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[71] D i r e c t I n c o r p o r a t i o n o f B i o t i n into D N A

By

M. ROTHENBERG, EDWARD A. BAYER

M E I R W I L C H E K , JEFFREY

AvI REISFELD,

and

As shown in other chapters of this volume, biotin is currently the nonradioactive probe of choice. For gene probes, biotin-containing mono-, oligo-, and polynucleotides have been prepared by various enzymatic and chemical means. Nevertheless, the utility of biotin as a probe can be further increased by introducing other biotin-containing reagents that react on the DNA at different positions which are not important for hybridization. In the following discussion, we describe the use of two classes of such reagents, namely, hydrazide derivatives of biotin and pdiazobenzoylbiocytin (DBB). Biotin hydrazide (BHZ) and biocytin hydrazide (BCHZ) label cytidine residues in a one-step transamination reaction (Fig. 1), 1 and DBB labels guanidine residues at position 8 (Fig. 2). 2 Neither of the labeling procedures interferes with subsequent hybridization, since an NH group remains on the cytidine, while position 8 of guanidine is not involved in hybridization. Biotin-dCTP and biotin-dGTP have also been prepared and were shown to be suitable substrates for DNA polymerases. Both derivatives can be introduced into DNA by nick translation.

Interaction of BHZ with DNA Reagents Denatured 3 or single-stranded DNA preparation Biotin hydrazide 4 (BHZ), 10 mg/ml in 0.1 M sodium acetate buffer (pH 4.5) 1 A. Reisfeld, J. M. Rothenberg, E. A. Bayer, and M. Wilchek, Biochem. Biophys. Res. Commun. 142, 519 (1987). For synthesis of BHZ and BCHZ, see M. Wilchek and E. A. Bayer, this volume, [13]. 2 j. M. Rothenberg and M. Wilchek, Nucleic Acids Res. 16, 7197 (1988). For synthesis of diazobenzoyl biocytin, see M. Wilchek and E. A. Bayer, this volume [13]. 3 The DNA preparation is denatured immediately prior to biotinylation by heating to 100° for 10 min followed by rapid cooling in an ice-NaCl bath. 4 To solubilize BHZ, the aqueous solution may be boiled. Owing to the relatively low solubility of BHZ, the reproducibility of the interaction can be improved by employing a more water-soluble hydrazide derivative, e.g., BCHZ. The extended lysyl chain in BCHZ would also serve to improve subsequent interaction with the avidin-based detection system.

METHODS IN ENZYMOLOGY,VOL. 184

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[71]

DIRECT BIOTINYLATIONOF DNA

609 O

0 -(CH2) 4 - CNHNH "- ( C H 2 ) 4 - CNH NH 2 -P ^,,j~,N~I

H 503 I R

I R

F]o. 1. Reactionof biotin hydrazidewith cytidinederivatives.

Sodium bisulfite Distilled water Tris-EDTA buffer [10 mM Tris-HC1 buffer (pH 7.6) containing 0.1 mM EDTA] Procedure. The DNA sample (50/~g in 50/xl solution) is brought to 0.5 ml with the biotin hydrazide solution, and sodium bisulfite is added to a final concentration of I M. The reaction is allowed to proceed for 24 hr, after which the solution is dialyzed against distilled water for 24 hr at 4°. The solution is changed 3 times during the dialysis. The contents of the dialysis bag are collected, placed into an Eppendorf tube, and concentrated to dryness in a SpeedVac Concentrator (Savant Instruments Inc., Hicksville, NY). The DNA is resuspended in the original volume of TrisEDTA buffer and stored at 4°.

0 HN~"~H

0

,~

HN~T~N~ +

0

~J ~ II N2~CNH-CH

COzH

I

0

H~

II I \ - (CH2)4 NH-C-(CH2)4""" S )

HzN J~ N ,v..-.-N

R

l

pH 9.0

N= N-~/'~

0

C NH- CH- (ell 2 )4 NH-C - (CH2 )¢"", S ')

H 2N ,,,~ N ,>'.,..,N I R

FIG. 2. Reaction of p-diazobenzoylbiocytinwith guanidinederivatives.

H

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Interaction of DBB with DNA Reagents Denatured 3 or single-stranded DNA preparation, 10 /~g in 10 /zl Diazobenzoylbiocytin (DBB), 5 8.8 mg/ml (14.6 raM) in 260 ~1 of 0. I M borate buffer (pH 9) 3 M Sodium acetate buffer (pH 5.5) Ethanol Tris-EDTA buffer Procedure. The DNA sample is added to the DBB solution, and the reaction is allowed to proceed for 30 min at room temperature. The DNA is precipitated by adding successively 0. I volume of acetate buffer and 2 volumes of ice-cold ethanol. The sample is kept at - 2 0 ° (1 hr to overnight), and the DNA is collected by a 15-min centrifugation in an Eppendorf microfuge. The pellet is then treated with 70% cold ethanol, and the washing procedure is repeated 4 times to remove excess reagent. The D N A is then resuspended in Tris-EDTA buffer (I0/xl) and stored at - 2 0 °.

