Nucleic Acids Research, Vol. 18, No. 11 3175

Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers S.Sauvaigo, B.Fouque, A.Roget, T.Livache, H.Bazin, C.Chypre and R.Teoule* Laboratoire des Sondes Moleculaires, CIS BIO International, Departement de Recherche Fondamentale, Laboratoires de Chimie, Centre d'Etudes Nucleaires de Grenoble, 85X 38041 Grenoble Cedex, France Received March 19, 1990; Revised and Accepted May 4, 1990

ABSTRACT

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32p) or gamma (1251) counting or by colorimetric revelation. The effect of a dilution step between the two amplifications is studied to obtain optimal sensitivity and specificity. This test is used to detect Human Papillomavirus types 16 and 18 in cells and biopsies and for the specific colorimetric detection of HIVI in extracted DNA. INTRODUCTION Nucleic acid hybridization assays have become a powerful analytical tool in biology research laboratories and are widely used in studies of gene structure and function. In conventional methodologies the nucleic acid samples are usually immobilized onto filters, and then detected after hybridization to a labelled probe by dot blot or Southern blot procedures (1). The major disadvantages inherent to this time consuming technology are the lack of sensitivity and simplicity. The problem of sensitivity has been overcome by the Polymerase Chain Reaction (2,3). Using this method, small amounts of crude DNA from biological samples can be enzymatically amplified. The use of a thermostable DNA polymerase simplifies the procedure, improving the sensitivity and allowing the reaction to be performed automatically (4). The PCR technology will find wide application in routine medical diagnosis and attempts have been made to find suitable formats for mass screening. In order to perform convenient diagnostic tests, new techniques have been proposed. Oligonucleotides can be immobilized onto supports via a polythymine tail and subsequently used as probes to detect enzymatically amplified fragments labelled with biotin (5). This methodology simplifies the assay for the detection of multiple alleles in the human genome. Oligonucleotides bearing biotin, *

To whom correspondence should be addressed

fluorophores or haptens are used to permit the incorporation of different ligands into the amplified DNA (6). The amplified products can then be detected after their binding on an affinity matrix (7,8,9). Moreover modified oligonucleotides can be conveniently prepared on a DNA synthesizer using phosphoramidite technology (14). The procedure described herein provides a sensitive and very simple detection system for routine DNA amplification, using either radioactive or colorimetric detection and allows the full automation of the procedure. MATERIALS AND METHODS Materials Bovine serum albumin fraction V (BSA), salmon sperm DNA, RNAse, polynucleotide kinase and 4X174 DNA were purchased from Boehringer-Mannheim. NHS-LC-biotin came from Pierce. Avidin, anti-dinitrophenyl (DNP) antibody, phosphatase alkaline conjugate and p-nitrophenyl phosphate (PNPD) were from Sigma. Polystyrene tubes (maxisorp startubes) were obtained from Nunc. Chloramine-T came from Serva and sodium metabisulfite from Ega-Chemie. Iodine-125 S4 (200 mCi/ml) was a product of CisBioindustries and gamma 32P-ATP (10 mCi/ml) of Amersham. Hybridization membranes (Nytran NY 13) came from Schleicher & Schull. Nick Columns were purchased from Pharmacia. HPV16 infected CaSki cells were a gift of B.Dutrillaux (Institut Curie, Paris); HPV18 infected Hela cells, BJAB cells and biopsies from patients with genital condylomas came from J.M. Seigneurin (Laboratoire de Virologie, Grenoble). HIV negative and HIV1/LAV DNA were a gift of C. Desgranges (INSERM, Lyon). All solvents were of analytical grade. Dichloromethane stabilized with 2-methyl-2-butene was used for phosphoramidite synthesis. 1H-NMR (200MHz) was recorded on an AC 200 Brucker spectrometer and 31P-NMR (101MHz) on a Brucker WM 250. Mass spectra were recorded on a Kratos M 50. Short column chromatography was run on G 60 silica gel (Merck), pyridine or triethylamine (c.a 0.5%) was added to the elution solvent to prevent loss of MMT or DMT groups. Merck silica gel 60 F254 pre-coated plastic sheets were used for monitoring

3176 Nucleic Acids Research, Vol. 18, No. 11 TLC in the following solvent systems: (A) chloroform-methanol (95:5;v/v); (B) chloroform-methanol (90: l0;v/v) and (C) dichloromethane-ethyl acetate-triethylamine (50:45:5;v/v).

