[go: up one dir, main page]

WO1991000868A1 - Polynucleotides immobilises - Google Patents

Polynucleotides immobilises Download PDF

Info

Publication number
WO1991000868A1
WO1991000868A1 PCT/GB1990/001040 GB9001040W WO9100868A1 WO 1991000868 A1 WO1991000868 A1 WO 1991000868A1 GB 9001040 W GB9001040 W GB 9001040W WO 9100868 A1 WO9100868 A1 WO 9100868A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
immobilised
target
pcr
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB1990/001040
Other languages
English (en)
Inventor
Philip John Royle Day
John Edward Fox
Matthew Robert Walker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Research Development Corp UK
Original Assignee
National Research Development Corp UK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB898915607A external-priority patent/GB8915607D0/en
Priority claimed from GB898921327A external-priority patent/GB8921327D0/en
Priority claimed from GB909008274A external-priority patent/GB9008274D0/en
Application filed by National Research Development Corp UK filed Critical National Research Development Corp UK
Publication of WO1991000868A1 publication Critical patent/WO1991000868A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • This invention is in the field of assays for polynucleotides, principally DNA, and relates to the immobilisation of polynucleotides on a support material.
  • PCR polymerase chain reaction
  • the process comprises the steps of denaturing a duplex to produce two single-stranded (ss) molecules, synthesising complementary DNA to each ss DNA by oligonucleotide priming synthesis, thereby producing a duplex from each ss DNA molecule, denaturing the duplex to produce two ss DNA molecules (one of the original length, one of length governed by the position at which priming began), using each of these molecules as the template for a further primed synthesis and so on for as many cycles as desired.
  • the DNA polymerase enzyme used for the primed synthesis is preferably one which is stable at the denaturing temperature, in order to avoid addition of fresh polymerase at each cycle.
  • a heat-stable polymerase from Thermus aquaticus enables the PCR amplification of DNA (directly from DNA, or indirectly from RNA) to be carried out at temperatures ranging from 30 to 100°C, at various stages of the process and dependent on the nature of the sequence to be amplified, in automated equipment.
  • the PCR is potentially of immense value in diagnostics since it enables either the analyte nucleic acid or the duplex produced upon hybridisation of the analyte to a labelled probe to be amplified and thereby very small amounts of nucleic acid to be detected. An amplification of 10 20 within 4-6 hours is possible. Hybridisation of native and/or synthetic DNA to complementary
  • DNA/RNA sequences is critical to the performance of PCR and many other DNA/RNA assays. Detection of analyte sequences with or without amplification is also dependent on hybridisation of complementary sequences to a labelled probe. Most of these present techniques require immobilisation of the analyte or probe sequences (e.g. to nylon membranes) prior to hybridisation.
  • Matrices commonly used have been control pore glass [S.S. Ghosh and G.F. Musso, Nucleic Add Res. 15 , 5353-5390
  • oligo-dT cellulose is linked to an oligonucleotide via a polynucleotide chain by the steps of (1) poly dA-tailing the 3'-ends of a double-stranded oligo, (2) hybridising the poly dA-tailed DNA to the oligo-dT cellulose, (3) filling 1n the gap between the last dT of the oligo-dT cellulose and the 5'-end nucleotide complementary to the
  • nucleotides can be linked to a support via a dithlo (-S-S-) linkage, which results in exceptional stability of linkage.
  • the supported polynucleotide can participate in a PCR reaction carried out at elevated temperature.
  • the dithlo linkage is formed by coupling a thiol (-SH) group or a reactive derivative thereof on the polynucleotide with a thiol group or reactive derivative thereof on thfi support.
  • polynucleotides thus linked to beads can be used in the PCR. They are also useful for a variety of purposes in heterogeneous assays of nucleic acids.
  • an important feature of the invention consists in an immobilised polynucleotide material comprising a support bound to a 5'-end of at least one polynucleotide through a linkage which includes a dithio (-S-S-) group.
  • polynucleotide encompasses DNA, RNA, oligonucleotides and any modified or labelled form of conventional nucleic acid in which pairable bases are arranged in helical chains.
  • An important subsidiary feature comprises an immobilised polynucleotide material comprising a support bound to a 5'-end of at least one polynucleotide which contains a "c-target" base sequence complementary to a "target” base sequence of interest, said support being bound through a linkage as defined above and which is effective to distance or space the support from the "c-target” sequence so as to permit the c-target sequence to hybridise to the target sequence.
  • the invention further provides a method of carrying out a polymerase chain reaction (PCR) in which an immobilised polynucleotide material of the invention is substituted for either or both of the oligonucleotide primers used in the PCR and the amplified product of the PCR is separated on the solid phase.
  • PCR polymerase chain reaction
  • the invention includes a method of heterogeneous assay for an analyte nucleic add, which comprises subjecting the immobilised polynucleotide material of the invention, having a c-target sequence, to hybridisation with the target sequence of analyte nucleic acid present in a liquid phase analyte, separating the phases and determining the occurrence or extent of hybridisation.
  • the analyte nucleic acid is labelled either directly or by hybridisation at a second site thereof with a labelled complementary polynucleotide.
