AU667846B2 - Solid phase amplification process - Google Patents

Description

*to a OPI DATE 07/06/93 AOJP DATE 05/08/93 APPLN. ID 28723/92 PCT NUMBER PCT/AU92/00587 11111111 11111111111111111111111111111111 AU9228723

PCT)

(51) International Patent Classification 5 (11) International Publication Number: WO 93/09250 C12Q 1/68 Al (43) International Publication Date: 13 May 1993 (13.05.93) (21) International Application Number: PCT/AU92/00587 (74)Agent: R.K. MADDERN ASSOCIATES; 345 King William Street, Adelaide, S.A. 5000 (AU).

(22) International Filing Date: 30 October 1992 (30.10.92) (81) Designated States: AT, AU, BB, BG, BR, CA, CH, CS, Priority data: DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, PK 9224 1 November 1991 (01.11.91) AU MN, MW, NL, NO, PL, RO, RU, SD, SE, US, European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, SE), OAPI patent (BF, BJ, CF, (71) Applicants (for all designated States except US): ADE- CG, CI, CM, GA, GN, ML, MR, SN, TD, TG).

LAIDE CHILDREN'S HOSPITAL [AU/AU]; UNI- VERSITY OF SOUTH AUSTRALIA [AU/AU]; 72 King William Road, North Adelaide, S.A. 5006 Published With internaonal search report (72) Inventors; and With amended clatms Inventors/Applicants (for US only) MORRIS, Charles, Phillip [AU/AU]; 72 King William Road, North Adelaide, S.A. 5006 HARRIS, Raymond, John [AU/ AU]; University of South Australia, North Terrace, Adelaide, S.A. 5000 (54) Title: SOLID PHASE AMPLIFICATION PROCESS LCR (Ligame Chain Reaction) AMllificatio (57) Abstract A

B

An assay and method of use thereof for amplification and de- w tection of target nucleic acids in patient samples, which is rapid, simple and accurate is described. The assay and method utilizes any one of a variety of amplification methods, in combination with primers im- ci B= bt-ia.pnmar m i3 cry a mobilized onto either the container containing the patient sample, T apoiwtg such as a microtiter well plate, or a dipstick which is readily immersi- r r ble into the container. Amplification occurs in the presence of both the first, immobilized primer and a second, labelled primer which hybrid- izes to the target nucleic acid in the opposite direction to the first. Ex- nasa- amples demonstrate the effectiveness of the method in detecting a spe- cific species of mycoplasma, as well as the gene characteristic of cystic fibrosis. ,mgbte DNA ha dAa d o(LCRd l CY I Dw :404SC &B.

r r r 3" c I I I/dO(LL3.lo2 Md o LC cyls PCT/AU 9 2 IU 0 b 8 RECEIVED 17 AUG 19 -1- SOLID PHASE AMPLIFICATION PROCESS BACKGROUND OF THE INVENTION The present invention is generally in the field of methods for the detection of nucleic acid sequences through amplification and hybridization to labelled probes.

Hybridization methods are widely utilized in testing for the presence of particular nucleic acid sequences, identifying and/or quantifying such sequences. Hybridization occurs when there is sufficient complementarity of two nucleic acid sequences to effect non-covalent binding between the two molecules.

Various improvements and modifications have been introduced tD increase the specificity and sensitivity of the reaction. In particular, polymerase chain reaction (PCR) techniques have been used to amplify the target nucleic acid sequence. In a recent development, two consecutive sets of PCR cycles ("nested PCR") have been used to both amplify and label the target nucleic acid sequence. The labelled sequence is then immobilized on a solid phase carrier, and testing is carried out using a reagent specific to the label. (See DJ Kemp, et al., "Colorimetric detection of specific DNA segments amplified by polymerase chain reactions", Proc. Natl. Acad. Sci. USA 86, pp.

2423-2427, 1989).

W089/11546 by Paulsen, et al., describes a method using PCR with DNA .primers, one of which is soluble and the other which is bound to superparamagnetic particles, allowing greater ease of separation of the amplified product which facilitates detection.

While the nucleic acid is bound to the particle, however, amplification via the beads is still taking place in a soluble phase, ie the beads are suspended in a liquid phase, increasing the time and complexity of the reaction, and requiring equipment for isolation of the superparamagnetic particles.

I IPESUBSTITUTE S~ii7' PcT/Au 9 2 /0 0 5 8 7 RECEIVED 1 7 AUG 1993 -2- AU A-47144/89 by Molecular Diagnostics, Inc. also reports a method for increasing detection of nucleic acids using PCR to amplify sequences, where one or both of the primers are in solution or immobilized or immobilizable, for example, to Sephadex, to facilitate detection. The process can be used to yield immobilized target nucleic acid.

However, prior art methods for amplifying nucleic acid sequences have all suffered from problems associated with carrying out reactions in the liquid, rather than solid, phase.

