Nucleic Acids Research, Vol. 20, No. 19 5061-5065

A rapid chemiluminescent method for quantitation of human DNA P.Sean Walsh, Joseph Varlaro and Rebecca Reynolds Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA 94501, USA Received July 10, 1992; Revised and Accepted August 31, 1992

ABSTRACT A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, DI 7Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.

INTRODUCTION Molecular biology techniques such as PCR (1) and RFLP (2) have resulted in an increasingly detailed analysis of genetic diversity in human DNA samples. In particular, the specificity and sensitivity of PCR has resulted in the widespread use of this technique in the fields of forensic science (3), studies of ancient DNA samples (4), analysis of genetic diseases (5) and in studies of population genetics (6). PCR has been particularly useful in the field of forensic science, where many biological evidence samples contain either extremely small quantities of DNA, or degraded DNA. In forensics, the quantity and quality of DNA in a sample can be important factors

in deciding how to proceed with the analysis of that sample. For example, RFLP tests require relatively large quantities (typically greater than 50 ng) of non-degraded DNA. Therefore, samples containing only a few nanograms of degraded DNA are more suitable for analysis by PCR methods. In general, the efficiency of a PCR amplification is influenced by the quality, purity and total quantity of DNA in a sample. Lack of amplification is usually due either to insufficient DNA of appropriate molecular weight, or to the presence of PCR inhibitors in the DNA extract (3). Knowledge of the quantity of DNA in a sample can help distinguish between these two possible causes, and influence the course of action when a sample does not amplify. Also, in order to maximize the number of PCR tests that can be performed on a given sample, it is generally desirable to consume only as much DNA sample as is necessary to obtain a result. Determination of DNA quantity is particularly important in the PCR amplification of VNTR loci, where optimal specificity is obtained only with a relatively narrow range of input DNA (7). Current methods for quantitation of DNA include UV spectroscopy, fluorometry (8) and semi-quantitation by agarose gel electrophoresis. However, these methods require nanogram quantities of non-denatured DNA for analysis, and are not specific for human DNA. A very sensitive quantitation method based on DNA binding proteins and detection of pH change catalyzed by urease has also been described (9), but this method is not specific for human DNA. Determination of the quantity of human DNA is important in the analysis of forensic samples, which sometimes contain bacterial or fungal DNA. Recently, Waye et. al. reported a method for quantitating sub-nanogram quantities of DNA using a hybridization method that gives signals at least fifty times more intense for human DNA than for equivalent amounts of nonhuman DNA (10). However, this method requires the use of radioactivity and takes several hours to obtain results. In the present paper, we report a simple method for quantitation of human genomic DNA that can be performed in 1.5 hours and can detect less than 150 pg of human DNA. This quantitation method is based on the hybridization of a biotinylated oligonucleotide probe to sample DNA immobilized on nylon membrane. The probe is complementary to a primate specific alpha satellite DNA sequence on chromosome 17 (D17Z1), which is estimated to be present in 500 to 1,000 copies per chromosome 17 (11). The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol based reagent (12). The oxidation of

5062 Nucleic Acids Research, Vol. 20, No. 19 luminol by the horseradish peroxidase enzyme results in the emission of photons, which is detected on standard autoradiography film. The intensity of the signal on film is a function of DNA quantity. Because of the linearity and lack of background with the quantitation method described, the film results can be scanned and analyzed by computer to obtain very accurate quantity estimates of DNA samples. A method is also described for the very sensitive determination of quantity and quality of human genomic DNA on Southern blots; this approach is particularly useful for the analysis of denatured DNA, which is not readily detected with ethidium bromide stained agarose gel methods.

