Anal. Chem. 1995, 67, 2644-2649
Time-Resolved Fluorometric Hybridization Assays with RNA Probes Synthesized from Polymerase Chain Reaction-Generated DNA Templates Paula Radovich, Susan Bortolin, and Theodore K. Christopoulos* Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, Ontario N9B 3P4, Canada
DNA templates suitable for direct synthesis of RNA probes are produced by the polymerase chain reaction. The nucleic acid sequence of interest is amplified using a downstream primer carrying the "7 RNA polymerase promoter sequence. The modified primer is incorporated into the amplitied DNA, which is subsequently used for RNA probe synthesis in the presence of T7 RNA polymerase and a hapten-labeledribonucleotide (digoxigeninUTP). As a model, we prepared RNA probes specitic for the BCR-ABL mRNA characteristic of chronic myelogenous leukemia. The probes are used in time-resolved fluorometrichybridization assays. Mixtures of BCR-ABL positive and negative cells, as well as whole blood samples, are analyzed. The sample mRNA is amplified using a biotinylated upstream primer. The amplified product (target DNA) is captured onto streptavidin-coated wells and hybridized to the RNA probe. The hybrids are detected with an alkaline phosphatase (ALP)-labeled antibody. ALP hydrolyzes the phosphate ester of fluorosalicylic acid, and the fluorosalicylate produced forms highly fluorescent ternary complexes with Tb3+-EDTA, which can be quantified by measuring the Tb3+ fluorescence in a time-resolved mode. As low as 0.4 fmol of target DNA can be detected. Also, a single leukemic cell may be detected in the presence of 0.5 million "normal" cells. Nucleic acid hybridization has become a fundamental analytical technique for detecting specific DNA or RNA sequences and is used extensively in research and (more recently) in routine laboratories.' The principle of nucleic acid hybridization is based on the ability of labeled DNA or RNA probes to bind with high afiinity and specifcity to their complementary nucleic acid sequences (targets). Major areas of application of hybridization assays include the analysis of inherited mutations, deletions or insertions associated with genetic disease, the detection of viral or bacterial nucleic acids, the analysis of mutations and gene rearrangements associated with malignancies, and DNA lingerprinting for identification of individuals by hybridization with DNA minisatellite probes (forensic science).Is2 The most common method for labeling probes has been the incorporation of the radioisotope 32P. However, the short halfMe of 32P,the health hazards and problems associated with its (1) Landegren, U.; Kaiser, R.; Caskey, C. T.: Hood, L. Science 1988,242,229-
237. (2) Kricka, L.J. Nowisotopic DNA Probe Techniques; Academic Press Inc.: San Diego, CA, 1992.
2644 Analytical Chemistry, Vol. 67, No. 75, August 7, 1995
use and disposal, and the long exposure times (many hours to days) required for detection have placed limitations on the routine use of such hybridization assays in the clinical laboratory. In recent years, research efforts have focused on the development of nonisotopic alternatives? Nonisotopic labeling of DNA or RNA probes can be accomplished by two strategies. In the direct labeling approach, the label (e.g., fluorescein, alkaline phosphatase, horseradish peroxidase) is covalently attached to the pr0be.4.