Application Note 113

Gender Determination by Amelogenin Specific PCR* and Product Analysis by DNA Chromatography
Kaoru Kobayashi and Karl H. Hecker, Transgenomic Inc., San Jose, CA 95035
nalysis of PCR* products is traditionally performed by labor-intensive gel electrophoresis. We report here the use of ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) DNA chromatography using the WAVE Nucleic Acid Fragment Analysis System, for automated gender determination with amelogenin specific PCR* products. Analysis by DNA chromatography is fully automated. No sample preparation after PCR* is required. Purified samples can be quantified by peak integration and collected by peak capture for downstream applications. Unlabeled and fluorescein labeled primers were separated and purified. One primer labeled with an isomeric fluorescein mixture was resolved. Primer, labeled with a single isomer, was purified for amelogenin specific PCR*. Amplification of a segment of the X-Y homologous gene amelogenin yields a 212-bp Xspecific and a 218-bp Y-specific PCR* product. Products are well resolved by DNA chromatography in the presence and absence of a fluorescein tag.
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Introduction
Sex determination is an integral part of the characterization of forensic samples containing genomic DNA. PCR* offers an efficient and sensitive method for sex determination by amplifying gender specific sequences, which result in different size PCR* products depending on the gender of the donor (6, 11). Sizing of PCR* products is routinely performed by agarose gel electrophoresis (6, 11), denaturing polyacrylamide gel electrophoresis (PAGE) (1, 2), or capillary electrophoresis (CE) (10). Electrophoresis techniques are time consuming methods requiring considerable manual effort. Here, we introduce an automated technology, with minimal manual intervention, for gender identification using DNA chromatography after amplification of a section of the X-Y homologous gene amelogenin. The WAVE System is suitable for accurate and rapid sizing of doublestranded (ds) DNA fragments (4, 5, 7). Under non-denaturing conditions ds DNA fragments/PCR* products are separated in a sequence independent manner by DNA chromatography (IP RP HPLC) on a DNASep cartridge (Transgenomic, San Jose, CA). The stationary phase in this cartridge is a nonporous alkylated poly (styrene-divinylbenzene) matrix. Chromatography is performed using a two-eluent system. Eluent A is aqueous 0.1 M triethylammonium acetate (TEAA) at pH 7.0 and eluent B is aqueous 0.1

M TEAA at pH 7.0 containing 25% by volume acetonitrile (ACN). Aliquots from PCR* are injected directly, without the need for sample preparation, e.g.: purification or mixing with loading buffer. Ds DNA is eluted in a sequence independent, but size dependent manner with a gradient increasing in eluent B. Thus, short DNA fragments elute at a lower ACN concentration and with a shorter retention time (RT) than longer fragments. Chromatograms are recorded at either 260 nm (UV) or at the emission wavelength of the fluorescent dye used for labeling. Integration of peak areas provides a means of quantification (8). Gender identification on human genomic DNA samples can be performed by amplifying a segment of the X-Y homologous gene amelogenin using the GenePrint Sex Identification System from Promega (1, 2). The amelogenin specific primer set in this system amplifies a 212-base pair (bp) Xspecific and a 218-bp Y-specific product (6). The GenePrint Sex Identification System is available for silver detection (2) and for fluorescence detection (1). The latter contains a set of amelogenin specific primers in which one of the primers is labeled with fluorescein. Both systems are supplied with the appropriate amelogenin ladder. Here, we show that the WAVE System is suitable for the analysis of PCR* products from both GenePrint Sex Identification Systems.

