Anal. Chem. 1999, 71, 3974-3976
Gender Identification by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Nelli I. Taranenko,† Nicholas T. Potter,‡ Steve L. Allman, Valeri V. Golovlev,† and Chung H. Chen*
Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6378
A rapid, simple, and reliable gender determination of human DNA samples was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Detection sensitivity reached 0.01 ng or less for DNA samples. Gender identification of human DNA samples is often an essential component for the correct forensic DNA analysis and prenatal diagnosis of X-linked inherited disorders. To date, most DNA-based gender tests have relied on the polymerase chain reaction (PCR) amplification of the amelogenin loci (AMEL) on the X and Y chromosomes. As the X- and Y-derived amplicons differ in size, the detection of one or two distinct PCR products is generally interpreted as indicating the absence or presence of Y-derived sequences.1-3 Recently, this methodology has been further refined to include coamplification of AMEL with the sex-determining region Y locus (SRY) to decrease the risk of incorrectly genotyping a sample due to the presence of a recently described deletion polymorphism on the Y-derived copy of the amelogenin gene.1 In an attempt to develop rapid, reliable, and inexpensive DNA analysis technologies, we have validated the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the analysis of PCR products generated from the coamplification of the AMEL and SRY genes. With this approach, the time of sample analysis (excluding sample preparation and the single PCR reaction) is less than 1 s as compared to hours for conventional agarose or polyacrylamide gel electrophoresis. In addition, unlike capillary or polyacrylamide gel electrophoresis, no fluorescent dye or radioactivity is required. Furthermore, the sensitivity of MALDI-TOF-MS was at least 1 order of magnitude greater than what has been previously reported for fluorescentbased analyses.4 Since the development of MALDI time-of-flight mass spectrometry, many research groups including ourselves have succeeded in analyzing various protein and DNA products.5-10 The † Postdoctoral Fellows. Oak Ridge Associated Universities, Oak Ridge, TN 37831. ‡ Present address: University of Tennessee Medical Center, Knoxville, TN 37920. (1) Sullivan, K. M.; Mannucci, A.; Kimpton, C. D.; Gill, P. Biotechniques 1993, 15, 636-641. (2) Faeman, M.; et al. Nature 1997, 385, 212-213. (3) Saiki, R. K.; et al. Science 1988, 239, 487-497. (4) Mannucci, A.; Sullivan, K. M.; Ivanov, P. L.; Gill, P. Int. J. Leg. Med. 1994, 106, 190-193.
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most difficult problem in the mass spectroscopic analysis of large bioorganic molecules is maintaining the integrity of the analyte molecules. Recent developments clearly indicate that laser desorption mass spectrometry can be used for large DNA and RNA analysis. It was also discovered that only ion peaks that correspond to the molecular weight of single-stranded DNA were obtained, even though double-stranded DNAs are put into the samples for MALDI.11 In addition to the measurements of the sizes of DNAs, MALDI has also been used for sequencing short oligonucleotides.12-18 Disease diagnosis based on the detection of point mutation,19 base deletion,20 and trinucleotide repeat expansion21 has also been demonstrated. In this work, we extend the application of MALDI to DNA-based gender identification. EXPERIMENTAL SECTION Preparation of DNA Samples. Genomic DNA was extracted from peripheral blood leukocytes utilizing Puregene DNA isolation (5) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (6) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 436439. (7) Zhao, S.; Somajajula, K.; Sharkey, A. G.; Hercules, D. M.; Hillenkamp, F.; Karas, M.; Ingendoh, A. Anal. Chem. 1991, 63, 450-453. (8) Tang, K.; Allman, S. L.; Jones, R. B.; Chen, C. H.; Araghi, S. Rapid Commun. Mass. Spectrom. 1993, 7, 435-439. (9) Wu, K. J.; Steding, A.; Becker, C. H. Rapid Commun. Mass Spectrom. 1993, 7, 142-146. (10) Nelson, R. W.; Thomas, R. M.; Williams, P. Rapid Commun. Mass Spectrom. 1990, 4, 348-351. (11) Tang, K.; Allman, S. L. Allman; Chen, C. H.; Chang, L. Y.; Schell, M. Rapid Commun. Mass Spectrom. 1994, 8, 183-186. (12) Koster, H.; Tang, K.; Fu, D.; Braun, A.; van der Boom, D.; Smith, C. L.; Cotter, R. J.; Cantor, C. R. Nature Biotechnol. 1996, 14, 1123-1128. (13) Mouradian, S.; Rank, D. R.; Smith, L. M. Rapid Commun. Mass Spectrom. 