~In the indirect labeling approach, a ligand is attached to the probe, and the hybrids are detected by using a specific labeled binding protein. Thus, biotin can be used as a ligand in combination with labeled (strept)aVidins6Also, haptens (e.g., 24Nacetoxy-N-acetylamino)fluorene,5bromodeoxyuridine, and digoxigenin) have been used with labeled antibody molecules.7~8 The most common method for synthesis of RNA probes involves the insertion of the DNA sequence of interest into a suitable transcription vector (usually a plasmid) which contains a multiple cloning site located downstream of an RNA polymerase promoter (e.g., T3, "7, or SP6 promoter)? The vector is then linearized by restriction enzyme digestion in a position downstream of the DNA insert and subsequently is transcribed in the presence of a labeled ribonucleotide. The RNA polymerase starts the transcription downstream of the promoter and, using the DNA insert as a template, catalyzes the synthesis of several molecules of labeled RNA probes. The drawback of this procedure is that it requires long and tedious steps, such as digestion, purification, and ligation of the DNA template into a plasmid, transfection and culturing of bacteria, and purification of plasmid DNA. In this paper, the synthetic power of the polymerase chain reaction (PCR) lo is employed in order to prepare a DNA template suitable for direct synthesis of RNA probes. A remarkable property of PCR is that the 5' ends of the primers can be modified without significantly compromising the yield of the reaction (as long as the 3' ends remain intact). Thus, small molecules such as fluorophores, haptens, and ligands may be attached to the 5' ~
~~~
~
~~~
~~
(3) Diamandis, E. P. Clin. Chim. Acta 1990,194,19-50, (4) Pollard-Knight, D.; Read, C. A; Domes, M. J.; Howard, L. A; Leadbetter, M. R; Pheby, S. A; McNaughton, E.; Syms, A: Brady, M. A W. Anal. Biochem. 1990,185, 84-89. (5) Jablonski, E.; Moomaw, E. W.; Tullis, R H.; Ruth, J. L. Nucleic Acids Res. 1986,14, 6115-6128. (6) Christopoulos. T. IC;Diamandis, E. P.; Wilson, G. Nucleic Acids Res. 1991, 29,6015-6018. (7) Tchen, P.; Fuchs, R P. P.; Sage, E.; Leng, M. PYOC.Natl. Acad. Sci. U.S.A. 1984,81, 3466-3470. (8) Holtke, H. J.; Kessler, C. Nucleic Acids Res. 1990,18, 5843-5851. (9)Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning. A Laboratoy Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY,1989. (10) Mullis, K. B.; Faloona, F. A Methods Enzymol. 1987,155, 335-350.
0003-2700/95/0367-2644$9.00/0 0 1995 American Chemical Society
BCR ABL ends for subsequent capture and detection.11$12As well, DNA 3' sequences corresponding to restriction sites or to speciiic promot5' ers may be added for subsequent cloning,13t r a n ~ c r i p t i o n , ~or~ J ~ c \ s b d translation16 of the amplified DNA As a model system, we synthesized RNA probes complementary to the characteristic BCR-ABL mRNA produced from the Philadelphia (Ph) chromoFigure I. The BCR-ABL mRNA with the location of the oligonucleotides used as primers for the reverse transcription and the polysome. A suitable DNA template containing the T7 promoter was merase chain reactions. Primer a is used in reverse transcription of first prepared starting from a Ph chromosome positive cell line the BCR-ABL mRNA. Primers a and b are the downstream and (K562 cells). Cellular mRNA was isolated, and after reverse upstream primers for the first amplification of the BCR-ABL cDNA. transcription, the BCR-ABL sequences were amplified by PCR, Primer c carries the T7 promoter sequence at its 5' end. Primers c with a downstream primer canying the T7 promoter at its 5' end. and d are used for the second amplification, which produces the T7 promoter-containing DNA template. Primer f is biotinylated at the 5' The DNA template was used directly for transcription in the end. Primers e and f are the inside primers for the nested PCR of presence of digoxigenin (Dig)-UTP, thus producing Diglabeled the sample BCR-ABL cDNA, which produces the biotinylated target RNA probes. Moreover, the DNA template was immobilized on DNA. magnetic beads and used repeatedly for probe synthesis. The target DNA (the analyte) was prepared from the samples by reverse transcription polymerase chain reaction, using a nucleotides a