Barbara E. Llewellyn, M.S., Virginia Fristoe, M.S., Tara Savage, M.S., Michelle Squyars, M.S., Jeff Ban, B.S.
Virginia Division of Forensic Science, Richmond, VA 23219

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INTRODUCTION

The benefits of the polymerase chain reaction (PCR) technology to the forensic DNA community are many and diverse. This technique offers significant advantages over RFLP analysis such as obtaining results faster and more economically. The PCR technology also allows for the analysis of low concentrations of DNA because of increased sensitivity and the ability to tolerate partial degradation. The primary types of PCR analyses conducted in the forensic DNA laboratory include sequence polymorphisms (HLA DQA1 and Polymarker), AMFLPs (amplified fragment length polymorphisms) and STRs (short tandem repeats). STR loci are informative polymorphic loci that consist of short, repetitive sequences of 3 to 7 base pairs (bp) in length. These repetitive sequences are polymorphic due to variation in the base pair length among individuals, thus allowing for the discrimination between individuals. STR DNA analysis offers the advantage of simultaneous analysis of several highly polymorphic loci by multiplexing the amplification and typing of more than one locus at a time. The focus of this study was to optimize the established parameters for DNA analysis at the F13A01, FES/FPS, F13B, and LPL (FFFL) loci using the GenePrint ™ STR System Kit (Promega) and visualizing the amplified product with the FMBIO^®-100 Fluorescent Method Image Analyzer (Hitachi). This study was conducted in conjunction with the FBI’s STR Standardization Project.

Fifteen whole blood samples were obtained from individuals within the Virginia Division of Forensic Science laboratory system. These samples represented as many different alleles as possible at the F13AO1, FES/FPS, F13B, and LPL (FFFL) loci. DNA was organically extracted from these samples and amplified using the FFFL GenePrint™ STR System Kit (Promega). All samples were amplified in a Perkin-Elmer 9600 thermal cycler using 2.5 ng of DNA template following Protocol 9 as recommended by Promega (1). Amplification of the DNA was verified using a 3% NuSieve agarose (FMC) gel. The amplified fluorescently tagged alleles were separated on a Gel-Mix^®6 polyacrylamide denaturing gel (Gibco BRL) that
was run.

using a Gibco BRL model SA32 vertical gel apparatus. The samples were then visualized using the FMBIO^®-100 Fluorescent Method Image Analyzer.

All of the studies conducted were designed to optimize the amplification and running conditions for the FFFL kit using the FMBIO^®-100 Fluorescent Method Image Analyzer. These studies included the following:

1. Addition of BSA — Prior to amplification, 0.5 µl of 60 µg/ml BSA (Fraction V, Sigma) was added to the master mix along with the other reaction mixture components of the FFFL
   GenePrint ^TM STR System Kit, in order to determine if this would increase the efficiency of the amplification process.
   
2. Chelex-extracted samples v. Organically-extracted samples — This study was designed to evaluate the amplification of DNA extracted using the Chelex method (2). Another purpose of this study was to determine if the addition of BSA (Fraction V. Sigma) increased the efficiency of the amplification process. For each sample analyzed, 0.9µl of 60µg/ml BSA was added to the master mix along with the other reaction mixture components of the GenePrint™ STR FFFL System Kit, prior to amplification.
   
3. Dinucleotide Concentration — The concentration of dinucleotides (dNTPs) was varied from the recommended 2mM (1) to 0.5, 1.0, 4.0, and 5.0mM. Reagents used to make the master mix included: 10X Thermophilic Buffer - magnesium free (Promega), 25mM MgC12 (Promega), 100mM each dNTP (Promega), and Taq polymerase (Promega). The FFFL primer set was the only reagent used from the GenePrint^TM STR FFFL System Kit.
   
