Over 200 blood samples were collected at random from patients who visited the King Khaled University Hospital and Security Force Hospital in Riyadh in Saudi Arabia. 100 blood samples from the University Hospital were collected in EDTA tubes, transferred onto cotton swatches and dried at room temperature. 100 blood samples from the Security Force Hospital were spotted on FTAÔ stain cards then shipped to the United States for analysis. Upon arrival at the Indianapolis-Marion County Forensic Services Agency, the 100 cotton swatches were stored at -20°C until extraction procedures were performed.

After extraction, these 100 samples were coamplified for DQA1 and AmpliTypeÔ PM then typed at Indianapolis-Marion County Forensic Laboratories. All 200 DNA samples (Cotton swatches and FTAÔ stain cards) were amplified and typed for STR loci (CTTv and FFFL) at ReliaGene Technologies, Inc., in New Orleans, LA. The PCR genotypes for all samples analyzed were then subjected to Hardy Weinberg Equilibrium analysis at the Forensic Science Research and Training Center at the FBI Academy in Quantico, VA. The findings of this analysis revealed no significant deviations from Hardy Weinberg Equilibrium. Linkage analysis indicated that the markers were independent and therefore the multiplication rule can be used with this database. The allelic frequencies for the STR markers analyzed were similar to the published Caucasian population databases. This Saudi Arabian database should prove useful in PCR casework which may involve individuals of Saudi Arabian heritage.

This study was conducted after a literature search revealed that no DNA data on the Saudi Arabian population has been reported thus far. This study should provide useful scientific data for subsequent forensic and paternity analysis in the Saudi Arabian population.

Genetic information can be obtained from DNA in biological samples by amplification of target sequences using the polymerase chain reaction (PCR). Currently, there are several genetic loci that are amenable to PCR that can be analyzed using commercially available kits. Some of these are AmpliTypeÔ PM PCR amplification kit (Perkin-Elmer, Roche Molecular Systems, Inc., Branchburg, NJ) and GenePrintÔ Fluorescent STR System PCR amplification kit (Promega Corp., Madison, WI, USA).

To estimate the frequency of a multiple PCR-based DNA profile for parentage analyses or forensic casework, it is necessary to collect allele and genotype frequencies from relevant populations. Using the AmpliTypeÔ PM and DQA1 PCR Amplification kit and the GenePrintÔ STR Systems PCR amplification kit, such data was generated from 200 unrelated random Saudi Arabian individuals. The data was subsequently subjected to Hardy-Weinberg analyses. The comparative studies on the allele frequencies on Saudi Arabian, as well as Black and Caucasian, populations was also carried out.

Part of these studies was also used to validate the GenePrintÔ Fluorescent STR kits and the Genomyx fluorescent scanning system in our laboratory.

DNA was extracted from the cotton swatches by organic extraction (phenol/chloroform/isoamyl alcohol), followed by ethanol precipitation. The extracted DNA was then quantitated by Quantiblot utilizing the D17Z1 probe. All samples were then subjected to PCR amplification using approximately 2 to 5ng of DNA.

Genomic DNA was isolated from the blood stained FTAÔ card (6mm punch) using FTAÔ purification reagent (FITZCO Inc. Maple plain,MN) in 1.5 ml eppendorf tubes. The FTAÔ paper punch was washed twice with TE (10mM Tris HCl, pH 8.0, 0.1 mM EDTA) and was allowed to air dry at room temperature. Purified genomic DNA remains bound to the paper punch.

The DNA obtained from cotton swatches was amplified for DQA1 and AmpliTypeÔ PM using Perkin-Elmer’s AmpliTypeÔ PM + DQA1 kit (Part # N808-0094, Perkin-Elmer, Roche Molecular Systems, Inc., Branchburg, NJ). The amplification conditions were followed according to the protocols provided in the kit.

Amplification of loci for quadriplex 1 (CSF1PO, TPOX, TH01, vWA) and quadriplex 2 (F13A01, FESFPS, F13B, LPL) was completed with reagents and protocols provided by Promega Corporation’s GenePrintÔ Fluorescent STR kit DC6301 and DC6310. The procedures are described in the Fluorescent STR systems GenePrintÔ Technical Manual (Part # TMD006, Promega Corporation, Madison, WI). A master mix containing the fluorescent multiplex primers, 10X STR buffer, sterile water and Taq DNA polymerase (Cat# M1665, Promega Corporation) is added to the dried FTAÔ paper punch contained in a thin-walled amplification tube. Two different volumes of 25l and 50ml of master mix were attempted. The DNA was amplified in a Perkin-Elmer thermocycler model 480. Typical amplification conditions for both the quadriplexes consisted of a hot soak at 96°C for 2 minutes; 10 cycles of 94°C for 1minute, 60°C for 1 minute and 70°C for 1.5 minutes; followed by 20 cycles of 90°C for 1 minute, 60°C for 1 minute and 70°C for 1.5 minutes and a final extension at 60°C for 30 minutes. Positive control of K562 DNA and a negative control were run on each batch of amplified DNA. The success and efficiency of the amplifications was verified on yield gels which showed that consistent amplification was obtained with 50ml reaction systems.

The amplified products in the case of AmpliTypeÔ PM and DQA1 were detected by reverse dot blot hybridization.

In the case of fluorescent STR, amplified DNA products were separated by 6% polyacrylamide gel electrophoresis in denaturing conditions using Life Technologies model S2 electrophoresis apparatus. 3.0ml of amplified DNA was mixed with 3.0ml of Blue dextran dye (Cat# DV435A, Promega Corporation) and was heated at 95°C for 5 minutes. 2.5ml of allelic ladder was mixed with 3.0ml of Blue dextran dye.

Electrophoresis was performed at 40 Watts in 0.5X TBE (tris borate electrophoresis buffer) for 2 hours. After the electrophoresis was completed, the glass plates containing the gels were cleaned with water followed by ethanol to remove excess pieces of acrylamide. The gel was mounted in the fluorescent gel scanner Model Genomyx SC (Genomyx Corporation, Foster City, and CA94404) to visualize the DNA bands. The fluorescent gel scanner automatically scans the gel and takes the photograph, which can be viewed on a computer screen for analysis. Amplified products were analyzed by comparing the product migration with the STR allelic ladder.

Approximately 200 Saudi Arabian blood samples were analyzed for eight STR loci while 100 of these samples were also analyzed for AmpliTypeÔ PM and DQA1 loci. In the case of fluorescent STR the 50ml reaction system for PCR amplification gave consistent results. The distribution of observed allelic frequencies for each of the loci are shown in the tables below. There was no deviation from expected values for these loci.

AF= Allelic Frequency

CONCLUSION

A Saudi Arabian population database has been established for the eight STR loci, AmpliTypeÔ PM and DQA1 loci that can be amplified and typed using commercially available PCR amplification and typing kits.