and b are not labeled and serve as the outer PCR biotinylated upstream primer. The amplified fragments were then primers.I2 Oligonucleotide c is S-AAT'ITAATACGACI'CA(XATcaptured on streptavidin-coated wells and hybridized to the DigA G G G A T C A G A C C C X A G G C Y , a 47-mer which conlabeled RNA probes. The hybrids were detected with an alkaline sists of two parts: the underlined sequence at the 5' end phosphatase-labeled anti-hapten antibody and fluorosalicyl phoscorresponds to the T7 promoter, and the rest is complementary phate as substrate. The fluorosalicylate produced forms fluoresto ABL exon 2. This oligonucleotide serves as the downstream cent ternary compexes with Tb3+-EDTA (PH 12.5) which are primer for the PCR-mediated incorporation of the T7 promoter. measured by time-resolved f l u o r ~ m e t r y . ~ ~ , ~ ~ Oligonucleotides d and e are not labeled and serve as the PCR inner p r i m e r ~ . ~The ~ J ~sequence of oligonucleotide f is identical EXPERIMENTAL SECTION to that of primer d, but it is biotinylated at the 5' end. Biotin-lCdATP, 0.4 mmol/L, and Polymorphprep (a solution The mRNA was isolated from 1 million cells and reverse containing 13.8% sodium metrizoate and 8% dextran-500) were transcribed as described previously.12 All PCRs were set as in obtained from Gibco Laboratories Life Technologies (Gaithersref 12. The temperature profile for each cycle was as follows: burg, MD). The phosphate ester of 5fluorosalicylic acid WAP) denaturation at 95 "C for 30 s, annealing at 60 "C for 30 s, was purchased from Cyberfluor Inc. (Toronto, Canada). T7 RNA extension at 72 "C for 60 s. Upon completion of cycling, the polymerase (50 units/pL) was from Stratagene (La Jolla, C&. Antireaction mixture was held at 72 OC for 10 min and then cooled at digoxigenin antibody (Fab fragments, from sheep) conjugated to 4 "C. alkaline phosphatase, blocking reagent for nucleic acid hybridizaSynthesis of the DNA Template. mRNA was isolated from tion and detection (Catalog No. 1096 176), digoxigenin-11-uridine 1million K562 cells and then reverse transcribed by using primer 5'-triphosphate (Dig-11-UTP, 10 mmol/L), ribonucleoside triphosa. The reverse transcription mixture was diluted 10 times with phates (rNTP, 100 mM), human placental ribonuclease inhibitor water, and a 2 pL sample (containing mRNA-cDNA hybrids from (40 units/pL), and terminal deoxynucleotidyl transferase (25 10000 cells) was added as a template for PCR PCR was units/pL) were all purchased from Boehringer Mannheim (Laval, performed for 25 cycles using oligonucleotides a and b as Canada). Formamide was obtained from Fisher Scientific Vordownstream and upstream primers, respectively. This ampliicaonto, Canada). Streptavidin and pUC18 DNA marker (HaeIII tion produced a 308 bp DNA fragment. The PCR mixture was digest, 0.81 g/L) were from Sigma (St. Louis, MO). Magnetic diluted 50 times in water, and a 5 pL sample was reamplified for polystyrene beads (Dynabeads M-280) coated with streptavidin 30 cycles using oligonucleotides c and d as the downstream and and the magnetic particle concentrator (MPC, Model E-1) were upstream primers, respectively. Because primer c cames the T7 purchased from Dynal AS. (Oslo, Norway). The Wizard PCR promoter sequence at its 5' end, a 225 bp amplitied fragment is Preps DNA purification system was purchased from Promega generated (DNA template) with the T7 promoter incorporated. Corp. (Madison, wr). The size and purity of the PCR product were confirmed by agarose The following oligonucleotides were used as reverse transcrip gel (2%) electrophoresis and ethidium bromide staining. tion and PCR primers (synthesized from Biosynthesis, Inc., Preparation of Immobilized DNA Template. A 100 pL Lewisville, nr). Their locations are shown in Figure 1. Oligoaliquot of the 225 bp DNA template, with