81st SEMI-ANNUAL SEMINAR (Spring 1993)
CALIFORNIA ASSOCIATION OF CRIMINALISTS
FLUORESCENT RESTORATION OF ALTERED CREDIT CARD NUMBERS
Quentin Nerida, Fingerprint Examiner, Contra Costa County Criminalistics Laboratory, 1122 Escobar St., Martinez, CA 94553
Original account numbers on an altered plastic credit card can be restored when viewed with an Alternate Light Source set at the proper frequency. The use of high quality barrier filters can enhance the photographic results. The ALS illumination and photography of results are nondestructive techniques which will not interfere with subsequent processing for latent print evidence. The value of a tunable frequency high intensity Alternate Light Source was realized and will be discussed in this presentation.
FIREARMS OPEN CASE FILES AND DATABASES
Ronald Nichols, Oakland Police Department, 455 Seventh St., Room 608, Oakland, CA 94607
The Oakland Police Department Criminalistics Laboratory has established several databases, two of which have been very useful. One is an Open Case File. This file is categorized and contains photographs of casing heads and the most prominent rifling impressions(s) of bullets which are submitted in cases which lack responsible firearms. All the necessary case and class characteristic information is either achieved by categorizing or is placed directly on the photograph. Casings, bullets and firearms recovered in future cases are compared against the file in an attempt to find a link. We have been successful in making several such hits both on casings and bullets. The second is a Breechface Characteristics Database. This database contains information on the class characteristics of over one hundred different manufacturers and models of firearms. Included with this information is a photograph of the casing head after firing. By comparing evidence casings against this database, it is often possible to provide the investigator with a more useful list of possible weapons.
NO EVIDENCE TOO SMALL
Susan Johnson, LAPD Crime Lab, 555 Ramirez Street, Space 270, Los Angeles, CA 90012
An unidentified male, John Doe, was struck and killed by a hit and run vehicle. Broken glass was recovered from the accident scene. Subsequently, glass exemplars were collected from broken windows on a suspect vehicle. Also collected was the vehicle's broken side-view mirror which was located on the passenger floorboard. Acting on a request from the investigating officer, an attempt to associate the suspect vehicle to the accident scene was made. Initial examination of the broken glass from the scene revealed two small pieces of mirrored glass. The potential for a physical match of these fragments to the broken side-view mirror was immediately recognized. After first reconstructing the broken side-view mirror, a place where the two broken pieces, recovered from the scene, may have originally fit was located. Although a classic physical fit was not apparent, further examination focused on 'hackle' marks present on the broken edges. These 'hackle' marks are unique features which occur when glass breaks. Using comparison microscopy, the hackle marks on both mirrored glass fragments and corresponding areas on the broken mirror matched; thereby conclusively placing the vehicle at the accident scene.
THE USE OF THE ALTERNATE LIGHT SOURCE TO DETECT AEROSOL PAINT OVERSPRAY TRACES
Diane M. Bowman, BS, Mary M. Gibbons, M.Crim., Curfis Sato, BS, Pamela
Sartori, BA, Lansing Lee, BS, Alan Keel, BS and Anthony Camacho; Oakland Police Department Criminalistics Section, 455 Seventh Street, Room 608, Oakland, CA 94607
In a recent homicide, a key piece of evidence was message written in orange spray paint applied to the side of the abandoned vehicle in which the deceased was found. We found that traces of the paint overspray could be rendered observable by excitation using an Omnichrome Omniprint 1000 alternate light source. The paint exhibited a brilliant orange fluorescence at 525 nm when viewed through an orange barrier filter. In a search of the scene from which the vehicle had been recovered and removed, the ALS disclosed the presence of minute beads of occult orange paint overspray on the ground in a linear arrangement that corresponded with the spray painted lettering on the driver's side of the vehicle. From the amount of overspray on the ground, it appeared likely that occult overspray would have gotten on the clothing of the painter, as well. In a garment-by-garment search of the suspect's wardrobe at his home, the ALS disclosed the presence of traces of paint on a pair of shoes, blue jeans and gloves. Subsequent analysis by PLM and SEM/EDS confirmed that the paint samples recovered from the vehicle, scene and clothing of the suspect were indistinguishable in their pigment and vehicle compositions. The ALS enabled us to rapidly find traces of paint, 0.001 to 0.003 inches in diameter. The search of the vehicle, scene and numerous clothing articles was accomplished within 48 hours following the discovery of the body.
