A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA.

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs)--generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets--enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.

DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR.

Forensic laboratories are increasingly confronted with problematic samples from the scene of crime, containing only minute amounts of deoxyribonucleic acid (DNA), which may include polymerase chain reaction (PCR)-inhibiting substances. Efficient DNA extraction procedures, as well as accurate DNA quantification methods, are critical steps involved in the process of successful DNA analysis of such samples. The phenol-chloroform method is a sensitive method for the extraction of DNA from a wide variety of forensic samples, although it is known to be laborious compared with single-tube extraction methods. The relatively high DNA recovery and the quality of the extracted DNA speak for itself. For reliable and sensitive DNA quantitation, the application of real-time PCR is described. We modified a published real-time PCR assay, which allows for the combined analysis of nuclear and mitochondrial DNA, by introducing 1) improved hybridization probes with the use of minor groove binders; 2) an internal positive control (for both nuclear and mitochondrial DNA) for the detection of PCR inhibitors; and 3) different amplicon lengths for the determination of the degradation state of the DNA. The internal positive controls were constructed by site directed mutagenesis by overlap extension of the wild-type mitochondrial and nuclear DNA target with the advantage that no additional probes, which are cost-intensive, are required. The quantitation system is accomplished as a modular concept, which allows for the combined determination of the above-mentioned features (quantity/inhibition or quantity/degradation) depending on the situation.

A common nonsense mutation in the repetitive Kringle IV-2 domain of human apolipoprotein(a) results in a truncated protein and low plasma Lp(a).

LPA, the gene coding for apolipoprotein(a) [apo(a)], is the major determinant of lipoprotein(a) [Lp(a)] plasma levels, which are associated with risk for coronary heart disease (CHD) and stroke. It is not completely understood how variation in LPA relates to Lp(a) concentrations. One type of variation related to Lp(a) levels is the number of Kringle (K) IV-2 (g.61C>T; GenBank L14005.1) repeats in LPA, but sequence variation may also contribute. Human apo(a) contains from two to >40 nearly identical K IV-2 repeats of genomic size 5.5 kb, which makes it difficult to detect mutations. To elucidate the genetic variation of the apo(a) K IV-2 domain, we isolated a single "nonexpressing" apo(a) allele with 26 K IV-2 repeats, followed by PCR, cloning and sequencing of 96 clones, resulting in an average coverage of each K IV-2 repeat of approximately four-fold. The previously described K IV types 2A and 2B (K IV-2A and K IV-2B) were detected in 74% of the clones. In addition, a new type designated 2C (K IV-2C) was present. A nonsense mutation in the first exon of K IV-2 (g.61C>T) predicted to result in a truncated protein (p.R21X) was found in nine clones on a K IV-2A background. The presence of this mutation was confirmed by analysis of genomic DNA and was shown to represent the rare allele (frequency 0.02) of a SNP. Immunoblot analysis of apo(a) from plasma confirmed the presence of a truncated apo(a) isoform in the index individual and family members. Our data show that SNPs affecting Lp(a) plasma concentrations also exist in the apo(a) K IV-2 domain.

Systematic study on STR profiling on blood and saliva traces after visualization of fingerprint marks.

This paper describes a systematic study of the influence of optical, physical, and chemical methods used for fingerprint enhancement on subsequent DNA analysis of biological stains. Latent fingerprints as well as fingerprints in contact with blood and saliva on different surfaces were treated with dactyloscopic methods. As a general finding, subsequent STR profiling of the blood/saliva traces led to good results after all the enhancement methods included in this study. Concerning blood enhancement procedures, the airbrush technique showed deleterious effects on subsequent STR analysis in some cases. We therefore recommend the implementation of the layer technique, as it brings advantages for fingerprint enhancement as well. It could also be shown that, as can be necessary in practical casework, two enhancement methods can be performed on a single stain without having influence on STR profiling. In terms of methodological variety, this paper reflects a comprehensive study performed on STR profiling after fingerprint enhancement methods, including rare methods and variations of techniques, which can be a useful alternative in certain case scenarios.