Sometimes Forensic Casework items consist of less than 100 picograms (pg) of template DNA which, if we assume 3.5 pg of DNA per haploid cell, can be equated to approximately thirty haploid cells or fifteen diploid cells are termed as LCN DNA (low template DNA or LT DNA). (Gill 17)
Such low copy number (LCN) samples exist due to several factors like damaged or degraded DNA, oligozoospermia or aspermic perpetrators or extended interval post coital samples, where there is loss of sperm over time because of the effects of drainage or host cell metabolism. From crime scenes, Trace biological evidence which arises from casual handling of objects (‘touch DNA’) is increasingly being recovered and due to the low amount of DNA recovered many of such “touch DNA” samples are classified as LCN samples. Using standard STR testing, recovery of genetic profiles from LCN samples becomes difficult as the tests performed often result in recovery of partial profiles or total failure. (Gill 17)
Commercial STR kits have been developed to produce good quality, balanced profiles with 1ng of DNA with 28-30 polymerase chain reaction (PCR) cycles. Interpreting the resulting DNA profile from LCN samples where the results are below the stochastic threshold for reliable interpretation require more considerations than interpreting single-source or mixed DNA profiles generated using higher amounts of DNA. Consequently, special LCN DNA testing procedures have been developed to increase assay sensitivity; commonly this includes increased PCR cycles (e.g., 28 cycles increased to 31 or 34 cycles) to allow balanced and complete genetic profile recovery from limited quantity samples.
Many new methods have been discovered and perfected to balance and complete the profiling of LCN samples including entire genome analysis, increased cycle number and nested Polymerase Chain Reaction. Among the available methodologies forensic scientists and investigators have adopted ICN as the standard approach but also use other methods like DNA amplification volume reduction, desalting of sample before electrophoresis, increase in injection timing in electrophoresis etc. Whether defined using a quantitative, qualitative or a technical definition LCN usually refers to any case where the amount of DNA to be amplified is limited by a tiny sample size or other factors like DNA degradation, PCR inhibition etc.
However, as is usual with every new technique, LCN methods need improvement to increase their reliability. As observed over the years, there are many issues with LCN which are:
1) Greater room for occurrence of errors exists as LCN generates genetic profiles from samples where the quality and/or the quantity of genetic material present is not enough to give results when compared to other techniques
2) Due to the available assay being overly sensitive and sample types tested, LCN profile may turn out to be irrelevant to that particular case..
3) Guidelines about proper evidence collection and protocols regarding the handling of such samples are not established in a proper and official manner.
4) Due to large variations in shutter peak, drop-in and drop outs of alleles and peak imbalance interpretation of the profiles generated can contain a lot of errors.
5) The profile obtained from LCN cannot be used as legal evidence in a court of law. (Budowle et. al. 2001)
Numerous fundamental issues arise due to the analysis of sub-optimal amounts of DNA template in a PCR and as the amount of template is reduced, these issues become even more complicated. In addition, mixture interpretation has yet to be well – established. The basic issues are:
1. Stochastic effects
2. Profile interpretation
3. Detection threshold
4. Allele drop-out and heterozygote peak imbalance
5. Replicate analysis
6. Application limitations
7. Appropriate controls
8. Contamination
9. Stutter
Types of LCN
1) Increasing PCR cycle number; (Gill P. et al., 2000; Gill P., 2001)
2) Nested PCR;
3) Reducing the volume of the PCR;
4) Whole genome amplification prior to the PCR;
5) Enhanced fluorescent dye signal;
6) Use of higher purity formamide in sample preparation for capillary electrophoresis;
7) Post-PCR clean-up to remove ions that compete with DNA during electrokinetic injection;
8) Increasing Injection time (Budowle et. al. 2001)
Let us now look at some cases which will throw some light on certain aspects and uses of LCN techniques in practical cases. Presented here are three different cases in which LCN techniques were used.
