A densitometric nondestructive microassay for DNA quantitation

ANALYTICAL

BIOCHEMISTRY

14,

A Densitometric

15-19 (1985)

Nondestructive

SHARON Biology

Department,

P. MGGRE*

Brookhaven

National

Microassay

for DNA Quantitation’

AND BETSY M. SUTHERLAND Laboratory,

Upton,

Long

Island,

New

York

11973

Received February 2 I, 1984 A nondestructive assay for small quantities of nonradioactive DNA has been developed. Submicroliter volumes of DNA samples of concentrations as low as 10 &ml in 10 mM Tris, pH 8, containing IO &ml ethidium bromide are photographed in l-pi microcapillary tubes on a near-uv transilluminator. The negatives are scanned with a densitometer, producing a sharp peak for each capillary; samples whose peak heights show apparent linearity with DNA content are compared to the linear portion of a standard curve constructed from DNA samples of known concentrations. The samples may be recovered undamaged, as introduction into capillaries and illumination for photography do not introduce nicks into the DNA. The DNA samples may be of heterogeneous molecular weight, but all DNAs should be of similar base composition and in the same buffer. Traces of phenol do not interfere with the determination. 0 1985 Academic

Press, Inc.

DNA quantitation; ethidium bromide fluorescence; microcaps; densitometry; microassay; nondestructive. KEY

WORDS:

Agarose gel analysis of genomic DNAs of heterogeneous molecular weight requires accurate DNA concentration determination. For DNA samples available in extremely limited quantities [for example, the analysis of pyrimidine dimer content in DNAs from human skin (l)] the quantitation process should permit the recovery of the treated aliquot undamaged by the measurement. Widely used methods of DNA quantitation include an ethidium bromide spot test (2) gel electrophoresis and photography (2-5) spectrophotometry (2), or fluorometry (6-8). However, these methods involve estimation, inefficient recovery of high-molecular-weight DNA, or the use of large sample volumes. We have developed an assay which requires a small sample and is accurate and nondestructive. DNA samples or dilutions of 1 ~1 or less are mixed with ethidium bromide. The solutions are introduced into microcap-

illary tubes by capillary action; the capillaries are attached to transparent plastic rulers, placed on a near-uv transilluminator, and photographed using Polaroid positive-negative film. The resulting negatives are scanned with a densitometer, yielding a series of sharp peaks. DNA standards of known concentration whose peak heights increase linearly with concentration are used to construct a standard curve. DNAs of unknown concentration yielding peak heights which show linearity proportional to DNA content, and also which fall within the linear portion of the standard curve, are selected for analysis. The method yields DNA concentrations within 10% of those determined by spectrophotometry. Samples may be readily recovered and are undamaged by the measurement procedures. MATERIALS

AND

METHODS

Herring sperm and calf thymus DNA from Sigma were further purified by extraction with redistilled phenol and dialyzed against Buffer B (50 RIM Tris, pH 8, 10 mM EDTA,

’ This research was supported by NIH Training Grant T32 CA09121, HSS Grants CA 26592 and CA 23096, and the U. S. Department of Energy. * To whom correspondence should be sent. 15

0003-2697185 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved

16

MOORE

AND SUTHERLAND

10 mM NaCl). Clostridium perfringens and Micrococcus luteus DNAs (9) (Sigma) were extracted with phenol and dialyzed against Buffer C (10 mM Tris, pH 7.0, 0.1 mM EDTA, 50 mM NaCl). r$X 174 RF I DNA was obtained from Bethesda Research Laboratories. Determination of DNA concentration. DNA concentration standards (0- 123 &ml) and dilutions of DNA to be assayed (test DNA) were prepared in 10 mM Tris, pH 8, containing 10 &ml ethidium bromide (Solution A). Each solution was drawn up in three l-p1 microcaps which were then affixed to two plastic rulers by double-sided cellophane tape (Fig. 1A). The rulers were placed on a uv Products transilluminator and photographed using Polaroid Type 55 film. The negative was scanned with a Joyce-Loebl microdensitometer, yielding a sharp pen deflection corresponding to the ethidium fluorescence within each microcap. The three replicate pen deflection measurements for each DNA standard solution were averaged and plotted as a function of the corresponding DNA concentration to yield a standard curve. The concentration of the test DNA was determined by comparing the average of three replicate pen deflection measurements of a given dilution with the standard curve; only dilutions whose values fell within the linear portion of the standard curve were used.

