Spectral Measurements of Intercalated PCR-Amplified Short Tandem

Protocol 903223, PE-ABD, Foster City, CA). Master mixes of 30. μL were generated containing 83.5 mM KCl, 16.7 mM HCl, 2.5. mM MgCl2, 0.33 mM of each ...
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Anal. Chem. 1998, 70, 4514-4519

Spectral Measurements of Intercalated PCR-Amplified Short Tandem Repeat Alleles Michael A. Marino,*,† Joseph M. Devaney,‡ P. Ann Davis,† Jonathan K. Smith,† and James E. Girard‡

Operational Genetics Laboratory, Center for Medical and Molecular Genetics, Armed Forces Institute of Pathology, Washington, D.C. 20306, and Department of Chemistry, American University, Washington, D.C. 20016

Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNAdye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bisintercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration. The term intercalator was first used by Lerman in the 1960s to define a molecule containing a planar aromatic structure which inserts itself between two base pairs (bp) of double-stranded DNA (dsDNA).1 Theoretically, the double-helix structure saturated with intercalator should yield a binding stoichiometry of one intercalator molecule per base pair. Simple (monomeric) intercalator molecules such as EtBr and thiazole orange (TO), which have reduced steric constraints, could potentially intercalate with a ratio of 1/1 (dye/bp). However, solution studies determined a satura* Address correspondence to this author: Center for Medical and Molecular Genetics, Building 101, 1413 Research Blvd., Rockville, MD 20850 (Tel +301 319-0200; fax +301-295-9507; E-mail [email protected]). † Armed Forces Institute of Pathology. ‡ American University. (1) Lerman, L. S. J. Mol. Biol. 1961, 3, 18.

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tion maximum of one intercalator molecule per two base pairs. This observation led to the neighbor exclusion principle.2 This states that the intercalator can bind at alternate base pair sites on DNA with a maximum of an intercalator between every second site. The neighbor exclusion phenomenon gave rise to the development of bis-intercalators.3-5 These intercalators are two intercalating ring systems covalently attached to a linking chain of variable length. Bis-intercalators become more rigid upon insertion into the DNA helix. Intercalation dyes developed by Glazer and Rye are dimers of the intercalator compounds that have a higher binding affinity for DNA than the corresponding monomeric dye.3 The benzothiazolium-4-quinolinium dimer of thiazole orange (TOTO-1) and other bis-intercalators have demonstrated fluorescence enhancement upon intercalation.5,6 These intercalator dyes are used with many fluorescence techniques, such as flow cytometry,7 fluorescence microscopy,8 multiwell scanner fluorometers,9,10 and capillary electrophoresis laser-induced fluorescence detection (CE-LIF).6,11-15 An increase in the fluorescence emission upon binding makes these compounds ideal for nucleotide staining, quantitation, and probe applications.5 Studies of changes in fluorescence intensity of the intercalators and absorbance changes of the DNA have included samples containing calf thymus DNA5 and synthetic oligonucleotides.16,17 To determine the changes in absorbance and (2) Crothers, D. M. Biopolymers 1968, 6, 575-584. (3) Blackburn, G. M.; Gait, M. J. Nucleic Acids in Chemistry & Biology; Oirl Press: New York, 1990; Chapter 8. (4) Glazer, A. N.; Rye H. S. Nature 1992, 359, 859-861. (5) Haugland, R. P. Handbook of fluorescence probes and research chemicals; Molecular Probes: Eugene, OR, 1992; Set 31, pp 221-229. (6) Figeys, D.; Arriage, E.; Renborg, A.; Dovichi, N. J. J. Chromatogr. A 1994, 669, 205-216. (7) Product information, flow cytometry reference standards, Molecular Probes, Eugene, OR. (8) Smith, S. B.; Aldridge, P. C.; Callis, J. B. Science 1989, 243, 203-206. (9) Glazer, A. N.; Peck, K.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3851-3855. (10) Clark, B. K.; Mathies, R. A. Anal. Biochem. 1993, 215, 163-170. (11) Clark, B. K.; Sepaniak, M. J. J. Microcolumn Sep. 1993, 5, 275-282. (12) Skeidsvoll, J.; Ueland, P. M. Anal. Biochem. 1995, 231, 359-365. (13) Srinivasan, K.; Morris, S. C.; Girard, J. E.; Kline, M. C.; Reeder, D. J. Appl. Theor. Electrophor. 1993, 3, 235-239. (14) McCord, B. R.; McClure, D. L.; Jung, J. M. J. Chromatogr. A 1993, 652, 75-82. (15) Zhu, H.; Clark, S. M.; Benson, S. C.; Rye, H. S.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1994, 66, 1941-1948. (16) Larsson, A.; Carlsson, C.; Jonnsson, M. Biopolymers 1995, 36, 153-167. (17) Jacobsen, J. P.; Pedersen, J. B.; Hansen, L. F.; Wemmer, D. E. Nucleic Acids Res. 1995, 23, 753-760. 10.1021/ac980526q CCC: $15.00

