ANALYTICALBIOCHEMISTRY

185,84-89

(1990)

Nonradioactive Nucleic Acid Detection by Enhanced Chemiluminescence Using Probes Directly Labeled with Horseradish Peroxidase Denise Pollard-Knight,l Christopher A. Read, Malcolm J. Downes, Lesley A. Howard, Michael R. Leadbetter, Sally A. Pheby, Elizabeth McNaughton, Allan Syms,* and Michael A. W. Brady Corporate Research, Amersham International plc, White Lion Road, Amersham, Bucks HP7 9LL, United Kingdom; and *University Diagnostics Ltd., University College London, Gower Street, London, United Kingdom

Received

August

7,1989

The use of nucleic acid probes directly labeled with horseradish peroxidase for detection of single copy sequences on Southern blots of human genomic DNA by enhanced chemiluminescence is described. Of the target sequences, 6 X lo6 molecules (1 amol) have been detected on blue sensitive film using exposures of up to 60 min and probes of 0.3-6.1 kb. The chemiluminescent signal quantified using a cooled charge coupled device (CCD) camera is proportional to probe length for DNA probes in the range 50-3671 bases. The enzyme has no significant effect on the stability of a DNA/DNA hybrid formed with a 3571-base probe and target as determined by increasing the stringency of posthybridization washes by decreasing the concentration of a monovalent cation (NaCl) and by a Tm analysis. The kinetics of DNA hybridization have been analyzed by a cooled CCD camera to provide quantitative data. Ten nanograms per milliliter of probe may be used for an overnight hybridization. Southern blots can be reprobed using a DNA probe for the same or a different sequence without the necessity of stripping off the previously bound probe. 0 1990 Academic Press, Inc.

Nucleic acid hybridization is a powerful technique for detection of specific, complementary nucleic acid sequences and is increasingly being used for detection of viruses (l), bacteria (2), and genetic disorders (3). Most commonly the probe DNA is labeled with 32P nucleotides, however, there is an increasing demand for nonradioactive alternatives. Many alternative labeling approaches have been used to facilitate nonradioactive detection: ’ To whom 84

correspondence

should

be addressed.

(i) Labeling with biotin and detection by avidin/ streptavidin conjugates prepared with enzymes, fluorescent groups, or electron-dense particles/molecules. The biotin may be introduced into the probe as a biotinylated nucleotide triphosphate by nick translation, by random priming with hexamers (4), or by use of a photoactivatable analog, photobiotin, which crosslinks to DNA on irradiation (5). (ii) Labeling with other haptens, for example, digoxigenin or sulfone groups, and detection with enzymeanti-hapten antibody conjugates (6). (iii) Direct crosslinking of a detectable protein, for example, horseradish peroxidase, to the probe DNA using an enzyme-polyethyleneimine complex and glutaraldehyde (7). A frequent criticism of these directly crosslinked probes is their instability at temperatures greater than 45°C and interference of the protein with hybridization (8). Many of the nonradioactive labeling techniques employ calorimetric or fluorescent detection which lack the proper sensitivity required, for example, for single copy gene detection in human genomic DNA in the absence of the polymerase chain reaction amplification technique (9) or high levels of target sequence. The sensitive HRP2-catalyzed enhanced chemiluminescence reaction has recently been applied to immunoassays (lo), detection of DNA on dot blots (ll), and hepatitis B viral DNA in human serum (12). We have used nucleic acid probes directly labeled with HRP-polyethyleneimine (PEI) complexes via glutaraldehyde (7) and enhanced chemiluminescence to detect single copy genes in Southern blots of human genomic DNA on blue sensitive film. Ex2 Abbreviations used: HRP, horseradish peroxidase; eneimine; CCD, charge coupled device; SSC, standard SDS, sodium dodecyl sulfate. Copyright AU rights

0

1990 of reproduction

PEI, polyethylsodium citrate;

0003-2697/90 $3.00 by Academic Press, Inc. in any form reserved.