After each step of the reaction, the intermediate product must be isolated, before being reacted in the next step. Isolation steps are cumbersome, time consuming and often involve labile linkage reagents. In addition, no isolation step can be completely successful.

It is therefore an object of the present invention to provide a method which is rapid, simple, and provides an accurate, sensitive means for detecting small amounts of nucleic acid in a test sample.

It is a further object of the present invention to provide an assay which is relatively inexpensive, simple, rapid, and provides an accurate, sensitive means for detecting small amounts of nucleic acid in a test sample in clinical and research laboratories.

SUMMARY OF THE INVENTION The present invention utilizes the concept of amplification of a target nucleic acid by means of two primers, one of which is directly immobilized onto a sample container surface, and the other which is labelled for ease of detection.

By definition, both primers must be hybridizable with a target nucleic acid sequence. According to the method for determining the presence of a target nucleic acid sequence in a sample, the sample is placed in a container with the immobilized nucleic acid primer. The target sequence is then amplified using a second labelled primer or labelled polymerase substrate which, on SIPEAISUBSTITUTE

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i~Ec=rm~r~-rr=-as~rrrrr~i~ r~zlli II.. ICT/AU z i 0 0 5 8 7 RECEIVED 1 7 AUG 1993 -3incorporation, allows detection and quantification via the label on the solid phase amplified nucleic acid sequence.

Examples demonstrate the detection of the gene for cystic fibrosis and a specific type of mycoplasma in patient samples.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic of the PCR (Polymerase Chain Reaction).

Figure 2 is a schematic of the LCR (Ligase Chain Rection).

Figure 3 is a schematic of the NASBA (Nucleic Acid Sequence Based Amplification). Thin lines, RNA; thick lines, DNA; RT, reverse transcriptase.

Figure 4 is a schematic of the SPA method for amplification.

Figure 5a is a schematic of the t arget generation scheme for SDA (Strand Displacement Amplification).

Figure 5b is a schematic of automatically entering the SDA reaction cycle.

Figure 6 is a schematic of the reaction products of the modified SDA process.

DETAILED DESCRIPTION OF THE INVENTION The assay and method of use described herein have the following elements: a target molecule to be detected in any test or patient sample, a first primer which is immobilized to a solid surface suitable for containment of the patient sample and is hybridizable to the target molecule, a second primer which can be detected using a variety of different methodologies and is hybridizable to the target molecule, and means for amplification of the target sequence via the two primers.

A common problem in laboratories utilizing diagnostic methods based on nucleic acid amplification is the occurrence of false positives. This problem invariably arises due to the R;T contamination of the laboratory with amplified target molecules SPEA/SUBSTITUTE

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"191s31--1" I J SPT/AU 9 2 /00 5 87 RECEIVED 17 AUG 1993 -4- (amplicons). Of relevance here is the intrinsic high level of amplicon containment offered by solid phase amplification (SPA).

SPA requires very little manipulation of solution phase amplicons. Such amplicons are removed from SPA reactions by simple washes and can be readily destroyed. Additionally, the solid phase amplicons offer little or no contamination threat.

This level of containment is unmatched by any other amplification process. Thus SPA offers a high degree of amplicon containment.

The Target Molecule This method allows detection and identification of virtually any nucleic acid sequence, and thus allows the detection and identification of viruses, microorganisms and parasites, the detection of genetic diseases, either by detection of sequel ce variations (mutations) which cause or are associated with a disease or are linked (Restriction Fragment Length Polymorphisms or RFLP's) to the disease locus, and sequence variations which are associated with, or cause, cancer, and the detection and identification of nucleic acid sequences for forensic fingerprinting, tissue typing and for taxonomic purposes, namely the identification and speciation of microorganisms, flora and fauna, and for the production of solid phase nucleic acid templates for DNA and RNA sequencing. The methodology is also useful for other applications.

The following are a few examples of such applications 1. Pathogen Detection Detection of viruses (HIV, hepatitis viruses, papilloma).

Detection of miccoorganisms (Mycobacteria, Legionella, Mycoplasmas).

(Applications in clinical medicine, veterinary science, aquaculture, horticulture and agriculture).

4 pi jS PCAU 9 2 /00 5 8 7 RECEIVED 1 7 AUG 1993 2. Detection of sequence variations which cause or are associated with a disease or are genetically linked to a disease locus. Examples cover genetic diseases and cancer.

e.g. Point mutations sickle cell anaemia, phenylketonuria, Lesch Nyhan syndrome.

Small deletions Cystic fibrosis (AF508).

Large deletions thalassaemias, Duchenne muscular dystrophy.

Sequence reiteration Fragile X.

RFLP's Huntington's chorea, (Restriction thalassaemias, cystic Fragment Length fibrosis.

Polymorphisms) Population screening for carriers of serious genetic diseases, where the incidence of carriers is high and the number of mutations is low, e.g., cystic fibrosis, and Tay Sach's disease in Ashkenazi Jews.