AN113: Gender Determination by Amelogenin PCR and Product Analysis by DNA Chromatography

Results and Discussion Sex Identification with Unlabeled Primers

Amplification of a segment of the X-Y homologous gene amelogenin using the primers supplied with the GenePrint Sex Identification System yield a 212-bp X-specific and a 218-bp Y-specific product. PCR* products were analyzed on the WAVE System using the gradient shown in Table 1. Two peaks with retention times of 5.00 min (+/- 0.02 min) and 5.16 min (+/- 0.03 min) are characteristic for genomic DNA derived from male donors (Fig. 1A). Figure 1B shows at a RT of 5.00 min (+/- 0.02 min) a peak representative for the X-specific 212-bp PCR* product amplified from genomic DNA of a female. The retention times of the 212- and 218-bp fragments correlate well with the retention times of two external sizing standards (174 and 254 bp Fig. 1C) as well as with the amelogenin ladder (Fig. 1D) provided by Promega. It is apparent from Figure 1 (A, B) that the 212- and 218-bp PCR* products are sufficiently and reproducibly resolved on the DNASep cartridge. Thus, the WAVE System may be used for gender identification in combination with the GenePrint Sex Identification System. Also, peak areas may be integrated permitting quantification of amplified fragments (8). If the endpoint of PCR* amplification occurs in the exponential phase of the PCR*, the copy number of amplified genes in the genome may be inferred based on the intensity of the product peak.

Table 1 Gradient Program for Unlabeled PCR* Product Analysis

Time [min] Eluent A [%] 0.1 M TEAA 0.0 54 0.5 49 5.0 40 5.1 0 5.6 0 5.7 54 8.2 54 Eluent B [%] Flow Rate [ml/min] 0.1 M TEAA, 25% ACN 46 0.9 51 60 100 100 46 46

Figure 1. Sex Identification with Unlabeled Primers. (A) Amelogenin specific 212- and 218-bp PCR* products from a male (XY) subject (B) 212-bp PCR* product from a female (XX) subject (C) External sizing standard 174- and 257-bp restriction fragments from a HaeIII digested pUC18 plasmid (D) Amelogenin ladder (Promega, Madison, WI), 212and 218-bp fragments. PCR* reactions were performed on a PTC-100 thermoc ycler (MJ Research, Inc., Watertown, MA) using AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA). Approximately 100 ng of human genomic DNA was used per reaction. PCR* conditions: 2 min at 96C; (1 min at 94C, 1 min at 60C, 1.5 min at 70C) for 35 cycles; 7 min at 70C, 4 C soak. 5 L aliquots of the PCR* were loaded directly, without sample pr eparation, into the autosampler of the WAVE System. The gradient pr ogram used is listed in Table 1. The temperature for separation was 55C (non-denaturing conditions) and chromatograms were recorded at 260 nm.

Table 2 Gradient Program for Primer Analysis and Purification

Time [min] Eluent A [%] 0.1 M TEAA 0.0 95 0.5 80 5.0 47 5.1 0 5.6 0 5.7 95 8.2 95 Eluent B [%] Flow Rate [ml/min] 0.1 M TEAA, 25% ACN 5 0.9 20 53 100 100 5 5

Analysis and Purification of Fluorescein Labeled Primers

Analysis of Promegas amelogenin ladder unexpectedly revealed the presence of four DNA fragments with significantly different retention times (Fig. 2C). The same ladder analyzed by denaturing PAGE (1) exhibits only two bands corresponding to the expected 212- and 218-bp PCR* products. Therefore, it was suspected that the additional peaks
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in the chromatogram of the amelogenin ladder could be due to heterogeneity in the fluorescent label. Analysis of the primer pair supplied with the fluorescence detection kit, indeed, revealed the presence of two chromatographically distinct forms of the fluorescently labeled primer. While the chromatogram of the primer pair record at 260 nm (Fig. 3A) shows three peaks, the chromatogram recorded at the emission wavelength of fluorescein (528 nm) (Fig. 3B) shows only two

peaks. Absence of the peak with the shortest retention time (3.74 min) in the fluorescence chromatogram, as compared to the UV chromatogram, identifies this peak as the unlabeled primer. Thus, peaks at 4.85 and 5.21 min in the UV chromatogram represent two distinct isoforms of the labeled primer. The fluorescein labeled primer has a significantly longer retention time than unlabeled primer. This is due to the hydrophobic nature of the fluorescein molecule, which increases

