Polymarker, HLA-DQA1, and D1S80 allele frequency data in Chamorro and Filipino populations from Guam.

Allele frequencies were determined in sample populations of Chamorros and Filipinos from Guam at the loci LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1, and D1S80. Variable number tandem repeat alleles at the D1S80 locus were detected by silver staining following electrophoresis of amplified products in polyacrylamide. Allelic products of the other loci were detected by reverse dot blot hybridization following a multiplex amplification procedure. All loci, in both sample populations, are highly polymorphic and meet Hardy-Weinberg expectations, except for the D1S80 locus in the Chamorro population sample (p = 0.025). An interclass correlation analysis detected only one marginally significant departure from independence out of a total of 42 pairwise comparisons of the seven loci for both data sets (LDLR/HBGG in Chamorros, p = 0.048). The Chamorro and Filipino allele frequency data are similar to each other at six of the seven loci with only a marginally significant difference at the HLA-DQA1 locus (p = 0.049).

Simple protocols for typing forensic biological evidence: chemiluminescent detection for human DNA quantitation and restriction fragment length polymorphism (RFLP) analyses and manual typing of polymerase chain reaction (PCR) amplified polymorphisms.

Methods for identity testing are described that enable extraction of DNA from biological samples, determination of the quantity of human DNA, and genetic analyses of the materials using restriction fragment length polymorphism (RFLP) typing and/or amplified fragment length polymorphism (AMP-FLP) typing of PCR products. The salient features of the procedures are simplicity, manual typing, nonradioactive chemiluminescent assays or silver staining for detection, and low cost. Most application-oriented laboratories involved in forensic and/or paternity testing should be able to implement these procedures.

D1S80 typing of DNA from simulated forensic specimens.

The reliability of a D1S80 typing procedure has been evaluated using simulated forensic specimens. D1S80 alleles were detectable in DNA recovered from bloodstains exposed to sunlight for up to 20 weeks. However, D1S80 alleles were undetectable in semen stains after six weeks sunlight exposure. Analysis of blood and semen that had been deposited on a variety of substrates and examined over a twenty-week period, revealed no systematic influence of substrate on the ability to type D1S80. A study in which body fluids were exposed to household chemical substances, such as bleach, acids, oil, and gasoline, indicated that only HCl and bleach had a deleterious effect on the ability to type D1S80. In addition, personal care chemical products were without effect on D1S80 allele patterns derived from semen. Exposure of blood and semen to four different species of microorganisms resulted in no alteration of D1S80 genotype patterns in these body fluids. D1S80 genotypes could be reliably determined even when body fluids from different individuals were mixed. DNA from no animals other than humans and higher primates could be amplified at locus D1S80 when the DNA had been isolated through an organic procedure. These studies, in concert with the reports of others, indicate that the procedures for the amplification and detection of genetic variation at locus D1S80 are suitable for use on forensic evidentiary materials.

D1S80 population data in African Americans, Caucasians, southeastern Hispanics, southwestern Hispanics, and Orientals.

Allele frequencies for the locus D1S80 were determined in African American, Caucasian, Southeastern Hispanic, Southwestern Hispanic, and Oriental sample populations using the polymerase chain reaction and subsequent electrophoresis and silver staining of the amplified products. Due to the presence of anodal and cathodal electrophoretic variants (in reference to the steps in an allelic ladder), allele frequencies were established using a classification protocol based on the steps in the allelic ladder. All sample populations met Hardy-Weinberg expectations for D1S80. In addition, there was no evidence for association of alleles between the loci D1S80 and D1S7. The product of allele frequencies from the data from the sample populations in this study can be used in forensic analyses and paternity tests to estimate the frequency of a D1S80 DNA genotype.

Multigenerational amplification of a reference ladder for alleles at locus D1S80.

The interlaboratory typing of DNA specimens that have been amplified at the D1S80 locus necessitates the use of a standard allelic reference ladder. This communication describes a technique in which individual, amplified alleles are isolated, combined, and amplified by PCR to produce a functional reference ladder composed of many of the alleles that occur at this locus. The amplified ladder can serve directly as a template source for production of the next generation of reference ladder. This process, in which each amplified ladder serves as the template for the next has been carried through multiple generations.

Fixed-bin analysis for statistical evaluation of continuous distributions of allelic data from VNTR loci, for use in forensic comparisons.