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FIG. 1. (A) Photograph of I-p1 microcaps containing DNA of various known concentrations (standards) and dilutions of DNA to be assayed (test). All samples were mixed with ethidium bromide, resulting in ethidiumDNA fluorescence which increases in intensity in proportion to DNA concentration. (B) Densitometer trace of the negative corresponding to the photograph shown in (A). (C) Standard curve calculated from pen-deflection measurements in cm from microcap groups A-N from (B). Vertical dashed lines show concentrations of three dilutions of test DNA (microcap groups 6-8) derived by comparing their pen deflections with the standard curve.

RESULTS

Determination of DNA concentration. The method for ascertaining DNA concentration is illustrated in Fig. IA-C. Figure 1A shows the microcaps containing the DNA of standard concentration (standards) and dilutions of the test DNA (test). Figure 1B shows the densitometer tracing of the negative corresponding to the photograph shown in Fig. 1A. The standard curve (Fig. 1C) was constructed from pen deflections A-N. For the test DNA only those pen deflections which showed a linear increase in proportion to DNA content and which also fell within the linear portion of the standard curve were

chosen for analysis (6-8 of Fig. 1B). Replicate pen deflections were averaged. These values were compared to the standard curve and the resulting DNA concentrations were multiplied by their respective dilution factors. This calculation yielded three slightly different concentrations, which were averaged. The resulting concentration of the test DNA of Fig. 1 was determined by this method to be 36 &ml. The concentration of this DNA was determined spectrophotometrically to be 34 fig/ml. Properties of the assay. We wished to develop a nondestructive assay which allowed

DENSITOMETRIC

MICROASSAY

the rapid quantitation of numerous small DNA samples. To determine if this assay introduces nicks in the DNA, we chose a covalently closed supercoiled circle in which a single nick could easily be detected by alkaline agarose electrophoresis. $X 174 RF I DNA was diluted into Solution A. The sample was exposed to the uv transilluminator for 2 min in a microcap and then subjected to electrophoresis in an alkaline agarose gel (10) along with the following controls: (i) DNA taken up into microcaps but not exposed to uv or ethidium bromide, (ii) DNA in microcaps exposed to uv in the absence of ethidium bromide, (iii) DNA which had been mixed with ethidium bromide and drawn up into microcaps but not exposed to uv, and (iv) DNA exposed to neither uv nor ethidium bromide, and not taken up into a microcap. Electrophoresis was carried out in an alkaline gel consisting of 0.8% agarose, 0.06 M NaOH, and 0.006 M Na*EDTA. The gel was photographed and the negative scanned with microdensitometer. A nick in supercoiled DNA results in a closed circle and a linear strand. The percentage of supercoils was determined from the densitometer trace by cutting out the tracings, weighing on an analytical balance, and comparing the weights of the peaks (8,ll). The data are presented in Table 1. From these values, it TABLE 1 PERCENT SUPERCOILS OF $1X174 RF I DNA AFTER EXPOSURE TO ETHIDIUM BROMIDE, uv AND/OR MICROCAPILLARY TUBES

Treatment DNA in microcaps only DNA + ethidium bromide in microcaps (no uv) DNA + uv in microcaps (no ethidium bromide) DNA + uv + ethidium bromide in microcaps DNA only (no uv, no microcap, no ethidium bromide)

Percentage supercoils 85.8 89.7 88.5 88.5 88.1

FOR DNA QUANTITATION

17

appears that this method does not introduce a significant number of nicks in the DNA. The quantitation of numerous unknown samples in a single photograph is subject to spatial limitation. Space must be reserved for the microcaps containing the standard dilutions and sufficient space must be allowed between adjacent microcaps for distinct separation of pen deflections. In addition, “vignetting” limits the usable area of the photographic negative (3,4). These limitations may necessitate making several photographs if the number of samples to be assayed exceeds the accommodation of one photograph. Although standard dilutions must be present in each photograph, the same standard microcaps could be reused if photobleaching did not occur. Photodecomposition of ethidium bromide in electrophoretic gels following shortwavelength uv exposure has been reported (3,4). Therefore we tested the effect of prolonged uv exposure on the intensity of fluorescence. Herring sperm DNA was diluted into Solution A to final DNA concentrations of 30 and 12.5 pg/ml. The microcaps were exposed to uv from the illuminator and photographs were made immediately and after 2.8, 6.7, and 11.7 min. For Solution A controls and for two DNA concentrations (30 and 12.5 pg/ml) no significant changes in fluorescence were detected even after an uv illuminator exposure of approximately 12 min. Restrictions on the assay. We next determined if fluorescence intensity was affected by base content of the DNA, buffer composition, or traces of phenol. We tested the ethidium fluorescence of A4. luteus (72% G-C), calf thymus (41% G-C), and C. perfiingens DNA (3 1% G-C). These results are presented in Table 2 and show that base composition affects fluorescence; thus DNA chosen as the standard should be of similar base composition as the unknowns. To test the effect of traces of phenol in the buffer, T7 bacteriophage DNA was diluted into phenol-saturated 10 mM Tris, pH 8, precipitated with cold ethanol, and resolubil-