© 1998 American Chemical Society Published on Web 10/07/1998

Table 1. Characteristics of PCR-Amplified STR Loci locus

repeat sequence

no. of Alleles

size of alleles (bp)

range of AT% composition

TH01 F13A01 vWFA

AATG AAAG AGAT

5-11 3.2-20 11-21

154-178 181-247 127-167

47.4-51.1 68.3-69.7 66.7-69.9

fluorescence of the DNA/dye complexes, this study used polymerase chain reaction (PCR), amplified short tandem repeat (STR) alleles. These STR loci are highly polymorphic and commonly used for forensic identifications using electrophoresis techniques.18 Prior to quantitative analysis, the DNA fragments needed to be separated and purified. Ion-pairing reversed-phase (IPRP) high-performance liquid chromatography (HPLC) was used to separate and isolate alleles of the STR loci of THO1, F13A01, and vWFA31 before absorbance and fluorescence measurements were made.19 A spectrophotometer was used to quantitate the purified PCR products and determine the absorbance changes of the DNA after intercalation. The spectrofluorometer was used to make fluorescence measurements of the intercalation dyes at the appropriate emission wavelength. The fluorescence measurements were used to evaluate intensity changes upon binding to the DNA. In the discussion that follows, IPRP-HPLC-purified homozygote alleles of the loci THO1 (6,6), F13A01 (8,8), and vWFA31 (17,17) were mixed individually with the intercalators TOTO-1, TOPRO1, YOYO-1, and YOPRO-1. For each set of DNA-intercalator combination, absorbance and fluorescence measurements were performed, determining the maximum base pair-to-dye ratio for each STR allele. EXPERIMENTAL SECTION Samples. DNA was extracted from 300 µL of whole blood using the Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN) following the manufacturer’s protocol. The yields of purified genomic DNA isolated from nucleated cells fell within the range of 5-15 µg for 300 µL of whole blood. The samples of PCR-amplified STR loci were amplified according to the manufacturer’s instruction (ABI Prism STR Primer Set Protocol 903223, PE-ABD, Foster City, CA). Master mixes of 30 µL were generated containing 83.5 mM KCl, 16.7 mM HCl, 2.5 mM MgCl2, 0.33 mM of each deoxynucleoside triphosphate, 0.2 µM of each primer pair, 60 µg/mL bovine serum albumin, and 2 units of Taq DNA polymerase gold (Perkin-Elmer, Norwalk, CT). Added to the master mix was 20 µL of water containing 10 ng of genomic DNA (total volume of PCR, 50 µL). The samples were amplified using the Perkin-Elmer 9600 thermal cycler [10 min at 95 °C, 28× (45 s at 94 °C, 1 min at 54 °C, 2 min ramp, 1 min at 72 °C), 10 min at 72 °C, 4 °C soak]. Table 1 summarizes information on the three STR loci: TH01, F13A01, and vWFA31. Information detailing the entire STR fragment was obtained from GenBank (http:// www2.ncbi.nlm.nih.gov). The STR loci have alleles ranging in size from 127 to 247 bp, and the adenine and thymine (AT) content ranges from approximately 50 to 70% (see Table 1). (18) Lygo, J. E.; Johnson, P. E.; Holdaway, D. J.; Woodroffe, S.; Whitaker, J. P.; Clayton, T. M.; Kimpton, C. P.; Gill, P. Int. J. Leg. Med. 1994, 107, 77-89.

