An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA

Abstract

By increasing the PCR amplification regime to 34 cycles, we have demonstrated that it is possible routinely to analyse ,100 pg DNA. The success rate was not improved (without impairing quality) by increasing cycle number further. Compared to amplification of 1 ng DNA at 28 cycles, it was shown that increased imbalance of heterozygotes occurred, along with an increase in the size (peak area) of stutters. The analysis of mixtures by peak area measurement becomes increasingly difficult as the sample size is reduced. Laboratory-based contamination cannot be completely avoided, even when analysis is carried out under stringent conditions of cleanliness. A set of guidelines that utilises duplication of results to interpret profiles originating from picogram levels of DNA is introduced. We demonstrate that the duplication guideline is robust by applying a statistical theory that models three key parameters -namely the incidence of allele drop-out, laboratory contamination and stutter. The advantage of the model is that the critical levels for each parameter can be calculated. This information may be used (for example) to determine levels of contamination that can be tolerated within the strategy employed. In addition we demonstrate that interpreting one banded loci, where allele dropout could have occurred, using LR 5 1/2f was conservative provided a that the band was low in peak area. Furthermore, we demonstrate that an apparent mis-match between crime-stain and a suspect DNA profile does not necessarily result in an exclusion. The method used is complex, yet can be converted into an expert system. We envisage this to be the next step.

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

“…There was no cross-reactivity with other body fluids, except for occasional, often results in the appearance of foreign alleles ("allele drop-in") [29] and the same effects might also be observed with RNA multiplex systems. The identified blood specific mRNAs, while expressed abundantly in blood, are likely not to be completely absent in other cells.…”

Probabilistic expert systems for handling artifacts in complex DNA mixtures

“…It is worth emphasizing that our simultaneous analysis of a pair of mixtures does not require that an allele present in one mixture is also present in the other, even though we assume that the contributors to the two traces are the same. This is in contrast to the interpretation of duplicated STR analyses of low copy number (LCN) STR samples using the conventional consensus approach (Gill et al 2000).…”

“…This phenomenon will clearly affect the evidential interpretation of certain patterns of DNA mixture profiles. Several papers in the literature have dealt with genetic aspects of this, see for example Gill et al (2000). A possible explanation for a silent or null allele is sporadic failure of the apparatus to record the correct allele value or primer binding site mutations.…”

Typeability of DNA in Touch Traces Deposited on Paper and Optical Data Discs

“…Interpretation of DNA profiles may become challenging when analyzing low template (< 100 pg/μL) or compromised samples in terms of allele dropout, band stutter and allele gains [7].…”

“…Some of the scientific literature refers to the term LCN-DNA (Low Copy Number-DNA) to describe samples that contain low levels of DNA template where stochastic effects are present. LCN DNA was later accepted to define non-standard techniques used to improve the sensitivity of DNA profiling which include sample clean-up, increased PCR cycling and annealing time, and increased injection times [5][6][7][8][9], while Caddy et. al., introduced the term LT-DNA (Low Template-DNA) which refers specifically to samples that contain low levels of DNA [10].…”