18

MOORE

AND SUTHERLAND

EFFECTOFDNA BASECOMFQSITIONORDILUENTON

TABLE 2 PEAKHEIGHTSASAFUNCTIONOFDNA

DNA

DNA concentration WW

hf. luteus Calf thymus C. perfringens

O-36 O-36 O-36

G-C content = 72% G-C content = 4 1% G-C content = 31%

Herring Sperm

O-30

In 10 mM Tris,pH 8 potassium phosphate buffer pH 8 potassium phosphate buffer pH 7 potassium phosphate buffer pH 6 sodium phosphate buffer pH 8 sodium phosphate buffer pH 7 sodium phosphate buffer pH 6 In SSC, pH 7

Condition

(1Slope was calculated as peak height (cm) per DNA concentration (&ml) deviations are included. b Average of four experiments. ’ Average of two experiments.

ized in 0.15 M NaCl, 15 mM sodium citrate (SSC).3 Control DNA was diluted into 10 mM Tris, pH 8, without phenol, and was precipitated and resolubilized in SSC. Ethidium bromide was then added to the samples and the fluorescence was determined as described. DNA which had been exposed to phenol before precipitation showed a similar intensity of fluorescence to that which was precipitated in the absence of phenol. However, if the DNA was not precipitated, fluorescence in the presence of phenol was 2.6 times less than that showed by DNA in the absence of phenol. We also measured the fluorescence intensity of the same concentrations of DNA in different diluents: 10 mM Tris, pH 8, 10 mM potassium phosphate at pH 6, 7, or 8, 10 mM sodium phosphate at pH 6, 7, 8, and SSC. The results are presented in Table 2. These results indicate that fluorescence differs significantly as a function of diluent. Since RNA also fluoresces under the conditions of this assay, the sample should be as RNA free as possible. However, we have measured the fluorescence of equal weights 3 Abbreviation used: SSC, sodium citrate.

CONCENTRATION

Slope” 0.154 * 0.0046 0.173 k 0.004b 0.165 2 0.003b 0.34 0.31 0.33 0.32 0.34 0.32 0.36 0.40

* + tf + -t + +

0.00’ 0.01’ 0.01’ 0.01’ 0.01’ 0.01’ 0.01’ 0.01’

by least-squares analysis; standard

of tRNA and herring sperm DNA and found the fluorescence of the RNA to be only 7% of that of the DNA. Therefore a small amount of contaminating RNA should not greatly affect the accuracy of this assay. DISCUSSION

This assay has three principal advantages: sensitivity at low concentration, small sample size, and quantitative recovery of DNA. By filling the microcap only one-third full, we estimate that one can determine the DNA concentration of a 0.34 sample of minimum concentration of 10 &ml. The use of photographic enlargement may allow the quantitation of a sample of an even smaller volume. The sensitivity of the assay may be extended to lower DNA concentrations by using less ethidium bromide and appropriate photographic exposures as long as the intensity of the fluorescence is within the linear portion of the film response. Further, our results indicate that the measurement does not introduce nicks in the DNA. For optimum accuracy, we found that standards and unknown should be of similar base composition and diluted into the same buffer with, at most, traces of phenol. In addition, the

DENSITOMETRIC

MICROASSAY

DNA samples should be free of RNA, as RNA contamination will increase the intensity of fluorescence. This method cannot be used as a substitute for DNA quantitation of restriction enzyme fragments in electrophoretie gels, since the fragments are not separated in microcaps. General quantitative determinations of DNA from photographs of ethidium-DNA fluorescence on gels has been complicated by (i) a nonlinear film response (3-512) (ii) photobleaching of ethidium (3,4), (iii) differential diffusion of DNAs of varying molecular weights (3), and (iv) the problem of integrating over peaks of varying width (3-5). We have simplified DNA measurement by (i) choosing for analysis DNA dilutions in the range of apparent linearity, (ii) using a near-uv illuminator, (iii) measuring DNAs in solution, and (iv) using capillaries as uniform containers. ACKNOWLEDGMENTS We thank Kathleen P. Griffin for purification of C. perfringens and M. luteus DNAs and Alice Shih for .purihcation of calf thymus and herring sperm DNAs.

FOR DNA QUANTITATION

19

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