Dot-Blot Hybridization Reagents Target DNA Biotinylated DNA probe Prehybridization solution: 50% formaldehyde solution containing 5x SSC [Ix SSC is 0.15 M N a C I and 15 mM sodium citrate buffer (pH 7)], 2x Denhardt's solution [50× Denhardt's solution is 10 g poly(vinylpyrrolidone), 10 g Ficoll 70, and 10 g bovine serum albumin dissolved in 1 liter distilled water], 50 mM sodium phosphate, and 300/xg/ml shredded sonicated salmon sperm DNA Hybridization solution: same as prehybridization solution except that the solution is brought to 0.3% sodium dodecyl sulfate (SDS) and contains 500/xg/ml salmon sperm DNA Tris-EDTA buffer Direct Analysis of Biotinylated DNA. Nitrocellulose membrane filters are spotted with heat-denatured, biotin hydrazide-labeled DNA in sequential half-logarithmic dilutions. The filters are prehybridized for 4 hr at 42° in prehybridization solution. The filters are then subjected to enzymebased detection as described below. DBB is prepared in situ from the aminobenzoyl-biocytinprecursor immediately prior to use,

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Analysis of Target DNA. Nitrocellulose membrane filters are spotted with sequential half-logarithmic dilutions of target DNA samples. The filters are prehybridized for 4 hr at 42° in prehybridization solution and then hybridized with 0.5 /zg/ml of the biotinylated probe dissolved in hybridization buffer. The reaction is carried out for a minimum of 12 hr at 42 °, after which the filters are washed as described earlier. The filters are then subjected to enzyme-based detection as described below. Enzyme-Mediated Colorimetric Detection Reagents Quenching buffer: 0.1 M Tris-HCl buffer (pH 7.5) containing 0.15 M NaCI Quenching solution: 3% bovine serum albumin in quenching buffer Streptavidin-conjugated alkaline phosphatase 6 (BRL BlueGENE detection kit; Bethesda Research Laboratories, Gaithersburg, MD) diluted to 2/~g/ml in quenching buffer Substrate buffer: 0. I M Tris-HC1 buffer (pH 9.5) containing 0.1 M NaC1 and 50 mM MgC12 Substrate solution7: 5-bromo-4-chloro-3-indolyl phosphate (50 mg/ml in 33/~1 of dimethylformamide) and nitroblue tetrazolium (75 rag/ ml in 40/zl of 70% dimethylformamide) in I0 ml substrate buffer Procedure. The dried filters containing the biotinylated DNA samples are incubated with quenching solution for 1 hr at 65°. The filters are then incubated at room temperature with the conjugate for 10 min. The filters are washed twice in the quenching buffer and then incubated for 10 min in substrate buffer. The reaction with substrate solution is carded out for 1-3 hr in sealed plastic bags (Dazey Seal-a-Meal).

Comments Using the above-described procedures, we have succeeded in labeling picogram quantities of DNA on dot blots. The major problem with the two reagents described in this chapter, however, is their low solubility under conditions required to modify the DNA. Other biotin-containing reagents 6 Other avidin- or streptavidin-containing conjugates or complexes (see E. A. Bayer and M. Wilchek, this volume [18]) can be used in place of the streptavidin-conjugated alkaline phosphatase reagent used in this instance. 7 Other suitable substrate solutions may be used instead. For most of our blotting studies with alkaline phosphatase, we regularly use 10 mg naphthol AS-MX phosphate dissolved in 200/zl of dimethylformamide and mixed with a solution containing 30 mg Fast Red dissolved in 100 ml of 0.1 M Tris-HCl (pH 8.4).

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that are based on the same principles but designed to comprise elements of increased solubility in aqueous solution should be developed. The use of such reagents at higher concentrations would increase the incorporation of biotin, which will eventually lead to higher sensitivity than that achieved with the reagents described in this chapter.

[72] C o l o r i m e t r i c - D e t e c t e d D N A S e q u e n c i n g

By STEPHAN BECK Since their introduction in 1981,~ biotin-labeled polynucleotides have found widespread application as sensitive affinity probes for the detection of nucleic acids and have proved to be a real alternative to the convenient but hazardous detection using radioisotopes. 2 Their use for the detection of DNA sequencing bands, however, has been described only recently) Two problems, specific to the DNA-sequencing technique, have complicated its application. First, for DNA sequencing (for review, see Refs. 4 and 5), relatively large gels are used to separate the sequencing reactions (0.1-0.3 mm in thickness, 40-100 cm in length, and 20-40 cm in width). Since a biotin-based detection requires accessibility to the separated sequencing bands for the actual visualization reaction, the band pattern has to be transferred from the very fragile gels onto an immobilizing matrix. For this purpose, special techniques and equipment had to be developed beforehandY The second problem is a consequence of the nature of the biotinylated nucleotides. Traditionally DNA-sequencing bands are labeled by enzymatic incorporation of radioactive nucleotides, such as [32p]dATP or [35S]dATP, and are visualized by autoradiography. 8,9 Although a variety of biotinylated nucleotides have been synthesized L~° and are commerI p. R. Langer, A. A. Waldrop, and D. C. Ward, Proc. Natl. Acad. Sci. U.S.A. 78, 6633 (1981). 2 M. Wilchek and E. A. Bayer, this volume [2]. S. Beck, Anal. Biochern. 164, 514 (1987). 4 j. C. Moores, Anal. Biochem. 163, 1 (1987). 5 R. Wu, ed., this series Vol. 155. 6 G. M. Church and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 81, 1991 (1984). 7 S. Beck and F. M. Pohl, EMBO J. 3, 2905 (1984). s A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. U.S.A. 74, 560 (1977). 9 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977). 10 G. Gebeyehu, P. Y. Rao, P. SooChan, D. A. Simms, and L. Klevan, Nucleic Acids Res. 15, 4513 (1987).

METHODS IN ENZYMOLOGY, VOL. 184

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Direct incorporation of biotin into DNA.

608 APPLICATIONS [71] [71] D i r e c t I n c o r p o r a t i o n o f B i o t i n into D N A By M. ROTHENBERG, EDWARD A. BAYER M E I R W I L C H...
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