A systematic analysis of secondary DNA transfer.

The Nature letter by R. van Oorschot and M. Jones (1) addressed two topics: the primary transfer of DNA from person to person or to various objects, and the secondary transfer of DNA through an intermediary. Forensic scientists have described the primary transfer of DNA and other biological evidence for many years. However, the authors also reported detecting secondary transfer of DNA from an object to a person's hands, which could adversely affect DNA typing in the forensic context. The prospect of secondary transfer raises questions of interest to both the legal and forensic communities. Therefore, we sought to evaluate parameters potentially leading to secondary DNA transfer. Our data do not support the conclusion that secondary transfer will compromise DNA typing results under typical forensic conditions.

A systematic analysis of PCR contamination.

In light of the strict legal scrutiny surrounding DNA typing at this time, it has become necessary to systematically address the issue of PCR contamination. To precisely define the parameters affecting PCR contamination under casework analysis conditions, PCR amplification reactions were intentionally compromised by employing sub-standard laboratory technique and by introducing secondary sources of DNA. The PCR parameters considered for potential sources of contamination include amplification set-up, amplification product handling, aerosol DNA and storage. In addition, analyst technique was evaluated by modifying or eliminating standard safeguards. Under the circumstances normally encountered during casework analysis, PCR contamination was never noted. Significantly, using the dot blot detection method, contamination was never observed when nanogram quantities of genomic DNA were mishandled or aerosolized. Contamination occurred only when amplification product was carelessly manipulated or purposefully sprayed near or directly into open tubes containing water or genomic DNA. Although standard precautions should be employed during PCR-based DNA typing, our data indicates that contamination during amplification procedures is not prevalent when detected by dot blot analysis.

NaOH treatment to neutralize inhibitors of Taq polymerase.

The introduction of polymerase chain reaction (PCR) into the forensic field has greatly extended the ability to analyze DNA from small or degraded samples. However, one significant problem with PCR analysis is the sensitivity of Taq Polymerase to inhibitors found in many substrates commonly encountered with evidentiary materials. We hypothesize that the most problematic of these compounds intercalate into double stranded DNA (dsDNA) and have significantly less affinity for single stranded DNA (ssDNA). This study presents a comprehensive analysis of a novel method for the neutralization of Taq inhibitors by denaturation and washing with NaOH in Microcon-100 filtration units. The data show that DNA recovered following NaOH repurification routinely amplifies when other inhibitor neutralization techniques are unsuccessful. Genetic profiles have been obtained with both AmpliType PM + DQA1 and D1S80 systems. However, the NaOH protocol is not advised when the quantity of DNA is limited since the treatment results in significant loss of DNA.

Forensic applications of DNA typing: part 2: collection and preservation of DNA evidence.

The initial stages of physical evidence examination are pivotal to the successful resolution of criminal investigations. Recent cases clearly reinforce the notion that methods of evidence collection and preservation will continue to be rigorously scrutinized and challenged in court. This article reviews forensic applications of DNA typing, focusing on the collection and preservation of biological evidence. Topics addressed include physical evidence collection at the crime scene, the forensic laboratory, and the autopsy room. Specific concerns pertaining to different sources of DNA evidence are discussed, as are special collection methods associated with various substrates on which the evidence is deposited.

DNA extraction from liquid blood using QIAamp.

The implementation of convicted felon DNA databases by increasing numbers of forensic science laboratories has engendered the need for a quick, efficient, and cost-effective method for the isolation of DNA from liquid blood samples. Because of the large numbers of samples involved, the ideal method would combine high throughput capability with maximal yield, high quality, and minimal time. We have found that the QIAGEN QIAamp Blood Kit/Tissue Kit satisfy all of these requirements. This simple, low cost spin column procedure yields purified DNA of approximately 20-30 kb that can be used directly in PCR or other enzymatic reactions without further purification. We compared the QIAamp isolation procedure to the standard SDS-Proteinase K/organic extraction/microcon purification procedure currently used by many forensic laboratories. The QIAamp procedure consistently gave a two- to four-fold increased yield relative to the organic extraction procedure. The DNA obtained was of high molecular weight, exhibited little degradation, and was suitable for RFLP and PCR analyses. We have found QIAGEN's QIAamp DNA isolation procedure to be ideally suited for preparation of samples for DNA databasing.

A PCR-based strategy for ABO genotype determination.