3. Cancer detection of predisposing sequences, e.g., Philadelphia chromosomes in chronic myelogenous leukemia, antibody gene rearrangements, specific deletions; detection and monitoring of treatment, remission and relapse.

4. Tissue typing determination of HLA genotypes.

Forensic applications VNTR DNA fingerprinting of individuals and obtaining DNA fingerprints from forensic samples.

6. Maternity and paternity testing.

7. Foetal sex determination 8. Taxonomy Classification and speciation of organisms.

9. Quality control in agriculture and the food and pharmaceutical industries, assessment of authenticity of food products.

The assay and method of use described herein are equally applicable to RNA (ribonucleic acid) sequences, as well Sas DNA (deoxyribonucleic acid) sequences, although it may be E, A/SUBSTITUTE

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i. -I V L/ JU u 3 RECEIVED 1 7 AUG 1993 -6necessary to produce a cDNA copy of RNA sequences.

Primers The primers are synthesized to be complementary to the target molecule. In general these are approximately 15 to nucleotides in length and flank a portion of the target molecule.

One hybridizes to the 5' end of a portion of the target molecule, the other to the 3' end of a portion of the target molecule. All appropriate reagents and equipment are commercially available.

Solid Phase Support One primer is attached using known methodology, as described below, to a solid phase support. The advantages of the system described herein is that the solid support forms or is readily insertable into the container for the patient sample, eliminating the need for separate centrifugation, magnetic collection, filtration or other separation steps, as required by the prior art methods, for example, to separate superparamagnetic beads. A preferred material is the microtiter well plates that are commercially available.

The solid support may be any structure having a surface which can be derivatised with a nucleic acid primer, such that the primer can participate in solid phase nucleic acid amplification. In the preferred embodiment, the solid support is Sa-l-ana solid phase material, for example, the side of a microtiter well or a dip stick.

Specific preferred supports include 1. The inside surface of wells of any microtitre trays, Covalink trays Nunc.

2. Microtitre tray lids with protrusions which fit into microtitre trays, e.g. Fast Elisa dish Falcon.

3. Dipsticks for low volume applications, suburban and country medical practices.

These all offer the potential for high volume diagnostic applications for solid phase amplification (SPA), especially if isothermal (single temperature) amplification is used.

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PCT/AU 9 2 /005 RECEIVED 1 7 AUG 1993 -7- It is surprising that solid phase amplification of nucleic acids on the surface of a microtiter well produces a significant amount of amplified product due to the inherently less favourable kinetics of solid phase molecular interactions, versus those of the solution phase. The results demonstrate that, despite the slightly slower rate of amplification using a solid phase as compared with solution phase, or with bead-based amplification, which offers a vary large surface area, the system offers a rapid, sensitive, and clinically viable method of nucleic acid amplification.

Although described herein as preferentially involving binding of the first primer to the solid phase before initiation of amplification, in some cases it may be desirable to combine conventional liquid phase amplification techniques with the solid phase amplification (SPA) procedures. For instance, if the target nucleic acid molecule is only likely to be present in very small quantities, then it maybe beneficial to carry out an initial liquid phase amplification in the vessel to which the primer is bonded. This liquid phase amplification step may involve a small proportion of the first primer or it may involve other primers. When the target molecule is amplified to a sufficient level, then the SPA process takes over.

UTITU

i^TTA/SUBiUE SHEET PCT/AU 9 2 /00 5 87 RECEIVED 17 AUG 1993 -8- Anchorinr of the First Primer to Solid Phase Support.

Any method for linking nucleic acid sequences to solid supports may be used. For example, any of the following methods are suitable 1. Amide linkace of primer to a solid phase H O 0 I I II (2) X-N: -4 C (CH2) 6 C <-NH 2 Y pNlpN2p//N 2 0

CH

3 NHS NHS Secondary amine activated spacer synthetic oligodeoxyon the wall of a arm suberic acid nucleotide CovaLink plate bis(N-hydroxy- nucleotides long and succinimide) containing an added primary amino group) Derivatisation sequence The surface bound (solid phase) secondary amine (on walls of wells of CovaLink plates) is reacted with the activated spacer arm.

The solid phase activated spacer arm is reacted with the primary amino group of the synthetic oligo oxynucleotide, to form the product, which is a solid phase primer (amide linkage), as shown below.

0 0 I II X-N: C (CH 2 6 -C NH Y- pNlpN 2

CH

3 amide linkage IPEA/SUBSTITUTE SHEET] Pc/Au 9 2/ 0 058/' RECEIVED 1 7 AUG 1993 -9- 2. kmidafte l-inkacge of orimer to a solid phase A 5-phosphorylated oligodeoxynucleotide nucleotides long) is reacted wit~h a phosphate activati~ng compound, preferably a water-soluble carbodiimide such as EIDO (l-ethyl-3-(3dirnethylaminopropyl) -carbodiimide).

