it is applied it allows a biological sample found at a crime scene to be linked to the individual from whom it originated (Jackson and Jackson, 2011, p. 159). It is imperative to remember that when using DNA profiling to solve a criminal investigation you have to take into consideration that with the degree of amplification being used to analyse a DNA sample, it makes the method very sensitive. A single DNA molecule can give an easily detectable amount of DNA within only a few hours (Jackson and Jackson, 2011, p. 175). Due to the extreme sensitivity it must be noted that very great care must be taken when analysing these DNA samples in order to avoid contamination with other materials or forensic evidence.

Forensic evidence is a vital part of every criminal investigation. From the moment an officer enters a crime scene, up until the perpetrator is convicted any evidence found at the scene is crucial in determining the guilt or innocence of those who are convicted. DNA is one of the most common forensic evidence found at crime scenes and it could potentially still in some cases be the sole reason why a person gets convicted of a crime. Additionally, DNA is also significant in investigations as it can help exonerate the innocent, which is why anything found at a crime scene needs to be packaged in such a way that it cannot be contaminated or damaged. This is due to the fact that even DNA evidence itself can have its downsides since its analysis is subject to error and bias.

One of the most well-known cases where the contamination of DNA samples has led to a miscarriage of justice involved a man named Adam Scott, who was wrongly accused of raping a woman in Manchester due to faulty DNA evidence (Patrick Walter 2012). According to Peter Gill (2014) who was a professor of Forensic Genetics at University of Oslo, he stated that the Adam Scott case was a good example of confirmation bias, where inconvenient information that was given to the prosecution was either ignored or dismissed. Due to a technician at LGC Forensics not disposing a plastic tray after analysing it, which contained Adam Scott’s DNA from an unrelated matter a cross contamination occurred leading to a misidentification. This was due to the fact that the plastic tray that was supposed to get disposed was reused in the analysis of a swab taken from a victim of rape (Euroforgen Network of Excellence 2017). It comes to show how vital it is to make sure that all exhibits found are handled appropriately. Integrity has to be ensured at all times since it shows that an evidential item has been dealt with appropriately and demonstrates that there was no interfering, addition or loss of material that could have taken place whether it was intentional or accidental. In order to ensure integrity, the continuity of evidence has to be recorded, which commences from the moment that an item has been marked as potential evidence. From then on, its location and movement must be accountable and documented until it is presented before Court and its disposal is authorised. It is important to remember that the continuity and integrity of evidence must be proved beyond all reasonable doubt in Court. This is in order to avoid errors such as the one mentioned above being made, where Adam Scott’s DNA got mixed up with the genetic material taken from a rape Victim. The Technician should have disposed the plastic tray as soon as it was done being used which he did not, due to this human error the perpetrator was never found and Adam Scott spent 5 months on remand for a crime he never committed.

On the other hand, the OJ Simpson case (Linder 2018) is an example that shows how DNA evidence was not enough to prosecute someone. During the course of Simpson’s trail, many types of blood profile matches, extensive explanations about DNA profiling and how matches were found were put forward in court. The evidence given proved that there was a match between Simpson’s blood and the drops of blood that were found at the scene. There was even a match between the footprints at the crime scene to the pair of shoes that Simpson had. However, the jury was not impressed by the repeated prosecutorial demonstrations of blood profile matches, such as a match between Simpson’s blood sample and two drops of blood found at the crime scene alongside bloody footprints. This was due to the fact that the evidence given could not have proven beyond reasonable doubt that Simpson was the one who committed the crime, it could not prove that he in fact was there at the time of the murder. This could have been avoided if the defence did not solely relay on the DNA evidence, which is why it is important to remember that DNA evidence is used alongside other types of procedures. Another way of avoiding a situation like the one above from occurring is by thinking about the context of the evidence. It is important as an investigator to ask yourself could have OJ Simpson’s blood got there by other means? Could it have been transferred accidentally? Could someone possibly want to frame Mr Simpson? These are the kind of questions that should have been raised when carrying out that investigation.

Due to how complex DNA profiling is and how important it is to prevent innocent people from going to jail and convicting those who actually committed the crime, DNA alone cannot be the sole reason why a person gets convicted of a crime. According to a Forensic Science Regulator report (2012) even if DNA is found at a crime scene, it cannot be used to determine whether or not someone is guilty. DNA does not always convey how or when it got there or the body tissue it came from. Therefore, DNA needs to be perceived within a framework of additional evidence, rather than being the sole answer to solving a crime scene investigation. It is important to remember that knowing the background of any forensic evidence found at a crime scene is the key to solving any criminal investigation. DNA is unique to every individual and people leave a trail of it everywhere they go, meaning that in some cases DNA found at a crime scene might not necessarily have anything to do with the crime, which creates an issue. Thus, it is fundamental that while investigating a crime scene an investigator considers when and how that DNA might have gotten there. Asking such questions is crucial, since it helps to understand and possibly disclose information that could help eliminate any DNA that is not relevant to the case, or if its background DNA is the result of secondary transfer or contamination (Forensic Science Regulator 2012).

While DNA profiling can be helpful in an investigation, it needs to be noted that it should be used within a wider context along with any other evidence collected. A criminalist Paul Kirk (1953) said the following “However careful a criminal may be to avoid being seen or heard, he will inevitably defeat his purpose unless he can control his every act and movement so as to prevent mutual contamination with his environment, which may serve to identify him. Wherever he steps, whatever he touches, whatever he leaves – even unconsciously – will serve as a silent witness against him. Not only his fingerprints and his shoeprints, but also his hair, the fibres from his clothes, the glass he breaks, the tool mark he leaves, the paint he scratches, the blood or semen that he deposits or collects – all these bear mute witness against him. Physical evidence cannot be wrong; it cannot perjure itself; it cannot be wholly absent. Only in its interpretation can there be error. Only human failure to find, study and understand can diminish its value.” For an investigator this is important to remember since it goes to show how vital the context of forensic evidence is. With forensic evidence the following questions need to be addressed: How was the evidence recovered? Who was involved? What is the background or the circumstances of that evidence being found? More importantly, how could have the evidence got there in the first place? Could it have gotten there by other means? In terms of DNA, it is important to ask yourself whether the DNA found at the crime scene could have gotten there due to someone else picking up that DNA accidentally without even noticing and potentially carrying it into the crime scene. There are numerous questions that should be addressed and answered in order to carry out a good investigation.