Table 2. STR Alleles’ Size and Base Composition locus

no. of alleles

base pairs

% AT

THO1 F13A01 vWFA

6 8 17

158 199 151

48 69 68

Allele Purification and Isolation. The IPRP DNA purification was described previously.19 In brief, HPLC isolation was performed using a Dionex DX-500 poly(ether ether ketone) (PEEK) system with UV detection at 260 nm. The DNAsep column, 4.6 mm × 50 mm (Sarasep, Inc., Santa Clara, CA), and the 50-µL sample loop were heated to 55 °C using a Eppendorf model CH-30 oven (Madison, WI). The mobile phase consisted of HPLC grade acetonitrile (ACN) (EM Science, Gibbstown, NJ) and triethylammonium acetate (TEAA) (Applied Biosystems, Foster City, CA). The mobile phase’s pH was adjusted to 7.0 using glacial acetic acid (Mallinckrodt Specialty Chemicals, Paris, KY).20 Instrumentation. All individual STR alleles were sequenced using the ABI Prism 310 genetic analyzer (PE-ABD, Foster City, CA) (data not shown). The sequencing determined the exact base composition of the alleles used in this study (Table 2).19 Absorption and emission spectral measurements were acquired using the Beckman DU 65 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA) and model FP-777 spectrofluorometer (Jasco Corp., Tokyo, Japan), respectively. These systems were allowed to warm for 1 h before any measurements were made. Intercalation Dyes. The intercalator dyes were purchased from Molecular Probes (Eugene, OR). Dyes are chosen with excitation maxima near the argon laser wavelengths 488 and 514 nm. All dilutions of intercalators were made fresh daily. RESULTS AND DISCUSSION UV Spectral Measurements. DNA fragments of purified homozygote alleles were quantitated by UV absorbance measurements at 260 nm, using the DU-65 spectrophotometer (Beckman Instruments). A calibration plot of DNA concentration vs absorbance was generated using the low DNA mass ladder (LDML) (GibcoBRL, Gaithersburg, MD) (data not shown). The absorbance readings were plotted using the SPSS (ver. 6.1) statistical analysis program (SPSS Inc., Chicago, IL). The components of the line equation (slope and y-intercept) were used to calculate the concentration of the PCR product. The IPRP-HPLC-purified alleles’ concentrations ranged from 1 to 6 ng/µL, with collection volume ranging in size from 50 to 120 µL. To conserve PCR product and maintain maximum response, samples containing 4 ng/µL of DNA were used for the absorbance readings. Samples containing less than 4 ng/µL DNA were concentrated by Centricon 100 microconcentrator cartridges (Amicon Inc., Beverly, MA) or a Heto Spinvac (ATR Inc., Laurel, MD), spun to near dryness, and resuspended with deionized water. The alleles were prepared in stock solutions to ensure a uniform concentration of 4 ng/µL DNA for each sample. All (19) Marino, M. A.; Devaney, J. M.; Smith, J. K.; Girard, J. E. Electrophoresis 1998, 19, 108-118. (20) Oefner, P. J.; Bonn, G. K.; Huber, C. G.; Nathakarnkitkool, S. J. Chromatogr. 1992, 625, 331-340.

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Figure 1. UV absorbance of vWFA locus intercalated with TOPRO-1. The absorbance measurements are of allele 17 of locus vWFA intercalated with TOPRO-1. For each set of measurements, both samples and blanks were prepared. The following wavelengths were monitored: 260 nm ([) DNA and (]) blank, 280 nm (2) DNA and (4) blank, and 320 nm (b) DNA and (O) blank. Table 3. UV Absorbance at 260 nm of STR Loci locus

∞ 50/1 25/1 10/1 5/1 2/1 (bp/dye) (bp/dye) (bp/dye) (bp/dye) (bp/dye) (bp/dye)