Synthetic oligonucleotides Oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer and purified on a 20% polyacrylamide/8M urea gel. Oligonucleotides bearing biotin or DNP were prepared as previously described (10) with PAC phosphoramidites (11). Oligonucleotides bearing 4-hydroxyphenylethyl group (named tyraminylated oligonucleotides) were prepared using the compound 7 synthesized as described below. Oligonucleotide probes or primers (40 ng) were end labelled with T4 polynucleotide kinase and 40 4Ci of gamma 32P-ATP. Purification was performed on a Sephadex G25 Column (Nick Column). The specific activity obtained was approximatively 6.108 cpm/4Lg. The primer pairs and the probe used for the HPV 16 detection are described in table 1. They were selected in the virus E6 gene (GenBank database) and homologies with other sequences were checked by computer analysis. Oligonucleotides for the second amplification of HIV DNA were selected in the region delimited by the SK38 and SK39 primers (12). Synthesis of a fully protected tyraminylated nucleoside phosphoramidite (Figure 1) 3',5'-Di-O-(4-monomethoxytrityl)4-thiothymidine (2): 4-Thiothymidine 1 (13) (1.28 g, 5 mmoles) was dried by coevaporation with anhydrous pyridine (2 x 10 ml). It was dissolved in pyridine (50 ml). Then 4-methoxytrityl chloride (4.63 g, 15 mmoles) was added. The reaction was kept overnight at room temperature and 5 hours at 50°C. After completion (checked by TLC on silica plates) the reaction mixture was chilled, methanol (5 ml) was added and after 30 min diluted with chloroform (250 ml). This solution was washed with saturated aqueous NaHCO3 solution (3 x250 ml) and water (250 ml). The product was purified on silica gel column with a gradient of hexane-dichloromethane (10 to 0%). The product 2 was precipitated from hexane in order to eliminate residual methoxytritanol and obtained with a yield of 87%. Rf= 0.75 (system A). MS (FAB-): m/e = 801 (M-H).'H-NMR (CDC13) :6(ppm)= 7.6 (s, 1H) 6-H ; 7.38-7.11 (m, 24H) MMTr; 6.85 -6.77 (m, 4H) MMTr; 6.47-6.40 (dd, 1H) 1'- H ; 4.41 (m, 1H) 3'- H; 3.86 (m, 1H) 4'- H ; 3.80 (s, 3H) and 3.76 (s, 3H) MMTr; 3.26-3.21 (m, IH) 5'- Ha ; 2.90-2.84 (m, 1H) 5'- Hb ; 2.04-1.94 (m, 2H) H-2',2"; 1.6 (s, 3H) 5-CH3. 3', 5'-Di-O-(4-monomethoxytrityl)4-N-[2-(4-hydroxyphenyl)ethyl]-5-methyl-2'- deoxycytidine (3): To a solution of 2 (2.8 g, 5 mmoles) in absolute ethanol (50 ml) was added tyramine (2.74 g, 20 mmoles). The reaction was left 24 h at 60°C in a tightly stopped flask. After cooling and evaporation of the solvent, the residue was taken in chloroform (100 ml), washed with water (3 x 100 ml). The product was loaded on a silica gel column in chloroform-hexane: 80/20. After equilibration with chloroform the compound 3 was separated with a gradient of chloroform-methanol: 0 to 3 %. Yield 65%. Rf= 0.34 (system A). MS (FAB-): m/e = 904 (M-H). 'H-NMR (CDCl3): (ppm)m= 7.58 (s,1H) 6-H ; 7.4-7.12 (mi, 24H) MMTr; 6.94 (d, J=8.4 Hz, 2H) 2-H(arom) ; 6.85 (d, 2H) 3-H

(arom); 6.78-6.72 (m, 4H) MMTr; 6.58 (dd, IH)1'-H; 4.38 (m, IH) 3'-H ; 3.85 (m, 1H) 4'-H ; 3.78 (s, 3H) and 3.76 (s, 3H) MMTr; 3.65 (m, 2H) CH2; 3.22 (m, IH) 5'-Ha ; 2.84 (m, 1H) S'-Hb ; 2.75 (m, 2H) CH2; 2.21-2.12 (m, 1H) 2'-Ha; 1.92-1.85 (m, IH) 2'-Hb ; 1.36 (s, 3H) CH3.

3',5'-Di-O-(4-monomethoxytrityl)4-N-[2-(4-acetoxyphenyl)ethyl]-5 methyl-2'-deoxycytidine (4): Product 3 (3 mmoles) was dried by coevaporation with anhydrous pyridine and was treated with acetic anhydride (3 ml) in anhydrous pyridine (30 ml) for 4 hours at room temperature. The reaction was quenched with 3 ml of methanol and evaporated to dryness. The residue was taken up in chloroform (100 ml), washed with saturated aqueous NaHCO3 solution (3 x 100 ml), water (100 ml) and evaporated to dryness. The product was loaded on a silica gel column with a chloroform-hexane mixture (80/20). The elution was performed with a gradient of methanol in chloroform (0 to 2%). Product 4 was obtained with a yield of 82%. Rf= 0.46 (system A). MS (FAB+) : m/e = 970 (M+Na)+. IH-NMR (CDCl3): 6(ppm)= 7.55 (s, 1H) 6-H ; 7.38-7.15 (m, 24H) MMTr; 7.13 (d, J = 8.6 Hz, 2H) 2-H (arom) ; 7.02 (d, 2H) 3-H (arom) ; 6.76-6.71 (m, 4H) MMTr; 6.56 (dd, Jl',2'a = 5.5 Hz, Jl',2'b = 8.7 Hz, 1H) 1'-H; 4.37