The remains of a plane which crashed in 1965 are suspected to have been found in 1993 and the tiny fragments of human DNA found from the site makes it impossible to determine the identity of the pilot. In January, 2000 the largest bone sample fragment is sampled and submitted for DNA testing to be typed for mt DNA. The initial report reflects a western European lineage which supports the theory as the remains are supposedly of an American serviceman. Based on this information, as well as wreckage analysis and circumstantial evidence linking the crash site to this specific loss incident, a presumptive identification was established. However since no maternal reference is available, the investigators analyse the autosomal and Y markers of the DNA sample and compare them with those obtained from the wife and children of the deceased pilot. Both autosomal and Y STRs are found to match. The autosomal data further confirms the identity of the missing serviceman, resulting in a likelihood ratio of 9.9 billion to one, in support of the stated genetic relationship.
In 1943 an American B-24 D Liberator failed to return to base and the nine man crew manning the ship was presumed dead. In March 2002, some human remains were found amid the wreckage of a World War II Plane in the vicinity of the suspected crash site. The CIL took DNA samples and submitted in total 25 samples to AFDIL for mt. DNA testing along with maternal references for the nine missing individuals. .
On the basis of HVI⁄HVII sequence data, 15 of the 25 samples were confidently associated with five of the nine family references. The remaining 10 samples, possibly representing the remaining four unaccounted for servicemen, could not be completely distinguished with HVI⁄HVII sequence data alone. Four of these 10 evidentiary samples and two reference families matched the most common western European haplotype, and while two of these four samples could be distinguished by an HVII C-stretch length polymorphism, this difference alone provided weak evidence for sorting the four samples. Portions of the crash site critical to the origin of the samples in question had remained relatively undisturbed since the incident. Given that remains had already been sampled for mtDNA HVI⁄ HVII testing, additional genetic data was sought to permit the segregation of commingled remains sharing common mtDNA HV types. Additional testing outside of HVI⁄HVII, using both sequence data and coding region single nucleotide polymorphisms (SNPs), confirmed that four samples originated from two individuals. However, the remaining six case samples could not be resolved with available mtDNA data. As a result, LCN typing of Y and autosomal markers was conducted on all ten samples for further resolution.
Amplification success among skeletal elements varied significantly, with some elements producing no more than three or four confirmed alleles. However, the combination of LCN data from both the autosomal and Y markers provided enough information to confidently sort elements and establish that the 10 elements originated from four individuals, not three. For this particular case, a combination of mtDNA data and LCN autosomal and Y data contributed to the successful segregation of all nine missing servicemen.
In July 1918, during the combined French-American attack on German positions near Soissons, France, a young private was killed. His body was never recovered.
In August 2003, during a construction project near Soissons, a French national found human remains and artifacts and turned them over to the U.S. Army mortuary in Germany and then transported to the Joint POW⁄MIA Accounting Command’s Central Identification Laboratory (CIL) for analysis. Among the artifacts were a leather wallet bearing the soldier’s name and a military boot fragment consistent with sizes 5 or 5.5 were recovered. A presumptive identification of one of the skeletons was made based on the material evidence received associated with the recovery site (the wallet and the boot fragment), dental analysis of the remains, and the unusually small stature of the skeleton that was consistent with the personnel records for the soldier.
In order to acquire additional supporting evidence, a dental sample was submitted to the AFDIL in August 2004. Mitochondrial DNA testing was successfully executed on an extract from 170 mg of dentin. A sample of femoral bone (mass) was also taken from the second skeleton yielding a different mtDNA profile but unfortunately, a known maternal relative for the presumed missing serviceman could not be located and the mtDNA investigation stalled for lack of reference material. However, the recovered skeleton seemed to potray the gender of the deceased to be female rather than male. Taking this into consideration, the anthropologist analysing the skeleton was doubtful of the gender of the deceased. This analyst knew only the face that remains dated to the World War I era. He had not been informed of the name association to a specific casualty, nor was he aware of the dental evidence supporting the association of the remains to this man.
Later this issue was clarified when biological profile of the remains was compared to the service records of the individual in accordance with CIL Standard Operating Procedures. Also the size of the skeleton was in accordance with the casualty presumptively identified; he was simply a small, gracile male. However, a thorough investigation was demanded in order to confirm the sex so the CIL requested that AFDIL to conduct the same using DNA sample already recovered. The resulting STR profiles were unambiguous for amelogenin in the PowerPlex 16 system and almost complete for the Yfiler multiplex, confirming that the skeletal remains were indeed male. Data were recovered from all but one Y-STR locus, and 29 alleles were confirmed with PowerPlex 16. The DNA data produced from this set of remains were particularly notable both because the extract derived from such a small quantity of dentin and because the remains were significantly older than other cases that had produced far less data. It is likely that data recovery with this sample was facilitated by two factors: one, the moderate climate in which the remains lay for over 80 years and two, the protective anatomical environment that dental DNA enjoys, a factor that is highlighted by the more fragmentary profile recovered from the much larger osseous sample obtained from the second skeleton.