VISUALIZATION AND PHOTOGRAPHY OF UNTREATED LATENT FINGERPRINTS FROM SMOOTH NON-POROUS SURFACES UTILIZING THE EPISCOPIC VIEWING DEVICE MANUFACTURED BY ROFIN AUSTRALIA PTY. LTD.
Stephen M. Ojena, MS, Contra Costa County Sheriff's Crime Laboratory, 1122 Escobar Street, Martinez, CA 94553
The Rofin episcopic coaxial illumination device was used to visualize untreated latent fingerprints on smooth shiny non-porous surfaces, such as glass, polished metals, glossy paint and plastic. The light source was reflected from a semitransparent mirror. Both the incident light and reflected light were in a plane perpendicular to the surface. It appeared that the deposits from the ridges of the latent fingerprint diffused the incident light whereas the surface reflected, resulting in dark ridges against a light background. Latent fingerprints with remarkable detail were recovered using this device.
Christine Ong, Contra Costa County Criminalistics Laboratory, 1960 Muir Rd., Martinez, CA 94553
Identifying tablets and capsules that are submitted can be time consuming. In order to reduce the time used, information from various sources was consolidated into one identification guide. This guide has an alphabetical and a numerical section based on the pill markings. Each listing includes a drawing of the pill, its color, drug content, manufacturer and the source of the information. A description of the guide, as well as the computer techniques used to put it together, will be presented.
THE EFFECTS OF AEROSOL INHALERS ON PRE-EXISTING, PRE-DETERMINED BLOOD ALCOHOL CONCENTRATIONS USING THE INTOXILYZER 5000
Kristina L. Benson, Forensic Laboratory Services, SERI, 3053 Research Dr., Richmond, CA 94806
The objective of this research was to determine if aerosol inhalers compromise breath alcohol analysis. Subjects with a blood alcohol concentration (BAC) between 0.08% weight per volume and 0.12% weight per volume (w/v) used an aerosol, metered dosage inhaler; Alupent. Each volunteer then submitted to several breath tests using the Intoxilyzer 5000 breath analysis instrument. Immediately after the use of the inhaler and for the next 30 minutes, subsequent breath samples were given and the BAC recorded for each individual. The use of the Alupent, inhalers did not cause the BAC of the breath samples to rise in any of the subjects tested.
C6 PHENOTYPING BY IEF AND WESTERN BLOT DETECTION: A USEFUL GENETIC MARKER FOR FORENSIC APPLICATION
Shirley A. Miller and Moses S. Schanfield, Analytical Genetic Testing Center Inc., 7808 Cherry Creek S. Dr.#201, Denver, CO 80231
C6, the sixth component of complement, is polymorphic in plasma and serum and has proved to be a valuable genetic marker for paternity testing. This structural protein is a terminal product and is not converted or broken down to an activated complement component. Phenotypes have been obtained from post-mortem blood, dried bloodstains and products of conception, indicating that the system is potentially useful in forensics. This study describes a method using agarose isoelectric focusing pH 5-8, followed by a sensitive western blot detection. Gene frequencies for the common alleles C6*A and C6*B were 0.629 and 0.352 respectively for Caucasians (N=478), 0.618 and 0.370 for Amerindians (N= 166), 0.557 and 0.430 for Mexicans (N=263), and 0.520 and 0.429 for Blacks (N=324). Powers of discrimination are 0.631,0.626,0.640, and 0.691 respectively for the four population groups. An allele found in Blacks at a frequency of 0.039 was designated as C6*A1. Several other rare alleles were characterized and collectively grouped as variants.
DNA FROM ANCIENT TEETH
David DeGusta, Dept. of Anthropology; Charles Cook, Dept. of Geography; George F. Sensabaugh, Forensic Science Group, School of Public Health, U.C. Berkeley
Tooth pulp has been suggested as a good source of DNA for forensic testing. Unfortunately, pulp is absent in most archaeological samples. Dentin, however, is a hard tissue, similar to bone and should preserve at least as well as bone. In addition, dentin matrix contains more hydroxyapatite, a compound which binds DNA, than does bone, making it potentially a better source of ancient DNA. Mitochondrial DNA was extracted from the dentin of human tooth fragments estimated to be 100+ years old. A 1050 base pair segment was amplified using the polymerase chain reaction. Less than 0.05g of dentin powder was needed for successful extraction and amplification. In situ contamination is highly unlikely due to the protected location and impermeable nature of dentin. This technique should yield DNA even from poorly preserved and/or exceedingly old material, since the dentin is a hardy and well-protected tissue. This technique should also allow the amplification of longer segments of nuclear and mtDNA from available DNA sources than is currently possible.