0 11 P 0 NlpN 2 p//N 2 0 EDO -0 04 0 11 P-0

EDO

Interm. -,ate (activated oligodeoxynucleotide) 0 jNP12 CH3

EDO

Secondary amine in covaLink well Activated ol igodeoxyriucleot ide ,X-N:P 0 NipN 2 p//N 2 0

II

phosphorarnidate linkage p; Produc- solid phase primer (phosphorarnidate linkage) IJPEA/SUBSTITUTE

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PT/A 9 2 /00 5 87 RECEIVED 1 7 AUG 1993 3, Thioether linkage of primer to a solid phase The reaction sequence is as follows Bromoacetic acid NHS is reacted with the secondary amino group on the solid support.

The thiol group of the oligodeoxynucleotide is reacted with the bromomethyl group of the intermediate product formed in step H 0 I II X--N >C

CH

3

NHS

Br H S (2)

CH

2 S (CH 2 6 -NipN 2 p//N2o Secondary amine on wall of well of CovaLink tray NHS activated bromoacetic acid oligodeoxynucleotide with 5'-thiol group 0 X-N C CH 2 S (CH 2 6 -NIpN2p//N 2 0

CH

3 thioether linkage Product solid phase primer (thioether linkage).

4. Introduction of amino aroups onto the surface of microtitre tray wells Plates were nitrated with acetyl nitrate using the method of R. Taylor, "Electrophilic aromatic substitution" p. 269 Wiley and Sons 1990). Reduction was via SnC1 2 IPEA/SUBSTITUTE SHEE Sf. F J PCT/AU 9 2 0 0 5 8 7 RECEIVED 1 7 AUG 199 -11- Amplification Procedures Currently known methods of nucleic acid amplification include PCR (Polymerase Chain Reaction) LCR (Ligation Chain Reaction).

NASBA (Nucleic Acid Sequence Based Amplification), using RT/RNAse H/T7 polymerase, RT reverse transcriptase.

RNAse H ribonuclease H T7 polymerase T7 bacteriophage RNA polymerase.

QS replicase based amplification.

Strand displacement amplification (SDA).

The principles of some of these amplification methods are described below.

PCR (Polymerase Chain Reaction) Polymerase chain reaction procedures and reagents are commercially available and described in the literature, for example, by R.K. Saiki, et al., "Enzymatic Amplification of 6globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia", Science 230, pp. 1350-1354, 1985. Refer to Figure 1.

LCR (Lication Chain Reaction) The ligation chain reaction is well known and described in the literature, for example, by U. Landegren, et al., "A Ligase-mediated Gene Detection Technique", Science 241, pp. 1077- 1080, 1988. Refer to Figure 2.

SNASBA (Nucleic Acid Seauence Based Amplification) This method is also well established in the literature, as described, for example, by J. Cuatelli, et al., "Isothermal in Vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modelled After Retroviral Replication", Proc. Natl. Acad. Sci.

USA 87, pp. 1874-1878, 1990.

The strategy of the NASBA scheme is shown in Figure 3.

S The reaction depends on a continuous cycle of reverse IPEA/SUSSTITUTE SHEET PCT/AU 92/00587 RECEIVED 1 7 AUG 1993 -12transcription and transcription reactions to replicate an RNA target by means of cDNA intermediates. DNA targets can also be amplified. Primers 1 and 2 prime DNA synthesis and Primer 1 encodes the promoter sequence for the T7 RNA polymerase (black boxes). Steps 1-6 depict the synthesis of a double-stranded cDNA, which is a transcription template for T7 RNA polymerase.

Complete cDNA synthesis is dependent on the digestion of the RNA in the intermediate RNA-DNA hybrid (step 4) by RNase H.

Transcription-competent cDNAs yield antisense RNA copies of the original target (step These transcripts are converted to cDNAs containing double-stranded promoter on one end (steps 7- 12).

These cDNAs yield antisense RNAs, which can re-enter the cycle.

CHARACTERISTICS OF NUCLEIC AMPLIFICATION SYSTEMS One primer incorporated Two or more primers incorporated Doublestranded product Can be labelled at nonanchored end Can be internally labelled Requires temperature cycling 1. PCR 2. LCR 3. NASBA No RT/T7 42'only* 4. QE No SDA No Big technical advantage.

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C c~Crs;r~i=PL;r;L~iii---=fi-_Pii L PCAU 9 2 0 0 5 8 7 RECEIVED 1 7 AUG 199 -13- The process of solid phase amplification (SPA) of a target nucleic acid sequence, using PCR as the method for amplification, is illustrated in Figure 4.

One primer is immobilized to the wall of the plate, and the other is labelled with a fluorescent label. As shown by this diagram, generally 20-30 cycles are needed to amplify sufficient DNA for detection. The plate is then washed to remove unincorporated primer 2 and the plate read using a plate fluorimeter. A positive result indicates the presence of the target nucleic acid, a negative result indicates that the -arget nucleic acid is not present.

SDA (Strand Displacement Amplification) The following scheme is taken directly from G.T.

