A Study of PCR Inhibition Mechanisms Using Real Time PCR*,†

Abstract: In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co‐extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.     

A study on the effects of degradation and template concentration on the amplification efficiency of the STR Miniplex primer sets

In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called "Miniplexes" to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 microL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Finding the proper balance between freedom and justice: why we should not eliminate personal belief exemptions to vaccine mandates

Lantos and colleagues (this issue) propose to eliminate personal belief exemptions from school vaccine mandates, particularly for those vaccines that target deadly contagious childhood disease. They argue that not doing so would be unjust. In this counterpoint, we argue that, for reasons grounded in both health policy and morality, a just vaccine policy need not prohibit parents from claiming personal belief exemptions.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

Leisure Experience, Prison Culture, or Victimization? Sex Offenders Report on Prison Gambling

Opportunities for gambling in mainstream North American society have increased greatly over the past two decades. Despite the popularity of legalized gambling and attention it is receiving among social science researchers and economists, gambling within jails and prisons has been overlooked. This study utilized a semistructured interviewing procedure (Smith, 1995) to gain information about prison gambling from 11 sexual offenders on parole—all had spent significant time incarcerated. Qualitative analyses of responses suggest that offenders may gamble in various forms and for a variety of reasons, including a means of alleviating boredom, providing excitement and risk, and an opportunity to socialize. Prison gambling appears to be a complex phenomenon. A substantial amount of prison gambling seems to be associated with certain psychosocial benefits, while for a minority of cases there may be severe violence and victimization.     

When is 100% inspection not enough? An analysis of three pickup truck front wheel assembly failures

The loss of a cotter pin in the front wheel assembly of cars and trucks has been a problem for several years. This is shown by the fact that the automobile manufacturers have been committed to a 100% inspection of the process for installing them on cars and trucks. In this paper, three accident cases are presented, along with a method by which the forensic engineer can determine if a cotter pin has ever been installed on the front wheel assembly. Testing was done to show what the spindle would look like if the cotter pin had been installed properly and forcibly removed. Photographs show both the accident and test spindle for comparison.