J3',2'a

= 0 Hz, J3',2'b =5.5 Hz, 1H) 3'-H ; 3.87 (m, 1H) 3.78 (s, 3H) MMTr; 3.78-3.76 (m, 2H) CH2; 3.76 (s, 3H) MMTr; 3.23 (m, 1H) 5'-Ha ; 2.91 (m, 2H) CH2; 2.82 (m, 1H) 5'-Hb ; 2.28 (s, 3H) CH3 (acetyl) ; 2.12 (m,1H) 2'-Ha; 1.86 (m, 1H) 2'-Hb ; 1.32 (s, 3H) 5-CH3.

(m,

4'-H

;

4-N[2-(4-acetoxyphenyl)-ethyl]-5-methyl-2'-deoxycytidine (5): The product 4 (3 mmoles) was refluxed with 30 ml of 80% acetic acid during 15 min, evaporated to dryness and coevaporated with 50 ml of water (50 ml). The residue was taken up by 50 ml of water and washed by toluene (2 x 50 ml). The aqueous layer was evaporated to dryness and the resulting compound was used for the next step without further purification. 5'-O-Dimethoxytrityl4N-[2-(4-acetoxyphenyl)-ethyl]5-methyl-2'-deoxycytidine (6): Compound 5 obtained from compound 4 (3 mmoles ) was thoroughly dried by three coevaporations in anhydrous pyridine and was reacted with 4,4'-dimethoxytrityl chloride (3.3 mmoles) 16 hours at 4°C in anhydrous pyridine (30 ml). The reaction was checked by TLC on silica plates and after completion the excess of reagent was quenched by 1 ml of methanol and diluted by 200 ml of chloroform. This organic layer was extracted by saturated aqueous NaHCO3 solution (3x200 ml), water (200 ml) and evaporated to dryness. The residue was coevaporated two times with toluene and purified by silica gel chromatography. Compound 6 was obtained as a white foam with a yield of 69%. Rf= 0.4 (system B). Rf= 0.1 (system C). MS (FAB+) : m/e = 728 (M+Na)+. IH-NMR (CDCl3) : 6(ppm)= 7.7 (s, 1H) 6-H ; 7.42-7.21 (m, 9H) DMTr; 7.16 (d, J = 8.5 Hz, 1H) 2-H (arom) ; 6.98 (d, 1H) 3-H (arom) ; 6.98 (m ,4H) DMTr; 6.47 (dd, J = 6.4 Hz, 1H) 1'-H; 4.6 (m, 1H) 3'-H; 4.14 (m, 1H) 4'-H ; 3.74 (s, 6H) DMTr; 3.64 (m, 2H) CH2; 3.38 (m, 2H) 5'-Ha,Hb; 2.85 (m, 2H) CH2 ; 2.62 (m, 1H) 2'-Ha ; 2.25 (m, IH) 2'-Hb; 2.25 (s, 3H) CH3 (acetyl); 1.35 (s, 3H) 5-CH3. 13C-NMR (CDCl3): 6(ppm) = 169.5 (acetyl), 156.4 (C4), 135.7 (C-6), 130.4 and

34.5

(CH2-TYR),

21.1

121.6

(CH-TYR), 102.6

(acetyl),

12.3

(5-CH3)-

(C-5), 41.9 and

Nucleic Acids Research, Vol. 18, No. 11 3177 5'-O-Dimethoxytrityl4-N- [2-(4-acetoxyphenyl)-ethyl]2'-deoxycytidine-5-methyl-3'-O-(2-cyanoethyl)-N,Ndiisopropylphosphoramidite (7): Compound 6 (353 mg, 0.5 mmole) was dried by coevaporation in anhydrous acetonitrile and dissolved in dichloromethane (5 ml). To this solution were added diisopropylammonium tetrazolide (42 mg, 0.25 mmole), N,N,N',N',-(bis diisopropylamino) -2-cyanoethoxyphosphine (170 1l, 0.6 mmole). The reaction was kept 2 hours at room temperature protected from air and moisture. After completion, checked by TLC on silica plates, the reaction was diluted with dichloromethane (50 ml) and extracted by saturated aqueous NaHCO3 solution, water (50 ml), dried over Na2SO4 and evaporated to dryness. The residue was taken up by 5 ml of hexane and precipitated from 100 ml of cold hexane (-80°C) to give a white powder with a yield of 93%. Rf= 0.66 and 0.70 (system C). MS (FAB+) m/e = 906 (M+H)+, 928 (M+Na)+. 31P-NMR (CD3CN) 6(ppm)= 149.39