The detection of DNA polymorphisms by RFLP analysis is having a major impact on identity testing in forensic science. At present, this approach is the best effort a forensic scientist can make to exclude an individual who has been falsely associated with an evidentiary sample found at a crime scene. When an analysis fails to exclude a suspect as a potential contributor of an evidentiary sample, a means should be provided to assess suitable weight to the putative match. Most important, the statistical analysis should not place undue weight on a genetic profile derived from an unknown sample that is attributed to an accused individual. The method must allow for limitations in conventional agarose-submarine-gel electrophoresis and Southern blotting procedure, limited sample population data, possible subpopulation differences, and potential sampling error. A conservative statistical method was developed based on arbitrarily defined fixed bins. This approach permits classification of continuous allelic data, provides for a simple and portable data-base system, and is unlikely to underestimate the frequency of occurrence of a set of alleles. This will help ensure that undue weight is not placed on a sample attributed to an accused individual.

Effects of presumptive test reagents on the ability to obtain restriction fragment length polymorphism (RFLP) patterns from human blood and semen stains.

Some of the commonly used presumptive test reagents for identification of blood and semen could potentially affect the recovery of intact high-molecular-weight deoxyribonucleic acid (DNA) from evidentiary samples. Thus, the capability of performing restriction fragment length polymorphism (RFLP) analysis on evidentiary samples could be compromised. In order to investigate the potential effects of presumptive test reagents on the DNA present in these samples, bloodstains on cotton and glass were exposed directly to luminol, benzidine, phenolphthalein, o-tolidine, and leucomalachite green, while semen stains and vaginal swabs containing semen were exposed directly to bromochloroindolyl phosphate (BCIP) and sodium thymolphthalein monophosphate (STMP) reagents. The yield gels for DNA quality and quantity and RFLP results indicated that bloodstains exposed to luminol, benzidine dissolved in ethanol, and phenolphthalein, as well as semen stains and vaginal swabs exposed to BCIP and STMP yield RFLP patterns consistent with that of the uncontaminated control. Except for the phenolphthalein treatment, the quantity of extractable, high-molecular-weight DNA obtained was comparable with that of untreated stains. Therefore, evidentiary material purposely or inadvertently contaminated with these reagents can be successfully typed. However, stains exposed to benzidine dissolved in glacial acetic acid, leucomalachite green, and o-tolidine failed to yield high-molecular-weight DNA or to produce any RFLP patterns.

Tracking the violent criminal offender through DNA typing profiles--a national database system concept.

Implementation of standard methods for the conduct of restriction fragment length polymorphism analysis into the protocols of United States crime laboratories offers an unprecedented opportunity for the establishment of a national computer database system to enable interchange of DNA typing information. The FBI Laboratory, in concert with crime laboratory representatives, has taken the initiative in planning and implementing such a database system. The Combined DNA Index System (CODIS) will be composed of three sub-indices: a statistical database, which will contain frequencies of DNA fragment alleles in various population groups; an investigative database which will enable linkage of violent crimes through a common subject; and a convicted felon database that will serve to maintain DNA typing profiles for comparison to profiles developed from violent crimes where the suspect may be unknown.

Applying highly polymorphic variable number of tandem repeats loci genetic markers to identity testing.

The detection of alleles of variable number of tandem repeats (VNTR) loci by restriction fragment length polymorphism analysis has become an important aspect of genetic characterization for identity testing. Some VNTR loci are so polymorphic that the analysis of three to five genetic markers could potentially provide unique identity. However, the more informative a genetic marker is (i.e., high degree of polymorphisms), the better it is as an exculpatory tool. This approach currently provides the best avenue for excluding a falsely associated individual with a particular sample. When an analysis fails to exclude an individual as the source of the questioned material, a value (frequency of occurrence) should be placed on the VNTR profiles to assess weight to the inclusion in identity testing. Arbitrarily defined fixed bins were designed to accommodate quasi-continuous data and to provide a result that would not place an underestimation of the frequency of occurrence of a set of alleles attributed to an individual.

Hae III--a suitable restriction endonuclease for restriction fragment length polymorphism analysis of biological evidence samples.

Hae III has been selected by our laboratories as the restriction endonuclease of choice for restriction fragment length polymorphism analysis of forensic science samples. The enzyme is compatible with the D2S44 probe system and generates relatively small DNA fragments for that marker system. Similarly, Hae III is compatible with several other independent polymorphic loci, including D1S7, D4S139, D16S85, D17S74, D17S79, D14S13, and D20S15. Hae III is functional under a variety of adverse conditions for DNA digestion and is not affected by the methylation pattern in mammals. Finally, Hae III is a relatively inexpensive restriction endonuclease.

Modifications to improve the effectiveness of restriction fragment length polymorphism typing.

A streamlined and effective method for RFLP analysis of DNA has been developed. Southern transfers are accomplished by alkali blotting DNA onto positively charged nylon membranes. The prehybridization step has been eliminated. The hybridization solution is composed of three cost-effective reagents: 7% SDS, 10% PEG, and phosphate buffer. By using probes that hybridize to variable number of tandem repeat loci, and RFLP analysis of one to two micrograms of genomic DNA can be achieved within five working days under normal working hours. With longer autoradiographic exposures, as little as 20-100 ng of human genomic DNA is sufficient for analysis.