the T7 promoter incorporated through PCR, was first purified from the excess of (11) Kemp, D. J.; Smith, D. B.; Foote, S. J.; Samaras, N.; Peterson, M. G. Proc. Natl. Acad. Sci. U.S.A. 1989,86,2423-2427. primers using the Wizard PCR Preps purification system, accord(12) Bortolin, S.; Christopoulos, T. K. Anal. Chem. 1994,66,4302-4307. ing to the manufacturer's instructions. The puritied DNA frag (13) Kaufman, D. L.; Evans, G. A BioTechniques 1990,9, 304-306. ment was then tailed at the 3' end with biotin-dATP using terminal (14) Innis, M. A; Gelfand, D. H.; Sninsky, J. J.; White, T. J. PCR Protocols. A Guide to Methods and Applications; Academic Press, Inc.: San Diego, CA, deoxynucleotidyl transferase. The tailing reaction was performed 1990; Chapter 21. in a total volume of 20 pL and consisted of 0.2 mol/L potassium (15) Bales, K R; Hannon, K; Smith, C. IC;Santerre, R F. Mol. Cell. Probes 1993, cacodylate, 25 mmol/LTris-HC1 @H 6.6), 0.25 g/L BSA, 5 mmol/L 7, 269-275.
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(16) Kain, K C.; Orlandi, P. A; Lanar, D. E. BioTechniques 1991,10, 366374. (17) Evangelista, R A; Pollak, A; Gudgin-Templeton. E. F. Anal. Biochem. 1991, 197, 213-224. (18) Christopoulos, T.IC;Diamandis, E. P. Anal. Chem. 1992,64,342-346.
(19) Kawasaki, E. S.; Clark, S. S.; Coyne, M. Y.; Smith, S. D.; Chamblin, R; Witte, N. 0.; McCormick. F. P. Proc. Natl. Acad. Sci. U S A . 1988,85, 56985702.
Analytical Chemistry, Vol. 67,No. 15, August 7, 7995
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CoC12, 40 pmol/L biotin-dATP, 0.5 mmol/L dATP, 25 units of terminal deoxynucleotidyl transferase, and 9 pL of purified DNA template. The reaction mixture was incubated at 37 "C for 20 min. Afterward, the biotin-tailed DNA template was purified from the excess of biotin-dATP with the Wizard DNA purification system. Streptavidin-coated magnetic beads were prepared as follows: 200 pg of beads was placed into a microcentrifuge tube and washed once with 100 mmol/L NaOH, once with 50 mmol/L NaC1, and three times with a solution containing 10 mmol/L Tris-HC1, pH 8.0, 1 mmol/L EDTA, and 50 mmol/L NaCl (solution A). Finally, the beads were resuspended in 15pL of solution A. Next, 15 pL of biotin-tailed DNA template was added, and the mixture was incubated for 30 min at room temperature. Subsequently, the beads were separated from the reaction mixture and washed twice with solution A before being and used in the transcription reaction (described below). Synthesis of RNA Probes. RNA probes were synthesized either in solution or on the solid phase (magnetic beads). The transcription reactions were performed in a total volume of 20 pL containing 40 mmol/L Tris-HC1, pH 8.0, 6 mmol/L MgC12, 10 mmol/L dithiothreitol, 2 mmol/L spermidine, 10 mmol/L NaCl, 1 mmol/L ATP, 1 mmol/L GTP, 1 mmol/L CTP, 0.65 mmol/L UTP, 0.35 mmol/L Dig-UTP, 40 units of T7 RNA polymerase, 20 units of ribonuclease inhibitor, and 5 pL of the DNA template (for transcription in solution). The reaction mixture was incubated at 37 "C for 2 h, and then the reaction was terminated by adding 2 pL of 0.2 mol/L EDTA, pH 8.0. The probe was stored at -20 "C until use. For synthesis of RNA probe from immobilized DNA template, the transcription mixture (20 pL, prepared as above) was added directly to the beads, and the reaction was carried out for 2 h at 37 "C. The reaction was then stopped by adding 2 pL of 0.2 mol/L EDTA, pH 8.0, and the transcription mixture was separated from the beads. The beads were then resuspended in 100 pL of a solution containing 10 mmol/LTris-HC1, pH 8.0,l mmol/L EDTA, and 1 mol/L NaCl (solution B), and the mixture was stored at 4 "C until further use. To reuse the beads (for another synthesis of RNA probe), solution B was removed, and the beads were washed twice with solution