Samples preparation Biopsies were stored at -70°C and mechanically disrupted under liquid nitrogen. DNA samples from cultured cells and biopsies were prepared essentially as described in (14) by SDS cell lysis, proteinase K and RNAse digestions, phenol/chloroform extraction and ethanol precipitation. Crude cell lysates were obtained by denaturation 15 min at 95°C of 106 cells in 1 ml of H20. Cellular debris were eliminated by centrifugation.

Synthesis of BSA-biotin BSA-biotin was prepared by reacting NHS-LC-biotin (20 mg) with BSA (40 mg) in 10 mM sodium carbonate buffer pH 9 for 2 hours at 25°C. The reaction mixture was dialysed against 50 mM phosphate buffer pH 7.5 for 24 hours at 4°C. Final concentration was 5.3 mg of BSA-biotin per ml.

Coating of polystyrene tubes Polystyrene tubes were coated with 0.5 ml of BSA-biotin in 50 mM phosphate buffer pH 7.3 (10 ,tg/ml) for two hours at 25°C. The liquid was then discarded and avidin (5 jig/ml) in phosphate buffer was added. Non specific sites were saturated for 2 hours at 25°C with a solution of denatured sonicated salmon sperm DNA (20 ttg/ml) in the same buffer for at least two hours. The tubes could be stored with the DNA solution at 4°C for more than one week. Primer iodination Tyraminylated primers were iodinated by the chloramine T oxydation method (15). The reaction was performed in an Eppendorf tube containing 1 1l of iodine in 15 Al of 0.25 M phosphate buffer pH 8.5. 12 pmoles of primers in 10 A1 of phosphate buffer were added and then 10 ,al of freshly prepared chloramine T (5 mg in 1 ml of phosphate buffer). The tube was briefly mixed and 65 ,ll of sodium metabisulfite (2.4 mg in 1 ml of phosphate buffer) were added. The iodinated primer was purified by gel filtration using pre-packed column (Pharmacia NAP 10). The percentage of iodine incorporation was limited to 10%. The specific activity obtained was approximatively 3. 05 cpm per picomole of primer.

DNA amplification Step 1: PCR amplification was performed on 1 yg of genomic DNA or S 11 of cell lysate in 50-100 yd containing 10 mM Tris HCl pH 8.5, 50 mM KCI, 1.5 mM MgCl2, 0.01% gelatin, 200 ,aM each dATP, dCTP, dGTP, dTTP, 50 pmole each external primer and 2.5 U of Thermus Aquaticus (taq) DNA polymerase (Perkin Elmer Cetus). Thirty cycles of PCR were carried out: first denaturation 10 min 94°C, annealing at 50°C 1 min, extension at 72°C 1.30 min, denaturation at 94°C 1 min, final extension 5 min. Step 2: After a 200 fold dilution in H20, 2 yd of the step 1 PCR product were submitted to amplification with the internal biotinylated and label bearing primers. PCR was carried out in 25 IL containing 5 !d of iodinated primer, or 32p labelled primer (500 000 cpm) when necessary, 5 pmole of each internal primer, other reagents as described above and 1.25 U of taq. The PCR product was analysed after 16 cycles of amplification.

