BVd...... Analysis of LCN (low copy number) DNA, handy DNA or touch DNA, and also single cell DNA analysis, offer significant advances for forensic DNA profiling. Do you agree or disagree Justify your views

Traditionally, workers in a crime scene look for fingerprints. They also look for DNA. However, it is only now that scientists look for DNA from fingerprints, and forensic analysis of these samples are gaining credibility in the use in the courts of law. Used as a last resort for DNA samples with no results from standard DNA typing processes, analysis of low copy number (LCN) DNA indeed gives significant advances for forensic DNA profiling.

Low copy number DNA
Low copy number DNA or low-level DNA results when a person touches or comes into contact with an object, leaving behind trace amounts of biological substances on the surface. This can be a sample collected from a drinking cup, or from any surface with a partial fingerprint on it. When standard DNA testing is not enough to obtain a favorable result given a poor sample, LCN DNA techniques are often used. It implements several modifications to the standard method, allowing for DNA analysis from a small sample of cells, or from DNA in a poor state, to yield DNA profiles. LCN DNA analysis is defined by Wulff (2006) as the analysis of any results below the stochastic threshold for normal interpretation, indicating the microscopic amounts of DNA needed as samples, with 15-20 cells in a sample adequate enough (p.1).

Gill (2001) mentions that there are two general categories of evidence discrete (bone, hair) and non-discrete (blood stains). Discrete evidence is usually associated when using LCN, as bone samples are usually removed of its outermost layer, minimizing DNA contaminants, and hair samples can be washed with a detergent solution to remove extraneous DNA (p.230). Furthermore, a clear benefit of this procedure is in the event of an unusable fingerprint  smudged, partial, or unidentified, the fingerprints can still be used to give a name, or at least an increased factor with which to search for an identity.

In order to do this, modification of the PCR (polymerase chain reaction) amplification process is a simple and easy way to increase the sensitivity of standard DNA profiling methods. Standard commercial PCR needs 1 nanogram, or about 150-160 cells of DNA, to be successful low copy number DNA profiling needs only five to twenty cells to yield good results (Kanable, 2005, Getting in touch with touch DNA section). The standard PCR cycle goes for 28 cycles, but for LCN analysis, it is usually increased to 34 cycles, with fingerprints taken through 28-40 cycles, rootless hair shafts through 35-43 cycles (Wulff, 2006, p.2), and 38-43 cycles for ancient bones (Gill, 2001, p. 229), among others. Samples are aliquoted three times, with only two to be tested, as the third is set aside for further testing if the other two samples fail to give a good result (p. 230). As such, only if the results are present in two trials, indicating its reproducibility, are accepted. Statistical analyses follow, to show the possibility of the DNA results as a result of chance, contamination, or low-level samples. The results do not indicate about where the sample may have come from, but context may always be used to infer such information. Similarly, it cannot give details on the actions that caused the sample to be placed on an item (p. 231).

With this, there are several challenges facing LCN DNA processing. First is the increased risk of contamination, as greater PCR amplification causes background DNA to appear as well, which is DNA that can come from the people who collected, handled, and studied the sample, conflicting the primary source of the sample that is needed for investigation. Allele drop-out may occur, when only one allele of a heterozygote locus is selectively amplified in the PCR process, in addition to allele drop-in, where artificial short tandem repeat (STR) profiles may also appear but can be identified as such and eliminated as a contaminant. There is also the challenge of separating mixed DNA samples, especially if it is from same genders in contrast with a combination of male and female DNA (Wulff, 2006, p.2 Gill, 2001, p. 230).

Thus, ultra-clean dedicated laboratories are needed to ensure minimal to no contamination from another source. Some protocols include taking extra precaution to lessen the chance of coming into contact and contaminating a sample and pre-treatment of solutions and equipment to remove contaminant DNA. Laboratories operate under stringent requirements, as they have positive air pressure and special lighting and chemical treatments to prevent DNA contamination. This makes LCN typing a more expensive and less widely available process (Gill p. 232).

Nevertheless, even with these challenges that LCN DNA typing face, its most significant advantage can be seen in light of the reality that crime scene evidence sometimes has insufficient DNA to give a full or even partial DNA profile through standard forensic procedures. LCN typing can be used if the piece of evidence is critical to a case, and dealing with its limitations is worth the results.

Validation Studies
The judge of a well-known case involving LCN DNA analysis speaks of the value of validation studies. Mr. Justice Wier of the Omagh bombing case says that validation studies are important as it asks the scientific community to obtain the needed information to evaluate the ability of a method to get reliable results, set out the conditions under which such results can be obtained, and give limitations of the procedure. The validation process seeks the aspects of a procedure that are critical and must be carefully controlled. Without an agreed protocol for the validation of scientific techniques prior to their being admitted in court is entirely unsatisfactory. Since science is fundamentally an exoteric process, intended to be understood by the general public, it is the custom in empirical science that findings and data are independently replicated prior to widespread acceptance (Caddy, Taylor, Linacre, 2008, p. 16).