A. Preparation of Whole Blood Specimens. Four milliliters of fresh whole blood, with EDTA as anticoagulant, was layered over 4 mL of Polymorphprep and centrifuged at 400g for 30 min at room temperature. After centrifugation, two leukocyte bands were visible: the top band consisted of mononuclear cells, while the lower contained the polymorphonuclear cells. The polymorphonuclear band was removed and diluted with an equal volume of phosphate-buffered saline (PBS, 10 mmol/L sodium phosphate, 1.8 mmol/L potassium phosphate, 0.14 mol/L NaCl, 2.7 mmol/L KCl, pH 7.4). The cells were spun down (400g, 10 min at room temperature), and the supernatant was discarded. The cells were washed once more with PBS and finally resuspended in PBS and counted. One million cells were subsequently used for mRNA isolation and cDNA synthesis. The BCR-ABL sequences of interest were then amplified by nested PCR PCR I was carried out with 10pL of reverse transcription mixture for 25 cycles using primers a and b. The product was diluted 50 times in water, and a 5 pL sample reamplified by PCR 11. PCR I1 was performed for 30 cycles with primers e and f. During PCR 11, the biotinylated primer f is incorporated into the newly formed strands, so that 2646
Analytical Chemisty, Vol. 67,No. 75,August 1, 7995
the amplified product (200 bp) is labeled with biotin at the one 5' end. Hybridization Assay of the Amplified DNA with the RNA Probe. Opaque, polystyrene microtitration wells (Microlite 2; Dynatech Lab Inc., Chantilly, VA) were coated overnight with 100 pL of 1.4 mg/L streptavidin solution in PBS. Before use, streptavidin-coated wells were washed three times with wash buffer containing 50 mmol/L Tris, pH 7.4, 0.15 mol/L NaC1, and 0.1% (v/v) Tween-20. The PCR mixture was diluted 2@foldin PBS containing 0.1%Tween-20, and a 100pL sample was pipetted into each well, in duplicate. The wells were shaken for 30 min at room temperature and washed three times. Subsequently, the one strand was removed by adding 100 pL of 0.2 mol/L NaOH, incubating for 30 min, and washing three times. The wells were then blocked by a 15 min incubation with 250 pL of blocking solution consisting of 0.1%blocking reagent in 0.1 mol/L maleate and 0.15 mol/L NaCl, pH 7.5. The Dig-labeled RNA probe was diluted 13@fold in hybridization buffer containing 50% (v/v) formamide and 50%blocking solution. Hybridization buffer was made fresh daily and kept at 42 "C until needed. Hybridization was performed for 20 min at 42 "C with 100 pL/well of the probe solution. The wells were then washed three times, and the hybrids were detected by adding 100 pL/well of 750 units/mL alkaline phosphatase-labeled anti-digoxigenin antibody diluted in the wash buffer. After 30 min incubation, the excess of antibody was removed by washing the wells three times. Subsequently, we added 100 pL/well of substrate solution (1 mmol/L FSAP, 0.1 mol/L Tris-HC1, pH 9.1, 0.1 mol/L NaCl, and 1 mmol/L MgC12) and incubated the solution for 30 min. The reaction was stopped by adding 100 pL/well of a 0.4 mol/L NaOH, 2 mmol/L Tb3+,3 mmol/L EDTA, and 1 mol/L Tris solution, pH 12.5. The wells were shaken for 1min, and the fluorescence was measured with a time-resolved fluorometer (CFI 615, CyberFluor) . RESULTS AND DISCUSSION Figure 1 shows the position of the various PCR primers used in this work. Downstream primers are complementary to the ABL part of the BCR-ABL mRNA, whereas the upstream primers are homologous to the BCR part. mRNA from samples is amplified by nested PCR PCR I is performed with primers a and b and produces a 308 bp DNA fragment. Subsequently, this fragment is reamplified in PCR I1 using primers e and f. PCR I1 produces a 200 bp DNA, which is biotinylated at the one 5' end (target DNA). An aliquot of PCR I1 mixture is diluted appropriately, captured on