THO1 F13A01 vWFA blank

0.0499 0.0486 0.0498 0.0020

Intercalated with TOTO-1 0.0461 0.0545 0.0527 0.0422 0.0401 0.0475 0.0499 0.0605 0.0548 0.0075 0.0056 0.0071

0.0596 0.0551 0.0638 0.0110

0.0826 0.0715 0.0801 0.0281

THO1 F13A01 vWFA blank

0.0445 0.0396 0.0452 0.000

Intercalated with TOPRO-1 0.0440 0.0444 0.0441 0.0413 0.0383 0.0411 0.0447 0.0451 0.0448 0.0018 0.0029 0.0036

0.0457 0.0432 0.0464 0.0085

0.0532 0.0522 0.0540 0.0157

THO1 F13A01 vWFA blank

0.0397 0.0373 0.0403 0.0039

Intercalated with YOYO-1 0.0401 0.0429 0.0477 0.0508 0.0452 0.0475 0.0421 0.0461 0.0514 0.0023 0.0050 0.0117

0.0654 0.0578 0.0629 0.0168

0.0840 0.0888 0.0980 0.0501

THO1 F13A01 vWFA blank

0.0412 0.0402 0.0418 0.0014

Intercalated with YOPRO-1 0.0403 0.0419 0.0419 0.0389 0.0412 0.0429 0.0451 0.0433 0.0443 0.0035 0.0024 0.0055

0.0493 0.0440 0.0500 0.0071

0.0555 0.0552 0.0570 0.0192

samples and blanks were prepared at the same time to ensure consistent concentration of intercalation dye. Table 3 lists the UV absorbance data for the three STR loci investigated. Each set of analyses was performed in triplicate, and the averages were compiled. There was an increase in absorption at 260 nm, corresponding to the increase in the intercalation dye for each set of data. The increase was greater for the bis-intercalators TOTO-1 (THO1, ∆ ) +0.0327AU) and YOYO-1 (vWFA, ∆ ) +0.0577 AU). With each sample set, blanks containing everything except the DNA were also analyzed. After subtraction of the blank’s absorbance from the DNA-intercalator samples, the DNA absorption remained approximately constant. 4516 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

Thus, the increase in absorption reported by others21 was not due to the binding of DNA and dye, but rather was due to the increase in the number of absorbing molecules in each sample. The intercalation dyes may shift the absorbance of the DNA when complexed to it. Absorbance readings at 260, 280, and 320 nm were plotted for allele 17 of the vWFA locus intercalated with TOPRO-1 (Figure 1). Although the absorbance for the DNA averaged 0.007 AU less at 280 nm and 0.02 AU less at 320 nm than the 260 nm readings, the blank’s absorbance also demonstrated the same trend. The increase in absorption at the higher wavelengths also corresponds to an increase in the concentration of the intercalation dye. Fluorescence Measurements. The FP-777 spectrofluorometer (Jasco Corp.) was programmed with the following parameters: the excitation wavelength was set at 514 nm for TOTO-1 and TOPRO-1 and 488 nm for YOYO-1 and YOPRO-1. These parameters mimic the spectral lines of the argon ion laser and also correspond to the absorption maximum of each dye. Fluorescence scans of the STR loci THO1, F13A01, and vWFA31, from 500 to 650 nm, were performed using a concentration of 40 pg/µL HPLC-purified DNA. The free (not bound) intercalators had virtually no fluorescence. Figure 2 A is the fluorescence scan (500-650 nm) of the bis-intercalator YOYO-1 (3.65 × 10-8 mM). When the same intercalator is mixed with the STR locus vWFA, a dramatic increase in the fluorescence intensity is observed (Figure 2B). As with the UV measurements, all alleles were prepared in stock solutions, and aliquots of the samples were mixed with intercalator to ensure sample uniformity. The same base pair-todye ratios used for the UV absorbance experiments were analyzed for fluorescence intensity testing. The major difference between the two techniques (UV and fluorescence) was the concentration (21) Guttman, A.; Cooke, N. Anal. Chem. 1991, 63, 2038-2042.