Detection of amplified DNA Isotopic detection: 20 I1 of step 2 PCR amplification were diluted in 500 yl of 50 mM Tris HCI pH 9.5 NaCl 0.5 M and incubated in polystyrene coated tubes for 1 hour at 37°C. The tubes were washed twice with the same buffer for 5 min at room temperature and at 37°C. The bound radioactivity was directly measured by counting the tubes in a gamma counter. Filter detection: 20 and 4 1l of the first amplification products were slotted onto a nylon membrane and the DNA was fixed by UV irradiation. The membrane was prehybridized for 1 hour at 42°C in 6SSC, 5 x Denhardt, 1% SDS and 500 mg/ml denatured sonicated salmon sperm DNA (SSC 1 x is 0. 15M NaCl, 0.O1SM sodium citrate), and subsequently hybridized for 2 hours at 42°C in 50% formamide, Sx SSC, 0.8% SDS, 0. 12M phosphate buffer pH 7.5, 500 yg/ml denatured sonicated salmon sperm DNA, containing 40 ng of the labelled probe. Blots were then rinsed two times 5 minutes in 2 x SSC, 1 % SDS at room temperature to remove excess probe and washed two times 15 minutes in the same buffer at 42°C. Dried filters were then exposed to a Kodak X ray film for the night. Colorimetric detection of DNP: Tubes containing the tagged amplified products were incubated, after 30 minutes blocking with 3% BSA in PBS pH 7.5 at 37°C, for one hour with a rabbit antiserum to DNP diluted 1/800 with 4% Tween 20, 0.1% BSA, 0. 15M NaCl in PBS pH 7.2. The tubes were then rinsed three times 5 minutes with the same buffer and incubated with an alkaline phosphatase conjugated goat antiserum to rabbit IgG diluted 1/800 in the same buffer. The tubes were then washed as described above and the enzyme substrate was added. 5 mg tablet of PNPD were dissolved in 5 ml of buffer solution (prepared as follows: to 80 ml of H20 were added 100 mg of MgCl2, 6H20 and 9.7 ml of ethanolamine, pH was adjusted to 9.8 and the volume completed to 100 ml with H20) and 500 Il of the substrate solution was added to each tube. The reaction was terminated by the addition of 250 ju of 2N NaOH and optical density was measured at 405 nm.

RESULTS PCR amplification procedure The main outlines of the procedure are described in Figure 2. The PCR was performed as described by Saiki et al. (4), with a set of primers at the boundaries of the sequence of interest.

3178 Nucleic Acids Research, Vol. 18, No. 11 This led to the exponential accumulation of a specific target sequence in a typical run of 30 cycles. Despite the reliability of this method and the use of the Taq DNA polymerase, non-targeted products are also often synthesized because of random primer-target interactions. This phenomenon is especially common when the amplification is performed on crude preparations or on highly complexity DNA (16). Moreover, cell contaminants or biological inhibitors can remain in the solution leading to a loss of specificity and efficiency of the reaction. Detection at this stage of the procedure can result in low signal to noise ratio due to high background levels. Various ways have been used to improve the specificity in such cases,

allowing the rapid detection of scarce sequences among biological samples. It has been proven that size fractionation of template genomic DNA by restriction enzyme digestion could improve the specificity of the amplified products by reducing the numbers of non specifically amplified fragments (17). Efficiency of the PCR procedure is greater leading to the synthesis of an increased amount of the desired DNA. The main disadvantage of this interesting technique is that it must be performed on pure DNA samples with known restriction enzyme maps. Another approach to this problem is that of Mullis et al. (18) and Simmonds et al.(19). They proposed a ten-fold dilution of

H3C

JH

I

2

ii, iii

Reagents i : monomethoxytrityl chloride/pyridine ii: tyramine / ethanol iii: acetic anhydride/ pyridine iV acetic acid , water:. 8 :2, V/v V: dimethoxytntyl chloride/pyridine vi: 2- cyanoethoxy , NNbis diisopropyl amino phosphine/diisopropylammonium tetrazolide/dichloro methane.

H3C,

ivyv

H3C,

Dl vi

7

6

0

A

N

,P-OCH2CH2CN

Figure 1: Synthesis of a fully protected tyraminylated nucleoside phosphoramidite.

Nucleic Acids Research, Vol. 18, No. 11 3179 the first accumulated template, followed by another round of amplification with a second set of primers, located within the target sequences. Thus we used two rounds of PCR amplification, and we took advantage of the second PCR procedure to introduce biotinylated and marker bearing primers in the solution. This leads to the production of DNA hybrids carrying a biotin moiety at one 5' end and a 1251 or DNP label at the other end. Amplified products

then captured by their biotin moiety on an avidin affinity matrix and subsequently detected using a gamma counter or an enzyme antibody system.

are

Collection of target sequences Detection of amplified products is usually performed by fixation of the denatured target DNA onto nylon membranes and I ..

......~-

4-

4

Is

=

hybridization to a labelled probe. This general procedure is not fully adaptable to all cases, especially when different probes have to be tested, or when routine analysis are to be performed. Moreover the immobilization of target DNA onto nylon occurs at random along the DNA strand by covalent binding of the bases to the membrane. This results in a decrease in the accessibility of the target to the probe (20). To improve this methodology, Saiki et al. (5) proposed an interesting technique called 'reverse dot-blot'. The probe was immobilized onto the membrane via an homopolymeric tail and the target DNA, labelled during the PCR procedure with biotinylated primers, was subsequently hybridized to the tagged probe. Another proposal was to immobilize the biotinylated target DNA by affinity binding on an avidin-coated matrix and followed by the hybridization with the labelled probe (9). w