streptavidin-coated, wells and analyzed by hybridization with the Dig-labeled RNA probe. First, the binding capacity of the streptavidin-coated wells was tested by preparing dilutions of the PCR mixture and applying 100 pL/well. The results are presented in Figure 2A. The signal increases with the amount of PCR mixture applied per well. The solid phase is saturated at a 2@fold dilution of the PCR mixture, which corresponds to 2.5 pmol of biotinylated species (primer plus amplified product) /well. Although PCR mixtures from various samples may contain different amounts of amplified product, the total amount of biotin applied per well is constant since this is determined from the amount of biotinylated primer originally introduced into the PCR mixture. The T7 promotercontaining DNA template to be used for RNA probe synthesis was prepared from K562 cells by two rounds of amplification. The first round was performed with unlabeled primers (a and b) flanking the region of interest. The second
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Figure 2. (A) Study of the binding capacity of the streptavidin-coated solid phase. Biotinylated target DNA was produced by PCR from 100 K562 cells. Various dilutions of the PCR mixture were then captured on SA-coated wells and analyzed by hybridization with Dig-labeled RNA probe. (B) Optimization of the Dig-UTP/UTP ratio used in probe synthesis. RNA probes were prepared at various Dig-UTP/UTP ratios and then used in hybridization assays of target DNA produced (1) by PCR from 100 K562 cells and (2) by a 5-fold dilution of the PCR mixture used in (1). Hybridization assays were exactly as described in the Experimental Section.
round was carried out with primers c (containing the T7 promoter) and d. Although this approach involves two consecutive PCRs, it ensures that a single DNA template is always obtained (as seen by agarose gel electrophoresis of the amplified products). The DNA template is used directly (without prior purification) in the transcription reaction. The selection of the T7 promoter-canying primer determines the direction of the transcription reaction. If the downstream primer carries the promoter (as is the case here), the synthesized probe is complementary to the sense strand. If the promoter sequence is attached to the upstream primer, then a probe complementary to the antisense strand is produced. In order to study the effect of Dig-UTP concentration used in the labeling of the probe, we performed transcription reactions with identical concentrations of DNA template and NTP while varying the Dig-UTP/UTP ratio. The total concentration of DigUTP plus UTP was kept constant at 1 mmol/L (equal to the other NTP concentrations). The RNA probes were then used for hybridization to DNA target. The signal/background ratios obtained vs the Dig-UTP/UTP ratios used are presented in Figure 2B. The background is the fluorescence obtained when no target DNA is present in the well and is a measure of the nonspeciiic binding of the probe and the alkaline phosphatase-labeled antidigoxigenin antibody to the solid phase. With increasing Dig-
1200
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Dilution of PCR Mixture (x)
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60
Hybridirrtlon Time (min.)
Figure 3. (A) Effect of the probe concentration on the fluorescence (1) and the signallbackground ratio (2) obtained with hybridization assays of DNA target produced by PCR of mRNA corresponding to 10000 K562 cells with Dig-labeled RNA probe. (B) Study of the effect of the hybridization time on the signaVbackground ratios obtained in hybridization assays of target DNA produced (1) by PCR from 100 K562 cells and (2) by a 5-fold dilution of the PCR mixture used in (1). All other conditions were as described in the Experimental Section.
