Lawrence Kobilinsky

Characterization of Deoxyribonucleic Acid (DNA) Obtained from Teeth Subjected to Various Environmental Conditions

This study was designed to determine the effects of various environmental factors on the deoxyribonucleic acid (DNA) obtained from dental pulp. Extracted teeth were subjected to the following conditions: varying pH (3,7,10); temperature (4 degrees C, 25 degrees C, 37 degrees C, incineration); humidity (20%, 66%, 98%); various types of soil (sand, potting soil, garden soil); seawater; burying the teeth outdoors, and aging (one week to six months). In addition, teeth that had been extracted and held at room temperature for 16 and 19 years were also examined. Following isolation of DNA, the samples were analyzed on yield gels to determine the concentration and integrity of the recovered DNA. Restriction digestion with Pst I was followed by electrophoresis of the generated fragments, Southern transfer to nylon membranes, and hybridization to both human and bacterial probes. It was determined that, aside from soil, the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. Restriction fragment length polymorphic (RFLP) analysis of selected samples was performed. Dental pulp patterns were compared with bloodstain exemplars, revealing matching patterns, although an increase in band-shifting was observed with extended exposure to elevated temperatures.

Evaluation of Deoxyribonucleic Acid (DNA) Isolated from Human Bloodstains Exposed to Ultraviolet Light, Heat, Humidity, and Soil Contamination

This study was designed to analyze the effects of common environmental insults on the ability to obtain deoxyribonucleic acid (DNA) restriction fragment-length polymorphisms (RFLP) patterns from laboratory prepared specimens. The environmental conditions studied include the exposure of dried bloodstains to varying amounts of relative humidity (0, 33, 67, and 98%), heat (37 degrees C), and ultraviolet light for periods of up to five days. In addition, the effect of drying over a four-day period in whole blood collected with and without ethylenediaminetetraacetate (EDTA) was examined. The results of the study showed that, under the conditions studied, the integrity of DNA is not altered such that false RFLP patterns are obtained. The only effect observed was that the overall RFLP pattern becomes weaker, but individual RFLP fragments are neither created nor destroyed.

The Effects of Environment and Substrata on Deoxyribonucleic Acid (DNA): The Use of Casework Samples from New York City

This study was designed to analyze the effects of the environment and substrata on the quality of deoxyribonucleic acid (DNA) isolated from evidentiary specimens. The quality of DNA isolated from actual casework specimens was determined by measuring its size by agarose gel electrophoresis. The information obtained could be used to predict the suitability of the DNA in the samples for restriction fragment length polymorphism (RFLP) analysis. The evidentiary specimens chosen for DNA were classified according to substrate (scrapings, plastic bags, synthetics, denim, and carpet) and according to a subjective evaluation of the condition of the stain (soiled, damp, or putrefied) and to its size (small or large). The results show that DNA of sufficient quality and high molecular weight (HMW) can be reliably isolated from bloodstains deposited on evidentiary items which have an unknown environmental history and which have dried onto a variety of substrata. Subsequent RFLP analysis of a selected number of these samples verified that the DNA was suitable for this type of analysis.

A Physical Method for Separating Spermatozoa from Epithelial Cells in Sexual Assault Evidence

The analysis of genetic markers for the purpose of individualization of semen specimens is extremely important in cases of sexual abuse and assault. The serological analysis of sexual assault evidence can sometimes be complicated because stains are often composed of a mixture of spermatozoa, vaginal epithelial cells and white and red blood cells. A filtration method has been developed to cleanly separate spermatozoa from epithelial cells based upon differences in size and shape. Nylon mesh filters of the appropriate pore size can be used to separate the smaller oval shaped spermatozoal cells from the larger and flatter epithelial cells. The former pass freely through the membrane while the latter are retained on the filter. In this study, cell separation was demonstrated by (a) microscopic observation of stained cells, (b) amplified fragment length polymorphism analysis of DNA obtained from separated cells. The results of these analyses indicate that: (1) Approximately 70% of spermatozoa in the mixed cell sample will penetrate the 10 microns pore size filter, (2) Only about 1-2% of intact epithelial cells will do so, and (3) A small number of nuclei from spontaneously lysed epithelial cells will cross the filter. Experimental results using mixtures of spermatozoa and vaginal epithelial cells prepared in different ratios support the conclusion that the filtration process is an efficient and reliable method to separate spermatozoa from epithelial cells in casework specimens for subsequent DNA analysis.

Restriction Fragment Length Polymorphism and Polymerase Chain Reaction-HLA DQα Analysis of Casework Urine Specimens

DNA was isolated from casework urine samples previously submitted for toxicological analysis. The quality and quantity of DNA isolated was determined by spectrofluorometry and agarose yield gel electrophoresis. Hae III restricted samples were then resolved by analytical agarose gel electrophoresis, transferred to a membrane by Southern blotting and hybridized with a chemiluminescently-labelled (D2S44) probe. The DNA fragment banding patterns were indistinguishable from the DNA banding patterns of blood specimens collected from the same donor. Only 5 of 20 samples yielded banding patterns and the banding intensity relative to background was low. Genomic DNA was also obtained from casework samples by Chelex extraction, amplified by polymerase chain reaction (PCR) and then genotyped for human leucocyte antigen (HLA) DQ alpha. Of 20 specimens, 13 (65%) were typed correctly producing identical results for urine and blood specimens obtained from the same donor. Aging studies of casework samples and normal samples (from a non-drug using population) were also conducted with PCR-HLA DQ alpha analysis. Results of these studies indicate that amplification by PCR was more likely to produce positive results. Based on these findings, we conclude that PCR-initiated analysis is more suitable than RFLP analysis for individualization of urine samples.

