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Whole blood obtained by venipuncture was collected in EDTA vacutainer tubes from individuals attending Split Hospital, Split,Croatia. Blood was dried onto clean cotton cloth, sealed in individual plastic bags and transported to the United States for testing. Approximately 3 mm squares were extracted using Chelex (1). The DNA was not quantified. PCR amplification was performed using the AmpFlSTR Profiler Plus™ and Cofiler™ PCR amplification kits (PE-Biosystems, Foster City, CA) following the manufacturer's protocol using 2 µL of sample. The amplified products were separated and detected using the ABI Prism™ 377 DNA sequencer (PE-Biosystems, Foster City, CA). Allele frequencies and goodness of fit tests were performed using Excel spread sheets. Each locus (n = 13) was tested for the following: Hardy-Weinberg Global Chi-square goodness of fit using all genotypes with expected values greater than 1.0, all other genotypes were pooled to form a residual class (df = number of genotypes minus the number of alleles)( p1); T statistic testing θ = 0 recommended by NRC 2 (2)(p2); and the Chi-square comparing expected and observed total homozygosity and heterozygosity (df = 1)(p3). The Bonferroni corrected threshold was 0.0038. No loci exceeded the Bonferroni threshold; only one locus (D8S1179) was significant (p (0.05) for one of three tests of deviation from Hardy-Weinberg equilibrium due to an excess of heterozygotes and deficiency of homozygotes prior to the Bonferroni correction for multiple tests.

As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA.

The samples for database studies were obtained from unrelated males from the indicated population groups residing in the state of Connecticut and were anonymized before analysis. The DNA from blood samples was extracted using the QIAamp® DNA blood MiniKit (Qiagen, Valencia, CA) following the recommended procedures. The quantity of human DNA was determined by slot blot hybridization using the QuantiBlot kit (Applied Biosystems, Foster City, CA) and following the manufacturer’s recommended protocols. The Y-PLEX™6 system (ReliaGene Technologies, Inc., New Orleans, LA) was used for the amplification of the Y short tandem repeat (STR) loci. PCR amplification and the analysis of amplified product were performed as recommended by the manufacturer and as described in Sinha et al. (1).

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.

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.

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.

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.

The postmortem remains of sixty-one war victims were excavated from 6 mass graves in Bosnia and Herzegovina one and a half years after interment. Using standard identification methods, including the matching of medical and dental records, the recognition of distinguishing characteristics such as the use of clothing and belongings, and video superimposition, 35 persons were identified. For the remaining 26 persons identification efforts continue. DNA typing was performed at the HLA DQA1 locus and five PM system loci. Results from DNA typing were confirmed by other methods. DNA profiles of family members of 150 missing persons are now being developed using the 6 loci. These DNA profiles will then be compared with those generated from the bone and teeth remains of the unidentified victims.

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.

Human X and Y chromosome alpha-satellite sequences lying within higher order repeats were amplified by the polymerase chain reaction (PCR) in genomic deoxyribonucleic acid (DNA) isolated from blood, bone, and several other tissues and specimens of potential forensic science interest. X and Y sequences could be coamplified under some of the PCR conditions employed. Monomorphic sequences in the 3'-apolipoprotein B gene (designated "H") and in an alpha-satellite higher order repeat on Chromosome 17 (p17H8, D17Z1) were likewise amplified in the specimens. X and Y sequence amplification can provide information about the sex of origin. Amplification of the X, H, and D17Z1 sequences was found to be primate-specific among the common animals tested and can thus provide species of origin information about a specimen. The authors suggest that amplification of X and D17Z1 or H sequences might provide "relaxed" and "stringent" controls for appropriate PCR amplification tests on forensic science specimens. Testing was carried out using PCR protocols that employed Thermophilus aquaticus (Taq) and Thermus flavis (Replinase) thermostable DNA polymerases.

A combination absorption-elution, two-dimensional absorption-inhibition procedure was used to determine the ABH antigen composition of a series of human bone specimens of known ABO type that had been aged up to nine months under dry and humid conditions at ambient temperature, 37°C, and 56°C; at ambient temperature in dry and wet soil; and buried in soil outdoors. Grouping data for the separate elution and inhibition testing, as well as for the combination procedure, are given. The combination method was found to be a highly reliable procedure for bone tissue ABH typing. Some data on microbial contaminants of human bone specimens aging in soil, and their effects on ABH typing results, are presented. No direct correlation between the properties of microbial contaminants and specific changes in the ABH antigenic composition of aging bone tissue specimens could be ascertained. Data on IGH antigen determination and on the quantitation of immunoglobulin G (IgG) in human bone tissue extracts indicated that immunoglobulin levels were typically too low to expect routinely successful Gm antigen testing results. However, these factors can sometimes be determined in fresh bone tissue extracts, particularly if the extracts are concentrated.

Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a "per milligram of starting tissue" basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different single-locus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQ alpha phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3' to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.

Restriction fragment length polymorphism analysis of human deoxyribonucleic acid (DNA) using two probes, pYNH24 and CMM101, was performed on the BIOS Timeframe™ system following the Federal Bureau of Investigation (FBI) Laboratory protocol and some variations of it. Comparable results were obtained by the different methods used.

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.

Simple, reliable procedures for the assay of pepsin and rennin-like enzyme activities are described as a means of identifying gastric fluid-containing samples in forensic science laboratories. These samples are usually vomitus, or stomach contents originating from wounds that perforate the stomach. They may be encountered at scenes or on articles submitted for examination, in fresh form or as dried stains. The pepsin activity assay is based on proteolytic activity with bovine albumin as substrate and the rennin-like activity assay is based on the coagulation of milk protein.

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.

Various types of cooperative arrangements between the forensic science programs of colleges and universities are discussed as possible means of increasing student exposure to faculty expertise in specialized subject areas and of using more effectively the comparatively scarce resources of individual programs.

Sixty-eight different commercially available blood grouping antisera and lectins with ABH, MN, and Rh D, C, E, c, and e specificities were serologically evaluated for their applicability to bloodstain antigen determination. The characteristics of the antisera were determined with red cells, with fresh bloodstains, and with series of aging bloodstains. The Rh antisera were tested under a variety of serological conditions and with bloodstains on various substrata. Additionally, studies on optimization of absorption-elution procedure variables were carried out, and some data on the storage characteristics of red cells and blood grouping antisera were gathered.

This book is a revised, updated, and somewhat expanded version of the original report light Characteristics and Stain Patterns of Human Blood” submitted to the LEAA (Law Enforcement Assistance Office). Those of us who have used the original report will be pleased with this updated and expanded edition. About half of the book is devoted to basic concepts in blood pattern interpretation and characteristics of liquid and dried blood. The other half consists of figures, diagrams, photos, and an appendix. New references were added. Of particular value are those court decisions on bloodstain evidence.

Some areas of common interest and potential cooperation between casework forensic science laboratories and university and college based forensic science programs are discussed. These include internships, joint research, the use of laboratory personnel as instructors in programs, and the use of faculty as consultants for the casework laboratory.

All published and unpublished population frequency data that could be located for U.S. populations are tabulated and presented for the blood group and secretor systems. Results obtained by combining data for comparable racial/ethnic groups are also presented. The results obtained with combined data may give better information on frequencies for the U.S. population at large than is obtainable from studies conducted in restricted geographic areas.

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.

All published and unpublished population frequency data that could be located for U.S. populations are tabulated and presented for the serum group systems haptoglobin (α-chain), group specific component, and transferrin and for the common β-chain variants of hemoglobin. Results obtained by combining data for comparable racial/ethnic groups are also presented. Some evidence is presented to indicate that the results obtained from the combined data may give better information on frequencies for the U.S. population at large than is obtainable from studies conducted in restricted geographic areas.

A new method has been developed for determination of the species of origin of denatured bloodstains. Antiserum with known titer is incubated with the denatured bloodstain. If the bloodstain has the same origin as the antiserum, a substantial amount of antibody will be absorbed by the stain, resulting in an attenuation of the antiserum titer. By reacting the supernatant with a known serum by using immunodiffusion it is possible to detect any appreciable attenuation of the antiserum titer. We have been able to determine the species of origin of various denatured bloodstains using this inhibition method.

All published and unpublished population frequency data that could be located for U.S. populations is tabulated and presented for the isoenzyme systems phosphoglucomutase, esterase D, adenylate kinase, acid phosphatase, glyoxalase I, adenosine deaminase, 6-phosphogluconate dehydrogenase, glutamic-pyruvic transaminase, carbonic anhydrase II, and glucose-6-phosphate dehydrogenase. Results obtained by combining data for comparable racial/ethnic groups are also presented. The results obtained with combined data may give better information on frequencies for the U.S. population at large than is obtainable from studies conducted in restricted geographic areas.