1996, 10, 1475-1478. (14) Shaler, T. A.; Tan, Y.; Wickham, J. N.; Wu, K. J.; Becker, C. H. Rapid Commun. Mass Spectrom. 1995, 9, 942. (15) Roskey, M. Y.; Tuhasz, P.; Smirnov, I. P.; Takach, E. J.; Martin, S. A.; Haff, L. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4774. (16) Taranenko, N. I.; Chung, C. N.; Zhu, Y. F.; Allman, S. L.; Golovlev, V. V.; Isola, N. R.; Martin, S. A.; Haff, L. A.; Chen, C. H. Rapid Commun. Mass Spectrom. 1997, 11, 386. (17) Taranenko, N. I.; Allman, S. L.; Golovlev, V. V.; Taranenko, N. V.; Isola, N. R.; Chen, C. H. Nucleic Acids Res. 1998, 26, 2488. (18) Fu, D.; Tang, K.; Braun, A.; Reuter, D.; Darnhofer-Demar, B.; Little, D. P.; O’Donnell, M. J.; Centor, C. R.; Koster, H. Nature/Biotechnol. 1998, 16, 381-384. (19) Taranenko, N. I.; Matteson, K. J.; Chung, C. N.; Zhu, Y. F.; Chang, L. Y.; Allman, S. L.; Haff, L.; Martin, S. A.; Chen, C. H. Gen. Anal.: Biomol. Eng. 1996, 13, 87-94. (20) Chang, L. Y.; Tang, K.; Schell, M.; Ringelberg, C.; Matteson, K. J.; Allman, S. L.; Chen, C. H. Rapid Commun. Mass Spectrom. 1995, 9, 772-774. (21) Taranenko, N. I.; Potter, N. T.; Allman, S. L.; Golovlev, V. V.; Chen, C. H. Genet. Anal.: Biomol. Eng. 1999, 15, 25-31. 10.1021/ac990150w CCC: $18.00
© 1999 American Chemical Society Published on Web 08/12/1999
Table 1. Sex-Typing Markers for Human Genomic DNA gene
primer names
primer sequence
product size (bp)
ref
amelogenin
AMEL-A AMEL-B F11 R7
5′-CCCTGGGCTCTGTAAAGAATAGTG-3′ 5′-ATCAGAGCTTAAACTGGGAAGCTG-3′ 5′-ATAAGTATCGACCTCGTCGGAA-3′ 5′-GCACTTCGCTGCAGAGTACCGA-3′
X ) 106 Y ) 112 Y ) 93
1
SRY
kits (Gentra Systems, Minneapolis, MN). All samples were quantitated by UV spectrophotometry using standard procedures. The primer pairs for the amelogenin and SRY loci are provided in Table 1. Coamplification of selected genomic templates was performed essentially as described by Santos et al.,22 except the final reaction volume was increased to 25 µL. Following amplification, PCR products from 4 × 25 µL reactions pooled and DNA purified using QIAquick spin columns (Qiagen, Inc., Santa Clara, CA). The final product was then precipitated in alcohol, centrifuged, and dried under vacuum and then the pellet was resuspended in 2-5 µL of sterile, deionized water. The sample for mass spectrometry analysis was prepared by mixing 1 µL of aqueous analyte solution and 1 µL of matrix solution. A 1-µL sample of this mixture was spotted on a stainless steel plate and dried by a forced nitrogen gas jet at ambient temperature. The dried sample was loaded into the TOF-MS immediately without further exposure to the atmosphere. The matrix was a mixture of 0.3 M 3-hydroxypicolinic acid, 0.5 M picolinic acid, and 0.3 M ammonium fluorate (molar ratio 9:1:1). Facility for MALDI for Sex Determination Analysis. A linear time-of-flight mass spectrometer (Voyager, PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser for desorption and ionization was used in obtaining mass spectra of DNA samples. The typical laser fluence was measured between 45 and 65 mJ/cm2. The acceleration voltage was 28 125 V. The pressure of the TOF-MS vacuum chamber was typically 1.5 × 10-7 Torr. The signals of negative DNA ions were collected and digitized by a digital oscilloscope (Tektronix 520A) controlled by a laboratory computer. A delayed pulsed ion extraction device23-25 was installed to improve mass resolution. However, resolution improvement for large DNAs used in this work is not very significant. RESULTS AND DISCUSSION DNA has become broadly utilized in forensic analysis, disease diagnosis, paternity determination, and wildlife protection due to the fact that every individual’s DNA structure is nearly identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphism (RFLP).26 However, the RFLP analysis has limitations such as sensitivity and failure to type degraded samples, and it is time-consuming. With PCR amplification, the quantity of genomic samples needed for analysis can be significantly reduced. (22) Santos, F. R.; Pandya, A.; Tyler-Smith, C. Nat. Genet. 1998, 18, 103. (23) Christian, N. P.; Colby, S. M.; Giver, L.; Houston, T.; Arnold, R. J.; Ellington, A. D.; Reilly, J. P. Rapid Commun. Mass Spectrosc. 1995, 9, 1061-1065. (24) Brown, R. S.; Lenon, J. J. Anal. Chem. 1995, 67, 1998-2003. (25) Vental, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrosc. 1995, 9, 1044-1050. (26) Jeffrey, A. J.; Wilson, V.; Thein, S. L. Nature 1985, 314, 67.