Walker, et al., "Strand displacement amplification an isothermal, in vitro DNA amplification technique", Nuc. Acid Res.

pp. 1691-1696, 1992.

j

I

j CI C The target generation schema for SDA is shown in Figure This figure depicts the initial steps in an SDA reaction which transform the original target sequence into the amplification cycle depicted in Figure 5b. A target DNA is heat denatured. Four primers (BI, B2, S1 and S2), present in excess, bind the target strands at positions flanking the sequence to be amplified. Primers Sl and S2 have HinclI recognition sequences GTTGAC) located 5' to the target complementary sequences.

The four primers are simultaneously extended by exo-klenow using dGTP, dCTP, TTP and dATPS. Extension of B1 displaces the S1 primer extension products, S1 -ext. Likewise, extension of B2 displaces S2 -ext. B2 and S2 bind to displaced S1-ext. B1 and S1 bind to displaced S2-ext. Extension and displacement reactions on templates Sl-ext and S2-ext produce two fragments with a hemiphosphorothiote HinclI at each end and two longer fragments with a hemiphosphorothioate HincII site at just one end. HincII nicking and exo-klenow extension/displacement reactions initiate at these four fragments, automatically entering the SDA reaction cycle depicted in Figure 5b. Sense and antisense DNA strands are differentiated by thin and thick lines.

dlfj IPEA/SUBSTITUTE SHEET PCT/AU 92 /0 0 587 RECEIVED 1 7 AUG 1993 -14- HincII recognition sequences are depicted by (thin line- thick line-thin line).

The SDA reaction cycle is shown in Figure 5b. These reaction steps continuously cycle during the course of amplification. Present in excess are two SDA primers (Si and S2). The 3'-end of S1 binds to the 3'-end of the displaced target strand TI, forming a duplex with 5' overhangs.

Likewise, S2 binds T2. The 5'-overhangs of Sl and S2 contain the HincII recognition sequence GTTGAC) Exo-klenow extends the ends of the duplexes using dGTP, dCTP, TTP and dATPS, which produces hemiphosphorothiote recognition sites on S1.T1 and S2.T2. HincII nicks the unmodified primer strands of the hemiphosphorothiote recognition sites, leaving intact the modified complementary strands. Exo-klenow extends the 3'-end at the nick on S1.T1 and displaces the downstream strand that is equivalent to T2. Likewise, extension at the nick on S2.T2 results in displacement of TI. Nicking and polymerization/ displacement steps cycle continuously on S1.T1 and S2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because the strands displaced from SiT1 serve as target for S2 while strands displaced from S2.T2 serve as target for S1. Sense and antisense DNA strands are differentiated by thin and thick lines. Intact and nicked HincII recognition sequences are depicted by (thin line-thick line-thin line) and (thin line-thick line, space, thick line-thin line), respectively. The partial HincII recognition sequence 5' GAC and its complement 5' GTC are present at the 5'-and ends of displaced strands as represented by (thick line-thin line) and (thin line-thick line).

The following modifications of SDA (see Walker et al., 1992 Nuc. Acid Res 20, 1691-1696) are required to allow implementation of SPA.

These modifications should be read in conjunction with Figure 1A Primers Sl and S2 are chemically prepared to contain HincII recognition sequences which are resistant to cutting by this enzyme, i.e., primers Si and S2 are chemically prepared to IPEA/SUBSTITTE SHEET PT/AU 9 2 /00 5 8 7 RECEIVED 1 7 AUG 1993 contain phosphorothioate nucleotides using known methodology.

1B Primer S2 carries a detector tag, introduced during chemical synthesis, for example, a biotin group or 2 Only standard dNTPs are then required for amplification dATPS are not be used).

With regard to the SPA version of SDA the process proceeds as follows Primer S1 is covalently bound via the 5'-end to the microtitre well surface.

Biotinylated primer S2 is free in solution.

SPA-SDA isothermal amplification is followed by a simple wash to yield a final solid phase double stranded product as shown in Figure 6.

The label is then used to detect the bound target molecule. For example biotin would then be detected colorimetrically via avidin-horseradish peroxidase or fluorimetrically via avidin- Europium.

It is also possible to improve the amount of solid phase product produced by the inclusion of a small quantity of primer S1 in the solution phase to increase the quantity of the target in solution.

Detection Labels Any suitable method for labelling nucleic acid sequences can be used, fluorescent labelling, biotin labelling or enzyme labelling. The label is introduced into the anchored amplified nucleic acid product either via labelled primer 2 or via labelled nucleotide substrates.

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PCTr/AT 9 2 /0 0 5 8 7 RECEIVED 1 7 AUG 1993 -16- Exemplary detection systems include: Fluorescent labelling and detection Source of fluorescent label Fluorescent primer 2, fluorescein-primer 2.

(ii) Fluorescent derivatives of ribo or deoxyribo-NTPs (substrates for RNA and DNA polymerases, respectively).

