Relevance of empirical optimization for cross-linking DNA by using an ordinary ultraviolet-light source

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GATA 10(3-4): 95-98, 1993

Relevance of Empirical Optimization for CrossLinking DNA by Using an Ordinary Ultraviolet-Light Source IQBAL PARWEZ, SUBBIAH KUMAR, SHASHI BALA, and SHER ALl

For cross-linking DNA onto nitrocellulose or nylon membranes by using ordinary ultraviolet ( UV) light, empirical optimization with respect to exposure time is crucial for obtaining complete hybridization signals. Longer than optimal UV exposure reduces the signal intensity particularly in the larger DNA fragments, whereas shorter exposure due to insufficient immobilization produces incomplete signals. Relevance of using ordinary source of UV light with respect to fixing DNA on a large number of circular filters during library screening is discussed.

Introduction The discovery that the nitrocellulose filter strongly adsorbs single-stranded DNA [1] has led to capillary DNA transfer from gels to nitrocellulose membranes [2], which is now a widely used method in molecular biology. Following Southern transfer, DNA is immobilized on the membrane either by baking the same at 80°C for 2 h in a vacuum oven or by exposing it briefly to UV energy. The latter approach is less time consuming and produces better results [3, 4]. However, UV-induced DNA fixation is usually carried out with a commercially available UV oven, such as Strata Linker from Stratagene or GS Gene Linker from Bio-Rad, which is not always available in every laboratory. While the DNA immobilization by UV light is a well-established phenomenon and even a From the Division of Molecular Endocrinology(I.P.), Department of Zoology, Aligarh Muslim University, Aligarh; the Research and Development Division (S.K.), Forensic Science Department, Madras; and the National Institute of Immunology (S.B., S.A.), Aruna Asaf Ali Marg, New Delhi, India. Address correspondence to Dr. S. Ali, Staff Scientist, National Instituteof Immunology,ArunaAsaf Ali Marg, New Delhi 110 067, India. Received 15 April 1993; revised and accepted 23 June 1993.

UV 'transilluminator is used for this purpose, no systematic attempt has been made to evaluate the efficiency of ordinary UV light for cross-linking DNA onto nitrocellulose membrane (NCM) or nylon membrane (NM) with respect to optimal hybridization signals. In view of the extensive use of NCM or NM in Southern or Northern blot hybridization as well as plaque or colony lifts during library screening, we evaluated the efficiency of an ordinary UV source for overall DNA fixation by monitoring the intensity of the hybridization signals. Our results indicate that longer than optimal UV exposure reduces the signal intensity whereas shorter exposure, due to insufficient immobilization, produces incomplete hybridization signals. Relevance of empirical optimization for cross-linking DNA by using ordinary UV source is elucidated. Materials and Methods The ordinary UV light used for DNA immobilization was from our tissue culture hood fitted with a 30-W UV tube G30T8 (NIS Sankyo-Denki, Japan). The DNA membranes from different sources were immobilized by using this UV light, and the efficiency of the immobilization was assessed by hybridization followed by analysis of the sigaal intensity. Five samples of equal amounts of h DNA (200 ng each) predigested with HindlII enzyme were electrophoresed on 0.7% agarose gel, stained with ethidium bromide, and photographed under UV light. Prior to transferring the DNA, the gel was treated with 0.25 M HC1 for 15 min for depurination and denatured with 0.5 M NaOH-0.15 M NaC1 for 30 min, followed by neutralization with 0.5 M Tris-0.15 M NaC1 for 30 min, all at room temperature. The DNA was then transferred onto NCM (Schleicher and Schuell) by using 20 × SSC following standard procedures [2]. Completion of the DNA transfer was ascertained by restaining and checking the gel. Of the five lanes, 1-4 were exposed to UV light for 10, 15, 20, and 30 min, and the lane 5 was left unexposed. The blot was hybridized with end-labeled h DNA (HindlII digested) following the hybridization protocols mentioned elsewhere [5], and the signal intensity was quantified by densitometric analysis using Bio-Rad video-densitometer model 640. A similar strategy was followed for genomic DNA isolated from Indian catfish Heteropneustes fossilis (Bloch). DNA from four different blood samples was digested with HinfI (New England Biolabs) by following the supplier's specifications and then electrophoresed and transferred onto NCM as mentioned