To conclude, it is safe to say that within the past few decades the improvements made to DNA profiling and DNA analyses are substantial. DNA technology is important to ensure a good level of accuracy and justice in the criminal justice system. Even DNA profiling evidence from biological samples increases the chances of finding the perpetrators in cases where there is lack of evidence or if no witnesses stepped forward. However, no matter how accurate DNA profiling and DNA analyses is, a crime scene investigator must not forget about the context of forensic evidence. Questions such as, ‘when and how did the evidence get there?’ need to be raised and answered before presenting anything to the court. More importantly, any DNA evidence presented to the court needs to be obtained alongside other types of evidence and not as a sole piece of evidence. This is it to avoid cases like the OJ Simpson case from occurring. It is also crucial to note that any evidence sent to the lab need to packaged appropriately to avoid contamination. Once those reach the lab any forensic scientist that is in charge of those evidence has to take great care to reduce any errors from occurring by making sure their methods have been carefully tested and are performed properly using calibrated equipment. This then has to be followed by a list of well-controlled procedures to prevent cross contamination. Following each of those steps will ensure that any crime scene investigation is carried out correctly and would hopefully result in the conviction or the release of an innocent person.

Police And DNA

Are our privileges being dismissed when police use DNA databases, for example, 23andme to get suspects? Cases that started the discussion, for example, the Golden State Killer began the discussion regarding the matter. This has also led to the debates about if it is an ethical action to take and if it dismisses our rights to privacy. Will this lead to changes in or rights as citizens and laws will change to adapt to this newfound crime-fighting tool.

The Golden State Killer was a police officer who looted homes, committed assaults and in the long run murder. He was captured after a relative presented their DNA to be tried to discover their family line and starting point. Police utilized a DNA test taken from one of the crime scenes and it was a partial match to the submitter of the DNA. It later prompted the capture and progressing trial of the Golden State Killer.

At the point when people present their DNA to these sites they accept that their DNA won’t be utilized for anything, they didn’t consent for their DNA to be utilized to get a lawbreaker. Be that as it may, did they at any point think about that their DNA would be utilized, rather than just tested? Did the organizations that did the testing even think about it? Did they by any chance feel this could be an issue that should be considered? Will this lead to changes later on for organizations?

When sending your DNA to these organizations would you say you are surrendering your entitlement to security? You are offering it to an organization that you don’t think a lot about what they could do with your DNA? On the off chance that you willingly surrendered it to these organizations, for what reason can’t Police use it get dangerous culprits? Will this, in the end, lead to organizations changing their security approaches, or will this change the revision that ensures our entitlement to protection? Will our framework need to change to acknowledge this new type of DNA policing, which in the past innovations and convictions have brought change? Will this, in the long run, be an approach that the police can’t take or will it bring us into another era of capturing offenders. (Zhang, 2019).

The privilege of security shields us from utilizing our data that they don’t have the motivation to investigate. this fundamentally implies we are shielded from look without warrants, anybody investigating restorative records that don’t have our consent. The privilege to security essentially implies that we have command over our data, we get the opportunity to state who gets the chance to investigate that. Incomparable Court Justice Louis Brandeis expressed, ‘that the privilege to protection is known as the privilege to be disregarded’. (Kennett, 2019).

One milestone case that carried this to open light was the situation of the Golden State Killer. The Golden State Killer was active in California from 1974 to 1986, he is expected to have committed thirteen homicides, over a hundred robberies and fifty assaults in a twelve-year range. Police inevitably tracked Down the speculated Golden State executioner when a removed relative presented their DNA for testing with a free site.

It is contended that transferring a suspect’s DNA to a database, for example, 23andMe disregards their entitlement to protection. Does this damage the privilege of protection, if they were captured your DNA and fingerprints are taken and placed into a framework that can distinguish you? The police could simply place the suspect’s DNA into that database and turned out with the Golden State Killer just as simple as utilizing the free DNA database. It was later discovered that the site utilized that prompted the catch of the Golden State Killer was a site that you could make the profile accessible for anybody to see.

Presently, people can send away a DNA test for testing at a privately owned business and get a report with their predecessors ‘ nations of their origin and their potential for diseases. This can even transfer these test results to open-source sites to associate with others who might be identified with them. Law enforcement has perceived the incentive in this innovation and started transferring DNA tests from potential suspects to solve cold cases, including the ‘Golden State Killer’. Be that as it may, open-source DNA databases are not normal for law enforcement databases. In an open-source database, there is no assurance that a transferred DNA profile is secure, regardless of whether the client is as far as anyone knows. The DNA testing system used to test the DNA uncovers unmistakably more data about the individual. (Stokes, 2019).

According to Christi J. Guerrini, who is a published author in PLos Biology She stated that” The arrest of DeAngelo was widely celebrated as a brilliant police coup that could finally bring to justice a man who had terrorized citizens of California for years. However, as details of his arrest emerged, concerns about privacy were rapidly raised about police searches of the kind of personal genetic data used to capture DeAngelo. Law enforcement officers regularly search the ‘offender registry’ of the Combined DNA Index System (CODIS) maintained by the Federal Bureau of Investigation (FBI), which contains the genetic profiles of suspected felons, misdemeanors, and convicted offenders, for similar crime scene information.”(Guerrini,2018).

I can comprehend why individuals would be disturbed about this, yet if you are happy to post your profile move your DNA on to a site for anybody to see then for what reason can’t police use it to get dangerous individuals and close cold cases. In something where your DNA is being utilized that is amazingly close to home your DNA is your hereditary code and is remarkable to just you. As people, our DNA is 99% like that of the remainder of the human populace. just 1% over DNA is remarkable to just us. This one percent is the thing that police use to catch and get justice against dangerous individuals, for example, the Golden State killer.

As I would see it doesn’t make a difference to me if my DNA is being utilized to catch these risky people, it can make our public a lot safer. This can also be utilized in diminishing the acts of wrongdoing if people realize that police can utilize this strategy in catching them. If an individual feels they are bound to be captured they are less likely to commit the act itself. At the end of this could continue to happen. Imagine how many unsolved murders, sexual assaults would be solved and someone punished.

Are our privileges being encroached upon when police use DNA databases, for example, 23andme to get suspects? He was captured after a relative presented their DNA to be tried to discover their family line and starting point. It later prompted the capture and progressing trial of the Golden State Killer. Be that as it may, open-source DNA databases are not normal for law enforcement databases. This one percent is the thing that police use to catch and get justice against dangerous individuals, for example, the Golden State killer. The police could simply place the suspect’s DNA into that database and turned out with the Golden State Killer just as simple as utilizing the free DNA database. Police utilized a DNA test taken from one of the crime scenes and it was a partial match to the submitter of the DNA.