Figure 2. Fluorescence emission of STR locus vWFA31 intercalated with YOYO-1. Panel A is the emission scan of YOYO-1 with no DNA, and panel B is the scan of YOYO-1 with vWFA31 allele 17 (151 bp). Both spectra were obtained using the FP-777 spectrofluorometer (Jasco Corp., Tokyo, Japan) with a scan speed of 200 nm/min and a range of 100 fluorescence intensity (arbitrary units) (FI). The emission wavelengths were monitored from 500 to 650 nm, and excitation wavelength set at 488 nm. Both panels A and B contained the same concentration of YOYO-1. Panel B also contained 2.0 ng of the homozygote allele 17 of the locus vWFA.

of the DNA used for the analysis. Samples containing 40 pg/µL of an alleles were used for fluorescence versus 4.0 ng/µL used for the UV experiment. Considering the volume of sample for each analyses, UV required 200 times more DNA than the fluorescence analysis (40 0000 vs 2000 pg of DNA). The sample cuvette was rinsed with deionized water between samples, and water blanks were analyzed to ensure no intercalator contamination or carryover occurred. The DNA samples mixed with intercalation dyes were stored in the dark for a minimum of 1 h

prior to analysis. All alleles contained the same number of base pairs per sample. Each demonstrated a general increase in fluorescence as the number of dye molecules increased. The plot of fluorescence intensity data of TOPRO-1 (Figure 3) shows an increase of emission, until the base pair-to-dye ratio reaches 5/1. The emission of the bis-intercalator TOTO-1 was greater than the that of the mono-intercalator TOPRO-1. Also, the point at which the DNA is saturated with intercalation dye is at the bp/dye ratio of 10/1. This was most dramatic for TOTO1. In Figure 4, the fluorescence intensity of TOTO-1 was plotted vs bp/dye ratio. At the bp/dye ratio 10/1, all three loci’s emission intensities plateau, with only slight increases for the higher dye concentrations. The second set of monomer and dimer intercalation dyes was also plotted. YOPRO-1 (Figure 5) has a gradual increase in the emission intensity for all three loci. The most dramatic and intense fluorescence was seen with the bis-intercalator YOYO-1 (Figure 6). This was expected; the intercalator YOYO-1 has a fluorescence enhancement, when bound to DNA, greater than that of TOTO-1.5,6 But, there is a large increase in the emission at 540 nm and a distinct maximum emission at the bp/dye ratio of 5/1. This behavior is the result of the dye’s (YOYO-1) ability to bind to DNA in two different modes.5 In the first binding mode, at low bp/dye ratio, YOYO-1 intercalates into the DNA helix. When the concentration of the dye is higher, it can also bind externally as a grove binder. All intercalators except YOYO-1 demonstrated the same intensity pattern at bp/dye ratios 25/1 and greater. The fluorescence intensity grouping was as follows: F13A01 (highest), followed by vWFA (slightly less intense), and then THO1. The difference of dye emission between F13 and THO1 ranged from 16% for TOPRO-1 to 28% for YOPRO-1, and from 22% for YOYO-1 to 25% for TOTO-1. Their corresponding base compositions in % AT are F13A01 69%, vWFA 68%, and THO1 48% (Table 2). These

Figure 3. Fluorescence emission (540 nm) of STR loci intercalated with TOPRO-1. This plot contains the fluorescence intensity (FI) measurements at 540 nm of the samples containing the STR loci THO1 ([), F13A01 (9), and vWFA (2), all intercalated with TOPRO-1. FI measurements were plotted as a function of intercalation dye concentration expressed as base pair-to-dye ratio.

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Figure 4. Fluorescence emission (540 nm) of STR loci intercalated with TOTO-1. This plot contains the fluorescence intensity (FI) measurements at 540 nm of the samples containing the STR loci THO1 ([), F13A01 (9), and vWFA (2), all intercalated with TOTO-1. FI measurements were plotted as a function of intercalation dye concentration expressed as base pair-to-dye ratio.

