Matrix (multi-component) failure, sometimes referred to as 'pull up' is a result of the inability of the detection instrument to properly resolve the dye colors used to label STR amplicons. This phenomenon is due to spectral overlap. A peak of another color is 'pulled-up' or 'bleeds through' (see Figure 15.4) as a result of exceeding the linear range of detection for the instrument (i.e., sample overloading). A matrix failure is observed as a peak-beneath-a-peak or as an elevation of the baselines for any color. Matrix standards may need to be rerun with the latest set of conditions and a new matrix generated by the software to correct this problem.
Dye blobs (artifacts) occur when fluorescent dyes come off of their respective primers and migrate independently through the capillary. These peaks are fairly broad and possess the spectrum of one of the dyes used for genotyping (see Figure 15.4). Dye artifacts can be removed following PCR using filtration columns (Butler et al. 2003).
Air bubbles, urea crystals, or voltage spikes can give rise to a false peak in the ABI 310. These peaks are usually sharp and appear equally intense throughout all four colors (see Figure 15.4). These peaks are not reproducible and should not appear in the same position if the sample is re-injected onto the capillary.
Sample contaminants. Materials which fluoresce in the visible region of the spectrum (~500-600 nm) may interfere with DNA typing when using fluorescent scanners or one of the ABI PRISM systems by appearing as identifiable peaks in the electropherogram. In some early studies conducted by the Forensic Science Service (FSS), a number of fluorescent compounds were examined to determine their apparent mobility when electrophoresed in a polyacrylamide gel (Urquhart et al. 1994).
Figure 15.4 Hypothetical electropherogram displaying several artifacts often observed with STR typing
All of the compounds studied, which included antibiotics, vitamins, polycyclic aromatics, fluorescent brighteners, and various textile dyes, could be removed with an organic extraction (i.e., phenol/chloroform, as is commonly used to extract DNA from cells). Interestingly enough, the Chelex method of DNA extraction (see Chapter 3) failed to remove all of the contaminating fluorescent peaks (Urquhart et al. 1994). Fortunately, these interfering peaks were usually wide and possessed a broader fluorescent spectrum, which made it fairly easy to distinguish them from the fluorescent dye-labeled PCR products. The FSS researchers concluded that the use of appropriate substrate controls or negative controls in PCR should alleviate this potential problem.
Several possible forensic scenarios exist where sample contaminants may be possible (Urquhart et al. 1994):
1. Body fluid stains on dye materials from which the dye may leach during extraction;
2. Body fluid stains on plant material, from which chlorophyll may co-extract with DNA;
3. Blood or tissue samples from individuals with some pathological conditions, e.g., lead poisoning or some forms of porphyria, in which blood porphyrin levels are greatly elevated; and
4. Bone or tooth samples from individuals who were treated with tetracycline-group antibiotics in their youth as growing bones and teeth are known to incorporate and accumulate these antibiotics.
Was this article helpful?
This book discusses the futility of curing stammering by common means. It traces various attempts at curing stammering in the past and how wasteful these attempt were, until he discovered a simple program to cure it. The book presents the life of Benjamin Nathaniel Bogue and his struggles with the handicap. Bogue devotes a great deal of text to explain the handicap of stammering, its effects on the body and psychology of the sufferer, and its cure.