For the LCN DNA analyses, validation studies start with obtaining a large number of DNA samples of known profiles, which reflect the different alleles at the 10 different loci. The samples would then be serially diluted to provide masses of 10, 20, 40, 80, 100 and 200pg. Next, each of these would then be taken through the extraction procedure, and appropriate PCR amplification occurs, followed by capillary electrophoresis. The profiles yielded would then be compared with the known profiles of these samples and an evaluation made in respect of all the parameters discussed above. This entire procedure must then be repeated enough times to obtain a statistically robust measure of the reproducibility of the system. It often goes in logical succession of experiments to determine the limitations of their processes, these include, optimization of the capillary electrophoresis system, limits of detection, the reproducibility and evaluation of stochastic effects, drop in and out and inhibition in a way very similar to the pattern of experiments the review advocates above (p.17 - 18).

Caragine et al. (2009) performed an experiment on the reliability and reproducibility of known LCN DNA samples. Amplification resulted to the aforementioned problems, such as allelic dropout and separation of mixed samples, but by following interpretation guidelines and stringent protocols, they conclude that LCN DNA testing is reliable and robust (p. 250). Similarly, Kloosterman  Kersbergen (2003) undertook a validation study of amplification of extremely low DNA samples using 34 cycles. Facing the same challenges, they nevertheless conclude that LCN typing allows for reanalysis of crime samples that do not result in a full profile, and the addition of 6 PCR cycles gives a reliable alternative if there is too little of the original sample for performing a reanalysis (p. 795). Alessandrini et al (2003) subjected 374 fingerprints left on various substrates for quantitative and type analysis. They found that DNA recovered form fingerprints amounted to only 31.8 of complete profiles (with an average of 25 pg of sample), but partial profiles amounted to 54.5 (p.4). It is confirmed that DNA can be recovered from fingerprints, though it is dependent on the shredder status of the donor and most DNA is still lost during harvesting and extraction steps (p. 6). Lastly, Smith and Ballantyne (2007) performed post-PCR purification of PCR products obtained from 100 pg of DNA sample and run for 28 cycles. Although the number of cycles remains standard, it is the possibility of using LCN DNA to obtain a full profile that was considered. Encountering allele dropout and contamination as well, they ultimately state that DNA profiles can be obtained from low copy number samples, as long as stringent protocols are followed (p.820).

These studies all end with the recommendation that low count number DNA is a promising technique for use in forensics. Precautions must be taken to assure meticulous and proper analyses, limiting the chances for extraneous factors to affect the results.

We now consider the instances when LCN DNA profiling played a very significant role in determining how law cases turn out.

Forensic Application
The Forensic Science Service is a leading provider of forensic services to police forces in England and Wales. They pioneered the implementation and expansion of the use of DNA technologies in forensics, establishing the worlds first DNA database in April 1995. In 1999, they developed the use of DNA techniques that enabled the possibility of low copy number DNA to produce DNA profiles that were unattainable before. They cite several case studies on how useful LCN DNA typing has been to the closing of cold cases. It all started under Operation Phoenix of Northumbria Police, where unsolved sexual offenses between 1985-1999 were reviewed and reopened. FSS scientists studied more than 200 of these cases where past technology just was not enough to garner substantial results. LCN DNA techniques were used in handling frozen swabs or old DNA extracts on slides or tapings (FSS, n.d., History section).

The 1977 attack, sexual assault, and finally, murder of Mary Gregson remained opened for a few years. During that time, DNA profiling was unavailable, and semen stains found were subjected to blood grouping tests only. In 1985, single locus profiling (SLP) was performed on the sample, but this was unsuccessful as the DNA was insufficient to give a profile. Advances in 1995 allowed more sensitive tests to be performed, but these remained unsuccessful. It was only in 1999 that samples were brought out again that possibly contained enough DNA, sent to the forensic science service for LCN DNA testing in the facilitys most sensitive technique as of the time, finally resulting to a full male profile. In 2000, the police finally found the suspect, closing the murder case of more than thirty years of age (FSS, n.d., Mary Gregson section). Another case study by the FSS presents the rape and strangling of 14-year-old Marion Crofts in 1981. There was a single microscope slide that contained samples collected by the FSS from the teens body after the murder, that was intentionally untouched for 20 years. Scientists were aware that there was a great risk of losing the evidence unless they waited for better DNA typing techniques to be available. In 1999, through the novel LCN DNA technology the FSS developed, they took the microscope slide and obtained a full profile of the murder suspect. Twenty-one years after the murder, the teenagers rapist and murderer was incarcerated for life in 2002 (FSS, case studies section). The approach as employed by Northumbria Police can be a framework that can be used by other sectors as well (FSS, n.d., Marion Crofts section).