One milestone case that carried this to open light was the situation of the Golden State Killer. Be that as it may, did they at any point think about that their DNA would be utilized, rather than just tested? You are offering it to an organization that you don’t think a lot about what they could do with your DNA? In an open-source database, there is no assurance that a transferred DNA profile is secure, regardless of whether the client is as far as anyone knows. The DNA testing system used to test the DNA uncovers unmistakably more data about the individual.

The DNA testing wasn’t basic when April Tinsley was killed in 1988. It was unwieldy, tedious, costly, and the tests required significantly more physical proof—semen, blood, and spit—than is normal today. DNA tests would just get de rigueur in the mid-1990s when the convention changed and specialists could now enhance limited quantities of DNA taken from natural liquids, yet from objects like weapons, garments, and devices.

The issue was, even as Fort Wayne police stayed aware of advances in testing, the DNA they had from the scene where April’s body was found essentially wasn’t sufficient to deliver a convincing outcome. By 2015, although the affectability of DNA testing had improved enough to identify DNA at the degree of a solitary nanogram—a grain of salt is around 58,000 nanograms—the proof still didn’t coordinate anybody in the FBI database.

In June 2015, Fort Wayne police discovered that Parabon NanoLabs, a biotech organization headquartered in Reston, Virginia, was offering a new technology called Snapshot, in which a working representation of a criminal suspect could be created straightforwardly from infinitesimal measures of DNA. Parabon’s foundations were in bioinformatics, and their systems, including Snapshot, which was created and trademarked by the organization in 2012, were initially intended for use in restorative research.

Snapshot utilizes a procedure known as DNA phenotyping to decide a few particular physical attributes—eye shading, hair shading, nose shape—to create PC produced representations of a suspect. The portrayals of April Tinsley’s executioner, produced in May 2016, indicated what the suspect resembled in 1988 and what he may resemble 30 years after the fact: dim hair, hazel eyes, unmistakable nose, with dark specks inside his sideburns in the age movement. In spite of the exact nature of the representations, which showed up all through Allen County on the neighborhood news and were circulated utilizing the police division, they didn’t prompt a recognizable person to capture.

Will this lead to changes in or rights as citizens and laws will change to adapt to this newfound crime-fighting tool. It later prompted the capture and progressing trial of the Golden State Killer. At the point when people present their DNA to these sites they accept that their DNA won’t be utilized for anything, they didn’t consent for their DNA to be utilized to get a lawbreaker. Will this, in the long run, be an approach that the police can’t take or will it bring us into another era of capturing offenders.

Are our privileges being dismissed when police use DNA databases, for example, 23andme to get suspects? At the point when people present their DNA to these sites they accept that their DNA won’t be utilized for anything, they didn’t consent for their DNA to be utilized to get a lawbreaker. The police could simply place the suspect’s DNA into that database and turned out with the Golden State Killer just as simple as utilizing the free DNA database.

Presently, people can send away a DNA test for testing at a privately owned business and get a report with their predecessors ‘ nations of their origin and their potential for diseases. The DNA testing system used to test the DNA uncovers unmistakably more data about the individual. I can comprehend why individuals would be disturbed about this, yet if you are happy to post your profile move your DNA on to a site for anybody to see then for what reason can’t police use it to get dangerous individuals and close cold cases.

Are our privileges being encroached upon when police use DNA databases, for example, 23andme to get suspects? The police could simply place the suspect’s DNA into that database and turned out with the Golden State Killer just as simple as utilizing the free DNA database. The DNA testing system used to test the DNA uncovers unmistakably more data about the individual.

The issue was, even as Fort Wayne police stayed aware of advances in testing, the DNA they had from the scene where April’s body was found essentially wasn’t sufficient to deliver a convincing outcome. By 2015, even though the affectability of DNA testing had improved enough to identify DNA at the degree of a solitary nanogram—a grain of salt is around 58,000 nanograms—the proof still didn’t coordinate anybody in the FBI database. Snapshot utilizes a procedure known as DNA phenotyping to decide a few particular physical attributes—eye shading, hair shading, nose shape—to create PC produced representations of a suspect. These are just a few cases that were solved using this new technique. Do you feel like it is worth it? I do, these people deserve justice and the dangerous perpetrators need to be punished for taking their lives.

References

1. Guest, Christine. ‘DNA AND LAW ENFORCEMENT: HOW THE USE OF OPEN SOURCE DNA DATABASES VIOLATES PRIVACY RIGHTS.’ American University Law Review 68.3 (2019): 1015-52. ProQuest. Web. 1 Dec. 2019.
2. Kennett, Debbie. ‘Using Genetic Genealogy Databases in Missing Persons Cases and to Develop Suspect Leads in Violent Crimes.’ Forensic Science International (Online) 301 (2019): 107-17. ProQuest. Web. 1 Dec. 2019.
3. Schuppe, Jon. “Police Were Cracking Cold Cases with a DNA Website. Then the Fine Print Changed.” NBCNews.com, NBCUniversal News Group, 29 Oct. 2019, www.nbcnews.com/news/us-news/police-were-cracking-cold-cases-dna-website-then-fine-print-n1070901.
4. Stokes, Jim. ‘Technical Note: Next Generation Identification – A Powerful Tool in Cold Case Investigations.’ Forensic science international 299 (2019): 74-9. ProQuest. Web. 1 Dec. 2019.
5. Wamsley, Laurel. “After Arrest Of Suspected Golden State Killer, Details Of His Life Emerge.” NPR, NPR, 26 Apr. 2018, www.npr.org/sections/thetwo-way/2018/04/26/606060349/after-arrest-of-suspected-golden-state-killer-details-of-his-life-emerge.
6. Zhang, Sarah. “The Messy Consequences of the Golden State Killer Case.” The Atlantic, Atlantic Media Company, 2 Oct. 2019, www.theatlantic.com/science/archive/2019/10/genetic-genealogy-dna-database-criminal-investigations/599005/.