MATERIALS AND METHODS DNA extraction DNA was extracted from samples by the following methods: Samples 1A-1H, 2D-2H, and 3A-3E (Figure 1) were extracted by direct lysis using the Chelex method (13); a bloodstain (3 square millimeters), buccal scraping, or 1 cm hair root section were incubated in 5% Chelex at 560C, followed by boiling for eight minutes. Samples 2A-2C and the human DNA standard (Figs. 1 and 3) were extracted by a 'salting out' procedure (14). Human DNA probe SW49 is a 40 nucleotide long biotinylated probe of the following sequence: 5'- XTAGAAGCATrCTCAGAAACTACTTTGTGATGATTGCATTC- 3' where X = biotin. This probe was prepared by automated

synthesis using reagents and protocols obtained from Glen Research (Sterling, VA). DNA quantitation protocol Five 1l of each extracted DNA sample was added to 100 t4L spotting buffer (0.4N NaOH, 25mM EDTA). DNA standards were prepared by adding the following quantities of control DNA to 100 AL of spotting buffer: 10, 5, 2.5, 1.2, 0.6, 0.3, 0.15 ng. A blank was also prepared which contained no DNA added to 100 ytL spotting buffer. The Biodyne B membrane (Pall Biosupport, Glen Cove, NY) was pre-wet in distilled water and placed in a slot blot apparatus (The Convertible, 0.75 x7.5 mm, GIBCO BRL, Gaithersburg, MD). The entire volume of each sample was added to the wells and the vacuum applied. Two hundred ,uL of 15% hydrogen peroxide was added to each well with a sample and incubated for one minute before applying the vacuum. The membrane was immediately placed in 200 mL prehybridization solution (5 x SSPE, 0.5 % SDS) pre-warmed to 50°C and incubated in a shaking water bath for 15 minutes at 50°C. The membrane was then transferred to 30 mls of 5 x SSPE, 0.5 % SDS containing 15 pmoles of SW49 probe, and incubated in a shaking water bath for 15 minutes at 50°C. Following a brief rinse in 1.5 x SSPE, 0.5% SDS, the stringent wash and conjugation steps were carried out simultaneously; the membrane was placed in 30 mls of 1.5 xSSPE, 0.5% SDS containing 90 yL of SA-HRP (Perkin Elmer, Norwalk, CT) and incubated in a shaking water bath for 10 minutes at 50°C. The membrane was rinsed briefly in 1.5 x SSPE, 0.5% SDS and then washed in 200 mL of 1.5 xSSPE, 0.5% SDS on an orbital shaker for 15 minutes at room temperature. The membrane was rinsed in

0. 1M NaCitrate, pH 5. Detection of the DNA was carried out using ECL (Amersham, Arlington Heights, IL), which is a luminol based reagent used for enhanced chemiluminescent detection. Ten mL of ECL Reagent 1 was mixed with 10 mL of Reagent 2. The membrane was placed in the reagents and shaken for one minute at room temperature. The membrane was placed on a sheet of Benchkote (Whatman, Maidstone, England), covered with Saran Wrap and wiped free of excess moisture. To visualize the DNA, the membrane was exposed to Hyperfilm (Amersham, Arlington Heights, IL) or Kodak XAR5 film (Kodak, Rochester, NY) for 15 minutes at room temperature. Computer image analysis DNA quantitation was determined both by visual comparison of sample slot blot intensities to the DNA standard and by computer image analysis of the slot blot results on film. The computer analysis was performed as follows: Slot blot results on film (see Figure 1) were scanned into a Macintosh computer using an Abaton 8-bit flatbed scanner. The NIH computer program Image 1.41 (written by Wayne Rasband; NIH, Bethesda, MD) was then used to determine the mean density of the slot blot signals for all samples from the scanned image. Measurements were also taken next to each slot and subtracted from the mean density for each sample. The mean densities for the DNA standard dilution series were then plotted against the known quantities for the DNA standard, giving a standard curve. The equation describing the line for the standard curve was then used to estimate the quantity of DNA in the unknown samples based on their mean densities. DNA quality evaluation Human genomic DNA was diluted to 2 ng/4tL in both 5 % Chelex and glass distilled water, and then boiled for 0, 1, 3, 8 minutes in a boiling water bath. Seven ,uL (14 ng) of each sample was subjected to electrophoresis on a 1 % agarose gel containing 0.5 /tg/mL ethidium bromide in 1 x TBE for 30 minutes at 100 volts. The gel was photographed, soaked in 0.25M HCl for 15 minutes to depurinate the DNA and then soaked in 0.5N NaOH, 1.5M NaCl for 10 minutes to denature the DNA. The DNA was transferred to Biodyne B membrane using the Posiblot transfer system (Stratagene, La Jolla, CA). Transfer was performed at 75 mm Hg for one hour using lOx SSPE as the transfer buffer. The membrane was baked in a vacuum oven for 15 minutes at 80'C to fix the DNA. The membrane was wetted with 2 x SSPE before it was soaked in 15% hydrogen peroxide for 2 minutes. Hybridization and detection of bound SW49 probe was performed as described in the DNA Quantitation Protocol section above, except that the blot was exposed to film for 30 minutes.