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a5

posure times of less than 60 min are required to achieve this sensitivity. In this paper we describe these results and some of our studies on the characteristics of HRPPEI-labeled probes. These include a measurement of the Tm value of probes directly labeled with HRP in comparison with 32P labels, the effects of probe size and concentration on the detection limit of the system, and the stability of the labeled probe. Because of the difficulty in quantifying low levels of light with film (13) a cooled charge coupled device (CCD) camera has been used in these studies to directly quantify the light output from nitrocellulose blots. We also describe our results on reprobing Southern blots without the necessity of stripping the filter to remove the previously hybridized probe.

A 20mer and 50mer derived from the pBR322 sequence were synthesized on an Applied Biosystems 381A synthesizer using phosphoramidite chemistry (14) on 0.2-pm controlled pore columns. The remaining probes of up to 3571 bases were prepared by restriction digest of pBR322 with NarI as described previously (15) to generate fragments of 113,657, and 3571 base pairs.

MATERIALS

Hybridization

AND

METHODS

Preparation of Horseradish PeroxidasePolyethyleneimine Complexes HRP was from Boehringer-Mannheim, p-benzoquinone was from Aldrich, Sephadex G-25 was obtained in a prepacked PDlO column from Pharmacia. Polymin G35 (polyethyleneimine) was obtained from BASF. HRPpolyethyleneimine complexes were prepared as previously described (7) with the minor modification that the free PBQ was removed on a Sephadex G-25 column and not on a Sephadex G-100 column. Preparations were routinely analyzed by gel filtration on HPLC (TSK-3000 column). The complex formed is the DNA labeling reagent. This is available from Amersham (product RPN 2101). In analysis by gel filtration the labeling reagent was determined to be a mixture of HRP complexes in the form of monomers, dimers, and trimers.

Preparation

Labeled probes may be stored in 50% glycerol at -20°C for up to 6 months with no loss of activity. Probes were end labeled with 32P using Klenow and 32P dNTPs (Amersham International plc).

Preparation

of pBR322

Probes of Varying Length

Southern blots were prepared as described previously (16). Dot blots were prepared as follows. The target DNA was denatured by heating at 95°C for 5 min. Following rapid cooling on ice for 5 min a 2-~1 sample was spotted onto nitrocellulose (Amersham Hybond ECL) which had been soaked in 6X SSC for 10 min. The membrane was then baked at 80°C for 2 h. Nitrocellulose (Amersham Hybond ECL) containing immobilized DNA was prehybridized and hybridized using buffers in the Amersham ECL gene detection system (product 2102). Urea (6 M) is used as a denaturant. The labeled probe was added to hybridization buffer (containing 0.5 M NaCl) to a final concentration of 20 rig/ml DNA and hybridized overnight at 42°C unless otherwise stated. The blots were then washed in 6 M urea, 0.1% SDS, 0.5X SSC for 2X 20 min at 42°C followed by two rinses for 5 min in 2X SSC unless otherwise stated (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate).

of the Labeled Probes

Double-stranded DNA (0.2 pg/20 ~1) was denatured by heating (95°C 5 min) and rapidly cooling on ice for 5 min. An equivalent volume of DNA labeling reagent (0.5 mg/ml) and glutaraldehyde (5%) was then added, mixed, and incubated at 37°C for 10 min. The final concentration of the glutaraldehyde was 1.25%. The probe was then used directly without further purification. HPLC analysis shows that it is the trimer HRP complex which is preferentially labeling the DNA. Probes of varying length to unique sequences in human genomic DNA were obtained from the Amprobe range from Amersham International pk. Cloned inserts were separated from vectors by digestion with a restriction endonuclease and purification on agarose gels. Inserts separated from vectors in low melting point agarose gels can be labeled in the presence of agarose. Any insert purification method can be used, if the ionic strength of the final buffer containing the isolated insert is low.

Detection Sufficient detection reagents from Amersham’s ECL gene detection kit (product RPN 2101) were prepared to cover the blot. These are as described previously (11). Excess buffer from the washing step was drained from the blot which was then incubated in detection reagents for 1 min. Excess reagent was washed off and the blots were wrapped in Saran wrap and placed DNA side up in a film cassette for detection on blue sensitive film (Hyperfilm-ECL RPN 2103) or under a liquid nitrogen cooled CCD camera from Wright Instruments, Unit 10, 26 Queensway Road, Enfield, UK. RESULTS

Sensitivity

and Probe Length

Horseradish peroxidase modified with polyethyleneimine has previously been used to directly label DNA probes for detection of specific sequences by calorimetric