DQA1 GENETIC ANALYSIS BASED ON SEQUENCE DEPENDENT DNA CURVATURE AND POLYACRYLAMIDE GEL MOBILITY RETARDATION
M. D. McGinnis and D. L. Quinn, GeneType Corp., 400 East Horsetooth Rd, Fort Collins, CO 80525
We report an electrophoresis-based approach to analysis of the human DQA1 locus that eliminates the requirement for oligonucleotide probe typing. The method is based on sequence-dependent, intrinsic curvature of double stranded DNA molecules. Such curvature results in electrophoretic mobility retardation relative to non-curved DNA fragments on polyacrylamide gels. We demonstrate that polyacrylamide mini-gel analysis of PCR-amplified fragments can partially reveal the genetic polymorphism of the DQA1 locus. The 780 base pair amplification products (second exon plus approximately 520 base pairs of the flanking introns) are resolved into 5 distinct mobility polymorphisms which reflect the DQA1 allele supergroup organization: 01 (two distinct variants), 02,03, and 04. Fluorescent labeling of these fragments permits analysis on the ABI 672 Genescan system using sequencing-sized gels. The increased resolution and accurate gel mobility analysis of this system, based on internal size standards, allows reproducible resolution of 10 distinct sequence polymorphisms, several of which are genetically stable intron polymorphisms. Finally, since this approach is gel-based, it is compatible with automated analyses of STR loci which are becoming increasingly important in individuality studies.
UTILIZATION OF METAPHOR AGAROSE, FMC BIOPRODUCTS, FOR AMPLIFLP D1S80 GELS
Marc Scott Taylor, Technical Associates Inc., 952 Athens St., Altadena, CA 91001
Typing of AmpliFLP D1S80 alleles amplified using the polymerase chain reaction is commonly done by polyacrylamide gel electrophoresis or by using a acrylamide-like polymer gel such as the "GeneAmp" detection gel. An effective alternative to an acrylamide-type gel is an agarose gel prepared with "Metaphor" agarose from FMC BioProducts A 20cm*32cm*0.8mm gel of 2.5% "Metaphor agarose cast and run in IX TBE buffer results in separation of the largest D1S80 alleles by approximately 2mm when electrophoresed for 4 hours at 500V. Higher voltages can be used with this system to shorten the run time or increase separation. The DNA bands can be visualized by staining with ethidium bromide and/or silver (BIORAD, Silver Stain Plus). The complete protocol for preparation, running and staining of this gel system will be discussed and illustrated.
FORENSIC VALIDATION STUDIES ON THE APOB AMPLIFIED FRAGMENT LENGTH POLYMORPHISM
Moses S. Schanfield, David Latorra, Kevin Humphreys, and Thomas A. Wahl, Analytical Genetic Testing Center, Inc., 7808 Cherry Creek S. Dr. #201, Denver, CO, 80231
Boerwinkle et al.(1989) and Ludwig et al.(1989) described the amplification of VNTR alleles associated with the Apolipoprotein B (APOB) locus using PCR; they termed these "Amplified Fragment Length Polymorphisms" (AFLP). A typing system was established using a biotinylated primer system with detection of PCR products non-isotopically after transfer to nylon membranes. Allele assignments are done using an allele ladder. Validation studies include the testing of non-probative and simulated evidence (blood and semen stains, vaginal swabs, oral swabs, placental tissue and hair) by student trainees and laboratory staff, the evaluation of inorganic extraction methods and testing of non-human specimens. The results show that accurate APOB typings can be obtained with minimal training. Inorganic extraction with differential digestion yields clean male fraction DNA amenable to PCR amplification. DNA obtained from different tissues from the same individual always produced the same APOB type. Non-human DNA does not appear to be a complicating factor in the amplification of forensic evidence. Preliminary studies indicate that both APOB and DQA1 can be co-amplified in the same reaction mix, and that APOB amplifies better than D1S80 but not as well as DQA1.