RESULTS Determination of DNA quantity Prior to the analysis of genetic markers by RFLP and PCR-based methods, DNA typically is extracted from fluids and tissues ranging from blood and semen to bones and mummified remains. These types of forensic samples vary greatly in size and condition. Consequently, the quantity of DNA extracted from biological sources can be highly variable. Since the success of most DNA analysis methods is dependent on the amount of DNA used, it is beneficial to quantitate the DNA in individual extracted samples. Ideally, the quantitation method would be specific for human DNA, sensitive, rapid and non-radioactive.

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Figure 2. Standard curve for determination of DNA quantity. The NIH computer program Image 1.41 was used to determiine the mean density of the slot blot signals for the DNA standard from a scanned image of the film. The data points accurately fit an exponential relationship between mean density and DNA quantity (R = 0.99). The equation describing the line for the standard curve was then used to estimate the quantity of DNA in the unknown samples based on their mean densities.

DNA extracts from several blood, hair and buccal samples applied to nylon membrane in a slot blot format, and hybridization with SW49 probe and chemiluminescent detection were performed as described in Materials and Methods. A serial dilution of human DNA applied to the same membrane served as a standard for estimating the quantity of DNA in the extracted samples. The slot blot results shown in Figure 1 indicate that were

Quantity Result (nanograms)

Figure 1. DNA quantity slot blot results. Sample DNA was immobilized on nylon membrane, and hybridization was performed with a probe complementary to a human alpha satellite repeat sequence at locus D17Z1. Bands were visualized using chemiluminescent detection with a 15 minute exposure to film. Column 'S' is a titration series of a human genomic DNA standard of known concentration. Quantities for the unknown samples (columns 1-3) are estimated by comparison to the DNA standard. Only 5 ytL of each sample DNA extract (equivalent to 2.5% of the total extract volume) was used for quantitation. The sources of the extracted DNA for the samples above were as follows: IA- lE are bloodstains; 1F-1H and 2A-2C are whole blood; 2D-2H and 3A-3B are single hairs; 3C-3E are buccal samples; 3F is 1 jg of cow DNA; 3G is 1 yg of mouse DNA; no sample was added to 3H.

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2.24 0.29 1.07 0.88 1.74 0.83 1.09 7.17 6.32 1.32 2.33 0.28 0.94 0.52 0.68 2.49 6.34 1.64 1.46 1.49 1.00 0 0

The values in the right column indicate the quantity of DNA in the 5 uL of each sample that was tested. These results were obMined by using the equation describing the standard curve to compute DNA quantity, given the mean density of the slot blot signal for each sample. Note that cow and mouse DNA give no signal.

150 pg of DNA standard was detected after only a 15 minute exposure to film. Ten to twenty picograms of DNA can be detected with a three hour exposure (data not shown). Quantitation can be performed visually by comparing slot intensities for the samples to slot intensities for the DNA standard. For example, the extracted bloodstain sample in slot IA in Figure 1 has an intensity which is just less than 2.5 ng of control DNA. Figure 1 also indicates that 1 ztg of cow or mouse DNA (positions 3F and 3G, respectively) gives no detectable signal. One microgram quantities of rat, pig, chicken, dog, cat, yeast and E. coli DNA have also been tested and found to give either no signal, or a signal much less intense than that obtained with 150 pg of human DNA (data not shown). In order to obtain a more objective and potentially more accurate estimate of DNA quantity, the slot blot results shown in Figure 1 were scanned into a computer using a standard flatbed scanner, as described in Materials and Methods. The NIH computer program Image 1.41 was then used to determine the mean density of the slot blot signals from the scanned image of the film. The mean densities of the DNA standard dilution series were plotted against DNA quantity (on a log scale), giving a standard curve (Figure 2). The data points shown in Figure 2 closely fit an exponential relationship between mean density and DNA quantity (R = 0.99). The equation describing this standard curve is as follows: Y = 0. 1787 3- e(0.03083 X), where X is mean density and Y is DNA quantity, in nanograms. This equation was then used to determine the quantity of DNA in the extracted samples based on their mean densities. This method provides a more objective and accurate estimate of DNA quantity than simply comparing slot blot intensities visually. The quantity results for all of the blood, hair and buccal samples are shown in Table 1. These results indicate that samples of approximately the same size can contain very different amounts of DNA. For