Human renal carcinoma: asparagine independence with asparaginase susceptibility in culture.

A human renal carcinoma cell line (Caki-1) was examined for asparagine (Asn) dependence and susceptibility to Escherichia coli asparaginase. Because this enzyme hydrolyzes glutamine (Gln) as well as Asn, even though at only 2-3% the rate, Asn- Gln+ and Asn- Gln- media were prepared. Only the former supported Caki growth. The Asn- Gln- medium was then repleted with Asn, Gln, or both. Although Asn repletion failed to promote growth, addition of Gln alone or the combination supported growth as well as complete medium. With [3H]leucine and [3H]mannose incorporation to indicate protein and glycoprotein synthesis, respectively, the Gln repleted medium supported these processes as well as complete medium. Asparaginase added to complete medium was highly toxic to the Caki cells, but this is a reflection of Gln depletion rather than Asn depletion.

Presumptive screening of suspected semen stain in situ using cotton swabs and bromochloroindolyl phosphate to detect prostatic acid phosphatase activity.

A novel approach to the presumptive screening of questioned semen stains has been developed which enables the rapid identification of stains which are devoid of semen. Questioned semen stains can be swabbed with a moist cotton swab, and the prostatic acid phosphatase (SAP) activity transferred to the swab identified through assay with 5-bromo-4-chloro-3-indolyl phosphate (BCIP). Controlled laboratory studies revealed that the BCIP swab procedure was as sensitive as the semiquantitative SAP test currently employed in the FBI Laboratory for the presumptive screening of semen stains. A validation study of the BCIP swab procedure in parallel with the current procedure using 4305 case evidence stains indicated that the BCIP swab procedure was as effective as the current procedure in identifying those questioned stains which lack semen. The advantage of the BCIP swab procedure is that it can be performed on questioned stains in situ and thereby avoids the requirement of removing and extracting the stain before assay of SAP activity.

Secreted blood group substances: distributions in semen and stabilities in dried semen stains.

A sensitive microplate hemagglutination-inhibition technique has been used to ascertain the distributions of secreted blood group substances (BGS) in a population of 176 semen specimens and to characterize the stability of these substances in dried semen stains. The BGS concentrations in semen were found to vary throughout a wide range of titer. Despite this latitude of variation, the titers for the component BGS within the blood groups could be described by a log-normal distribution function. Studies of a number of sequential semen specimens obtained from the same donors revealed that the intraindividual variation in BGS titers was much more limited than the interindividual BGS titers. Attempts to correlate variations in titers between A and H in Group A semen or B and H in Group B semen indicated that the levels of these component substances vary independently. Studies of the stability of BGS in Groups A and O semen suggested that these substances were stable when the semen stains were stored at -20 degrees C, 4 degrees C, or at ambient laboratory temperature in a dry state. In contrast, stains stored at 37 degrees C under humid conditions suffered a dramatic loss in BGS titer, with the half-life of the BGS being on the order of 30 days.

Interaction of antigens with dimethyldioctadecylammonium bromide, a chemically defined biological response modifier.

Dimethyldioctadecylammonium bromide (DDA) stimulates immune responses, primes (or activates) macrophages, and binds to antigens. Because relatively little is known about the binding of adjuvants to antigens, the nature of the interaction of DDA with soluble protein and cellular antigens was investigated. Dose-dependent, stable complexes are formed between cells and the lipoidal cation of DDA. Since the interaction is independent of negatively charged sialic acid residues of the cell membrane and little DDA binds to intracellular structures, it is suggested that binding occurs primarily at the cell membrane, probably through hydrophobic interaction with lipids. The idea of membrane perturbation is supported by the leak of macromolecules (lactic dehydrogenase) from treated cells. Reaction of varying amounts of DDA with a constant amount of ovalbumin was also dose dependent. Because of a minimal effect of ionic strength on the reaction, it is concluded that ionic interaction may make a minor contribution to product formation. Complexes of DDA and antigen are articularly effective in eliciting a delayed hypersensitivity reaction, which has been postulated to be desirable for an antitumor effect.

Identification of autoanti-Thi-1 antibody in the sera of BALB/c mice immune to P1798 lymphoma.