4. Primer Volume — The volume of the FFFL primer set from the GenePrint^TM STR FFFL System Kit added to the master mix was varied from the control volume of 2.5 µl per sample (1) to 1µl, 2µl, 3µl and 4µl per sample.
5. Taq Polymerase Volume — This study was designed to vary the volume of Taq polymerase added along with the other reaction mixture components of the GenePrint^TM STR FFFL System Kit to determine the effects on amplification. The amount of Taq polymerase was varied from the control volume of 0.20µl per sample (1) to 0.05µl, 0.10µl, 0.15µl, 0.40µl, and 0.50µl per sample.
6. MgCl₂ Concentration — Different concentrations of MgCl₂ (10mM, 12.5mM, 17.5mM, and 20mM) were added to the master mix which consisted of: 10X Thermophilic Buffer - magnesium free (Promega), 25 mM MgCl₂ (Promega), 100 mM each dNTP (Promega), and Taq polymerase (Promega).
7. Template Amount — This study was conducted to evaluate the results obtained from amplifying various amounts of DNA template. Samples were amplified using the GenePrint^TM STR FFFL System Kit with varying concentrations (0.5ng, 1.0ng, 4ng, and 5ng) of DNA template.
8. Cycle Number — In order to determine if additional cycles of the PCR would improve
   the yield of amplified template DNA, the parameters were varied by adding either 2 or 6 additional cycles to the recommended Protocol 9 (1).
   
9. Annealing Temperature — The purpose of this study was to determine the effect of varying the annealing temperature on the efficiency of the PCR. The annealing temperature during the PCR was varied from the recommended 60^oC in Protocol 9 (1) to 56^oC, 58^oC, 62^oC, and 64^oC.
10. AmpliTaq Gold^TM DNA Polymerase — This study was conducted to compare the efficiency of AmpliTaq Gold^TM DNA Polymerase (Perkin-Elmer) with that of Taq polymerase (Promega). Samples were amplified using AmpliTaq Gold^TM DNA Polymerase in place of Taq polymerase using the GenePrint^TM STR FFFL System Kit. Approximately 1.5 µl of a 25 mM Tris, pH 7.5 (3) was added to the GenePrint^TM STR FFFL System Kit master mix along with 0.15µl of AmpliTaq Gold^TM DNA Polymerase.

BSA has been shown to increase the efficiency of the PCR when inhibitors are present. As expected, BSA had no effect on the amplification results because pristine samples were utilized for this study. Therefore no differences were observed between samples amplified with the addition of BSA and the control samples which did not contain BSA (data not shown).

Non-specific amplification products were observed in all chelex-extracted samples, whether or not BSA was added before amplification. In addition, locus drop-out was observed at the F13AO1 locus for the same two chelex-extracted samples regardless of whether BSA was added. Locus drop-out at the F13B locus was observed for one chelex-extracted sample and one chelex-extracted sample did not amplify (data not shown). All samples organically extracted and amplified gave good typing results. More studies may be needed to optimize conditions when using chelex-extracted samples with the GenePrint^™ STR System Kit for the FFFL loci.

Non-specific amplification products and stutter bands were observed at the LPL locus when dNTP concentrations of 0.5mM or 1.0mM were used. Three of the 15 samples demonstrated amplification at the LPL locus with a dNTP concentration of 4.0mM. The remaining 12 samples did not amplify or demonstrate product at any of the FFFL loci. When a dNTP concentration of 5.0mM was used, no amplification was observed for any of the samples (data not shown). At the recommended dNTP concentration of 2mM (1) good amplification and typing results were obtained. Therefore, it is recommended that the dNTP concentration remain at 2 mM as suggested by Promega (1).

Allele intensities were weak at the FES/FPS locus for 8 of the 15 samples when only 1µl of the primers was added to the master mix instead of the 2.5µl used in the control samples. The remaining 7 samples did not amplify. Three of the 15 samples demonstrated weak allelic intensities at the F13AO1 locus when 2µl of the primers was added to the master mix. When 3µl of the primers was used, non-specific amplification products and stutter bands were observed for 3 samples at the F13B and F13AO1 regions of the gel. Nine of the 15 samples did not amplify when 4µl of the primers was added to the master mix. The remaining six samples demonstrated sporadic amplification at either the F13AO1, F13B, or LPL locus and no amplification at the FES/FPS locus (data not shown). All samples amplified using the manufacturer’s recommended volume of 2.5µl of primer (1) gave good amplification and typing results.

Non-specific amplification products were observed when the larger volumes of Taq DNA Polymerase (0.40 and 0.50µl/ sample) were used. Only two of the 15 samples exhibited amplification when 0.05µl/sample volume of Taq DNA Polymerase was used. The 0.10µl volume of Taq DNA Polymerase exhibited good results however, some of the allele intensities were light. Results comparable to the manufacturer’s suggested 0.20µl (1) of Taq DNA Polymerase were obtained with a volume of 0.15µl.