AN113: Gender Determination by Amelogenin PCR and Product Analysis by DNA Chromatography

the overall affinity of the labeled, as compared to the unlabeled, oligonucleotide for the hydrophobic matrix of the DNASep cartridge. Similar observations have been reported in the literature for other fluorescent labels (9). In order to conduct gender identification using the fluorescein labeled primer, it was necessary to purify the primers supplied by Promega before use in PCR*. In brief, a large amount of primer mix (50 L) was separated by DNA chromatography and peaks at 3.74 and 5.21 min were collected manually. Peak fractions were then dried and resuspended in TE buffer, checked for purity by DNA chromatography, quantified, and used in PCR*. Oligonucleotides labeled with the fluorophores 5-carboxy-fluorescein, 2,7-dimethoxy-4,5-dichloro-6carboxyfluorescein, N,N,N,Ntetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine (FAM, JOE, TAMRA, and ROX) yield single peaks for labeled oligonucleotide and the corresponding PCR* products and should be considered as alternatives to fluorescein for labeling (9, 3).

Figure 2. Sex Identification with Purified Labeled Primers. (A) Amelogenin specific 212and 218-bp PCR* products from a male (XY) subject (B) Amelogenin specific 212-bp PCR* product from a female (XX) subject (C) Fluorescein labeled amelogenin ladder from Promega with peak doubling due to the presence of two isomers of the fluorescein label. PCR* r eactions wer e performed with IP RP HPLC purified primers under the conditions described in Figure 1. The gradient program used is listed in Table 3. The temperature for separation was 55C (non-denaturing conditions) and chromatograms were recorded at 528 nm.

Table 3 Gradient Program for Labeled PCR* Product Analysis

Time [min] Eluent A [%] 0.1 M TEAA 0.0 54 0.5 49 6.5 42 6.6 0 7.1 0 7.2 54 8.7 54 Eluent B [%] 0.1 M TEAA, 25% ACN 46 51 58 100 100 46 46 Flow Rate [ml/min] 0.9

Sex Identification with Purified Labeled Primers

Amplification of the X-Y homologous gene amelogenin using the purified primers yielded the expected 212-bp X-specific and the 218-bp Y-specific PCR* products (Fig. 2A, B). A single peak with a retention time of 7.53 min (+/- 0.09 min) (Fig. 2B) represents the fluorescein labeled X-specific 212-bp PCR* product amplified from genomic DNA of female donors. Peaks with retention times of 7.52 min (+/0.05 min) and 7.74 min (+/- 0.06 min) represent the 212-bp and 218bp fluorescently labeled X- and Yspecific PCR* products obtained after amplification from genomic DNA of male donors. Chromatograms A and B in Figure 2 clearly show that the fluorescein labeled PCR* products, differing in size by only 6 bp, are sufficiently and reproducibly resolved by DNA chromatography on the WAVE System.
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Figure 3. Analysis and Purification of Fluorescein Labeled Primers. UV chromatogram recorded at 260 nm (top) and fluorescence chromatogram recorded at 528 nm (bottom) of the fluorescein labeled amelogenin specific primer pair. Peak (1) unlabeled primer, peaks (2) and (3) fluor escein labeled primer. Due to the presence of two isomers of fluorescein with different chromatographic properties the signal for the fluorescein labeled primer is split into two peaks. Serial arrangement of UV (first) and fluorescence detector (second) results in a relative shift of retention times in the fluorescence chromatogram of +0.3 min, e.g.: void volume between the detectors is 270 L. The gradient program used for primer analysis is listed in Table 2. Column temperature for separation was 50C.