3 -o

_

_

2

.. _

6

/3

OR g COUNTING

5

..-

IMMUNOENZYMATIC DETECTION Figure 2: Outlines of the method. The target DNA was submitted to a standard PCR procedure (1). The products formed were diluted 200 folds (2) and were subjected to another PCR procedure with modified inner primers (3,4). The resulting amplified DNA beared a biotin moiety at one end and a label at the other end. The detection occured after capture of the hybrids onto an avidin affinity matrix (5) by beta or gamma counting (6) or immunoenzymatic detection. Table 1: Genomic location of primers and probe. HPV16A1, HPV18A1, HPV16Ad, HPVl8Ad, SPO12 are complementary to the coding strand of the virus; other primers are complementary to the non-coding strand. SK38 is complementary to the minus strand of HIVI; SK39, HIVlAd are complementary to the positive strand. The Ad oligonucleotides represent the detection primers labelled either with tyramine residue or with DNP group. The Ac oligonucleotides represent the capture primers labelled with biotin. Numbers in parentheses represent the location of the oligonucleotides in HPV 16, HPV18 and HIVI (BRU isolate).

primer HPV16A1 HPV16A2

HPV16Ad HPV16Ac

HPV18A1 HPV18A2 HPV18Ad HPV18Ac

8K38 SK39 HIViAd HIVIAO

sequence AAGGGCGTAACCGAAATCGGT

virus HPV 16

CACATACAGCATATGGATTCC GAAAGTTACCACAGTTATGC CACGTCGCAGTAACTGTTGC

region

amplified

(26-293) E6 (132-224)

CACTTCACTGCAAGACATAGA GATTCAACGGTTTCTGGCAC GATTCAACGGTTTCTGGCAC ATTCAGACTCTGTGTATGGAG

HPV 18

ATAATCCACCTATCCCAGTAGGAGAAAT TTTGGTCCTTGTCTTATGTCCAGAATGC

HIV 1

(170-444) E6

(366-444)

(1090-1204) gag

(1090-1176)

GGTAGGGCTATACATTCTTACTATTTTA ATAATCCACCTATCCCAGTAGGAGAAAT

probe SPOI,2

GCAAACAACTATACATGATATAATATTAGAATGTGTGTA

HPV 16

(160-205) E6

3180 Nucleic Acids Research, Vol. 18, No. 11 Table 2: Detection of HPV16 in biological samples; comparison of the double amplification procedure using 1251 with a classical PCR/dot blot procedure. 100 ng and 10 ng of CaSki DNA (CIO, C100), 1 lAg of DNA extracted from four biopsies (I,IV,V,VI) were assayed for the presence of HPV16 using the double PCR procedure with 1251 labelled primers. Hela (100 ng) and cIX174 (1 jig) DNA were used as negative control. Absence of contamination was checked with a template free solution (control). The radioactivity bound was the mean of duplicated amplifications. Total activity was 400 000 cpm/tube. The percentage of fixation was defined as (radioactivity bound/total radioactivity present in the tube) x 100. Samples giving a percentage of fixation below 3 steps the background value were considered negative. The biopsy VI was suspected positive using the AmpliCis test, and negative with a classical PCR/dot blot procedure. Other results were well correlated. C100

OX 174

I

IV

V

VI

control

69866

47955

383

5958

72682

468

1507

173

17.80

12.80

0.10

1.60

19.40

0.12

0.39

0.05

Hela

CIO

161

| fixa10.05

DNA

radioactivity bounded

_-

conclusion

PCR/dot blot

|

-

| +++

.+++

These two techniques are difficult to incorporate into a simple automation procedure due to the need for a denaturation step before hybridization. Kemp et al. presented a novel technique based on a two round amplification procedure where the amplified DNA is captured by a DNA binding protein (21). Hence there is no need for hybridization with a labelled probe, but the main disadvantage of this procedure is that the protein used to tag the amplified products is not easily obtainable. We tried to provide a universal and very simple detection system, by first taking advantage of the high affinity of biotin for avidin (22). After the two-step amplification, the biotinylated formed hybrids were easily and strongly captured by an avidin matrix, consisting of polystyrene tubes, with no need for extensive washes. Preliminary investigations had shown that the binding capacity of the avidin matrix for biotinylated oligonucleotides was higher than 10 picomoles and that the binding efficiency was 80% in two hours (data not shown). Biotinylation of primers was also easily performed during the oligonucleotide synthesis without further chemical modification (10). Hence, this procedure offered an easy way to tag the specific amplified sequences on a solid support before performing their detection.