86

POLLARD-KNIGHT

ET

AL. TABLE

Probes Used in Successful in 2 pg of Southern Blotted scribed in Fig. 1

1

Detection of Single Copy Genes Human Genomic DNA as De-

Probe

Length

mos proto-oncogene k-ras (intron) proto-oncogene N-myc proto-oncogene raf-1 proto-oncogene fm.s proto-oncogene obl proto-oncogene fos proto-oncogene sis proto-oncogene N-ros proto-oncogene

2.75 0.6

1.0 1.6 0.9 0.45 3.1

1.0 1.5 5.1 1.3

7~22"

met H” Human

FIG. 1. Detection of the single copy genes: (a) abl proto-oncogene; (b) mos proto-oncogene; (c) N-ras proto-oncogene in human genomic DNA. Tracks 1,2, and 3 are 10,5, and 2 pg loadings of genomic DNA, respectively. The probes were 0.45,2.75, and 1.5 kb, respectively, and represent approximately 1 amol of target sequence in the 2-rg track. Human genomic DNA was restricted with EcoRI and Southern blotted onto nitrocellulose filters. The immobilized DNA was then prehybridized and hybridized as described under Materials and Methods using the probe labeled with HRP at a concentration of 20 rig/ml. Hybridized probe was detected by enhanced chemiluminescence as described under Materials and Methods using a 60-min exposure to the film.

methods (7). To assess the sensitivity of enhanced chemiluminescence for nucleic acid detection using HRP-directly labeled probes, human genomic DNA was restricted with EcoRI and probed for single copy sequences. Some of the results are shown in Fig. 1 where target sequences represent approximately 1 am01 using probes of 0.45-2.75 kb and detected on blue sensitive film with an exposure time of 60 min. Table 1 shows those sequences detected successfully and the probe size. One attomole is probably higher than the true target levels since it is reported that only lo-20% of the target DNA remains bound to a nitrocellulose membrane under hybridization conditions (17). These results were achieved with several different batches of the DNA labeling reagent. The effect of probe length on signal intensity was determined by preparation of several DNA probes ranging from 50 to 3571 bp which were complementary to the plasmid pBR322. These were labeled with the DNA labeling reagent and hybridized to dot blots containing lo1000 pg (3.5-350 amol) of the plasmid and control dots of calf thymus DNA. Following incubation in detection reagents the light output was analyzed on a cooled CCD camera. Figure 2 shows the probe length versus light output integrated over the area of the dots at three target DNA concentrations. The signal intensity is propor-

growth

hormone

(kb)

gene

0.5

Note. These represent detection of approximately sequence. o Cystic fibrosis gene linked marker.

1 amol

of target

tional to the probe length in the range 50-3571 bases. This method can therefore be used to label probes only 50 bp long. We have tried unsuccessfully to label 20 bp probes. In other experiments we have shown that, on average, there is approximately one active HRP molecule per 50-100 bp so these results are predicted (data not shown).

iOooPL7 * 4-

Lc

1% X

low + intonratinn

3-

2-

l-

0, 1.5

2.0

2.5

3.0

Log probe length (bpl

FIG. 2. The effect of probe length on signal intensity. The light output was integrated over the area of the dot blot on the cooled CCD camera. Dot blots of the plasmidpBR322 (4363 bp) containing 10,100, or 1000 pg (3.5-350 amol) DNA were prepared on nitrocellulose as described under Materials and Methods. These were prehybridized and hybridized with probes complementary to pBR322 of length given in the graph and labeled and detected as described under Materials and Methods. The light output over the integral area of each dot was quantified using a cooled CCD camera.

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FIG. 3. Detection of the single copy gene mos proto-oncogene in 10, 5, and 2 fig (tracks 1, 2, and 3, respectively) of human genomic DNA using a DNA probe labeled with HRP that has been stored in 50% glycerol at -2O’C. The probe is 2.75 kb and represents approximately 1 amol of target sequence in the 2-pg track. Experimental details are described in Fig. 1 and Materials and Methods.

One of the advantages of this direct labeling system is that the labeled probes can be stored in 50% glycerol at -20°C for up to 6 months without activity loss. A Southern blot probed for the human mos proto-oncogene using a labeled probe which had been stored for 6 months is shown in Fig. 3.