The addition of 10mM MgCl₂ resulted in 3 samples exhibiting stutter bands at the LPL and FES/FPS loci. Non-specific amplification products were observed above the F13B region of the gel for one sample. When a MgCl₂ concentration of 12.5mM was used, non-specific amplification was observed in the LPL locus region of the gel. Three of the fifteen samples exhibited locus drop-out at the F13B locus. At a MgCl₂ concentration of 17.5mM one sample demonstrated non-specific amplification in the F13AO1 locus region. When a concentration of 20mM MgCl₂ was used, 3 samples exhibited stutter bands at the LPL and FES/FPS loci; non-specific amplification products were observed below the F13B locus region of the gel (data not shown). The best results obtained were from those samples amplified using the 15mM MgCl₂ concentration recommended by Promega (1).

The best results for this study were obtained from the 1.0ng concentrations of DNA template and the manufacture's recommended 2.5ng (1) (Figure 1). However, even though the allele intensities obtained with the 0.5ng were very weak, they could still be correctly typed. Non-specific amplification at the LPL locus was observed with 4.0ng and 5.0ng of DNA template. The DNA template at a concentration of 5.0ng exhibited stutter bands at the LPL locus and non-specific amplification below the F13B region of the gel. Therefore, a DNA template concentration above 0.5ng to approximately 2.5 ng gives optimal results.

H. Cycle Number

The addition of two cycles to Protocol 9 (1) resulted in locus drop-out at the F13AO1, FES/FPS, and F13B loci for four of the 15 samples. Non-specific amplification and stutter bands were observed at the LPL locus for all samples. When six cycles were added to Protocol 9, all samples demonstrated non-specific amplification above and below the F13B region and at the LPL and F13AO1 loci (data not shown).

When the annealing temperature was changed from the recommended 60^oC (1) to 56^oC, locus drop-out was observed at the F13AO1 locus for approximately 5 of the 15 samples. When a 58^oC annealing temperature was used 12 of the 15 samples showed locus drop-out at the F13AO1 locus. Only one sample showed amplification at the FES/FPS locus and non-specific amplification products and stutter bands were observed at the LPL locus. Locus drop-out at the F13B locus was observed in 12 of the 15 samples when the annealing temperature was changed to 64^oC. Three samples did not amplify. When the annealing temperature was changed to 64^oC, locus drop-out was observed for all the samples except the positive control at the F13B locus. Only one sample and the positive control demonstrated amplification at the LPL locus and two samples showed locus drop out at the FES/FPS locus (data not shown).

When the AmpliTaq GoldÔ DNA Polymerase was used in place of Taq polymerase good results were obtained. However, these results were only obtained when 1.5µl of a 25mM Tris buffer, pH 7.5 was added to each sample (3) and 0.5ng of template DNA was used (Figure 2). A DNA template amount above 0.5 ng created extra bands at the LPL locus. AmpliTaq Gold^TM DNA Polymerase is optimized to work at a pH of 8.3 or less. With the GenePrint^™ STR System Kit it was necessary to add 25mM Tris buffer to the amplification reaction mixture to reduce the pH and thus allow the AmpliTaq Gold^™ DNA Polymerase to work. The advantage of using the AmpliTaq Gold^™ DNA Polymerase is that it allows for the set up of many more samples to be amplified at one time because the AmpliTaq Gold^™ DNA Polymerase does not become active until it reaches 95^oC.

The results obtained from these studies suggest that the manufacturer’s recommendations: 2.5µl volume of primer pairs, 2 mM concentration of dNTPs, 15mM concentration of MgCl₂, an annealing temperature of 60^oC, and the number of cycles to be used during the PCR (1), should be followed. Varying these parameters resulted in the observation of non-specific amplification and stutter bands, and therefore did not improve the results. However, better results were obtained with 0.15µl of Taq polymerase versus the manufacturer’s recommended 0.20µl (1). The amount of template DNA should be above 0.5ng to 2.5ng to obtain the best results. The results obtained for those samples extracted using chelex showed non-specific amplification products. Therefore, it is not recommended that chelex-extracted samples be used with the GenePrint™ STR FFFL System Kit. Overall, the manufacturer’s protocol for the GenePrint^™ STR FFFL System Kit works well and can be used in conjunction with the FMBIO^®-100 Fluorescent Method Image Analyzer to obtain good results.

REFERENCES

Figure 1. Template DNA