Still, some question must remain over the authenticity of the profile obtained from the dentin, given that it was based on a single extract. In favor of authenticity are the clean negative controls and reagent blanks, the presence of a positive but different profile in the sample from the second skeleton and the absence of any mixtures in either sample. It remains possible, although we maintain unlikely, that a specific contaminant on the dentin sample resulted in the recovery of an exogenous profile with little or no evidence of endogenous DNA. Typically such a question mark would be addressed through replication of the initial result; a second dentin sample (or an osseous sample) from the same skeleton, independently sampled and processed could significantly narrow the room for doubt. However, at the point that the LCN analysis was completed, the circumstantial case for identification had been assessed and was found compelling. As a result, the identification was already in hand independent of any DNA data.
A set of skeletal remains was unilaterally turned over by the Vietnamese government in 1989 as part of a large series of remains repatriated over several months that year. Some of these cases had very little associated information, while others were associated with erroneous information. The case in question had no data concerning the geographic location of the recovery but the remains returned were allegedly those of an American. With no circumstantial or material evidence to consider, the only meaningful data came from the anthropological examination which suggested that in the absence of definitive skeletal indicators of sex, the size and gracility of the remains most likely pointed to a female skeleton. Subsequently, mtDNA testing was successfully performed on a cranial sample. However, comparisons to the maternal references for all three female American casualties that remain unresolved as a result of the war Southeast Asia resulted in exclusions. The mtDNA sequence data were also inconsistent with the hypothesis that the casualty was indigenous to Southeast Asia. The mtDNA lineage was of western European origin (haplogroup H) and thus it was unlikely that the skeleton represented a Vietnamese female whose remains had been inadvertently turned over to the United States. There are however an unknown number of third country national females, deceased in Vietnam, both during the colonial period and the subsequent conflict whose remains might have been inadvertently included with the materials repatriated by the Vietnamese authorities. In order to verify the suspected sex, typing of amelogenin and subsequently Y-chromosomal STRs was conducted on a femoral sample. LCN analysis established that the remains were, in fact, male. Duplicate extractions were performed in this case, but LCN typing was only conducted using the extract with the higher DNA concentration. Mitochondrial DNA testing was conducted on both extracts. The mtDNA data were concordant and the haplotype was unique in the SWGDAM database of 4839 individuals. This information redefined the list of potentially associated casualties by providing key data for this case, which had previously hit a dead end. The case remains unresolved, but has been reinvigorated by the genetic typing. Identification efforts will now proceed on the basis of the supposition that the remains could represent and unaccounted for casualty who is a small male.
Conclusion
Taking a look at the various cases quoted in this paper and various others which exist but given the paucity of space have been left out, we can safely conclude that LCN techniques have various limitations and may not be developed to the fullest possible extent and accuracy that can eliminate any doubt in the minds of the critics of these techniques. However, given the positive attributes of LCN it is not wise to ignore a technique which raises hope for family members and provides researchers clues for completing their investigation and finding solutions to other wise unsolvable puzzles.
Works Cited
Gill, P., J. Whitaker, C. Flaxman, N. Brown, and J. Buckleton. “An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA.” Forensic Science International 112.1 (2000). 17–40. Print.
Gill, P. “Application of low copy number DNA profiling.” Croatian Medical Journal 42.3 (2001): 229–32. Print.
Budowle, Bruce, Arthur J. Eisenberg, and Angela van Daal. “Validity of Low Copy Number Typing and Applications to Forensic Science.” Croatian Medical Journal 50.3 (2001): 207–217. Print.
Jodi A. Irwin, M.S, Mark D. Leney, Odile Loreille, Suzanne M. Barritt, Alexander F. Christensen, Thomas D. Holland, Brion C. Smith, and Thomas J. Parsons. “Application of Low Copy Number STR Typing to the Identification of Aged, Degraded Skeletal Remains.” Forensic Science International 52.6 (2010): 1322-1327. Print.