UTP/UTP ratios, the signal/background ratio increases. A plateau is reached at a ratio of 0.5. The effect of the probe concentration was tested by preparing various dilutions of the probe in hybridization buffer and using them for measurement of amplified DNA corresponding to 10 OOO K562 cells. The results are presented in Figure 3A. As the probe Concentration was increased, a continuous increase of the fluorescence signal was observed (line 1). However, the signal/ background ratio (line 2) shows a peak and then decreases as the probe concentration becomes higher. This is because at high probe concentrations, the increase in the nonspeciiic binding of the probe to the solid phase is such that the signal/background ratio drops. The time required for completion of the hybridization reaction was studied in the range of 10-60 min, with two target DNA concentrations (see Figure 3B). It was found that the best signal/ background ratio is obtained after a 15-20 min incubation period. After 15 min, the signal is stabilized (hybridization is complete), and there is a gradual increase of the background as the probe remains in contact with the solid phase for a longer time. The short incubation time is a signiilcant advantage of the proposed system over the classical Southern or Northern blots, where hybridization takes place on membranes (nitrocellulose or nylon) and requires hours for completion and long washing steps following hybridization. Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
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A
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u t
.d
= I
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I
Table 1. Data Showing the Reusability of Immobilized DNA Template
signal/background ratio day
1cellu
100 cellsa
1 2 4 5 8 29
2.6 2.7 2.1 3.7 2.8 3.5
16.5 16.2 12.3 18.4 14.7 18.0
Number of K562 cells in the presence of 500 000 HL60 cells.
im 1
.1
Amplified
10
im
DNA (fmoler)
4a
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1 m
.I
1
IO
im iao imrrimao
Number of K562 cells Flgure 4. (A) Plot of fluorescence vs the number of femtomoles of biotinylated target DNA applied per well. Biotinylated target DNA was quantitated densitometrically, and various dilutions (in PBS-0.1 YO Tween-20 buffer) were analyzed by hybridization as described in the Experimental Section. (B) Study of the variation of the fluorescence with the number of K562 cells. Mixtures containing mRNA from 1 to 10 000 K562 cells in the presence of 500 000 HL-60 cells were subjected to PCR (see Experimental Section), and the amplified fragments were analyzed by hybridization.
In order to assess the sensitivity of the optimized hybridization assay, biotinylated ampliied products (200 bp) from K562 cells were pooled and subjected to agarose gel electrophoresis (on a 2%minigel), followed by ethidium bromide staining. Pictures of the stained gels were taken under UV excitation, and negatives were produced. The target DNA was then quantitated by densitometry; (GS670 densitometer, BioRad Laboratories Ltd., Mississauga, Canada) using pUC18 restriction fragments as standards. Various dilutions of target DNA in PBS-0.1% Tween 20 were then prepared, and 100 pL aliquots were analyzed, in duplicate, by hybridization. In Figure 4A, the fluorescence is plotted vs the amount of amplified DNA. As low as 0.4 fmol of amplified DNA may be detected (signal/background ratio, 1.5). The ability of the proposed system to detect BCR-ABL mRNA from a few K562 cells in the presence of a large excess of Ph chromosome negative cells (HL60cells) was tested by preparing and analyzing samples containing mRNA corresponding to 0, 1, 10, 100, 1000, and 10 000 K562 cells in the presence of 500 000 HL60 cells. In Figure 4B, the fluorescence is plotted vs the number of K562 cells. Amplified BCR-ABL mRNA corresponding to a single K562 cell can be detected with a signal/background ratio of 2. The background here is the fluorescence obtained in 2648 Analytical Chemistry, Vol. 67, No. 15, August 1, 1995