5064 Nucleic Acids Research, Vol. 20, No. 19 Chelex 1

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in either 5% Chelex or in H20 for 0, 1, 3 or 8 minutes, amd then subjected to electrophoresis on a 1% agarose gel in 1 xTBE (panel A). The DNA was then transferred to nylon membrane and hybridization withi SW49 probe and chemilumniescent detection were performed as described (palnel B); this procedure greatly increases the sensitivity of detection, particularly fc )r the boiled samples which represent denatured DNA. Note the double band ftor the sample in Chelex for 1 minute, which presumably corresponds to non-denatured DNA. After 3 to 8 minutes of boiling in Ch4 elex, the DNA shifts completely to the denatured state, and remains high molecular weight. The DNA boiled in H20, however, shows slight degradation after 3 minutes of boiling, and significant degradation after 8 minutes of boiling.

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Determination of DNA quality The molecular weight of human genomic D NA (i.e., DNA quality), is conventionally evaluated by electri ophoresis of the DNA in agarose gels followed by ethidium brormide staining. In this analysis, high molecular weight DNA runIs as a relatively tight band at about 20 kb relative to an approlpriate molecular weight marker, and degraded DNA appears as;a smear of DNA below 20 kb in molecular weight. Because ethiditam bromide does not readily stain denatured (single stranded) E)NA, evaluation of DNA quality is difficult for samples that are extracted using

methods that require heating or boiling steps or alkaline treatments. For example, the Chelex method of DNA extraction for PCR requires boiling for cell lysis (13). Also, some DNA extraction methods require heating to 95°C for inactivation of proteinase K (15). The sensitivity and specificity of DNA quality analysis can be improved by electrophoresis of the DNA in agarose, transfer of the DNA to a nylon membrane, and then chemiluminescent detection using SW49 probe, as described in Materials and Methods. The experiment presented in Figure 3 is an example of DNA quality evaluation applied to the analysis of denatured and degraded DNA. In this experiment, control DNA was boiled in either H20 or in 5% Chelex for up to eight minutes. The samples were then subjected to electrophoresis on a 1 % agarose gel and stained with ethidium bromide, as shown in Figure 3A. The DNA was then transferred to nylon membrane, and hybridization and chemiluminescent detection were performed as described (Figure 3B). The results in Figure 3A show weak or absent band intensities for the boiled samples using ethidium bromide detection, particularly for the H20 samples. However, the increased detection sensitivity achieved by blotting and probing (Figure 3B) allows for a greatly improved ability to evaluate the effects of boiling on DNA quality. Specifically, DNA boiled in Chelex remains relatively intact, whereas boiling in H20 leads to slight degradation after 3 minutes and significant degradation after 8 minutes of boiling. These results are consistent with previous reports that Chelex has the effect of protecting DNA from degradation during boiling, presumably by chelating metal ions that would otherwise catalyze the degradation of DNA at high temperatures (13). These results indicate that the methods described here for the evaluation of DNA quality give improved detection sensitivity, particularly for the analysis of denatured DNA. This approach also allows for the analysis of the quality of human DNA in a potential background of bacterial or other non-human DNA. DISCUSSION

This paper describes a simple, accurate and sensitive procedure for quantitation of human genomic DNA. This method is based

probe hybridization to a human alpha satellite locus, D17Z1 (11). The entire procedure can be performed in 1.5 hours, and