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96 GATA 10(3-4): 95-98, 1993

above. After Southern transfer, the membrane was cut into four equal strips, each containing four lanes of individual DNA samples. These strips were then exposed independently to the UV source for 10, 15, 20, and 30 min and subsequently hybridized with a 36-base-long synthetic oligodeoxyribonucleotide probe (OAT36) comprising nine repeats of a 5'-GACA-3' motif. The origin, synthesis, and purification of the oligo probe have been reported elsewhere [5, 6], and the probe labeling and hybridization conditions have been described elsewhere [6, 7]. The third experiment, based on the outcome of the previous ones, was carried out to ascertain whether the optimal time of UV exposure that we had observed was adequate for the circular filters of library plaque lifts. During the screening of the bovine genomic library constructed in EMBL3 (Clonetech), duplicate as well as single plaque lifts were made on 82-mm circular filters (Schleicher and Schuell). These filters were subjected to denaturation and neutralchization steps [8] and were exposed to the abovementioned UV source for 15 min. The duplicate circular filters were hybridized with end-labeled oligo probe (OAT36) and single lifts were hybridized with a 1.8-kb nick-translated [9] KpnI-repetitive fragment of the cow Bos taurus genomic DNA following standard hybridization procedures [8].

Results and Discussion The h DNA hybridization results revealed signals of varying intensity in all of the lanes, including the one that was not exposed to UV light (Figure 1A). Unexposed lane 5 showed a distinctly reduced signal, which was attributed to insufficient DNA fixation. Signals in lane l, exposed to UV light for 10 rain, were not considered to be optimal since the overall signal intensity was found to be relatively reduced compared with that of lane 2. Similarly, progressively longer UV exposure reduced the overall hybridization signals, with a marked effect being observed in the high molecular weight range (see Figure 1A, lanes 3 and 4). Lane 2, exposed to UV light for 15 min, showed optimal signals both in the low as well as the high molecular weight range. The increased level of UV energy has been suggested as a cause of pyrimidine dimer formation in the DNA [10]. The observation that longer exposure reduces the signals, preferably in the higher molecular weight DNA, suggests that larger fragments are probably more prone to dimer formation. To substantiate our visual assessment of the signal intensity, we carried out a densitometric analysis

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B Figure 1. (A) Autoradiogram of HindItI-digested h DNA used as probe and target substrate. Lanes 1-4 were exposed for 10, 15, 20, and 30 min, respectively. Note the maximal signal intensity in lane 2 and a gradual loss of signals observed with the progression of the exposure time in lanes 3 and 4. Lane 5, unexposed to ultraviolet light, shows faint signals. (B) Densitometric analysis of the autoradiogram of h DNA. Panels from top to bottom correspond to lanes 1-5 of A. Numbers 1-8 represent the signal intensity of DNA fragments ranging from 23.1 to < 0.56 kb. Note the diminishing peaks with the progressively longer ultraviolet exposure.

© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

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GATA 10(3-4): 95-98, 1993

Empirical Optimization for Cross-Linking DNA

Table 1. Numerical Values of the Densitometric Scan of h DNA Relative signal intensity (area under the peak) (absolute values) DNA samples

Exposure time to UV (min)a

1

2

3

4

5

1 2 3 4 5

10 15 20 30 Unexposed

126.0 178.8 108.0 72.0 24.5

121.1 158.0 114.8 52.5 18.0

81.0 110.5 87.0 28.5 15.0

22.0 42.0 20.0 3.5 7.5

137.5 163.5 23.0 20.0

Peak numbers 6

162.0 c 22.5 18.8 20.6 17.5

7

8

96.5 160.0

69.5 290.8 525.0" 580.5" 468.8c

Cumulative intensityb 678.1 1100. I 1037.1 780.6 571.3

~UV, ultravioletlight. bCumulativesignal intensityof all the peaks. 'Cumulative values of merged peaks 5 and 6 and 7 and 8.