The FSS also dealt with the infamous Omagh Bombing case in 2007, which mainly focused on the validity of LCN DNA analyses as a robust and valid technique. The defence criticized it in terms of contamination issues, reproducibility, absence of quantification, and interpretation and the setting of guidelines (Woffinden, 2010). The Crown Prosecution Service ordered for the re-examination of cases currently going through the courts after the judge Mr. Wein, who cleared Sean Hoey, the only man charged with the 29 Omagh bombing murders, questioned its reliability. In Hoeys trial, the prosecution used LCN DNA techniques to link him to some of the explosive devices used as evidence, however, its truthfulness was question when a sample taken from a car bomb was wrongly linked to a 14-year-old schoolboy in Nottingham. Mr Justice Weir of the Belfast Crown Court also mention that the process is only acceptable as evidence in New Zealand and the Netherlands, and that an internationally accepted standardized system has to be set before the process can be considered accurate. This was the cause for the importance of the analysis technique to be validated (Gill, 2007). Because of this case, validation of the use of LCN DNA in forensic investigations increased, setting the parameters of LCN DNA analysis (Caddy et al, 2008, p.22).

The Forensic Science Service allows for people, who escape their crimes, to be justified and put in jail, all with the help of advancements in technology. As of 2008, the company has used the LCN DNA technique is more than 20,000 cases, garnering convictions and admissions of guilt, particularly for cold cases (Randerson, 2008).

A Review of the LCN DNA Process
The sensitivity of LCN DNA samples are affected by first, the recovery of samples from crime scenes, and second, the recovery of samples by the scientists in the laboratory. Appropriate training must be given to those responsible for the recovery of LCN DNA samples by scene of crime officers, seeking advice if needed, to ensure that proper precautions have been done to avoid contamination. As not all are aware of the sensitivity of LCN analysis, this training is vital to also clarify the occasions when LCN DNA is used, that is, as a last resort. Use of DNA-free equipment, particularly swabs and containers, is an important aspect. With this, having a database of the personnel working for supply companies may also be useful in elimination purposes (Caddy et al., 2008, p. 8).

Recovery of DNA samples in the laboratory first starts with the decision of what analysis technique will be performed on a sample. If LCN DNA is at hand, only specialized laboratories can handle the sample. This includes pressurized laboratories, UV irradiation of benches, and constant monitoring of contamination levels (p. 10). Quantification of DNA is usually the initial step taken, as it enables a better guess of possible inhibition, and also allows for a fully analysis in duplicates up to a third if needed. PCR amplification follows, with either increased cycles, or post-PCR clean-ups (p.13). It depends on the knowledge of samples and techniques, and the ability to perform modifications that guarantee good results. Problems that are usually faced is the loss of alleles, addition of alleles or stutter-bands, and detection of low levels of contaminants. Other issues are inhibition with certain dyes, chelating agents, and heparin that are hard to remove, and excessive DNA may also appear, affecting the resulting DNA profile (p.14).

In order to further the use of LCN DNA despite these problems commonly faced, it is important to give more details to these issues to dispel wrong preconceptions. Drop-in alleles are random and infrequent, usually linked with contamination from equipment, crime scene officers and laboratory personnel. To answer this, DNA profiles from a database of people in the industry (manufacturers, police, and laboratory) are usually provided (p.15). Allelic dropout caused by stochastic effects is a common event, but the lack of a comparison samples alleles from a mixture does not immediately indicate exclusion. Thus, scientists must be knowledgeable in inferring whether the observed alleles are what can be expected if an individual contributed to the mixture. The quantity of DNA that undergoes applification, the count of the contributors, the empirically defined loci characteristics, and the length of the repeats should be taken into consideration (Caragine, Mikulasovich, Tamariz, Bajda, Sebestyen, Daum, Prinz, 2009, p.265).

On concerns on the transfer of cellular material from person to person to object and the possibility of DNA to be handed off to a place a person has never stepped into. It should be noted that transfer of material is subjected to time, pressure, and nature of surfaces, and thus, transfer to secondary surfaces will not be 100. Cellular material is also subjected to cellular enzymatic actions, degrading this material once it has been transferred. Temperature and moisture affects this, and in addition to other numerous factors that degrade cellular material and successively, DNA, LCN DNA analyses avoids these associations unless samples can be associated with discrete evidence such as teeth and bones (Caddy et al, 2008, p. 20-21).

The most common problem that cases which use LCN DNA typing face is the belief that it is unreliable and brings too many doubts with its results. Improper handling of evidence by police and investigators of evidence can easily give false profiles, and shaking hands can be a simple way for someone elses DNA to transfer to another, after which the persons hand can plant that DNA on any surface. Still, the important factor to consider is that technology and techniques are continuously improving, and with this, reliability is also increasing. Improvements on the methods of collection, extraction, quantification and amplification, are endless. Its use does not intend to replace the cheaper and simpler fingerprint matching techniques, but it can be a vital tool when all other techniques have been exhausted. Ramotowski, of the International Association for Identification, puts it simply, this is something that wed like to have in our toolbox and take out when needed (Kanable, 2005, Will DNA from fingerprints be used more in the future).


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