HLA DQA1 TESTING OF NON-HUMAN DNA
Thomas A. Wahl and Moses S. Schanfield, Analytical Genetic Testing Center Inc., 7808 Cherry Creek South Drive #201, Denver. CO 80231
In the course of validating the Cetus HLA DQA1 AmpliType Kit for forensic use in our Laboratory, bacterial, non-primate and non-human primate samples were subjected to amplification using this equipment. 21 species of non-primates (some phylogenetically similar to each other), 12 species of non-human primates and 2 species of bacteria were tested. DNA was extracted from all samples inorganically and confirmed by yield gel analysis. DNA quantitation was performed using both UV spectrometry and quantitative yield gels. l0 ng DNA from each sample were amplified per the AmpliType Kit, using a Bios Model BSC 1000 Thermal Cycler. Amplification products were detected on mini-gels; samples which exhibited product bands were hybridized with AmpliType strips. All the non-human primate DNA samples produced bands in the HLA DQA1 region. These samples hybridized with allele- specific oligonucleotides (ASOs) to exhibit an array of typing patterns, some consistent and some inconsistent with human typing patterns. The non-primate DNA samples did not produce any product band in the HLA DQA1 region. Although 5 of the 21 samples yielded product bands outside that region, these samples did not hybridize with the ASOs. The bacterial DNA samples yielded no products.
THE ELEMENTAL COMPOSITION OF BULLET WIPINGS ON CLOTH AND ITS RELATIONSHIP TO SUSPECTED AMMUNITION: A CASE STUDY
Faye Springer, California Dept. of Justice, 4949 Broadway, Sacramento, CA 95820
In 1989, an Immigration and Naturalization officer was shot and killed while questioning suspects involved in the buying and selling of illegal aliens as laborers. The fatal bullet entered the victim's back and exited through the lower abdomen. Numerous shots were fired by both law enforcement personnel and the suspects. During the shooting investigation, a question arose as to who fired the fatal shot. The bullet wipings around the margin of the bullet hole were examined with scanning electron microscope/energy dispersive X-ray. The appearance and the elemental composition of the wipings were compared to test wipings made with six possible combinations of weapons and ammunition. Significant differences were noted in the relative abundance of elements such as lead, barium and antimony between some types of ammunition. Furthermore, bullets with heavy lubrication transfer the lubricant to the wipings. A conclusion was made in this case as to which of the six possible combinations of ammunition/weapons fired the fatal round.
INCREASING THE YIELD OF SPERMATAZOA FROM STAINS AND SWABS
Marc Scott Taylor, Technical Associates, Inc., 952 Athens St., Altadena, CA 91001
The ability to extract spermatozoa from semen stains and swabs is dependent on the nature of the stain and the type of substrate involved. A significant increase in the number of spermatozoa released from most substrates can be achieved by reagitating the substrate after it has been processed through an initial "epithelial cell" digest with Proteinase K. One to five times the number of sperm in the initial extract can often be recovered by this post-digestion agitation. The increase in the numbers of sperm and subsequent increase in sperm DNA in various samples will be presented and the protocol in use at Technical Associates Inc. laboratories will be discussed.
DQα AMPLIFICATION IN THE PRESENCE OF BOVINE SERUM ALBUMIN (BSA)
Susan Swarner, Contra Costa County Forensic Laboratory, Martinez; Norah Rudin, Lance Gima and Lisa Barcellos, California Dept. of Justice, Sacramento
Some samples, including those containing dyes and natural pigments, amplify weakly or not at all. One common approach to overcome this problem is the addition of Bovine Serum Albumin (BSA) to the amplification mix; the BSA presumably binds polymerase-inhibiting factors. This approach has been tested on various kinds of samples and, in the majority of cases; samples that had previously failed to amplify were successfully amplified. No erroneous DQα types were obtained.
LIGHT AND ELECTRON MICROSCOPY OF INORGANIC PAINT CONSTITUENTS
Richard S. Brown, Thomas H. Hopen and Tim B. Vander Wood, MVA Inc., 5500-200 Oakbrook Parkway, Suite 200, Norcross, GA 30093
The identification of inorganic paint constituents is a necessary step in the examination and comparison of paint fragments in the crime laboratory. A combination of polarized light microscopy (PLM) and scanning electron microscopy -energy dispersive X-ray spectrometry (SEM-EDS) can be used to identify most inorganic paint pigments, fillers and extenders. After the paint fragments have been examined for a physical fit, the exemplar paint can be examined in a refractive index liquid (n=l.55) to characterize and identify any inorganic particles that may be present: eg. mica, talc, diatoms, calcium carbonate. A second sample of the exemplar is cross-sectioned and analyzed by SEM-EDS, then low temperature-ashed in an oxygen plasma to free the inorganic constituents from the paint resin and re-analyzed by SEM-EDS. Multiple cross-sections can be prepared for further PLM analysis. PLM and SEM-EDS are complementary techniques and should be used in any forensic examination. PLM is used to identify those constituents that the SEM-EDS system alone is unable to identify and some particles which are too small for the PLM to sufficiently characterize for identification will require examination by SEM-EDS. Morphology and EDS data can then be correlated with the PLM analysis to fully identify the inorganic paint constituents that are present.
