Henry C. Lee

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 persons 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.

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 QIAGENs QIAamp DNA isolation procedure to be ideally suited for preparation of samples for DNA databasing.

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.

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.

DNA typing in forensic science. I. Theory and background.

In the last few years, DNA typing procedures have become increasingly important in the fields of forensic science and forensic medicine. This paper reviews background information on DNA and human genetics, and addresses how molecular techniques such as restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analysis have been used to detect genetic polymorphism in human populations. The systems discussed include single locus RFLP, HLA DQ-alpha, amplified fragment length polymorphism (AMP-FLPs), short tandem repeats (STRs), and mitochondrial DNA typing. Several DNA typing methods have been thoroughly validated for forensic use. With proper control measures, DNA analysis should be considered reliable. At this time, DNA evidence/testimony is generally accepted by the courts and greatly assists in the resolution of criminal and civil investigations.

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.

ABH Antigen Typing in Bone Tissue

Results obtained from the ABH grouping of bone tissues by the absorption-elution procedure and by a recently described two-dimensional absorption-inhibition procedure are reported. Neither the elution nor the inhibition procedure alone yielded uniformly correct results. A combination procedure consisting of the use of both absorption-elution and two-dimensional absorption-inhibition is proposed for bone ABH grouping. When elution and inhibition were used in combination, specimens yielding concordant results with both techniques were correctly grouped.

Two-Dimensional Absorption-Inhibition

A novel inhibition procedure, called two-dimensional absorption-inhibition, is described. The theory underlying this technique is developed based on a review of and comparison with existing inhibition methods. Two-dimensional inhibition takes advantage of the best features of inhibition-titration and titration-inhibition, and is shown to be more sensitive than either of them. Results obtained using all the inhibition methods on secretor saliva, semen, urine, urine stain, and perspiration stain specimens show that the new technique is especially powerful in correctly determining the ABH antigens in secretor body fluids having lower concentrations of soluble blood group antigens. A two-stage version of the two-dimensional procedure that makes it a practical casework method is described as well.

Evaluation of antisera for bloodstain grouping. II. Ss, Kell, Duffy, Kidd, and Gm/Km.

Thirty-one different examples of commercially available blood grouping antisera specific for the S, s, K, k, Fya, Fyb, Jka, and Jkb antigens and anti-human globulin sera were serologically evaluated with red cells and in absorption-elution tests to determine their applicability to bloodstain antigen determinations. Nineteen examples of commercially available antisera specific for various Gm and Km antigens and their corresponding anti-D reagents were likewise evaluated in inhibition tests with sera and bloodstains. Elution tests with the blood grouping antisera and inhibition tests with the Gm/Km antisera on a series of aging bloodstains on cotton cloth, and on bloodstains on a number of different substrata, demonstrated that properly evaluated commercial antisera are useful reagents for bloodstain grouping in forensic serology.