The specificity was established for the anti-thymocyte autoantibody which appeared in BALB/c mice (BAA) during immunization to the P 1798 lymphoma. The results indicate that BAA is specific for the Thy-1 alloantigen of murine T cells. By immunoprecipitation of 125I-labeled A/J thymocyte surface antigens from freeze-thaw lysates and electrophoretic resolution of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels, the BAA and congenic anti-Thy-1.1 or 1.2 were shown to be specific for determinants on the same antigens. Specificity of BAA for Thy-1.2 was shown also quantitative absorption studies. Coating P 1798 lymphoma cells with either BAA or anti-Thy-1.2 reduced the absorptive capacity of these cells for the same or the reciprocal antiserum. Coating P 1798 with an antiserum to P 1798 which was indifferent to the Thy-1.2 alloantigen did not diminish the absorptive capacity of these cells for either BAA or anti-Thy-1.2. These studies have provided firm evidence that BAA is specific for determinants on the Thy-1 alloantigen.

Immunogenicity of solubilized tumor antigen extracted from P1798 murine lymphoma cells or isolated from tumor-bearer ascites fluid and reactivity with anti-thy-1.2 antiserum.

Solubilized antigen was prepared from P1798 lymphoma cells by sonication, 3 M KCI extraction, or isolated from the ascites fluid of syngeneic tumor-bearing BALB/c mice. Antigen was detected and quantitated by its ability to block activity of anti-P1798 serum raised in syngeneic mice, as assayed by cytotoxic and indirect immunofluorescence tests. It was established that the reaction was immunologically specific as the P1798 antigen did not inhibit the binding to L1210 lymphoma cells of antisera raised against L1210 in syngeneic DBA/2 or allogeneic BALB/c mice. Vaccination of BALB/c mice with different subcellular fractions of sonicated antigen or with ascites fluid resulted in protection against a live P1798 challenge with results comparable to those obtained using iodoacetamide-modified tumor cells. Solubilized antigen prepared by each of the three methods eluted from a Bio-Gel A5m agarose column exclusively in an early peak that had a molecular weight estimated to be greater than 2 X 10(6). This column-fractionated antigen was shown to cross-react with antiserum raised against Thy-1.2 antigen, which is present on P1798 cells. The purified P1798 antigen sedimented at 200,000 g and was shown to protect syngeneic mice in immunoprophylactic tests.

Anti-thymocyte autoimmunity in BALB/C mice accompanying immunization to a syngeneic lymphoma.

As immunization of BALB/c mice to the syngeneic P1798 lymphoma is effected by administration of iodoacetamide-modified P1798 cells, serum antibodies appear which are reactive with P1798 and normal BALB/c thymocytes, splenocytes, and peripheral blood lymphocytes. Anti-P1798 serum also cross-reacted with thymocytes from AKR, DBA/2, and C3H mice as well as the allogeneic lymphoma 6C3HED. Anti-P1798 serum was unreactive with the Thy-1 deficient L1210 lymphoma. Multiple absorptions of anti-P1798 serum with normal BALB/c thymocytes or brain or P1798 removed antibodies to P1798 and thymocytes commensurately. Normal BALB/c liver and kidney did not absorb antibody activity. Treatment of a BALB/c splenocyte suspension with anti-Thy 1.2 serum and complement removed the population of spleen cells which were capable of reaction with anti-P1798 serum. The data suggest that antibodies to P1798 and thymocytes are the same and that specificity may be directed toward a Thy-1 related structure but without distinguishing Thy-1.1 and Thy-1.2.

The influence of glutamine, its decomposition products, and glutaminase on the transformation of human and mouse lymphocytes.

The extent of blast transformation for human and BALB/c mouse lymphocytes has been examined over a wide range of glutamine concentrations with several agents which initiate blastogenesis. Maximum [3H] thymidine incorporation was seen at 0.5 mM glutamine for lymphoid tissues stimulated in the following manner: human and BALB/c splenic and peripheral blood lymphocytes with phytohemagglutinin, BALB/c splenic lymphocytes with lipopolysaccharide, and BALB/c vs C3H/HeJ two-way mixed lymphocyte cultures. The inhibition of blastogenesis exerted by glutamine concentrations greater than 0.5 mM could not be reversed by washing and reculturing the cells at 0.5 mM glutamine. To elucidate the reason for inhibition by higher glutamine concentrations, the products of spontaneous glutamine decomposition, L-2-pyrrolidone-5-carboxylic acid and ammonia were tested for their in vitro influence on BALB/c splenocyte blastogenesis. Pyrrolidone-carboxylic acid, in concentrations up to 5 mM, was without effect. In contrast, ammonia concentrations exceeding 1 mM became increasingly more inhibitory. The genesis of inhibitory levels of ammonia in culture medium was confirmed and has been considered as primarily responsible for inhibiton by high glutamine. Addition of Escherichia coli glutaminase (pH optimum 4.9) to cultures of BALB/c splenocytes or human peripheral blood lymphocytes had no effect on either the extent of blastogenesis of these tissues or the glutamine levels in their culture medium.