Forensic DNA Analysis: Strengths And Limitations

Mitochondrial DNA (mtDNA) analysis is becoming more common in criminal investigations to characterize forensic biological specimen. This paper will examine mtDNA analysis in the forensic field, the expertise and training required and its strengths and limitations. The strengths of mtDNA analysis are the following: mtDNA has a high copy number, it provides an alternative option when nuclear DNA (nucDNA) is not viable, better recovery from degraded samples, confirms maternal relatedness and some discriminatory power using hypervariable regions. The limitations of mtDNA analysis are that it does not provide definitive identification, heteroplasmy, risk of contamination, paternal leakage and is time-consuming and expensive. Lastly, this paper will discuss the development of next generation sequencing (NGS) technologies and its implications on future cases.

Forensic mtDA Analysis

Forensic mtDNA analysis allows analysts to develop a DNA profile from samples that cannot yield results of nucDNA. One mitochondrion contains 2-10 copies of mtDNA and there are thousands of mitochondria in each cell (Hameed, Jebor & Kareem, 2015). Mitochondria are energy-producing organelles and are often referred as the powerhouse of the cell (van Oven & Kayser, 2008). mtDNA is a circular, double-stranded molecule and has 16,569 base pairs, which encode 37 genes for 22 tRNAs, two rRNAs and 13 mRNAs (Anderson, 2017; Hameed et al., 2015). Also, mtDNA haplotypes are genetic information that is uniparentally inherited from the mother to offspring through the egg cell; accordingly, individuals from the same maternal lineage have identical mtDNA (Parson, 2014). For this reason, the assumption is that all mtDNA types can be traced back to a common maternal ancestor; mtDNA sequence variation evolved as a result of mutations (van Oven & Kayser, 2008). There is more sequence divergence in mtDNA than nucDNA (Hameed et al., 2015). Mutations can occur during mtDNA replication; consequently, rate of change of mtDNA is five times faster than nucDNA (Hameed et al., 2015).

Expertise and Training

There are several steps necessary to become a forensic biologist. Although the minimum requirement for entry level forensic analyst is a three-year diploma, most analysts have Bachelor of Science (BSc) degrees and some also have Master’s degrees (Anderson, 2007). Forensic lab personnel have a strong science background; analysts can specialize in any biological science, such as Biology, Biochemistry, Chemistry, Molecular Biology or Forensic Biology (Anderson, 2007). Once hired, applicants undergo an understudy period of 9-18 months and receive on-going training (Anderson, 2007). Additionally, forensic analysts must be competent in their written and oral communication skills, as a large proportion of their duties require them to provide expert testimony in court (Anderson, 2007). The analysts must be able to effectively communicate relevant scientific knowledge in lay terms for those without a scientific background, such as the jury (Anderson, 2007).

Strengths

One significant strength of mtDNA is that it has a high copy number in each cell. Compared to only two copies of nucDNA in a cell, thousands of mtDNA copies are present in each cell, increasing the likelihood of obtaining viable DNA (Alzarez-Cubero et al., 2012). The mtDNA copies are distributed throughout the cytoplasm of cells, which increases its sensitivity and chances of detection (Sampath & Jagannathan, 2014). Also, mtDNA is present in red blood cells, whereas nucDNA is not due to absence of nuclei (Sampath & Jagannathan, 2014). In hair samples, mtDNA can be detected anywhere along the hair follicle, including the hair shaft; in comparison, nucDNA is only present in the hair root (Budowle, Allard, Wilson & Chakraborty, 2003). As expressed, mtDNA is present in more areas of the body and in higher quantities.

Related to a high copy number, mtDNA analyses can be used when only small quantities of DNA are available. When obtaining nucDNA is not possible, sequencing of mtDNA is an alternative option (Nilsson, Andréasson-Jansson, Ingram & Allen, 2008). Samples, such as hairs, teeth or bone often contain low amounts of DNA (Nilsson et al., 2008). Collecting mtDNA from dental tissue is relatively easy due to large pulp chambers and resistance to decomposition (Sampath & Jagannathan, 2014). Hair samples as small as 0.2 cm have a high probability (86%) of obtaining partial or full profiles; however, thick and pigmented hairs produce the best results (Melton, Dimick, Higgins, Lindstrom & Nelson, 2005). As a result, mtDNA analysis is preferred when forensic analysts only have access to small quantities of DNA.

Forensic mtDNA analysis is optimal for old or degraded samples, as mtDNA is resistant to harsh environmental conditions. Mitochondria’s have a strong protein coat that protects mtDNA from bacterial enzymes (Hameed et al., 2015). Conditions where mtDNA analysis may be ideal are for fragmented skeletal remains, bones or teeth that have been exposed to long durations of acidity, high temperature or humidity, fingernails, shed hairs and samples that were unsuccessful for nucDNA detection (Alaeddini, Walsh, & Abbas, 2010; Kumar, Kumar, Honnugar, Kumar & Hallikeri, 2012). Also, improperly stored biological materials may not yield a nucDNA profile but may yield results for mtDNA (U.S. Department of Justice, 2002).

Remains from the World Trade Center attack, Tsar Nicholas II (remains from 1918) and Neanderthal bones have been characterized using mtDNA analysis (Budowle et al., 2003). Since these remains are heavily degraded and the latter two are significantly older, it is unlikely that obtaining nucDNA is possible. For Tsar Nicholas II, mtDNA was extracted from the putative bones and was matched with living maternal relatives (Budowle et al., 2003). In another case, mtDNA samples were extracted from the mouth, small fragments of the brain, feet and hand bones of bodies that were severely burned; this demonstrates that mtDNA can survive even in severely degraded and charred remains (Ricci, 2015).

Matrilineal inheritance of mtDNA allows living maternal relatives to be a reference sample to compare with unidentified remains (e.g. missing persons and mass casualties). There are large databases with DNA profiles of convicted offenders, human remains, crime scene samples and maternal relatives of missing persons (Alvarez-Cubero et al., 2012). In missing persons case where nucDNA samples cannot be obtained of the victim, living maternal relatives are able to submit their mtDNA to determine if the samples match (Alvarez-Cubero et al., 2012). Also, since there is a lack of recombination, maternal relatives several generations down can also serve as reference samples (Alvarez-Cubero et al., 2012). Many missing persons database of unidentified remains and maternal relatives automatically compare genetic data to find similar matches; this is a useful tool and has been successful in identifying remains (Alzarez-Cubero et al., 2012). Lastly, mtDNA discovered at crime scenes can potentially be an exculpatory tool to reduce wrongful convictions and potential candidates, consequently, redirecting resources and increasing efficiency of investigations (Alzarez-Cubero et al., 2012).