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FIG. 4. Hybridization of an RNA probe (K-ro.s intron) labeled with HRP to a Southern blot of homologous DNA. The loadings are 1, 10, and 100 pg (0.35-35 amol, tracks 3,2, and 1, respectively). Experimental details are as described in Fig. 1. The blot was exposed to blue sensitive film for 60 min.

Maximum signal at 10 rig/ml of probe occurs after 6-8 h hybridization. Hybridization for 16 h gives little, if any, increase in signal. Unlike other nonradioactive detection systems reported to date, these directly labeled

lpg target *

RNA Probes

Log CCD

counts:

500

1opg +

1oopg x

set integration

4

An RNA probe for K-ras intron (600 bp) generated from a pSP64 construct (Amersham SP6 system) was labeled with HRP as described under Materials and Methods for DNA. As an indication of sensitivity the labeled probe was hybridized with Southern blots of 100-l pg (35-0.35 amol) of homologous DNA. As shown in Fig. 4 the sensitivity is approximately 1 pg. Hybridizations using other RNA probes to probe human genomic DNA indicate that the sensitivity depends on both copy number and transcript size. Kinetics

and Stringency

There are many factors which can affect the rate of hybridization of a probe to target DNA bound on a solid support. The effect of varying hybridization time and probe concentration at variable target concentrations on Southern blots of plasmid DNA (pBR322) has been determined using the CCD camera and is shown in Fig. 5.

X

xyx

3-

/ X +

+---+ 2-

+/*--*.-.*

i-

0

,k, 0

Hybridization

87

DETECTION

5

10

Hybrdizatlon

,

,

15

20

time

(hours)

FIG. 5. The effect of varying hybridization time and probe concentration at variable target concentrations. Southern blots of pBR322 with 1, 10, or 100 pg (0.35, 3.5, or 35 amol) of immobilized DNA were prepared on nitrocellulose. The dot blota wara prehybridixed, hybridized with labeled probe DNA (total plasmid, 4363 bp) at 10 &ml, and detected as described under Materials and Methods. The light output over the integral area of the dot was quantiied using a cooled CCD camera

88

POLLARD-KNIGHT

ET

AL.

washes was kept constant (2X SSC, 6 M urea, 0.1% SDS) and the temperature was varied between 42 and 80°C. Dot blots of target plasmid DNA pBR322 (1 ng, 350 amol) were hybridized to labeled homologous plasmid probe DNA as described under Materials and Methods. The percentage of melted hybrid was determined using the 32P label by Cerenkov counting the filters. The results for decreasing the SSC concentration are shown in Fig. 7. There are no significant differences between the two probes, indicating that the hybrids have similar stability. This has been confirmed by the analysis of varying temperature in which a Tm value of 68°C was observed for the probe labeled with 32P only compared with a Tm of 69°C for the dual labeled probe (data not shown). Due to enzyme instability the stringency of hybridization cannot be controlled by temperature increases above 42°C. When enhanced chemiluminescent detection is used the stringency must therefore be controlled by varying the SSC concentration. DISCUSSION Chemiluminescent detection of nucleic acid probes directly labeled with horseradish peroxidase provides a rapid reliable method for quantification of specific sequences in a sample. The sensitivity is superior to colorimetric detection (7) and is sufficient to detect single copy sequences in human genomic DNA. The light emission can be quantified on a cooled CCD camera or a luminomFIG. 6. Reprobing a Southern blot of an EcoRI digest of human genomic DNA (10,5, and 2 pg loadings, tracks 1,2, and 3, respectively) for the single copy gene N-m-s proto-oncogene for the third time. The blot was nrenared. prehvbridized. hvbridized with labeled N-ras, and detected as described udder Materials and Methods. A probe concentration of 20 rig/ml was used in all hybridizations. The film was exposed for 60 min. Previously bound labeled probe was not removed from the nitrocellulose filter prior to reprobing.