SPA Product fluorescently labelled anchored nucleic acid.

Detection system fluorescent plate reader or other fluorescence detection system.

Biotin label Biotinylated primer 2.

(ii) Biotinylated dNTPs, e.g. biotinylated dATP.

SPA product Biotin labelled anchored nucleic acid.

Detection systems Europium labelled avidin, with quantification of Europium by time resolved fluorescence spectroscopy.

(ii) Avidin horseradish peroxidase, with quantification of the resulting coloured product by spectroscopy plate reader).

(iii) Avidin alkaline phosphatase, with quantification of the resulting coloured or fluorescent product by standard or fluorescent spectroscopy.

Other labels include radiolabels, other enzyme systems, and dyes, as well as nucleotides with altered emission spectra.

rU ELj iVS9A/IUBSTITUTE SHEEF rPT/Au 9 2 /00587 RECEIVED 1 7 AUG 1993 -17- The present invention is further demonstrated by reference to the following non-limiting examples.

Example 1: Affigel anchored Solid Phase Amplification (SPA) Linkage of primer to Affigel

T

M

OH O I 11 Sepharose -O-CH 2

-CH-CH

2 -NH- (CH2) 5 -C--NH 2 -X-pNipN 2 p//N 2 0 bead HS

(NHS

primer 1 spacer arm activating group SDMSO (solvent) OH 0 I II Sepharose O-CH 2

-CH-CH

2 -NH- (CH 2 )5 NH-XpNIpN 2 p//N 2 0 bead amide linkage Product: Primer 1 bound to Affigel T M beads (amide linkage).

The DNA sequence to be amplified was a portion of the CFTR (cystic fibrosis transmembrane conductance regulator) gene.

Amplification was by the polymerase chain reaction (PCR).

The PCR conditions were as follows mM Tris-HCl (pH 8.3 at 250C), 2.5 mM MgCl2, 50 mM KC1, 0.1% v/v Triton x 100, 0.04% w/v gelatin, 0.4 mM dNTP's, .Primer 2 250 ng, normal human DNA, 0.5Lg (note dried blood spot on paper diameter 3.0 mm was used for some reactions). Taq polymerase 2.5 units. Cycling conditions 30 sec 949C, 60 sec 72QC, 30 sec 559C, 30 cycles.

In this particular embodiment, the SPA product was labelled with biotin, rather than a fluorescent label.

IIPEAI8UBSTITUTE SHEETI tl 1- PCT/AU 92 /00587 RECEIVED 17 AUG 1993 -18- Detection of SPA Product The Affigel T bound product was detected by use of Europium labelled avidin and time resolved fluorescence spectroscopy. The results are shown in Tabl 1.

Table 1: Results of CFTR gene Test.

Affigelprimer 1 (11) Target Test Denatured 8 e Counts Control v ruI t-a ruurrrv Ig DNA Jg DNA gg DNA g DNA Lg DNA Control Control 3 mm blood disc 3 mm blood disc boiled extract of 3 mm blood disc boiled extract of 3 mm blood disc No DNA No DNA 35,000 74,000 550,000 300,000 400,000 60,000 42,000 112,000 180,000 54,000 83,000 6,400 3,800 22,000 110,000 150,000 6,000 4,800 8,000 12,000 12,000 34,000 Test counts Control Counts 12 9 8.8 14 4.6 The SPA product can be treated at pH 9.8 to dissociate the biotin labelled DNA strand from the primer anchored DNA strand.

Conclusion: 5-10 il of AffigelT-primer 1 is an efficient solid support for SPA for either purified DNA or DNA of 3 mm blood discs.

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PCT/AU 9 2 0 0 5 8 RECEIVED 17 AUG 199 -19- Example 2: SPA Detection of the CFAPhe 508 mutation The amplification reagents and conditions were as follows: Primers: Common primer carrying 5'-biotinyl group GGC ATG CTT TGA TGA CGC TTC-3'.

Primer for detection of A F 508 mutation A F 508 5'-(T)10 GGC ACC ATT AAA GAA AAT ATC ATT GG-3' Normal primer for detection of normal sequence TTT TTT GGA TCC GGC ACC ATT AAA GAA AAT ATC ATC TT-3'.

Each SPA 50 [l reaction contained 67 mM Tris HC1 pH 8.8 at 259C, 16.6 mM ammonium sulphate, 0.45% Triton X-100, 200 g/ml gelatin, 200 pM each of dATP, dCTP, dGTP, TTP, 3.0 mM MgC12 250 ng of 5' biotin primer, 2.5 units Taq polymerase.

SPA was performed in Costar microtitre wells previously nitrated with acetic anhydride/nitric acid and reduced by tin chloride to give wells which carried amino groups covalently attached to the well walls.

The normal or AF 508 primers were covalently linked to amino groups on the well walls via the amidate linkage method.

Product detection was via avidin-Europium and time resolved Sfluorescence quantification and colorimetrically by avidinhorseradish peroxidase.

