Forensic Casework Analysis Using STRs, DQa, and PM in Combination

Charlotte J. Word, PhD, Anjali A. Ranadive, MFS, Melisa A. Weber, MFS, and Robin W. Cotton, PhD
Cellmark Diagnostics, 20271 Goldenrod Lane, Germantown, MD 20876

Analysis of polymorphic genetic loci using the polymerase chain reaction (PCR) enables results to be obtained from small and/or degraded samples. PCR analysis of multiple polymorphic loci has significantly enhanced the amount of information obtainable from biological evidence. The AmpliType™ HLA DQa Forensic DNA Amplification and Typing Kit and the AmpliType™ PM PCR Amplification and Typing Kit allow typing of 6 polymorphic loci using a reverse dot format. The recent availability of reagents to amplify short tandem repeat (STR) loci such as the HUMCSF1P0, HUMTPOX, and HUMTHO1 triplex (CTT) using the GenePrint™ STR systems adds additional loci which can be analyzed using the PCR. Although validation studies have been conducted using the CTT triplex, limited data was available on the efficiency of amplification of the CTT triplex in comparison to the DQa and PM systems using casework samples. Therefore, STR analysis at Cellmark Diagnostics was initially implemented only on evidence samples where a sufficient amount of DNA was extracted to complete DQa and/or PM analysis which was then followed by STR analysis.

To date (September 1995) we have analyzed and reported 32 cases using this approach. DNA extractions were done using phenol/chloroform or Chelex® following Cellmark Diagnostics' standard operating procedures. DNA quantitation using the QuantiBlot™ kit was performed on all samples analyzed with STRs.

Samples containing as little as approximately 0.1 ng of DNA, as measured by slot blot, produced results in all three systems. All samples which produced results using DQa and PM also produced results using the CTT triplex. Additionally, as would be predicted from validation studies, equivalent sensitivity for detection of mixtures was found in both the PM and STR systems. Use of the two detection methodologies together helped to confirm the presence of minor mixture components.

Thus far, all inclusions on single-source samples using DQa and PM have also been inclusions using the CTT triplex. Based on a database of 105 Caucasians and 98 African-Americans the most common frequencies expected for these combined systems is 1 in 46,000 for Caucasians and 1 in 156,000 for African-Americans. The STR CTT triplex is as robust as the DQa/PM systems. The use of the two types of detection systems provides additional information for the interpretation of mixed samples.

Polymerase chain reaction (PCR)-based DNA testing has become the method of choice in forensic cases where the DNA is too limited in quantity or too degraded to do restriction fragment length polymorphism (RFLP) testing or where results are needed rapidly. There are several commercially available PCR tests that are commonly used in forensic laboratories for casework, which include the AmpliType™ HLA DQa Forensic DNA Amplification and Typing Kit, the AmpliType® PM PCR Amplification and Typing Kit and the AmpliFLP® D1S80 PCR Amplification Kit. Test systems that permit the amplification and typing of several short tandem repeat (STR) sequences are now commercially available from Promega. The CTT GenePrint™ STR Multiplex system permits the co-amplification and typing of the three DNA loci HUMCSF1P0, HUMTPOX, and HUMTHO1. Here we report a summary of observations from 32 forensic cases tested at Cellmark Diagnostics using the CTT STR system from November of 1994 to September of 1995.

DNA was extracted from forensic casework samples using standard protocols at Cellmark Diagnostics. The DNA was extracted using either an organic (phenol/chloroform) extraction procedure (1) or Chelex® (2). For mixed stain samples containing sperm, a differential lysis procedure (1) was used to separate non-sperm and sperm fractions. In some cases DNA that had been extracted in other laboratories was tested. The DNA was quantitated using the QuantiBlot™ quantitation system.

To obtain DQa and PM results, the extracted DNA was amplified using the AmpliType® PM PCR Amplification and Typing Kit (Perkin Elmer) according to the recommendations of the manufacturer. To obtain results for the CTT triplex, 1-5 ngs of extracted DNA was amplified using the CTT GenePrint™ STR Systems (Promega). The amplification reaction mixture first used at Cellmark Diagnostics contained the sample DNA in a mixture of the HUMCSF1P0, HUMTPOX and HUMTHO1 primers and reagents which had been purchased separately and combined at Cellmark, 5 units of Taq polymerase (Perkin Elmer), and 160 µg/ml bovine serum albumin (BSA) (Sigma) in a final volume of 50 µl. The latter case samples were amplified using the CTT GenePrint™ STR multiplex, 1.25 units of Taq polymerase and 160 µg/ml BSA in a final volume of 25 µl. Product gels of 4% NuSieve™ 3:1 agarose were run to determine if amplified products were present and to determine the amount of STR amplified product to run on the analytical gel.

The amplified PM products were used to obtain both PM and DQa results using the protocols provided with the PM test kit. The amplified STR products were typed by running 1-4 ml of the products on a denaturing 4% acrylamide gel in 0.5X TBE following the procedures recommended by Promega and silver staining the gel to visualize the products. The types were determined using the allelic ladders provided with the CTT STR kit which were run in a lane adjacent to each sample on the analytical gel.

DNA isolated from blood from mothers and/or fathers submitted for paternity testing and DNA from blood from several laboratory personnel were amplified and typed for DQa (including 4.1 and 4.2/4.3 alleles), PM and STR genotypes using the procedures above. The genotypes for 105 Caucasians and 98 African-Americans were determined.