HUMAN HAIR INDIVIDUALIZATION II: HAIR TYPES AND ARCHETYPES
Robert R. Ogle, Jr., Forensic Scientist, P.O. Box 5267, Vallejo, CA 94591
This paper addresses the underlying foundation for all efforts at individualization - the "type" and its frequency in the evidence class under consideration. The various hair "types" and their taxonomic correlate, the archetypes, are defined with a view toward providing a more scientific basis for forensic individualization attempts, especially as individualization applies to human hair. Without the delineation and characterization of the hair "types" and knowledge about their frequency in the population of interest, any opinion about the probative value of a hair "match" is an unscientific opinion; i.e., one hazards a guess for the rarity of the questioned hair and shrouds the guess in scientistic patois.
HUMAN HAIR INDIVIDUALIZATION III: STATISTICAL INTERFERENCE IN HUMAN HAIR: THE OLD SHELL GAME IN A NEW FORMAT
Robert R. Ogle, Jr., Forensic Scientist, P.O. Box 5267, Vallejo, CA 94591
The attempts at providing probability answers to the questions posed by the task of human hair individualization are discussed. The concept of "GIGObytes" is introduced to illustrate the futility of using "large" amounts of data to guesstimate the probative value of a human hair "match." "GIGObytes" are large amounts of data derived from meaningless experiments. The GIGObytes provide a numerical base for statistical maneuvering to arrive at majestic numbers, which, nevertheless, are trivial in the effort to determine the probative value of a hair match. Experiments which ignore the concept of the hair type can produce only GIGObytes of data which cannot be used to support a scientist's opinion with regard to the interpretation of a hair match in forensic work. The concept of the hair type must be developed and characterized so that the frequency of each hair type in the relevant populations can be determined by appropriate survey techniques. Once the frequency of a questioned hair's type in the population under consideration is known, the examiner will be able to offer a scientific opinion regarding the probability of the hair matching any individual other than the one to which the hair has been "matched."
STUDIES ON THE POLYMORPHISM AT THE HUM-FABP AND HUM-TH0I LOCI
M. Savill(1) and G.F. Sensabaugh(2);
(1) Institute of Environmental Health and Forensic Sciences, Christchurch Science Centre, Christchurch, New Zealand; (2) Forensic Science Group, School of Public Health, University of California, Berkeley
Short tandem repeat (STR) polymorphisms at the human fatty acid binding protein (HUM-FABP) and tyrosine hydroxylase (HUM-THOI) loci has been described by Edwards et al. (Amer. J. Hum. Genet. 49:746, 1991). FABP contains a trinucleotide repeat (AAT) and THOI contains a tetranucleotide repeat (AATG). Our typing method entails amplification by PCR using end labeled primers; the PCR products are separated on 6% sequencing gels and the results are visualized by autoradiography. A Ml 3 sequencing ladder is used to provide absolute sizing standards. Over 120 samples drawn from 7 different populations have been typed; the population pool includes Maoris and Samoans. Allele frequencies in New Zealand Caucasians are similar to those reported by Edwards, et. al. Maori and Samoan exhibit comparable heterozygosity but with some differences in allele frequency distributions. One individual was observed with three alleles at the FABP locus; the flanking region sequences of each were identical suggesting a gene duplication. The possibility of hidden sequence variation in the repeat and flanking regions of FABP was investigated by sequencing 14 alleles; no sequence variation was seen.