Although mtDNA does not provide positive identification, there are distinctive differences in the hypervariable regions of mtDNA that have considerable discriminatory power. Individuals have unique differences in two hypervariable regions of mtDNA: hypervariable region 1 (HV1) and hypervariable 2 (HV2) (Anderson, 2017). HV1 and HV2 contain approximately 610 bases of information; these control regions are highly variable due to a high evolutionary rate of mtDNA (Parson & Coble, 2001).

Gonçalves, Fridman & Krieger (2011) evaluated the discriminatory power of HV1/HV2 variables using 290 unrelated participants. The results showed that 77% of the participants could be discriminated using the HV1/HV2 types (Gonçalves et al., 2011). Although 23% of participants could not be distinguished using HV1/HV2 types, the study showed a large number of unique haplotypes. Parson & Coble (2001) found majority of the mtDNA types vary and 982 of 1175 mtDNA types are unique. It is likely that newer technology will be able to detect more distinguishing features (Gonçalves et al., 2011).

Limitations

One significant limitation of mtDNA is that it does not provide definitive identification. Although mtDNA is a class characteristic, it is a single linked molecule and the probabilities of individual polymorphism cannot be multiplied (Parson & Coble, 2001). mtDNA only reveals that the unknown sample shares the same maternal lineage as the reference sample (Parson & Coble, 2001). Parson & Coble (2001) claim that mtDNA should be treated as supplementary circumstantial evidence to assist in criminal proceedings. Additionally, because all maternal relatives share the same mtDNA, they are indistinguishable from one another on this basis. In cases where multiple family members are missing or died in a mass casualty, mtDNA analysis will have low probative value and will not help identify the unknown sample. Furthermore, studies have found some common HV1/HV2 types in the population. Parson & Coble (2001) found that about 7% of U.S. Caucasians share the most common type of HV1/HV2 type and Americans with African and Hispanic descent share similar mtDNA sequences as Caucasians. As a result, there is a chance for a random match to occur between known and unknown samples (Parson & Coble, 2001).

Another limitation for forensic mtDNA analysis is heteroplasmy. Heteroplasmy is the presence of more than one mtDNA type in an individual (Budowle et al., 2003). Heteroplasmy can manifest in three ways: multiple mtDNA type in a single tissue, heteroplasmic in one tissue and homoplasmic in another, or one mtDNA type in one tissue and a different type in another tissue (Budowle et al., 2003). Although rare, when heteroplasmy is observed, it is usually found in a single base of the mtDNA (Budowle et al., 2003). Budowle et al. (2003) report that one individual had some hairs that were homoplasmic and others were heteroplasmic; these results challenge the value of mtDNA analysis in forensic cases. mtDNA analysis is premised on the assumption that mtDNA is identical across maternal lines; the findings suggest that DNA databases and forensic analysts are potentially administrating false negatives.

Heteroplasmy is frequently observed in hair samples because of mitochondrial bottleneck (Budowle et al., 2003). Bottlenecking is a developmental process of cell-to-cell variability from a mutation; cell-level selection eliminates cells with high heteroplasmy and cells with low heteroplasmy are retained (Johnson et al., 2015). Samples with low heteroplasmy create an obstacle for mtDNA analysis, as they are difficult to detect. In cases where heteroplasmy is observed in a known sample and not in the recovered sample (or vice versa), additional samples should be collected to determine if they are a match (Bär et al., 2000).

There is an increased risk of contamination in forensic analysis of mtDNA. In highly degraded remains, there is often minute amounts of mtDNA for extraction (Wurmb-Shwark, Heinrich, Freudenberg, Gebür & Schwark, 2007). Particularly in cases of historical remains, it is difficult to be confident that the mtDNA extracted is from the victim, rather than modern DNA, such as from those that handled the sample (Wurmb-Shwark et al., 2007). When two objects are in contact with one another, there is an inevitable exchange of material, called Locard’s exchange principle (Anderson, 2017). Contamination can occur during evidence collection at crime scenes, evidence recovery or during forensic analysis (Wurmb-Shwark et al., 2007). Also, contamination can occur by inexperienced civilians that initially found and reported the remains (Wurmb-Shwark et al., 2007). Contamination concerns can be addressed by collecting known samples of those that came in contact with the DNA for elimination purposes; however, not all people that came in contact with the specimen will be known (Wurmb-Shwark et al., 2007). Also, contamination prevention measures should be taken, such as wearing a laboratory coat, disposable gear (e.g. gloves, caps, sleeves) and face mask, maintaining a clean and controlled workspace and monitoring a negative control (Bär et al., 2000).

Interestingly, recent research suggests that paternal mtDNA inheritance may coexist with maternal inheritance. Luo et al. (2018) found three unrelated multigenerational families, including 17 individuals, had high levels of mtDNA heteroplasmy (24-76%) and showed evidence of paternal leakage. Patients with diseases caused by mtDNA mutations were tested and revealed copies of paternal mtDNA, in addition to maternal mtDNA (Luo et al., 2018). From the 17 individuals, 13 members directly inherited paternal mtDNA and four inherited paternal mtDNA from previous generations (Luo et al., 2018). Also, mtDNA analyses were administered independently in at least two different laboratories with new technicians and new blood samples to increase the validity of the study; the results were replicated and showed no sign of contamination (Luo et al., 2018). The findings challenge the assumption that mtDNA is exclusively inherited by the mother. In regard to forensic cases, this raises concern as missing persons database only contain mtDNA from maternal relatives. However, evidence of biparental mtDNA inheritance is unusual, suggesting that majority of humans inherit mtDNA through maternal lineage.

Lastly, an important limitation of mtDNA analysis is that it is very time-consuming and costly. Many countries do not have mtDNA analysis laboratories and depend on private service providers (Melton, Holland & Holland, 2012). Forensic mtDNA analysis is expensive and tedious, as analysts need to work meticulously to avoid contamination of degraded samples (Melton et al., 2012). The cost of private testing of mtDNA is a significant barrier and law enforcement agencies infrequently use mtDNA laboratories (Melton et al., 2012). Also, many cases in mtDNA laboratories require analysis of a large number of samples; on average, each case requires about four samples but can require as many as 200 samples for a single case (Melton et al., 2012). Therefore, it is possible that mtDNA analysis laboratories experience back-logs, thus, decreasing its efficacy. Melton et al. (2012) report that majority of cases in their laboratory involve mtDNA analysis of samples from old or cold cases, crime scene hairs less than 10 mm and nonhuman samples; this demonstrates the irregularity of mtDNA analyses in criminal investigations. Furthermore, mtDNA testing laboratories have large costs due to “training, quality control, quality assurance, accreditation, proficiency testing and casework costs” (Melton et al., 2012, p.8). For these reasons, mtDNA analysis is conducted only when nucDNA is not viable and DNA is essential to the case.