4
Figure 1. Negative ion mass spectra of sex-specific coamplification (AMEL X/Y plus SRY) locus for (A) female, (B) male, and (C) female and male samples, respectively, with a concentration of 10 ng. The molar ratio of female plus male samples is 1:1. The laser wavelength was 337 nm. The laser fluence was 65 mJ/cm2.
DNA-based sex determination is an important application in areas such as forensics, archaeology, and in some cases prenatal genotyping. The appropriate conditions allowing coamplification of both amelogenin and SRY genes, DNA sequencing was determined using DNA from whole blood samples taken from unknown individuals. Coamplification of genomic templates are listed in Table 1. Male DNA generates three products of masses M1 ) 28 737 (93 bp), M2 ) 32 754 (106 bp), and M3 ) 34 608 (112 bp). Coamplification of female DNA generates only one product of the mass M2 ) 32 754 (106 bp). As shown in Figure 1, genotyping of male and female samples is unambiguous. Figure 1 shows examples of mass spectra of PCR products for (A) female gender, (B) male gender, and (C) mixture of male and female gender. Data in Figure 1 are presented for samples loaded with 10 ng of DNA species. Figure 2 demonstrates the sensitivity of conventional polyacrylamide gel electrophoretic detection of the AMEL Y and SRY amplicons. In both instances, the detection limit for malespecific sequences was 0.1 ng. A special interest in forensic analysis is detection of a trace amount of male-specific gender in the female sample. Such detection can be performed by demonstrating the presence of male-derived products using the male-specific products 93 and Analytical Chemistry, Vol. 71, No. 18, September 15, 1999
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Figure 2. A 10% polyacrylamide gel analysis of PCR products obtained after the amplification of a mixture of female and male DNA with the AMEL and SRY primer pairs (A) or the SRY primer pair alone (B). PCR conditions were as described above. For each gel, 15 µL of PCR product was visualized after ethidium bromide staining and UV transillumination. (A) lane 1, blank; lane 2, molecular weight markers; lane 3, blank; lane 4, 10 ng of female DNA + 10 ng of male DNA; lane 5, 10 ng of female DNA + 1 ng of male DNA; lane 6, 10 + 0.1 ng; lane 7, 10 + 0.01 ng; lane 8, 10 + 0.001 ng. (B) Same lane designation as in (A).
112 bp. The MALDI spectrum of a composite sample containing male and female species is shown in Figure 3. The amount of female DNA in the PCR was 10 ng while the amount of malespecific DNA was 0.01 ng. Figure 3 shows that as little as 0.1% of male DNA in the female DNA sample can be detected. It also indicates that detection sensitivity can reach 0.01 ng or less which is about 1 order of magnitude higher than gel electrophoresis. CONCLUSION In this work we evaluated MALDI sensitivity for the detection of SRY and AMEL Y coamplification products for DNA gender identification. MALDI was found to be more sensitive than the conventional polyacrylamide gel electrophoretic detection. Comparing with gel electrophoresis, MALDI also provided better dynamic range, thus demonstrating better performance for detecting male-specific coamplification products in a female sample.
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Figure 3. Negative ion mass spectrum of PCR amplified product of female (10 ng) DNA with 0.01 ng of male DNA. The laser fluence was 65 mJ/cm2.
ACKNOWLEDGMENT This research is sponsored by the National Institute of Justice, National Institute of Health, and the Office of Biological and Environmental Research, U.S. Department of Energy under Contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corp. The loan of a Voyager mass spectrometer by PerSeptive Biosystems is also acknowledged. N.T.P. acknowledges the funding provided by the State of Tennessee, Departments of Health and Environment, and Mental Health and Mental Retardation. Thanks to Darlene Holt for manuscript preparation. Received for review February 10, 1999. Accepted June 25, 1999. AC990150W