GENETIC STRUCTURE OF THE HUMAN RED CELL ACID PHOSPHATASE (ACP1) LOCUS: GENETIC TYPING OF THE *A, *B, AND *C ALLELES AT THE DNA LEVEL
K.D. Ayer Lazaruk(1), G.F. Sensabaugh(1), and J. Dissing(2);
(1) Forensic Science Group, School of Public Health, University of California Berkeley; (2) Institute for Forensic Genetics, University of Copenhagen, Denmark
The polymorphism of human red cell acid phosphatase (ACP1) is determined by three common alleles, ACP1*A, ACP1*B, and ACP1*C. Each allele codes for two distinct isozymes, fast (f) and slow (s), which vary in proportion according to genotype. The fast isozymes from each allele differ in a 34 amino acid "signature region" spanning residue positions 40-73. DNA sequencing shows the ACP1 locus to contain at least six exons; the two signature regions are encoded in separate, alternatively spliced exons. The *A sequence differs from the *B and *C sequences at a minimum of two sites: a silent C to T transition in the s signature exon, and an A to G transition at codon position 105 which gives rise to the Glu to Arg mutation that defines the A isozymes. The latter creates a TaqI restriction site that can be used to distinguish the *A allele from the *B and *C alleles. The *C sequence differs from the *A and *B sequences at a minimum of one site: a C to T transition in the exon. This substitution deletes a CfoI restriction site, allowing the *C allele to be distinguished from the *A and *B alleles. It is thus possible to type ACP1 at the DNA level.
SPECIES IDENTIFICATION FROM MITOCHONDRIAL CYTOCHROME B SEQUENCES
C. Cook and G.F. Sensabaugh, Forensic Science Group, School of Public Health, University of California, Berkeley CA 94720
We illustrate an approach for species identification based on the use of the polymerase chain reaction and direct sequencing of a 300-base segment of the mitochondrial Cytochrome B gene. This approach is demonstrated with the identification of vertebrate bloodmeals ingested by the mosquito Culex tarsalis. Species identification can be achieved in many cases by comparing the unknown sequences with published sequences; the library of vertebrate Cytochrome B sequences is extensive and contains representatives from most major vertebrate groups. If the unknown sequence is not found in the sequence library, it can be subjected to phylogenetic analysis to place it in the group which contains the most closely related species. Identification of mosquito blood meals was possible from samples collected up to four days after feeding, from dried smears of mosquito bloodmeals, and from mosquitoes which had been frozen for three decades. Amplification and sequencing for reptilian, avian, and mammalian mitochondrial sequences were performed using the same conserved primers; sequence differences between species allowed unambiguous identification of each sample. This example illustrates the robustness and great sensitivity of this methodology.
THE CASE OF THE LAUNDERED RESULTS
Lisa Barcellos and Norah Rudin, Ph.D. , California Department of Justice, 626 Bancroft Way, Berkeley, CA 94710
The effect of various chemical contaminants on blood and semen was investigated. The blood and semen samples were from the same individual and were unpreserved. Experiments were performed on both liquids and dried stains but not all contaminants were paired with both blood and semen or both liquids and stains. Contaminants included were: Vaseline, Spermicide, K-Y jelly, Douche, Drano, Dish Soap, Tide detergent, Bleach, Windex, Pinesol, 409, Luminol, various carpet cleaners and several laundering conditions. Both RFLP and PCR-DQα were performed on aliquots of the same organically extracted sample. Aliquots for PCR-DQα analysis were further purified by dialysis through a Centricon-100. Liquid blood, but not liquid semen treated with nonoxynol 9 gel spermicide showed extreme distortion from the yield gel through the analytical gel in RFLP analysis. Drano, essentially a strong base, severely affected the yield of DNA. Very weak bands were detected from the liquid blood and none from the bloodstain. Dawn dish soap and Tide detergent inhibited restriction digestion of bloodstains, while 409 inhibited digestion on liquid blood samples, but not bloodstains. Liquid blood exposed to bleach produced no results, although a very weak signal is sometimes visible from bloodstains. Bloodstains exposed to Capture carpet cleaner gave very weak signals on the final autorad, although the usual indicators of quality and quantity of DNA gave no initial clue to this result. Both Drano and Capture carpet cleaner treated bloodstains were inhibited for PCR-DQα amplification. The addition of BSA restored results in the Carpet cleaner sample, but not for the Drano sample. Both blood stains and semen stains were exposed to several laundering protocols:
- wash and dry with Tide detergent
- wash with Tide after treating with Spray and Wash.
- wash with Tide after treating with Spray and Wash and dry.
Even in the worst case (3), a weak signal was sometimes visible on an autorad after RFLP analysis. Older stains were more resistant to the treatments than fresh stains. Semen was more labile than blood. All samples produced PCR-DQα results.