Future of mtDNA Analysis

NGS technologies are being adapted to increase accuracy, reproducibility, speed and cost efficiency for mtDNA analysis. NGS technology can detect heteroplasmy at the whole mitochondrial genome level (Yang, Xie & Yan, 2014). As mentioned earlier, heteroplasmy is problematic for mtDNA analysis; more than one haplotype is present within a single person or tissue and these mutations occur with relative frequency. Current NGS technology has largely focused on nucDNA because mtDNA mutations are often present at very low heteroplasmy levels, making them difficult to detect (Bosworth, Grandhi, Gould & LaFramboise, 2017). However, newer NGS technologies can detect mtDNA of very low frequency molecules (less than 1%) (Just, Irwin & Parson, 2015). NGS technologies simultaneously conduct analyses of large number of samples and determine the base composition of single DNA molecules (Yang et al., 2014). Also, NGS technologies can recover very short mtDNA fragments, old, poor quality or low quantity specimen (Just et al., 2015).

The NGS technologies have led to three major improvements in DNA analysis. Firstly, the new technology does not require bacterial cloning of DNA fragments; they depend on the formation of NGS libraries in a cell-free system (Yang et al., 2014). Secondly, NGS technologies can parallelize thousands to millions of sequencing reaction, compared to hundreds seen in previous technologies (Yang et al., 2014). Thirdly, sequencing outputs are detected without the use of electrophoresis (technique to separate DNA molecules) (Yang et al., 2014). For these reasons, it is likely that NGS technologies will replace older mtDNA analysis techniques.

Bosworth et al. (2017) use MitoDel (computation procedure) to detect mtDNA deletions using NGS data, which was generated from human brain tissue of a previous study. Since MitoDel focuses exclusively on mtDNA, which is significantly smaller than nucDNA, it reduces the burden of mapping (Bosworth et al., 2017). The results of MitoDel were efficacious in detecting deletions present in mtDNA below 1% heteroplasmy levels, with a low false positive rate (Bosworth et al., 2017). NGS methods are relevant to forensic cases because they produce larger volumes of mtDNA sequence data at a lower cost (Just et al., 2015).

One concern for NGS technologies is that the increased sensitivity for highly degraded specimen can mistakenly detect nuclear mitochondrial DNA segment (NUMT), transposition of mtDNA into the nucDNA and confound heteroplasmy detection (Just et al., 2015). Although NUMT detection does not pose as a significant challenge, laboratory-based and bioinformatic modifications need to be made for optimal efficiency (Just et al., 2015). Also, NGS technologies need to develop mechanisms to authenticate heteroplasmy to reduce risk of contamination and false positives (Just et al., 2015). Furthermore, current forensic mtDNA analysis primarily detect polymorphisms within a hypervariable region; NGS technology has the potential to increase the discriminatory power of identification by incorporating additional polymorphic loci (Yang et al., 2014).

NGS technologies have implications on the field of forensic science and future criminal cases. Firstly, a high resolution of the full mitochondrial genome will help detect heteroplasmy. Secondly, NGS technologies will contribute to larger mtDNA databases; these databases will improve haplotype frequency estimates and provide a greater reference sample (Butler, 2015). Thirdly, and perhaps most importantly, NGS technologies significantly reduce the time and cost for mtDNA analysis. It is likely that NGS technologies will be available more widespread and more frequently used by law enforcement. Familial DNA can significantly aid investigations of missing persons, identify victims, eliminate potential candidates and redirect resources accordingly.

Overall, mtDNA analysis is a useful tool in forensic science. mtDNA has several advantages in comparison to nucDNA; mtDNA analysis is optimal for small, degraded or old samples, an alternative when nucDNA does not yield results and for reference samples of maternal relatives. Although mtDNA does not provide positive identification and has issues with heteroplasmy, paternal leakage and contamination, it is beneficial when nucDNA is not possible. The development of NGS technologies is significant for forensic mtDNA analysis; it is likely that mtDNA analysis will more commonly used in the future by law enforcement agencies.