Cellmark Diagnostics began accepting forensic casework for CTT STR testing in November 1994. Our acceptance policy required that the samples be typed first using another PCR-based typing method either in our laboratory or in another forensic laboratory. By September of 1995, we had tested and reported CTT STR results in 32 forensic cases. DNA from several different kinds of samples containing blood, semen, saliva or other biological samples was isolated, amplified and typed for CTT. All samples from forensic casework that had been previously amplified and typed with another PCR-based system also gave results using the CTT STR system. A list of the 106 samples which successfully amplified and typed for CTT are shown in Table 1. A listing of the other PCR-based tests that were performed on the casework samples are listed in Table 2. With the exception of one case, all of the cases had DQa and PM tests performed. We were therefore able to compare the test results obtained using the DQa and PM systems to the results obtained using the CTT STR system. For single-source samples, all of the 20 inclusions reported using DQa and PM results remained inclusions when CTT STR results were obtained. Similarly, all exclusions reported using DQa and PM test results were also exclusions with CTT STR testing. In no case did STR results exclude someone who had been included by DQa and PM test results in a single-source sample. For samples that clearly contained a mixture of two or more individuals by DQa and/or PM testing, testing with the CTT STR system permitted the exclusion of 7 individuals (generally boyfriends or husbands) some of whom had been included as possible donors using the DQa and PM systems. For 15 of the mixed samples, an inclusion with DQa and PM remained an inclusion with CTT STR testing. In some cases, statistical frequencies were reported for the major contributor in the mixed samples.

For the cases where we reported statistical frequencies using DQa, PM and CTT STR data, we compared the statistical frequencies obtained using the three CTT STR loci alone versus the frequencies obtained using all nine loci (DQa, PM, CTT). The results are shown in Table 3. For CTT STRs alone, the frequencies ranged from approximately 1 in 130 to 1 in 6300 for Caucasians and from approximately 1 in 600 to 1 in 25,000 for African-Americans. The frequencies reported using the DQa, PM, and CTT STR data in combination are generally three or more orders of magnitude more rare than the frequencies for the STR results alone, with the most common frequency being approximately 1 in 1 million for both Caucasians and African-Americans.

We found that cases were submitted for STR testing for a variety of reasons. These included: (1) obtaining more data for single-source samples and to improve the statistical frequencies, (2) confirming that a sample is a mixture of DNA from two or more individuals, (3) determining if an individual who was included in a mixed sample by DQa and PM testing was still included or could be excluded with CTT STR testing, (4) performing STR testing on a case with other strong data and evidence for future admissibility purposes, and (5) for independent testing by a second laboratory.

Several interesting cases where CTT STR testing was useful in resolving questions regarding mixed samples are briefly presented in Tables 4, 5 and 6. For the case shown in Table 4, the evidentiary sample was a semen stain on panties. The DQa and PM results indicated that the sperm fraction from the panties contained DNA from two or more individuals and the victim, suspect and boyfriend were all included as possible sources. Testing with the CTT STR system, however, excluded the boyfriend as a possible source at the HUMTHO1 locus. For the case presented in Table 5, the evidentiary sample was a stain from fingernails. DQa and PM results indicated that the DNA from the fingernails originated from two or more individuals. Neither the victim nor the suspect could be excluded as possible sources of the DNA. However, if there were only two sources of DNA obtained from the stain from the fingernails, the DQa and PM results were not consistent with both individuals being sources; that is, either the victim could be a source but the suspect was not, or the suspect could be a source but the victim was not. The CTT STR test results excluded the suspect as a source of the DNA. For the case presented in Table 6, the evidentiary sample was a vaginal swab. The DQa and PM results indicated that the sperm fraction contained DNA from two or more individuals and the victim was included as a possible source. The suspect was included as a source if there were two sperm donors, but was not a source if there was only one sperm donor and the victim was one source of DNA. CTT STR results excluded the suspect as a source of the DNA in the sperm fraction.

As of September 1995, Cellmark had testified twice in court to STR results. The results were not challenged in either of these cases. As of February 1996, Cellmark has testified in at least seven cases to STR results including three admissibility hearings. The STR results were admitted in all cases to date where rulings have been made.

Walsh P.S., Metzger D. and Higuchi R. (1991) Chelex® 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10:506-513.

Sample Types	Number of Samples
Blood/stains (knowns)	55
Vaginal swabs	11
Other swabs (semen)	2
Semen stains	15
Blood stains (fabric, carpet)	6
Wood	1
Tissue	1
Esophageal tube (known)	1
Hair (1 known, 1 unknown)	2
Cigarette butt	1
Stain from fingernail	1
DNA from other labs	10

Other PCR Text Performed	Number of Cases*
DQa - other lab	1
DQa/PM - Cellmark	21
DQa/PM - other lab	6
DQa/PM/D1S80 - other lab	1
DQa/PMRFLP - Cellmark	3

Total	32

* Number of forensic cases where CTT STR results and other PCR-based results were obtained.

	Caucasians	African-Americans
CTT STRs	130-6300^a	600-25,000
DQa/PM/CTT	1 x 10⁶-7 x 10⁸	3 x 10⁶-3 x 10⁸

^a The numbers shown are the range of frequencies for that population group given as 1 in that number of individuals.

	DQa	PM	STR
Victim	Included*	Included*	Included
Suspect	Included*	Included*	Excluded
Boyfriend	Excluded	Included	ND

* If there are only two sources of DNA, the data are not consistent with a mixture of DNA from the victim and the suspect.
ND = not done.

* If there are two sperm donors, the suspect is included. If there is only one sperm donor and the second source is the victim, then the suspect is excluded.