Cecelia Crouse, Ph.D., Debra Glidewell
Palm Beach County Sheriff's Office Crime Labroratory, 3228 Gun Club Road, West Palm Beach, Florida 33406

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INTRODUCTION

Palm Beach County has approximately 950,000 permanent residents and 150,000 temporary or transient residents. According to the 1994 Annual Report, Crime in Florida, 93 homicides and 788 forcible sex offenses were reported in Palm Beach County. The Palm Beach County Sheriff’s Office (PBSO) Crime Laboratory has been committed to the application of PCR-based DNA technology since 1991, when the request for DNA analysis was first submitted. In 1993, the genetic marker HLA DQA1 was introduced on casework. The addition of polymarker loci (PM) and the short tandem repeat (STR) multiplex CTAT (CSF1PO/TPOX/TH01 with Amelogenin) has been used on casework since September, 1995.

We will present a) a summary of CTAT validation studies prior to use on evidence, b) an update of the PSBO forensic cases in which the STR multiplex CTAT has been used, c) results from these cases, d) reaction by attorneys and the court system to STR technology, e) challenges that have been encountered when using STR technology, both technically and in court, and f) the future direction of the STR program at the PBSO Crime Laboratory.

MATERIAL AND METHODS

1. DNA extraction/quantitation: The laboratory uses a single-step DNA organic extraction method followed by microcon concentration. All evidence samples are quantitated using the Perkin-Elmer QuantiBlot kit. The extraction protocol must have a reagent control positive (P) and reagent control negative (N) sample.
   
2. Amplification: The amplification of CTAT loci (Promega Corporation) on DNA samples is as per manufacturer. BSA is added to the sample when the sample DNA concentration is low (about 0.5 ng) and/or when amplification may be inhibited by the substrate (jeans, burgundy-colored substrates, etc.) and BSA may be of benefit. All amplified samples must be electrophoresed on a 2.9% post-amplification agarose gel.
   
3. CTAT Typing: The polyacrylamide gel is denaturing 4% acrylamide:bis in 0.5X TBE buffer. Electrophoresis is done using a 0.5X TBE buffer on the BRL SA32 apparatus with constant power (40W) for 70 minutes. Post-electrophoresis, gels are stained using the Promega Silver Staining Kit. Upon drying, the gels are exposed to KODAK EDF film and developed in a Konica QX-70 automatic film processor.
   
4. Statistics: PBSO has population databases for Caucasian, African American, Hispanic, and Haitian populations. The DQA1, PM and CTAT loci have been determined across each individual in each population. Dr. Ranajit Chakraborty has evaluated the PBSO population data and has shown they fulfill the requirements of Hardy-Weinberg. PBSO has designed a spreadsheet which upon data input, automatically calculates expected genotype frequencies for each loci. Statistics are reported for the three major populations.
   

The PBSO Serology/DNA section conducts only PCR-based technology on casework and up
until the development of the STR multiplex systems, only reverse dot-blot typing was performed. The impetus for the development of STR technology at PBSO was the need to discern mixtures in biological stains, to conserve evidence by utilizing PCR multiplex systems and to provide statistical frequencies closer to virtual identity. The PBSO Crime Laboratory began investigating the feasibility of using STR technology on forensic casework in June, 1994 as participants in the European DNA Project. The purpose of this project was to determine if laboratories could identify alleles for the loci vWA, TH01, F13A1 and FES for five test samples. In the winter of 1995, PBSO was asked to participate in a study at Promega involving five laboratories including PBSO, FBI, California DOJ, Wisconsin Paternity Lab, and Promega. The purpose of this study was to evaluate STR multiplex systems for potential application to casework (JFS, July 1996).

In preparation for the initiation of STR technology on casework, the requisite validation studies were conducted including: single-source sensitivity studies, mixture sensitivity studies, amplification primer-annealing windows, species specificity, non-probative studies and regional population frequencies for all of the loci. In addition, two formal presentations were given to the Palm Beach County State Attorneys. The first seminar included an introduction to STR technology. The second included a series of handouts containing a) general PCR and STR information, b) computation of allele frequencies, c) copies of the visual aids to be used in court, and d) a generic approach to the presentation of STR data in the courtroom. In September, 1995, the Promega CTT multiplex system with the addition of Amelogenin (CTAT) was officially used on PBSO casework.

The Serology/DNA Section of the Sheriff’s Office currently has two analysts. Each analyst signs out evidence, identifies biological fluids, conducts DNA analysis on appropriate stains, interprets the typing results and prepares the final report. An additional analyst is currently in training for DNA analysis.

The PBSO Serology/DNA Section actually receives about 600 cases a year. Table 1 reflects only those cases in which DNA analysis has been conducted since PBSO began DNA typing (May, 1993 through August, 1996). Approximately one-third of the total number of cases have additional evidence submitted after DNA analysis has previously been initiated or completed.

Table 2 is a summary of the types of biological samples on which CTAT analysis has been conducted. It is mandatory that all amplified samples be electrophoresed on a post-amp gel to determine the approximate amount of amplified product. This information is necessary in order to decide how much amplified product to load onto the vertical gel. BSA is no longer routinely added to the amplification reaction as excess amplified n-1 bands have been observed.

To date, there have not been any court challenges to the reliability of the DNA testing methods employed at the PBSO Crime Laboratory. Over the past three years, the two analysts who conduct DNA testing at the Sheriff’s Office have testified to the DNA results in court 25 times, most of which include DQA1 testing only. Table 3 summarizes expert testimony that has been rendered by these same two analysts for those cases in which CTAT has been involved.

Figure 1 represents a typical CTAT analysis conducted on evidence associated with a sexual assault case. A young woman was jogging late one evening along a somewhat isolated road. The suspect was driving along that road when he spotted the jogger. He drove about a quarter-mile in front of the victim and pulled over onto the side of the road as if he was having car trouble. When the woman passed, the man grabbed her and dragged her into some nearby woods. The woman was vaginally assaulted. A condom was recovered at the crime scene. The suspect’s initial statement was that he had never before seen this woman and was not even in the area at the time of the assault. After DNA testing was completed, the suspect remembered the victim, and stated she was a prostitute and he did in fact pay for her services that evening in the woods.

The condom recovered from the crime scene was analyzed by swabbing the inside and outside surfaces and DNA profiles were obtained for each side. One side of the condom is consistent with the victim and the other side is consistent with the suspect. There is an obvious mixture in the NS fraction on one side of the condom.

The defendant was found guilty.

The PBSO laboratory routinely uses the CTAT multiplex on most casework samples in conjunction with the DQA1/PM typing strips. As expected, the greatest impact of the addition of the CTAT multiplex has been the ability to separate mixtures of biological stains. This is especially true with sexual assault cases where epithelial DNA and sperm DNA have not been completely separated. The most impressive feature of the CTAT system is not only the ability to discern mixtures, but also the fact that a) information for three loci are on the same film and b) a difference in the intensity of the alleles provides a way to assign major and minor DNA profiles to the individual standards.

Since the initiation of STR profiling on evidence in our jurisdiction, it is the perception of the State Attorney’s Office that there appears to be an increase in the number of sexual assault cases in which consent is now the issue. Although we have had some degree of n-1 band patterns, this has not been problematic in court. We routinely report rare DQA1/PM/CTAT profile frequencies. Additionally, amelogenin loci data has had an unexpected significant benefit to casework analysis. Both the prosecution and the defense have found the sex-typing information interesting and extremely informative.