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150 pg of human DNA can be detected with only a 15 minute exposure to fim. The procedure is highly specific for human DNA, which is important in the analysis of samples suspected to contain bacterial or other non-human DNA. Other advantages of this quantitation procedure include high throughput (up to 40 samples can be quantified per filter), lack of filter background, use of chemiluminescence instead of radioactivity, and the ability to quantitate denatured or non-purified DNA samples. In addition, a simple computer program can be used to create a standard curve from a scanned image of the slot blot results, thus providing objective and accurate quantitation results. The methods described can also be used for the very sensitive determination of DNA quality of human genomic DNA on Southern blots, making possible the analysis of denatured DNA. The analysis of human DNA samples by techniques such as RFLP and PCR often requires knowledge of DNA quantity. In the analysis of forensic evidence samples, the quantity and quality of DNA in a particular sample can determine whether that sample

Nucleic Acids Research, Vol. 20, No. 19 5065 is adequate for analysis by RFLP or is more suitable for PCR, which requires less input DNA. Also, determination of DNA quantity can allow for the practice of using only as much input DNA as is necessary to obtain a result, therefore maximizing the number of different tests or the number of repeat tests that can be performed on a given sample. Determination of DNA quantity has been found to be important for optimal specificity in the PCR amplification of VNTR loci (7). We have found that knowledge of DNA quantity can be valuable in distinguishing between insufficient DNA or inhibition as the mechanism for lack of PCR amplification. Frequently, samples that do not amplify but contain adequate quantities of DNA can be diluted before amplification to reduce the degree of inhibition (3). In general, DNA quantitation prior to PCR should increase confidence in negative PCR results in clinical or diagnostic tests, where insufficient target DNA could otherwise be interpreted as false negative test results. We have applied the DNA quantitation procedure to the evaluation of samples that generally contain very limiting quantities of DNA, such as single hairs, saliva samples, small bloodstains, and paraffin embedded and fixed tissue samples. This procedure has also been used to quantitate DNA in a 1,000 year old mummy sample. DNA quantitation and quality assessment by the methods described in this paper provide the best approach for the characterization of DNA extracted using protocols that result in denatured DNA and/or direct cell lysates, which may contain relatively high levels of protein or other cellular material. We expect these human DNA quantitation and quality evaluation procedures to be useful in a variety of other applications that require very sensitive and specific characterization of human DNA.

ACKNOWLEDGEMENTS We would like to thank Corey Levenson and his DNA Synthesis group for preparing the biotinylated probes. We also thank Henry Erlich, Nicola Fildes and Stephen Scharf for helpful comments, and Svante Paabo for providing samples.

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16, 9775-9787. 5. Gibbs, R.A., Chamberlain, J.S. and Caskey, C.T. (1989) In H.A. Erlich (ed.), PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, pp. 153- 169. 6. Helmuth, R., Fildes, N., Blake, E., Luce, M.C., Chimera, J., Madej, R., 7.

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Gorodezky, C., Stoneking, M., Schmill, N., Klitz, W., Higuchi, R., Erlich, H.A. (1990) American Journal of Human Genetics, 47, 515-523. Walsh, P.S., Erlich, H.A. and Higuchi, R. (1992) PCR Methods and Applications, vol.1, no. 4, 241-250. Brunk, C.F., Jones, K.C. and James, T.W. (1979) Analytical Biochemistry, 92, 497-500. Kung, V.T., Panfili, P.R., Sheldon, E.L., King, R.S., Nagainis, P.A., Gomez, B., Ross, D.A., Briggs, J., Zuk, R.F. (1990) Analytical Biochemistry, 187, 220-227. Waye, J.S., Presley, L.A., Budowle, B., Shutler, G.G. and Fourney, R.M. (1989) BioTechniques, vol. 7, no. 8, 852-855. Waye, J.S. and Willard, H.F. (1986) Molecular and Cellular Biology, vol. 6, no. 9, 3156-3165.

12. Whitehead, T.P., Thorpe, G.H.G., Carter, T.J.N., Groucutt, C. and Kricka, L.J. (1983) Nature, 305, 158-159. 13. Walsh, P.S., Metzger, D.A. and Higuchi, R. (1991) BioTechniques, vol. 10, no. 4, 506-513. 14. Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) Nucleic Acids Research, vol. 6, no. 3, 1215. 15. Higuchi, R. (1979) In H.A. Erlich (ed.), PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York, pp. 31-38.

A rapid chemiluminescent method for quantitation of human DNA.

A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite l...
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