(Figure 1B). The scan results clearly showed gradually diminishing peaks of high molecular weight DNA with increasing UV exposure time. The cumulative signal intensity (Table 1) for each densitometric scan with respect to UV exposure for various time intervals was highest (1100.1) in the DNA sample exposed for 15 min and was lowest in the unexposed one (see Table 1, sample 5). These results on the densitometric quantitation of the signal intensity agree with our visual assessment. Similar results were obtained with restriction-digested fish genomic DNA hybridized with the OAT36 probe where overall reduced signal intensity was noticed with progressively longer UV exposure (data not shown). The lanes exposed to UV light for 15 min showed maximal signals, indicating that optimal UV exposure is crucial for obtaining complete hybridization signals, and even slightly longer than optimal exposure may be counterproductive, which is also corroborated by our densitometric analysis. On this basis, in the subsequent experiment, we exposed all of the plaque lifts for 15 min and hybridized with the labeled probes. Clearly discernible Signals were obtained in all of the plaque lifts (Figure 2). The duplicate filters hybridized with the OAT36 probe showed identical signals (see the circles in Figure 2A and B), indicating that the 15-min exposure chosen for these filters was sufficient. The genomic DNA of cow Bos taurus digested with KpnI enzyme revealed a 1.8-kb fragment. We isolated this fragment by agarose gel electrophoresis, cleaned it with Gene Clean (Bio-101) and, after nick translation, used it as a hybridization probe. Although the KpnI fragment was isolated for a different experiment, we used this as the probe to detect signals in the circular filters. Clearly discernible hybridization signals were obtained in these filters. Figure 2C represents a single lift obtained in the

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D

Figure 2. Autoradiogram of plaque lifts from the bovine genomic library (EMBL3, Clonetech) exposed to ultraviolet light for 15 min. (A and B) Duplicate filters hybridized with oligo probe OAT36 (for details, see the text). Note the intense positive signals (encircled) in A and their corresponding duplicates (encircled) in B, (C and D) Hybridized with the 1.8-kb nick-translated KpnIrepetitive fragment from the cow Bos taurus genome. Note clearly discernible signals in the single lift obtained during the initial library screening (C) and very strong signals representing purified positive plaques (D).

initial screening of the library. Equally discernible signals were obtained in the other filter also obtained after several rounds of plaque purification (Figure 2D). This suggests that exposure of the circular filters to UV light for 15 min was sufficient, since all of the signals were recorded in duplicate as well as single plaque lifts. In the present work, target substrates as well as DNA used as hybridization probes were from different sources. The rationale is based

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98 I. Parwez et al.

GATA 10(3-4): 95-98, 1993

on the fact that we wanted to ascertain whether the inferences drawn on the basis of one experiment are corroborated by other experiments. It may be mentioned that, with this approach, genomic DNA as well as DNA from the library lifts may be crosslinked onto the membrane. The apparently optimal time reported here, however, may not be ideal with respect to other ordinary UV sources, since actual DNA cross-linking is based not only on exposure time but also on the total output of UV energy. An ordinary UV lamp has a limited life span and, with the passage of time, a natural shift in the energy output is to be expected. This type of ordinary UV source does not have an internal device to compensate for the shift in the energy output as compared with the commercially available UV ovens. Hence, it is appropriate to carry out the initial optimization of exposure time with any new source of ordinary UV energy for subsequent use for cross-linking DNA. The results reported here are based on the UV tube light, which has been in constant use for > 2 years, and this may provide basic guidelines. Thus, besides commercially available UV ovens, an ordinary source of UV light may be used and this may

prove to be an even more feasible approach for fixing DNA on a large number of filters during library screening.

Institutional core support for this work and financial assistance to I.P. in the form of a short-term inland visiting fellowship from Indian National Science Academy (INSA) are acknowledged. The authors are thankful to Dr. Anil K. Suri and Shri P. Sahai for densitometric tracings.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Noyes BE, Stark GR: Cell 5:301-304, 1975 Southern EM: J Mol Biol 98:503-517, 1975 Khandjian EW: Biotechnology 5:165-167, 1987 Church GM, Gilbert M: Proc Natl Acad Sci USA 81:19911995, 1984 Ali S, Mueller C, Epplen JT: Hum Genet 74:239-243, 1986 Ali S, Epplen JT: Indian J Biochem Biophys 29:1-9, 1991 Ali S, Gauri, Bala S: Anim Genet 24:199-202, 1993 Sch/ifer R, Ali S, Epplen JT: Chromosoma 93:502-510, 1986 Rigby PWJ, Dieckmann M, Rhodes C, Berg P: J Mol Biol 113:237-251, 1977 Lee H, Birren B, Lai E: Anal Biochem 199:29-34, 1991

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