Detection of target sequences Choice of labels: the high affinity of avidin for biotin is very convenient to anchor the target DNA onto an affinity support (7). 125 Iodine is a very sensitive isotopic label for precise measurement of the PCR products and dinitrophenyl group a nonisotopic one to offer an alternative to handling radioactivity. Among the available isotopes 1251 was chosen for its long half life (60 days) which allows storage of the radiolabelled primers for at least a few days without modifications. Radioiodination of DNA probes is usually done by randomly labelling the DNA bases along the DNA chain (23). Iodination gives rise to 5,6 saturated pyrimidines and modifies the hybridization properties of the probe. As an alternative, oligonucleotides bearing a free amino group are reacted in a postsynthetic step with a p-hydroxyphenyl-propionic acid derivative and subsequently iodinated (24). Our purpose was to avoid this post-synthetic step by the direct incorporation of a modified phosphoramidite reagent during the oligonucleotide synthesis. Then the oligonucleotide could be iodinated specifically on its modified base residue, using a very short procedure. A pyrimidine derivative bearing a phenol moiety as specific target

++

-

|

++

+*+

H

|

-

+

|

_

C1oo C10 0X

I

IV

V

VI

.-

iak.: .;:ii'

Z@pI

I

Figure 3: Analysis of PCR amplified fragments using a dot blot procedure. Fractions of the first amplification of Hela (H) cells, CaSki lOng (CIO), CaSki l00ng (C100), 4X174 (4$), Biopsies I, IV, V, and VI were applied onto a nylon filter (see 'materials and methods') and hybridized with a P-labelled probe, specific for HPV16. The signal is obtained after an overnight exposure.

was chosen for the iodination reaction. This phenol group was easily protected, in order to be compatible with the phosphoramidite chemistry used on the automated synthesizer. This phenol can then be iodinated specifically by a modification of the chloramine T method (15). The electrophoretic pattern of the 125I labelled primers showed specific incorporation of the isotope into the modified primer and no iodination on a non modified-nucleotide (data not shown). The 5'-end position of the tyramine derivative avoids any modification in the hybridization properties of the primers. Fragile ligand like DNP could be incorporated into the primer using the PAC phosphoramidite technology (10). Similar results were obtained using 32p labelled primers. The sensitivity observed was equivalent to that of 125I when the initial specific activities were identical. No diferences were observed in the incorporation behaviour of the primers labelled either with 1251 or 32p.

Analysis of biological samples Isotopic analysis: Two human cell lines (CaSki and HeLa), one phage DNA (4X174) and four biopsies were assayed for the presence of HPV16 DNA using our procedure with 125I. This technique was compared to a classical filter hybridization assay using fractions of the first amplification spotted onto nylon membranes.

Nucleic Acids Research, Vol. 18, No. 11 3181 Results obtained with the AmpliCis HPV16 procedure are shown in Table 2. Figure 3 shows the hybridization of amplified HPV16 DNA from biological samples to a specific radiolabelled probe, after one night exposure. Correlation between the two techniques is good; the procedure using 1251 seems a little more sensitive (Table 2) but the use of the support format offers a few more advantages. There is no need for extensive washing to eliminate the background due to the non-specific hybridization of the probe or due to cell impurities spotted onto the membrane. Preparation and long exposure of the filters is also avoided rendering the assay fast and convenient to perform. Table 3: Colorimetric detection of HPV18 and HIVI in biological samples. Amplifications were performed as described in 'materials and methods', using 1 yg of extracted DNA (HIV1/LAV DNA) for the detection of HIVI and 2 104 boiled cells (Hela, BJAB) for the detection of HPV18. Controls were performed with DNA free amplifications. Specificity was assayed using HIVI-negative DNA and BJAB HPV18-negative cells.

HIV 1

HPV 18

cen/lav DNA

HIVl-DNA

control

1.25

1.15

0.04 0.04

0.05 0.03

Hela cells

EBJAB

1.20

0.20

cells

control 0.08

The use of 125I allows the precise measurement of the amplified DNA, making the quantification of the procedure possible. Moreover, laboratory and hospital staff are used to handling this isotope, rendering the routine utilization of the procedure easier. Colorimetric analysis: Hela and BJAB cell lines were subjected to the PCR procedure for the detection of HPV 18. This test was also performed on HIVI uninfected DNA and on HIV1/LAV DNA. Values obtained for the colorimetric detection of amplified products are listed in table 3. It shows satisfactory results, as the detection of infected cells is specific. The presence of cell contaminants that could lead to high background was overcome by the dilution step, and the mean background value obtained was low. Although the technique could seem tedious, it offers a reliable non isotopic detection system that can be performed with routine laboratories equipment. This technique can easily be adapted to the detection of the amplified sequences using luminescent or fluorescent substrates providing a simpler non isotopic assay (Results will be given in a further article).

Dilution step Investigations to find the best signal to noise ratio lead us to study more precisely the influence of the dilution step on the second amplification phase.

z

0

30 .

H

0 cm,

C.