References

1. Alaeddini, R., Walsh, S. J. & Abbas, A. (2010). Forensic implications of genetic analyses from degraded DNA – A review. Forensic Science International: Genetics, 4(3), 148-157. doi: 10.1016/j.fsigen.2009.09.007
2. Alvarez-Cubero, M. J., Saiz, M., Martinez-Gonzalez, L. J., Alvarez, J. C., Eisenberg, A. J., Budowle, B. & Lorente, J. A. (2012). Genetic identification of missing persons: DNA analysis of human remains and compromised samples. Pathobiology, 79(5), 228-238. doi: 10.1159/000334982
3. Anderson, G. S. (2007). All you ever wanted to know about forensic science in Canada but didn’t know who to ask. Canadian Society of Forensic Sciences. Retrieved from https://www.csfs.ca/wp-content/uploads/2017/08/Forensic-Science-Career-Booklet-GSA- 2017-2nd-Edition-1-ilovepdf-compressed.pdf
4. Anderson, S. (2017). Crim 355 the forensic sciences. (11th ed.). Boston, MA: Pearson.
5. Bär, W., Brinkmann, B., Budowle, B., Carracedo, A., Gill, P., Holland, M.,…Wilson, M. (2000). DNA commission of the international society for forensic genetics: Guidelines for mitochondrial DNA typing. International Journal of Legal Medicine, 113(4), 193- 196. doi: 10.1007/s004140000149
6. Bosworth, C. M., Grandhi, S., Gould, M. P. & LaFramboise, T. (2017). Detection and quantification of mitochondrial DNA deletion from next-generation sequence data. BMC Bioinformatics, 18(12), 30-36. doi: 10.1186/s12859-017-1821-7
7. Budowle, B., Allard, M. W., Wilson, M. R., & Chakraborty, R. (2003). Forensics and mitochondrial DNA: Applications, debates, and foundations. Annual Review of Genomics and Human Genetics, 4, 119-141. doi: 10.1146/annurev.genom.4.070802.110352
8. Butler, J. M. (2015). The future of forensic DNA analysis. Philosophical Transactions B, 370(1674), 1-10. doi: 10.1098/rstb.2014.0252
9. Gonçalves, F. T., Cardena, M., Gonzalez, R., Krieger, J. E., Pereira, A. C., & Fridman, C. (2011). The discrimination power of the hypervariable regions HV1, HV2 and HV3 of mitochondrial DNA in the Brazilian population. Forensic Science International Genetics Supplement Series, 3(1), 311-312. doi: 10.1016/j.fsigss.2011.09.018
10. Hameed, I. H., Jebor, M. A., & Kareem, M. A. (2015). Forensic analysis of mitochondrial DNA hypervariable region HVII (encompassing nucleotide positions 37 to 340) and HVIII (encompassing nucleotide positons 438-574) and evaluation of the importance of these variables positions for forensic genetic purposes. African Journal of Biotechnology, 365- 374. doi: 10.5897/AJB2014.14090
11. Johnston, I. G., Burgstaller, J. P., Havlicek, V., Kolbe, T., Rülicke, T., Brem, G.,… Jones, S. (2015). Stochastic modelling, bayesian inference, and new in vivo measurements elucidate the debated mtDNA bottleneck mechanism. eLife, 4(e07464), 1-44. doi: 10.7554/eLife.07464
12. Just, R. S., Irwin, J. A. & Parson, W. (2015). Mitochondrial DNA heteroplasmy in the emerging field of massively parallel sequencing. Forensic Science International: Genetics, 18, 131- 139.
13. Kumar, V., Kumar, S., Honnungar, R. S., Kumar, A., & Hallikeri, V. R. (2012). Mitochondrial DNA – As a tool for identification. Medico-Legal Update, 12(2), 209-211.
14. Luo, S., Valencia, C., Zhang, J., Lee, N., Slone, J., Gui, B., . . . Huang, T. (2018). Biparental inheritance of mitochondrial DNA in humans. Proceedings of the National Academy of Sciences of the United States of America, 115(51), 13039-13044. doi: 10.1073/pnas.1810946115
15. Melton, T., Dimick, G., Higgins, B., Lindstrom, L., & Nelson, K. (2005). Forensic mitochondrial DNA analysis of 691 casework hairs. Journal of Forensic Sciences, 50(1), 73-80. doi: 10.1520/JFS2004230
16. Melton, T., Holland, C., & Holland, M. (2012). Forensic mitochondrial DNA analysis: Current practice and future potential. Forensic Science Review, 24(2), 102-122.
17. Nilsson, M., Andreasson-Jansson, H., Ingram, M., & Allen, M. (2008). Evaluation of mitochondrial DNA coding region arrays for increased discrimination in forensic analysis. Forensic Science International: Genetics, 2(1), 1-8. doi: 10.1016/j.fsigen.2007.07.004
18. Parson, T. J. & Coble, M. D. (2001). Increasing the forensic discrimination of mitochondrial DNA testing through analysis of the entire mitochondrial DNA genome. Croat Med J, 42(3), 304-309.
19. Parson, W., Gusmão, L., Hares, D. R., Irwin, J. A., Mayr, W. R., Morling, N.,…Parson, T. J. (2014). DNA commission of the international society for forensic genetics: Revised and extended guidelines for mitochondrial DNA typing. Forensic Science International, 13, 134-142. doi: 10.1016/j.fsigen.2014.07.010
20. Ricci, U., Carboni, I., Iozzi, S., Nutini, A. L., Contini, E., Torricelli, F., &… Norelli, G. A. (2015). Genetic identification of burned corpses as a part of disaster victim dentification effort. Forensic Science International: Genetic Supplement Series, 5, 447- 447. doi: https://doi.org/10.1016/j.fsigss.2015.09.177
21. Sampath, K. & Jagannathan, N. (2014). Role of DNA in forensic identification. Indian Journal of Forensic Medicine & Toxicology, 8(2), 43-47. doi: 10.5958/0973-9130.2014.00679.3
22. U.S. Department of Justice. (2002). Using DNA to solve cold cases. Retrieved from https://www.ncjrs.gov/pdffiles1/nij/194197.pdf
23. van Oven, M. & Kayser, M. (2008). The impact of DNA contamination of bone samples in forensic case analysis and anthropological research. Legal Medicine, 10(3), 125-130. doi: 10.1016/j.legalmed.2007.10.001
24. Wurmb-Schwark, N., Heinrich, A., Freudenberg, M., Gebür, M., & Schwark, T. (2008). The impact of DNA contamination of bone samples in forensic case analysis and anthropological research. Legal Medicine, 10(3), 125-130. doi: 10.1016/j.legalmed.2007.10.001
25. Yang, Y., Xie, B., & Yan, J. (2014). Application of next-generation sequencing technology in forensic science. Genomics, Proteomics & Bioinformatics, 12(5), 190-197. doi: 10.1016/j.gpb.2014.09.001

Human DNA Quantification And Forensics

Human DNA is present in every cell except RBCs and can be found in body fluids like saliva, blood, semen, vaginal fluids, bones, teeth, hair and sweat. DNA has its individuality and DNA typing methodologies are subjected to scientific and legal scrutiny. DNA has been used as unique investigation material in forensics since Alec Jeffrey introduced RFLP in 1985 for identifing the unique markers in the genetic material.[4]

DNA Quantification estimates the amount of DNA present in the source of DNA evidences. It focuses on establishing the DNA concentration and evaluating the PCR inhibitors.[2] DNA quantification saves time by evaluating the Concentration of DNA that avoids redoing the experiment. Besides, it helps to know whether the amount of DNA in the sample is sufficient for the experimentation or not. It informs, if DNA sample is diluted and also, that you are using right DNA species. This process assures that whatever results you are getting is referring to DNA without contaminating RNA or proteins in the sample.[13]

Methods of DNA Quantification

1)UV Absorbance

The UV absorbance was first proposed by Warburg, O. and Christian W.(1942) , used to obtain qualitative and quantitative details of DNA/RNA.[6]

The purine and pyrimidine bases in DNA absorb UV light of specific wavelengths but the composite absorption occurs at 260 nm. A DNA solution of concentration 50 p,g/ml will yield an absorbency at 260 nm. [7]

Pros:

• Fast, Easy, Reagents are not required.
• Determination of proteins and phenols are possible[10]

Cons:

• Limited sensitivity, contaminants cause inaccuracies
• Differentiating nucleic acid species and determining the extent of DNA degradation isn’t possible.[10]

2)Fluorescence Measurements

DNA does not have fluorescence. It can possess the property of fluorescence with two dyes Hoechst 33258 (Brunk et al. 1979) and ethidium bromide. The maximum excitation and emission for both dyes shifts after binding to DNA. Hoechst 33258 appears to bind to A-T base pairs rich regions; whereas ethidium bromide binds between the stacked bases (Watson et al. 1987). [7]