Development of a Radioimmunoassay Technique for the Detection of Human Hemoglobin in Dried Bloodstains

A sensitive radioimmunoassay for the detection of human hemoglobin in dried bloodstains for the purpose of forensic science species identification has been developed. Bloodstains from 13 animal species were tested and found to be negative for human blood. A minimum volume of 0.8 microL of fresh blood is required to produce sufficient stain for successful testing. Bloodstains prepared from newborn and sickle-cell bloods were determined to be human. Bloodstains ranging in age from 1 month to 6 years which had been maintained desiccated at 20 to 25 degrees C were also successfully tested. Positive results were obtained on human bloodstains stored at 24 degrees C with relative humidity ranging from 0 to 98% for a period of 3 weeks. Absolute counts per minute (CPM) decreased with increased humidity. Human bloodstains exposed to bacterial contamination (gram positive or negative species) under humid conditions for 2 weeks also tested positive. Bacterial contamination caused a decrease in CPM, but insufficient to result in an erroneous conclusion as to species of origin. Positive results were also obtained on human bloodstains stored for 6 weeks at various temperatures ranging from -16 to 37 degrees C. No significant decreases in CPM were noted for any of the temperature conditions described.

Individual characteristics of chemically modified human hairs revealed by scanning electron microscopy.

Hair that is treated with several different chemical reagents including those that are proteolytic, denaturing, or disulfide bond-reducing agents, undergoes structural alterations both internally and externally as revealed by scanning electron microscopic analysis. Some of these agents produce varying degrees of morphologic alterations in hairs obtained from different individuals. It would seem that this technique can be useful in the discrimination of human hairs from different individuals, since the chemically induced topological changes on the hair shaft apparently exhibit a high degree of intraindividual consistency.

A New Allele of the Short Tandem Repeat (STR) Locus, CSF1PO

CSF1PO is one of the thirteen core loci used for the CODIS database, and alleles reported for this short tandem repeat (STR) locus contain from 6 to 15 repeats of the tetranucleotide AGAT. Screening of DNA from 76 individuals by gel electrophoresis and silver stain detection yielded one sample that contained a rare, off-ladder CSF1PO allele; an allele larger than CSF1PO15 was detected in a heterozygote that also contained a CSF1PO10 allele. Capillary electrophoresis analysis using GeneScan software demonstrated that the variant allele contained four bases more than CSF1PO15. Following agarose gel electrophoresis to separate the two alleles of the heterozygote and cycle sequencing using dye terminators, sequence analysis showed that the variant, which was otherwise identical to the CSF1PO GenBank sequence, contained exactly 16 AGAT repeats. These results demonstrate the existence of an additional CSF1PO allele, a previously unreported size variant, CSF1PO16.

Detection of Hemagglutinins in Dried Saliva Stains and Their Potential Use in Blood Typing

Since 1928, hemagglutinins have been known to exist in saliva; however, they have not been utilized as evidence in criminal investigations because in the past, techniques for measuring them have not been sufficiently sensitive. In this paper we describe improved techniques for detecting salivary hemagglutinins and report initial results obtained with these methods. The stability of salivary hemagglutinins at several different temperatures was examined in liquid samples and in dried stains on filter paper, cigarette butts, and envelope flaps. Our observations indicate that salivary hemagglutinins may be sufficiently stable, over periods of one to several days at ambient room temperatures, to be of value to forensic science investigators. The results of the hemagglutinin assay are not affected by the age or sex of the sample donor. Because salivary hemagglutinins can be used to determine ABO blood type, analyses of this kind can serve as an important confirmatory test which the forensic serologist can use in conjunction with salivary agglutinogen determinations.

Simultaneous identification of alpha-L-fucosidase and phosphoglucomutase (PGM) subtyping in semen stains.

Seminal fluid and stains were analyzed by isoelectric focusing to determine the donor phenotype in the alpha-L-fucosidase (AlFuc) polymorphic system. The enzyme is found in both seminal fluid and spermatazoa. Three common phenotypes exist and can be identified in fluid specimens stored at 4 degrees C for more than a year. Untreated semen specimens display more than eight distinct bands of alpha-L-fucosidase activity with isoelectric points of pH 6.6 and below. Neuraminidase-treated specimens have enhanced banding patterns cathodally with a loss of activity in anodal bands making it easier to phenotype specimens. Semen stains maintained in dehumidified chambers at 25 or 37 degrees C retained activity for at least one month and could be accurately phenotyped. Activity was observed in semen specimens maintained at -20 degrees C in the dried state for a period of one year, whereas a complete loss of activity was observed after two weeks in similar specimens maintained at 25 or 37 degrees C under humid conditions. Of seventy-four semen stains analyzed, two had no apparent activity. Of the remaining seventy-two specimens 56, 32, and 12% were phenotyped as FUC 1-1, FUC 2-1, and FUC 2-2, respectively. Calculated gene frequencies are FUC1 = 0.72 and FUC2 = 0.28. Following analysis of alpha-L-fucosidase, the agarose gel can be chemically developed to reveal the PGM1 subtyping pattern. The ability to phenotype both systems in semen stains significantly improves the ability of the analyst to individualize this type of physical evidence. The probability of discrimination for these two combined systems is approximately 0.89.