probes can therefore be used at concentrations similar to those radioactively labeled. With this HRP labeling system reprobing a blot is possible without stripping off previously bound probes as shown in Fig. 6. We have shown that at least five reprobings can be performed on nitrocellulose. Reprobing can be performed using a probe for the same or an unrelated target sequence. Interference of protein/enzyme in hybridization is often cited as a concern in the use of directly labeled DNA probes. We have therefore compared the stability of hybrids formed with a 4363-bp probe 5’ end labeled with 32P to those formed with the same probe labeled with HRP and then 5’ end labeled with 32P in two ways. First, the effect of increasing the stringency by varying the SSC (NaCl) concentration in the posthybridization washes at constant temperature was determined. Setond, the SSC concentration in the posthybridization

32P

32PMRP

*

+

% hybrldlzed

100

so

-

+

*

-

1

:

70 -

I f

6050 -

I P

40 0

2

1

Salt

3

concentration c6SC)

FIG. 7. The effect of increasing the stringency of the posthybridization wash by decreasing the monovalent cation (NaCl) concentration on hvbrid stabilitv with a 32P-labeled and a 32P/HRP-labeled plasmid probe (pBR322). Dot blots of plasmid pBR322’DNA (1 ng, 350 amol) were prepared on nitrocellulose, prehybridized, and hybridized with labeled probe DNA as described under Materials and Methods. The SSC concentration was varied in the posthybridization wash (6 M urea, SSC, 0.1% SDS at 42°C) as indicated. The percentage melted hy bn ‘d was determined by Cerenkov counting the nitrocellulose filters. The maximum amount of hybrid formed was taken as 100%.

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eter (11) and detected on blue sensitive film to produce a hard copy of the result within 60 min of completing the posthybridization washes. It is well known that film does not respond proportionately to low light levels; this is known as low light reciprocity failure (13). Although this can be improved by preflashing the film, for quantitative studies a cooled CCD was used in our analyses. This gives low noise, a wide linear dynamic range, and good geometric resolution (J. Morse, unpublished work). Data processing facilities allow accurate quantification of both background and signal. The method of labeling probes with horseradish peroxidase described here can be used to label DNA probes from 50 to 3571 bases. The enzyme does not appear to change the stability of a hybrid formed with a 4363-bp labeled probe. We have determined this by variation of the stringency of posthybridization washes by decreasing the salt concentration and by a Tm analysis. This appears contradictory to other data in which a 10°C decrease in Tm was observed for an oligonucleotide end labeled with alkaline phosphatase (18). This conjugate, however, was composed of one enzyme molecule per 2126 bases with a mass ratio of protein to DNA of about 20. An oligomer of 26 bases was used in the Tm analysis. In the enzyme-DNA complexes reported here there is approximately one active horseradish peroxidase molecule per 50 bases and the experiments were carried out with a 4363-bp probe. From data on the effect of probe length on hybrid stability for probes of less than 50 bases (19) it is expected that an enzyme molecule conjugated to a probe will have a more destabilizing effect on a hybrid formed with a shorter oligonucleotide probe than with a probe longer than 100 bp. The discrepancy between the results can therefore be accounted for by the extent of labeling and the length of the DNA probes used in the experiments. It should also be noted that with the alkaline phosphatase labeled oligonucleotides the signal on hybridization at higher temperatures was not decreased to the extent expected from the Tm analysis (18). The Southern blots on nitrocellulose can be reprobed without stripping the filter. The ability to reprobe the same plot for a different sequence may be explained by inactivation of the horseradish peroxidase on the first probe. Reprobing the same blot for the same sequence may be the result of displacement of the first probe (hybridized to the target sequence) by the second probe (20). The labeled probes described here are stable for up to 6 months when stored in the presence of 50% glycerol. The stability and relative safety of the reagents used, the unexpected ability to reprobe without the necessity to strip the blot, the use of low probe concentration in hy-

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bridizations, and the short exposure times necessary should make this method especially valuable in routine work involving the detection of specific target sequences. The data presented here show the routine application to Southern blots using both DNA and RNA probes. In more recent work we are expanding the applications to include Northern blots and plaque and colony hybridization and detection on nylon membranes which will further increase the utility of the system. ACKNOWLEDGMENTS We thank Mrs. G. Seddon for the typing of this manuscript J. Morse for his advice and guidance on the CCD camera.

and Dr.

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Technology Acids

Ruth, (Woo,

J. L. S.,

Res. 9, 1905-