the presence of 500 000 HL60 cells only. The fluorescence increases with the number of K562 cells in the sample. At 10 000 cells, the characteristic “plateau effect” of PCR is observed. Here, the yield of PCR decreases because the accumulation of product results in an increased competition between reannealing of the amplified DNA strands and primer binding. The sensitivity achieved with the present method is similar to a previous assay12 that involves incorporation of a labeled primer during PCR. The contribution of the present work lies on the synthesis of RNA probes which can be used for detection of specific DNA sequences by hybridization. Whole blood samples from five patients were subjected to the preparation steps (described in the Experimental Section) for isolation of granulocytes, purification of mRNA, cDNA synthesis, and PCR ampliication. The amplification product was then analyzed by hybridization with the RNA probe. The negative represented the fluorescence obtained from 500 000 HL60 cells and gave a signal of 3356 & 503. Patients 1,2,4, and 5 had been diagnosed as Ph chromosome positive by cytogenetics of bone marrow aspirates and were undergoing treatment. Patient 3 was negative for the Ph chromosome. The analyses were performed in triplicate, and the results were as follows: 20 844 f 2111,14 510 f 132, 3602 f 99, 6931 & 663, and 40 311 f 2871, for patients 1-5, respectively. The higher the fluorescence, the greater the concentration of BCR-ABL mRNA copies present in the sample. This, in turn, means a greater number of leukemic cells in the sample and a higher likelihood of relapse. Finally, to further enhance the practicality of the proposed method, we immobilized the DNA template onto magnetic beads and used it for synthesis of RNA probes on the solid phase, thus allowing for a reusable DNA template. For immobilization, the DNA template was tailed at the 3’ end with biotin-dATP using terminal deoxynucleotidyl transferase. After the excess of biotindATP was removed, the DNA template was allowed to bind to streptavidin-coated magnetic beads. Subsequently, the beads can be stored in a solution of high ionic strength (1mol/L NaC1) that diminishes the repulsive forces between the negatively charged streptavidin and the DNA molecules. In order to test the reusability of the DNA template-coated beads, we synthesized RNA probes six times over a 1 month period and tested their ability to hybridize with amplification products corresponding to 1 and 100 K562 cells. The signal/ background ratios obtained are given in Table 1. The results suggest that the DNA template remains stable on the beads and the sensitivity of the system is maintained. DNA and RNA probes are used widely in the literature. However, RNA probes offer considerable advantages. The hybrids
involving RNA probes (RNA-DNA and RNA-RNA) are more stable than DNA-DNA hybrids and therefore produce higher signal in hybridization reactions than DNA probes of equal activity. Moreover, because the RNA probes are singlestranded, there is no competition between self-reannealingof the probe and probetarget hybrid formation (as is the case with doublestranded DNA probes). A disadvantage of RNA probes is the possibility of degradation by ubiquitous ribonucleases. However, if the general precautions required for RNA work are observed, the probes remain stable for a long period of time. For instance, we synthesized RNA probes and stored them for at least 4 months at -20 "C, and we found no loss in assay sensitivity. The use of PCR offers selective amplification of the sequence of interest from complex mixtures and simultaneous incorporation of the T7 promoter. Thus, a complete transcription unit (DNA template) is generated in only 4 h, as opposed to several days
required for cloning experiments. In addition, the DNA template can be immobilized on a solid phase and reused several times for RNA probe synthesis. ACKNOWLEWMENT This work was supported by grants to T.KC. from the National Science and Engineering Research Council of Canada (NSERC) . S.B. holds an NSERC Graduate Scholarship. We wish to thank Dr. H. Abu-Zahra from the Windsor Regional Cancer Center and Dr. D. Crisan from William Beaumont Hospital, Royal Oak, MI, for providing patient samples. Received for review March 24, 1995. Accepted May 18,
995.m AC950293S e Abstract
published in Aduonce ACS Abstracts, June 15, 1995.
Analytical Chemistry, Vol. 67, No. 15, August 1 , 1995
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