Pros:

• selective detection of nucleic acid species
• 10 times more sensitive [10]

Cons:

• Requires calibration with standards
• molecular weight of DNA and level of contamination cannot be estimated.[10]

3)Yield Gel Measurements

Yield gels are small agarose gels containing ethidium bromide. DNA fragments are introduced to a electrophoretic separation at relatively high voltage. After the completion ofelectrophoresis, the gel is placed under UV light and a photograph is taken. [7]

Pros:

• Approximate molecular weight and concentration can be obtained
• More rapid[7]

Cons:

• It has poor sensitivity, and does not result in a quantitation that is specific to human DNA.
• Results are relatively accurate. [1]

Yield measurements

4)Slot Blot Assay

In this process, the genomic DNA is denatured and a tiny amount of sample is spotted onto a nitrocellulose membrane. Then, the immobilization of sdDNA is done on a nylon membrane. The targeted sequence is revealed by hybridization with a labeled 40-nucleotide probe complementary to a primate-specific α−satellite DNA sequence at the D17Z1 locus. [9]

Pros

• Does not require DNA digestion[11]
• Easy to analyze [8]

Cons

• Not automatable, requires more efforts
• Semi-quantitative[8]
• Slot-Blot Assay

5)Quantitative PCR Assays

Based on the principle of PCR amplification, the amount of PCR product amplified correlates with the initial DNA concentration. There are two categories of quantitative PCR methods.

• A)End-point PCR methods measure the amount of synthesis of amplified product during PCR at the end of the reaction. Usually, the fluorescence is emitted by the dyes that intercalate into the dsDNA. The yield of amplified DNA is detected from the amount of fluorescence emitted by dyes.
• B)Real-time PCR methods can quantify the amplified DNA during the exponential phase of PCR.[9]

Exponential phase by RT-PCR

6)Real-Time Quantitative PCR

Real-time quantitative PCR was developed earlier in 1990s, and analyzes the cycle-to-cycle change in a fluorescence signal due to the amplification of a target sequence during PCR. A PCR is monitored by fluorescence reporter by increasing the molecules as products accumulate with each round of amplification. [9]

Pros:

• Sensitivity in picogram range
• Ability to design in sequence-specificity[10]

Cons:

• Requires calibration with standards
• Requires expensive reagents and instrumentation
• Time-intensive assay [10]

Forensic Significance

The quantitation of DNA plays a central role in all areas and applications of forensic DNA analysis such as a discrete windows of DNA concentration are allowed amplification of the 13 CODIS STR loci with the commercial kits that are used for forensic casework and databank genotyping.[1]

DNA quantification is needed in detecting cell-free fetal DNA in the maternal circulation and also for procedures like NGS, PCR, RT-PCR, qPCR, cloning, etc.[12]

References

1. Timket, M., Swango, K., Orrego, C., Chong, M., & Buoncristiani, M. (2005). Quantitation of DNA for Forensic DNA Typing by qPCR (quantitative PCR): Singleplex and Multiplex Modes for Nuclear and Mitochondrial Genomes, and the Y Chromosome.. Ncjrs.gov. Retrieved 2 October 2020, from https://www.ncjrs.gov/pdffiles1/nij/grants/210302.pdf.
2. Ricci, U., Marchi, C., Previdere, C., & Fattorini, P. (2006). Quantification of human DNA by real-time PCR in forensic casework [Ebook]. International Congress Series 1288 (2006) 750 – 752. Retrieved 2 October 2020, from https://www.isfg.org/files/67dcf7d7d9946629c70003d18d286e67abe87cfd.0600077x_613929581202.pdf.
3. Standard for Training in Forensic DNA Quantification Methods. (2018). [Ebook] (1st ed.). Retrieved 2 October 2020, from https://www.nist.gov/system/files/documents/2018/12/17/osac_draft_standard_for_training_in_forensic_dna_quantification_methods.pdf.
4. Nurun Nahar Sultana, G., & Zakir Sultan, M. (2018). Mitochondrial DNA and Methods for Forensic Identification. Juniperpublishers.com. Retrieved 2 October 2020, from https://juniperpublishers.com/jfsci/pdf/JFSCI.MS.ID.555755.pdf.
5. LaSalle, H., Duncan, G., & McCord, B. (2011). An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction. Forensic Science International: Genetics, 5, 185-193. Retrieved 2 October 2020, from https://cnso.nova.edu/forms/george_duncan_an_analysis_of_single_and_multi-copy.pdf.
6. Pachchigar, K., Khunt, A., & Hetal, B. (2016). DNA QUANTIFICATION [Ebook]. Retrieved 2 October 2020, from http://www.researchgate.net/publication/318744511_DNA_QUANTIFICATION.
7. Samuel Baechtel, F. THE EXTRACTION, PURIFICATION AND QUANTIFICATION OF DNA [Ebook]. Retrieved 2 October 2020, from https://projects.nfstc.org/workshops/resources/literature/Extraction/05_The%20Extraction,%20Purification%20and%20Quatification%20of%20DNA.pdf.
8. DNA Extraction & Quantitation for Forensic Analysts. (2008). [Ebook]. Retrieved 2 October 2020, from https://www.sjsu.edu/people/steven.lee/courses/c2/s2/DNA%20Extraction%20and%20Quantitation%20for%20Forensic%20Analysts.pdf.
9. Li, R. (2011). Forensic biology (pp. 211-221). CRC PRESS.
10. Choosing the Right Method for Nucleic Acid Quantitation. Promega.in. (2020). Retrieved 3 October 2020, from https://www.promega.in/resources/pubhub/choosing-the-right-method-for-nucleic-acid-quantitation/.
11. Blouin, J., Rahmani, Z., & Chettouh, Z. (2020). Slot Blot Method for the Quantification of DNA Sequence and Mapping of Chromosome Rearrangements: Application to Chromosome 21. American Journal Of Human Genetics, 46, 518-526. Retrieved 3 October 2020.
12. Co.KG, B. (2020). DNA quantification – Berthold Technologies. Berthold Technologies GmbH & Co.KG. Retrieved 3 October 2020, from https://www.berthold.com/en/bioanalytic/applications/dna-quantification/.
13. Nucleic Acid Analysis. Promega.com. (2020). Retrieved 3 October 2020, from https://www.promega.com/nucleic-acid-analysis/.