SPA cycle amplification conditions 1 min 959C j 1 min 579C 1 min 72°C Initial denaturation for 7 minutes at 956C and final extension for 10 minutes at 72QC.

IPEA/SUBSTITUTE SHEET P/Au9 2 0 0 5 8 7 RECEIVED 1 7 AUG 199 Table 2 Results of SPA AF 508 Mutation screen.

Template Wild type 500 bp PCR product AF 508 500 bp PCR product Wild type genomic DNA AF 508 genomic DNA Salmon sperm DNA (sonicated) No template TRF Counts 1,000,000 10,000 45,000 10,000 10,000 10,000 Absorbance at 450 1.8 0.2 0.8 0.15 0.15 0.2 Table 2 shows signals obtained with the wild type primer immobilized on the wells, following solid phase amplification of different templates. Additionally the signal with genomic DNA was increased to approximately 400,000 counts when 45 cycles were used. Additionally, when some of the solid phase primer was added in solution the signal was greatly increased from genomic DNA as a target.

For example, for detection of the normal Cystic Fibrosis gene, addition of normal primer to the solution gave the following results 2 ng ng ng ng normal primer in solution gave a product signal of normal primer in solution gave a product signal of normal primer in solution gave a product signal of normal primer in solution gave a product signal of 80,000 counts 1,000,000 counts 1,200,000 counts 1,200,000 counts i When the mutant AF 508 primer was immobilized signals obtained were similar to those in the table when AF 508 DNA was used as a template.

JIP BSITUTE SHEET RECEIVED 7 AUG 199 -21- Example 3 Detection of mycoplasma pneumoniae the major causative agent of atypical pneumonia.

Primers Solution phase primer 5'-biotinyl TCA AAA CAA CGA CAC- 3' corresponds to nucleotides 3863-3877 of the PI gene.

Solid phase primer 5'-TTT CAG AAA GTC GAC-3' corresponds to nucleotides 4114-4100 of the P1 gene, phosphorylated with polynucleotide kinase and ATP.

Preparation of DNA from M. Pneumoniae Various amounts (in colony forming units CFU) of whole M. pneumoniae cells were added to normal nasopharyngeal aspirate and were collected by centrifugation and the pellet was treated with proteinase K (30 Ll, 200 g/ml proteinase K, 10 mM Tris-HCl pH 8.3) at 37 9 C for 1 hour to liberate the DNA, then at to inactivate the proteinase K.

SDA reaction mix as for cystic fibrosis AF 508 detection except primers were as given above.

Microtitre Plates Plates derivatized with solid phase primer were prepared as for the cystic fibrosis AF 508 detection system.

Ampolification Conditions cycles of amplification using for 1 min 400C for 1 min 609C for 3 min The initial denaturation was 7 min at 95QC and the final extension was 10 min at 729C.

_IPEASUBSTITUTE SHEET "Z'QT011('

AC

PcT/Au 9 2 /0 0 5 8 7 RECEIVED 1 7 AUG 199 -22- Table 3 Product detection was resolved fluorescence via Avidin-Europium and time quantification of Europium M. Pneumoniae Counts

CFU

107 593,000 106 263,000 5 280,000 48,000 The following organisms did not give a positive signal (signals 14,000 47,000): M. buccale, M. fermentans, M.

genitalium, M. hominis, M. hyorrhinis, M. salivarium, A.

laidlawii A, A. laidlawii B.

Additionally five patients with confirmed M. pneumoniae infection with greater than 105 CFU/ml of respiratory secretion gave positive signals 121,000-330,000 counts.

The results demonstrate the surprising superiority of the assay where the first primer is immobilized to a solid planar support (Examples 2 and as compared with primer immobilized to beads (Example 1).

Modification and variations of the method and assay of the present invention will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.

I 4 V t IPEA/SUBSTITUTE

SHEET

Claims (14)