Figure 5. Fluorescence emission (540 nm) of STR loci intercalated with YOPRO-1. This plot contains the fluorescence intensity (FI) measuements at 540 nm of the samples containing the STR loci THO1 ([), F13A01 (9), and vWFA (2), all intercalated with YOPRO-1. FI measurements were plotted as a function of intercalation dye concentration expressed as base pair-to-dye ratio.

fluorescence data show the preference of the intercalation dyes to the AT base positions previously discussed by Gaugain et al.22 To investigate further the fluorescence emission between bp/ dye ratios of 10/1 and 1/1, a sample of THO1 with homozygote allele 6 was prepared. Samples of 40 pg/µL were used for evaluation of TOTO-1 (bis) and 20 pg/µL for TOPRO-1 (monomer) (22) Gaugain, H.; Barbet, J.; Capelle, N.; Roques, B. P.; Le Pecq, J.-B. Biochemistry 1978, 17, 5078-5088.

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intercalators. Samples were intercalated with the dyes at the following bp/dye concentrations: ∞ (blank containing DNA only), 50/1, 25/1, 10/1, 5/1, 4/1, 3/1, 2/1, and 1/1. Table 4 tabulates the fluorescence intensity with increasing dye concentration measured using the spectrofluorometer. The fluorescence intensity (FI) of the bis-intercalator increased until the base pair-to-dye ratio was 10/1. Then, after a concentration of 5/1, it decreased slightly and remained constant. The

Figure 6. Fluorescence emission (540 nm) of STR loci intercalated with YOYO-1. This plot contains the fluorescence intensity (FI) measurements at 540 nm of the samples containing the STR loci THO1 ([), F13A01 (9), and vWFA (2), all intercalated with YOYO-1. FI measurements were plotted as a function of intercalation dye concentration expressed as base pair-to-dye ratio. Table 4. Fluorescence Intensity of Intercalated THO1 (6,6) DNA ∞

50/1 25/1 10/1

5/1

4/1

3/1

2/1

1/1

TOTOa

1.84 24.50 28.12 31.78 28.74 24.20 26.51 26.98 26.70 TOPROb 1.93 8.56 12.23 11.94 12.80 15.86 11.43 15.18 15.02

a DNA sample concentration was 40 pg/µL in deionized water. DNA sample concentration was 20 pg/µL in deionized water. Intercalators were mixed with DNA and stored in the dark for 1 h before measurements.

b

monomeric intercalator samples were less concentrated (20 pg of DNA/µL) than the dimer sample, although a similar FI trend was observed. A maximum intensity was seen at 4/1, and this continued with the following concentrations with only slight variations. Therefore, little change occurs in the fluorescence emission of DNA-intercalation dye complexes above a bp/dye ratio of 5/1. CONCLUSION Alleles of the STR loci THO1, F13A01, and vWFA31 were PCR amplified and HPLC purified for use in this intercalation study. The alleles ranged in size from 151 (vWFA allele 17) to 199 base pairs (F13A01 allele 8) and, in percent AT, from 48% for the THO1 locus to 69% for the F13A01 locus. The single alleles of homozy-

gote individuals of each locus were mixed with two sets of intercalation dyes, each set consisting of a monomeric dye and the corresponding dimer dye. The DNA-dye absorbance data at 260 nm demonstrated that the increase in absorbance was due to addition of intercalator to the sample and not an increase in the DNA’s absorbance. The fluorescence emission measurements indicated a maximum intensity (plateau) at a base pair-to-dye ratio 10/1 for all three loci. Both monomeric intercalators (TOPRO-1 and YOPRO-1) had decreased fluorescence emission when compared to their corresponding dimeric dyes. The greatest emission was observed at 540 nm using the bis-intercalator YOYO-1 and the F13A01 allele (69% AT). The fluorescence was monitored at 540 nm to facilitate detection of DNA fragments using the in-house capillary electrophoresis laser-induced fluorescence detection system. The future direction of this research will be the evaluation of detection and resolution of dye-DNA complexes during electrophoresis. Disclaimer: The opinions or assertions herein are those of the author and do not necessarily reflect the views of the Department of Army of the Department of Defense. Received for review May 13, 1998. Accepted September 2, 1998. AC980526Q

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