AN113: Gender Determination by Amelogenin PCR and Product Analysis by DNA Chromatography

Conclusion
The WAVE System is suitable for size based separation of unlabeled and labeled ds DNA fragments that differ in size by only a few bp. Thus, this system may be used for product analysis in PCR* based assays that rely on the separation of PCR* products of different size. Also, PCR* products may be quantified by integration of peak areas and thus, permit conclusions about the original genomic copy

numbers of the gene fragments amplified. Compared with current gel-based methods, size-based separation of DNA fragments on the WAVE System omits sample preparation (mixing with loading buffer), gel preparation, pipetting, electrophoresis, visualization of DNA, photographic recording, and clean-up. Unlike analysis using gel-based technologies, product analysis on the WAVE System

requires only minutes of manual manipulation for up to 192 samples. Each sample can be analyzed in less than 10 minutes. Therefore, this technology provides a reliable, cost and labor effective alternative to PAGE and CE for sex identification with the GenePrint System and related applications that rely on size based separation of DNA fragments.

References
1 . GenePrint Fluorescent STR Systems Technical Manual, Promega Corporation, Madison, WI, USA, http:// www.promega.com/tbs/tmd006/ tmd006.html. 2. GenePrint STR System (Silver Stain Detection) Technical Manual, Promega Corporation, Madison, WI, USA, http:// www.promega.com/tbs/tmd004/ tmd004.html. 3. Hecker, K. H., Taylor, P. D., and Gjerde, D. T. (1999) Mutation detection by denaturing DNA chromatography using fluorescently labeled polymerase chain reaction products. Anal. Biochem. 272, 156-164. 4. Hecker, K. H., Turpie, B., and Kuklin, A. (1999) Optimization of cloning efficacy by pre-cloning DNA fragment Analysis. BioTechniques 26(2), 216.

5. Huber, C. G., Oefner, P. J., and Bonn G. K. (1995) Rapid and accurate sizing of DNA fragments by ion-pair chromatography on alkylated nonporous poly (styrenedivinylbenzene) particles. Anal. Chem. 67, 578. 6. Mannucci, A., Sullivan, K. M., Ivanov, P. L., and Gill, P. (1994) Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin. Int. J. Legal Med. 106(4), 190193. 7. Munson, K., Haefele, R., Kuklin, A., Gjerde, D., and Taylor, P. (1998) Sizing of DNA fragments with the WAVE DNA Fragment Analysis System. Transgenomic, Inc., San Jose, CA. Application Note # 107, http:// www.transgenomic.com/pdf/ AN107.pdf.

8. Munson, K., Haefele, R., and Taylor, P. (1998) Competitive quantitative PCR* on the WAVE DNA Fragment Analysis System. Transgenomic, Inc., San Jose, CA. Application Note #105, http:// www.transgenomic.com/pdf/ AN105.pdf. 9. Oefner, P. J., Huber, C. G., Umlauft, F., Berti, G. N., Stimpfl, E., and Bonn, G. K. (1994) High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids. Anal. Biochem. 223(1), 39-46. 1 0 . Pouchkarev, V. P., Shved, E. F., and Novikov, P. I. (1998) Sex determination of forensic samples by polymerase chain reaction of the amelogenin gene and analysis by capillary electrophoresis with polymer matrix. Electrophoresis 19, 76. 1 1 . Sullivan, K. M., Mannucci, A., Kimpton, C. P., and Gill, P. (1993) A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. BioTechniques 15(4), 636-638, 640-641.

* PCR is a process covered by patents issued and applicable in certain countries. Transgenomic does not encourage or support the unauthorized or unlicensed use of the PCR process. WAVE and DNASep are registered trademarks of Transgenomic, Inc. Transgenomic, Transforming the World, and the logo are trademarks of Transgenomic, Inc.

12325 Emmet Street Omaha, NE 68164 Tel 402.452.5400 Fax 402.452.5401 www.transgenomic.com
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2000 by DNA Chromatography AN113: Gender Determination by Amelogenin PCR and Product Analysis Transgenomic, Inc. Printed in the U.S.A.