QUALITATIVE AND QUANTITATIVE ASSAY OF ALPHA SATELLITE DNA
RM Thompson, TH Aulinskas, LJ Mendoza and HC Coleman, GeneLex, 2203 Airport Way, Seattle, WA
Alpha satellite DNA is a family of tandemly repeated monomers localized to human centromeric chromatin. D17Zl loci are the chromosome 17 specific alpha-satellite repeat sequences. Hybridization to D17Zl has been used to estimate human DNA quantities by "slot-blot" in forensic samples. We have extended this method to include qualitative assessment of DNA. Pre- and post-restriction "mini-gels" are Southern blotted and hybridized to 32P-labeled probe to D17Zl. Intact DNA appears as a single D17Zl hybridizing band, while Hae III digested DNA produces multiple bands. This procedure allows for a more rational choice of DNA analysis method than previously available. Casework data using this method demonstrates that the RFLP method can be used on some partially degraded DNA samples formerly considered as solely adequate for PCR. We have examined Hae III restriction site periodicities of the alpha satellites on chromosomes 17 and 7. A series of bands ranging in size and intensity were sized. The bands differ by multiples of ~171 bp, the fundamental repeat unit. Bands representing all 16 multiples were observed below 2.7 kb. Variations in copy number at individual bands were also observed. Size polymorphism, mainly in the region above 2.7 kb, was observed at both loci. Locus D7Z2 appeared to be a useful monomorphic marker.
SUMMARY OF HLA DQα FORENSIC CASEWORK AND EVALUATION OF AMP-FLP MARKERS FOR USE WITH FORENSIC SPECIMENS
Michael DeGuglielmo, MS, and John Rader, MS; Genetic Design, Inc., 7017 Albert Pick Road, Greensboro, NC 27409
The use of PCR in forensic testing has been validated in courts throughout the United States with HLA DQα which is currently employed in many forensic laboratories. Data will be presented summarizing our HLA DQα forensic casework experience as well as introducing the use of AMP-FLP markers for forensic casework. The use of AMP-FLPs with forensic specimens will extend the application of VNTR polymorphisms to specimens which were either too limited in quantity or too degraded for traditional RFLP Analysis. In this study we have evaluated the following four AMP-FLP markers: D1S80, YNZ22, Apo-B, and Col2Al for their use with forensic specimens. Data will be presented showing the population distribution of alleles for each AMP-FLP marker within major North American ethnic groups. Results of evaluation with forensic specimens will include optimization of extraction and amplification conditions for each marker. In addition, population distributions and specimen validation data for two STR (Short Tandem Repeat) Markers will be presented.
DEVELOPMENT OF MINISATELLITE VARIANT REPEAT (MVR) ANALYSIS FOR FORENSIC SCIENCE
Norah Rudin Ph.D. and Keith Inman M. Crim.; California Department of Justice, 626 Bancroft Way, Berkeley, CA 91710
One of the newest tools for use in DNA typing is MVR analysis. Non-random sequence variation between the repeats in a tandem array is detected using a application of the polymerase chain reaction (PCR). Because this system is based on sequence rather than length polymorphism, measurement imprecision associated with band sizing is eliminated. Discrete alleles obviate the need to statistically manipulate the data and the digital code that is generated nullifies any requirement for standardization of gel systems. The incorporation of PCR into the analysis carries with it all the advantages of analyzing samples of limited quality and quantity and chemiluminescent detection brings speed, safety and affordability. We have investigated the locus D1S8 (MS32) for use on forensic samples. In our system, 10ng of DNA gives a strong signal, leaving the possibility of analyzing even smaller amounts. We analyzed 4 families for inheritance patterns and found mendelian segregation, confirming previous results by others. 10 sets of nonprobative reference blood/epithelial cell samples were analyzed and the patterns easily correlated, confirming prior RFLP results. A non-probative sample set involving different tissue types was analyzed in duplicate. No matches were found, which confirmed the RFLP results. DNA samples were subjected to increasing amounts of UV irradiation. Signal loss began at 7200j/m2. The qualitative effect was fading of bands from both the top and bottom of the pattern, while the middle portion was affected last. Mixtures made from two known DNA samples were co-amplified in different proportions. In both directions, the minority component becomes undetectable at
less than 1/10 of the mixture. So far 32 different samples have been analyzed in our lab, all of which are differentiated by the 15th repeat. These data will be combined with others in order to produce a data base from which direct count statistics may be taken.