1. 2. A method for detecting a target nucleic acid sequence 2L comprising: providing a first primer hybridizing to the target 1 1 nucleic acid -equence, wherein the primer is immobilized on 41 San immobile solid phase support by a direct chemical linkage between the primer and the solid phase support, wherein the solid phase support forms a part of or is insertable into a container for the sample, providing a second primer hybridizing to the target nucleic acid sequence in the opposite direction, wherein the scond primer is labelled with a detectable label, reacting the first and second primers with a sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first and second primers in the container for the sample, and i rir~ 24 detecting the presence of bound second primer; steps and both being conducted in the container for the sample.
2. 3. The method of claim 1 or claim 2 wherein the container is a microtiter plate well.
3. 4. The method of claim 1 or claim 2 wherein the first primer is immobilized on a solid phase support which is immersible into the container. The method of claim 1 or acid sequence is amplified by
4. 6. The method of claim 1 or acid sequence is amplified by 0o: amplification. 0 7. The method of claim 1 or 0 2, acid sequence is amplified by oo amplification.
5. 8. The method of claim 1 or 4 acid sequence is amplified by o amplification.
6. 9. The method of claim 1 or acid sequence is amplified by amplification. claim 2 wherein the nucleic the polymerase chain reaction. claim 2 wherein the nucleic the nucleic acid sequence based claim 2 wherein the nucleic the ligase chain reaction based claim 2 wherein the nucleic the QS replicase based claim 2 wherein the nucleic the strand displacement The method of claim 1 or claim 2 wherein the nucleic acid sequence is selected from the group consisting of nucleic acid sequences specific to species of microorganisms, characteristic of a genetic disorder or disease, HLA types, an identity, sex or speciation of an organism. T." 25
7. 11. The method of claim 1 or claim 2 wherein the label is selected from the group consisting of fluorescent labels, biotin, radiolabels, and enzymes including those specific for chromogenic substrates and dyes.
8. 12. The method of claim 1 or claim 2 wherein the label is attached or incorporated during the amplification process.
9. 13. The method of claim 1 or claim 2 wherein the label is attached or incorporated after the amplification process.
10. 14. An assay system or kit for detecting a target nucleic acid sequence comprising: a first primer hybridizing to the target nucleic acid sequence, wherein the primer is immobilized on an immobile o+O solid phase support by a direct chemical linkage between the o0o primer and the solid phase support, 0 wherein the solid phase support forms a part of or is o0 000 insertable into a container for the sample, .t0: a second primer hybridizing to the target nucleic acid 0. sequence in the opposite direction, wherein the second primer is labelled with a detectable molecule, and 0°ooreagents for amplification of the sample containing nucleic acid sequences under conditions which allow amplification of the nucleic acid sequences that hybridize to the first and second primers in the container for the sample, and reagents for detection of the label on the bound second S primer. An assay system or kit for detecting a target nucleic acid sequence comprising: a first primer hybridizing to the target nucleic acid sequence, wherein the primer is immobilized on an immobile solid phase support by a direct chemical linkage between the prmer and the solid phase support, J0 0u 26 wherein the solid phase support forms a part or is insertable into a container for a sample, a second primer hybridizing to the target nucleic acid sequence in the opposite direction, wherein the second primer is labeled with a detectable molecule, and reagents for amplification of the sample containing nucleic acid sequence under conditions which allow amplification of the nucleic acid sequences that hybridize to the first and second primers in the container for the sample, and reagents for detection, in the container for the sample, of the label on the bound second primer.
11. 16. The assay system or kit of claim 14 or claim 15 wherein the container is a microtiter plate well, further comprising o the well plate. a 0 o oc S 17. The assay system or kit of claim 14 or claim 15 wherein -the first primer is immobilized on an immobile solid phase support which is immersible into the container.
12. 18. The assay system or kit of claim 14 or claim 15 wherein the reagents for amplifying the nucleic acid sequence are o those for use in the polymerase chain reaction.
13. 19. The assay system or kit of claim 14 or claim 15 wherein o o the reagents for amplifying the nucleic acid sequence are S0 those for use in the nucleic acid sequence based amplification. The assay system or kit of claim 14 or claim 15 wherein the reagents for amplifying the nucleic acid sequence are those for use in the ligase chain reaction based
14. 27- amplification. 21. The assay system or kit of claim 14 or claim 15 wherein the reagents for amplifying the nucleic acid sequence are those for use in the f replicase based amplification. 22. The assay system or kit of claim 14 or claim 15 wherein the reagents for amplifying the nucleic acid sequence are those for use in the strand displacement amplification i0 10 rocess. 23. The assay system or kit of claim 14 or claim 15 wherein the nucleic acid sequence is selected from the group consisting of nucleic acid sequences specific to species of microorganisms, characteristic of a genetic disorder or disease, HLA types, an identity, sex or speciation of an organism. 46 I 24. The method of any one of the claims 1 to 13 wherein an additional aliquot of the first primer or other primer(s) are initially added to the reaction, to allow nucleic acid sequence amplification in solution, concurrent with solid S phase amplification. It,' t 25. The assay system or kit of any one of claims 14 to 23 wherein an additional aliquot of the first primer or other primer(s) are initially added to the reaction, to allow nucleic acid sequence amplification in solution, concurrent with solid phase amplification. 26. A method according to any one of claims 1 to 13 and 24, substantially as described herein, and with reference to any one of the Examples. _1Y_ 28 27. An assay system or kit according to any one of claims 14 to 23 and 25, substantially as described herein, and with reference to any one of the Examples. Dated this 31st day of January, 1996. ADELAIDE CHILDREN'S HOSPITAL and UNIVERSITY OF SOUTH AUSTRALIA By their Patent Attorneys MADDERNS .'t o a o o 1 0 a| 0 00* 00 0 9 I It 0 0 f a Q u a o o 1 0 0 a 0 0 0l 4 a t« 0. 0 Ut 0 4 14 I I 4 tC 0< vy /r