The ABO blood group system has been widely used in forensic serology. Several techniques have been developed which detect ABH antigens. To overcome the problems associated with conventional methods such as bacterial contamination, extreme environmental conditions, antigen activity, non-secretor issues, and non-specific absorption, several new strategies have been employed to detect ABO genotypes by PCR. We have developed improved amplimers for the glycosyl transferase locus on chromosome 9 and examined the suitability of PCR-based ABO genotyping for forensic identification. We show that the ABO system is primate specific and that DNA extracted from various tissues commonly encountered in criminal cases can be quickly and reliably typed by ABO-PCR. The results indicate that ABO genotyping by PCR and restriction enzyme digestion of the amplified product is a useful procedure for forensic analysis that can provide additional discriminating power compared to conventional immunological methods.

Systematic analysis of antisense RNA inhibition of myosin II heavy-chain gene expression in Dictyostelium discoideum.

The powerful potential of antisense nucleotide inhibition of gene expression is presently being exploited in many biological systems. Although use of the technique is widespread, little is known about the variables that contribute to experimental success. We sought to define those variables that affect inhibition of myosin II heavy-chain (MIIHC) gene expression by stable nuclear-derived antisense RNAs in Dictyostelium discoideum. Different fragments of the MIIHC gene cloned in the antisense orientation into several transformation vectors were introduced into cells, and the accumulation of MIIHC protein and mRNA was examined. Inhibition of expression ranged from slight to virtually complete, and depended only on the specific gene fragment used to generate the antisense RNA. Fragments of the 3' end of the gene were the most effective and resulted in almost complete inhibition, whereas 5' fragments gave very little reduction. The severity of the morphological phenotypes associated with MIIHC depletion reflected the different levels of MIIHC in the transformants. Important implications for the design of antisense vectors suggested by these results are discussed.

Co-suppression of Dictyostelium discoideum myosin II heavy-chain gene expression by a sense orientation transcript.

We have previously demonstrated effective inhibition of Dictyostelium discoideum myosin heavy chain (MIIHC) gene expression after introduction of an antisense gene on a transformation vector into cells. We now report that transcription of a sense orientation MIIHC gene has a similar, though not as dramatic, effect on expression from the endogenous gene. Cells transformed with a sense fragment of the MIIHC gene are defective in cytokinesis and become giant and multinucleated when growing on a surface. This morphological phenotype is correlated with decreased expression of the endogenous MIIHC gene. Sense transformants accumulate less MIIHC protein and mRNA than control transformed cells, concomitant with accumulation of a vector-derived sense transcript. Expression of the introduced sequences seems to be required for the sense effect. The possible causes of this surprising phenomenon are discussed.

Variables affecting antisense RNA inhibition of gene expression.

We have sought to determine what variables affect the extent of inhibition of gene expression by stable nuclear-derived antisense RNAs. Myosin heavy chain gene II (MHCII) expression in Dictyostelium was used as a model system, because previous results have shown that nearly complete inhibition of expression can be achieved under appropriate conditions. Various fragments of the myosin gene were inserted into several transformation vectors in both sense and antisense orientation, and the effects on expression of protein and RNA from the endogenous MHC II gene were assayed. The results indicate that the critical factor was the particular fragment of the gene used to produce the antisense RNA. Some fragments produced complete inhibition of expression, whereas others gave only slight inhibition. The fragments that produced the greatest inhibition were from the tail region of the gene. In addition, cells were capable of overcoming the inhibition while still expressing the antisense RNA. We hypothesize that the three-dimensional topology of the antisense and sense RNAs determines their accessibility for interstrand hybridization. Transformation with sense fragments of the myosin gene also caused inhibition of the endogenous myosin gene. The inhibition seen with sense expression is not as dramatic as that for antisense, but it had the same phenotypic consequences for the cells. This phenomenon, which has now been documented in other systems, may be mechanistically similar to inhibition by antisense RNA in our system.

Identification and developmental expression of a novel embryonic myosin heavy-chain gene in chicken.

The developmental expression of an embryonic chicken myosin heavy-chain (MHC) gene homologous to the genomic clone pCM4.1 was examined by S1 analysis. Transcripts homologous to pCM4.1 are first detected at day 12 in ovo, and are maximally expressed between days 15-17 in ovo. No pCM4.1 transcripts are detected at earlier stages of embryogenesis or at high levels in posthatch stages. This unique pattern of expression has led to the proposal that pCM4.1 represents a previously uncharacterized MHC gene, which is confined in its expression to late embryogenesis. Genomic hybridization data, in addition to a comparison between the DNA and amino acid sequences of pCM4.1 and other characterized chicken MHC 3' end clones, provide further evidence for this proposal. We also present observations made during the sequence analysis of pCM4.1 that may be relevant to our understanding of the 3'-end processing of homologous primary transcripts, and of the mechanism controlling developmental MHC isoform transitions.