20

10

0

30 CYCLES

Figure 4: Effects of the dilution step on the amplification sensitivity and specificity. PCR was performed on HPV18 positive cells (Hela, empty symbols) and on HPV18 negative cells (BJAB, black symbols). The first step amplification was performed on crude boiled cells (2 I04) in a volume of 100 1l. After a 20-fold dilution (circles), a 200-fold dilution (triangles) or a 2000-fold dilution (squares), the samples were subjected to a second amplification procedure with the 1251 labelled primer and the biotinylated primer. The hybrids were collected and radioactivity was measured as described in 'materials and methods'. Total activity was 71000 cpm/tube. The value represented is the percentage of radioactivity fixed (see table 4).

3182 Nucleic Acids Research, Vol. 18, No. 11 Table 4: Values (in cpm) of the specific (S) and non-specific (NS) signals obtained for the determination of dilution step influence, on the HPV 18 detection.

DILUTION

CYCLE

1/200

1/20

1/2000

S

NS

S

8

3936

214

2195

70

259

12

6279

438

9375

236

1564

82

16

6473

745

14401

718

6759

164

20

7525

1066

19256

2853

14193

682

25 30

7219

1545

17555

7897

23564

5020

7768

2484

18952

10159

21939

13342

Preliminary experiments had shown that a ten-fold dilution could lead to high background with low signal after the binding of the hybrids to the affinity matrix, in a manner unrelated to the number of cycles used during the second amplification (data not shown). This could be explained by the following reasons: first, PCR is usually performed with a large excess of primers; after a ten-fold dilution, the amount of outer primers still present in solution is high. During the second round of amplification, there is competition between the outer and the inner primers leading to decrease in PCR efficiency. High concentrations of primers in the PCR solutions may also lead to primer dimer formation (25). Presence of cell contaminants can also lead to a partial inhibition of the reaction when PCR is performed on crude DNA samples and a ten-fold dilution is not sufficient to eliminate them. Moreover high levels of the initial template DNA are not good initial conditions to perform a PCR with correct yield. Influence of these factors on the sensitivity and specificity of the detection are described below. Background values were obtained by measurement of amplification of non-specific sequences. The specific signal was then defined by the number of counts of a specific amplification after subtraction of the background value. We estimate the sensitivity of the procedure by the measurement of the percentage of the specific signal fixation. Results are shown in Fig. 4 and table 4. Sensitivity of detection was plotted versus the numbers of cycles. This was done for each of three dilutions. A twenty-fold dilution results in limited incorporation of the modified primers during the second amplification and the stationary phase of the synthesis is rapidly attained. The highest specificity is obtained after a few numbers of cycles and thereby the signal detected is low (fig 4). As the dilution of the first amplification is increased (1/200, 1/2000), the copy numbers of the target sequence present in the second amplification are initially lower, resulting in a better PCR efficiency. Therefore the exponential phase of the PCR is also longer and the plateau is only reached after 20 and 25 cycles respectively. In comparison, the background starts to increase exponentially after 16 and 20 cycles respectively. At this stage, the incorporation of labelled primers is high and the best specificity is obtained with a very good signal.

NS

S

NS

The dilution step offers many advantages. First, this system is more sensitive than a classical filter hybridization assay and it allows the detection of scarce sequences in crude DNA samples. Problems related to cell contaminants or primer dimers formation are also overcome by this procedure. The main role of the dilution step is to permit the exponential amplification of a target sequence using the DNA accumulated in the step one. Therefore there must be no interference due to the presence of residual primers from the first amplification and the initial concentration of DNA must not be too high. Hence, the second amplification plateau is not rapidily reached and incorporation of modified primers is high. But, on the other hand, the concentration of starting material should not be too low otherwise too many cycles would be required to reach the exponential phase. Therefore a compromise must be made, depending on the sample to be analysed and on the target concentration at the end of the first amplification step.

CONCLUSION These experiments showed that 1500 copies of the target DNA present in the starting sample can be detected very rapidily, using a 1/200 dilution of the first amplification and performing 16 cycles during the second PCR procedure. Moreover the results appear clearly with numbered values using the 1251 label. The amplitude of the signal is very large (100-80000 cpm) and can be modulated by the amount of the radiolabelled primer present in the second amplification. The mean background is low (200-500 cpm) resulting in a signal to noise ratio that is very high (3-200). Another advantage of this procedure is that the amount of product necessary for the second amplification is very low. Hence many tests can be performiied using aliquots of a single sample obtained from the first amplification. The outer set of primers can be used to select a family of bacteria, viruses or genes, followed by testing for specific species, subtypes or point mutations using amplification with the inner set.This application may be useful in the diagnosis of inherited disorders due to point mutations or for the detection of bacteria whose subtype determination is important.

Nucleic Acids Research, Vol. 18, No. 11 3183

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Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detec...
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