  • Such an assay is preferably used in conjunction with a liquid phase PCR, to detect a product of the PCR. It can also be used in any nucleic acid assay context.
  • the invention further includes a method of producing an immobilised polynucleotide material of the invention which comprises reacting a 5'-(thiol-terminated) derivative of a polynucleotide with a support having a thiol group thereon, either or both of said thiol groups being optionally derivatlsed as co-reactive -S-S- linkage-producing derivatives, to form a said -S-S- linkage between said thiol groups.
  • Any chain atoms (a) between support and its pendant thiol group and (b) between 5'- nucleotide phosphate residue and its pendant thiol group are preferably thermally stable at the maximum temperature likely to be essential in the PCR reaction, normally 90oC-100oC.
  • the support needs to be distanced from the sequences which it is desired to hybridise.
  • the spacing is provided wholly by the linkage, that is to say between the 5'-end of the last nucleotide and the support.
  • it could be provided partly or wholly by attaching a sequence of irrelevant nucleotides to the last nucleotide of interest and providing the thiol termination on the last of the irrelevant nucleotides.
  • the spacing between the last nucleotide of interest usually a c-target nucleotide
  • the support can contain nucleotides.
  • Oligonucleotides having a thiol (SH) group pendant from the 5'-terminus are not, as a class, novel.
  • Connolly and Rider used these compounds in reactions to label the thiol-terminated oligonucleotides with the fluorophore, N-iodoacetyl- N'-(5-sulpho-1-naphthyl)ethylenediamine (AEDANS) or N-(1-anilino-4-naphthyl)maleiimide (ANM).
  • oligonucleotide "functional izing reagents” are described. These have a pendant thiol, amino or hydroxyl group linked to the 5'- phosphate group by a spacer chain of formula -(CH 2 ) 2 -(OCH 2 CH 2 ) n -CH 2 CHR*-O- where the oxygen atom shown is that of the phosphate group and R* represents H, CH 2 OH or tritylated CH 2 OH.
  • HRP horseradish peroxidase
  • the HRP derivative has a maleiimide termination, producing the same kind of linkage as described in the Connolly and Rider paper.
  • Figs. 1 and 2 are graphs in which the amount of hybridisation achieved by immobilised polynucleotides of the invention, assayed by heat-denaturing labelled hybrids, is represented by plots of amounts of labelled material released from the hybrids into solution against temperature of denaturation and compared with various controls;
  • Figures 3 and 4 are similar graphs but referring to immobilised polynucleotides of the prior art.
  • Figure 5 is a similar graph showing the effect of the inclusion of a spacer arm in the immobilised polynucleotides of the invention.
  • the invention can be used in the context of any procedure of immobilising or insolubilising a polynucleotide, especially a heterogenous assay procedure.
  • the invention is applicable in such assay procedures described in the above-mentioned Matthews and Kricka review paper.
  • the support material is not critical and can be any of those conventionally used.
  • One convenient material is beads which already have- thiol groups attached. These are available commercially for the purposes of affinity chromatography of proteins. See, for example, the above-cited "Affinity Chromatography - principles and methods", published by Pharmacia
  • CPG CPG
  • any other glass of high surface area can be used.
  • While the elements of the linkage can be provided in any order, it will be appreciated that it is desirable not to cause any reaction which might be non-specific. It will almost always be desirable to form the -S-S- bond last when preparing the immobilised polynucleotides.
  • a conventional aminoalkylating agent can be reacted with a support such as CPG to provide amino terminations.
  • the amino-functional support can then be reacted with cysteine in a peptide coupling reaction to provide the SH termination required.
  • preliminary indications suggest that such a cysteine link might not produce the desired high specificity of binding of the support to the DNA to be immobilised and at present a mercaptoalkyl termination is preferred.
  • a support such as CPG is preferably silanised by an ( ⁇ -mercaptoalkyl) trimethoxysilane, conveniently (3-mercaptopropyl) trimethoxysilane. This silanised support is then reacted with the thiol- terminated polynucleotide.
  • the linkage between the polynucleotide and the support will normally include a spacer arm between the polynucleotide and the -S-S- linkage, the -S-S- linkage and the support, or both.
  • the polynucleotide can itself contain a spacer arm, of one or more nucleotides, usually 3 to 20 and preferably 4 to 15, between its 5'-end and the nearest "relevant" nucleotide.
  • the polynucleotide is a sequence complementary to another sequence to which it is to be hybridised, the "relevant" nucleotide will be the last nucleotide of that complementary sequence, (the "c-target" sequence).
  • This nucleotide spacer arm consists of "irrelevant" polynucleotides which do not interfere with or participate in hybridisation. For the purposes of reckoning its length, the nearest “relevant” nucleotide is not counted in, but the 5'-end nucleotide is counted in.
  • the total length of spacer arm (whether in the linkage or the polynucleotide or both) will depend on the nature of the linkage and its spatial configuration. In general, a straight chain of from 5 to 27 atoms and nucleotides between the support and the relevant 5'-end nucleotide (inclusive of the two S atoms) is fuggested.
  • Too short a chain would tend to bring the support too close to the polynucleotide chain for successful hybridisation to occur, while if the linkage is too lengthy the support might dangle at such a distance as to wrap itself over the relevant 5' end nucleotide and its neighbours. This would probably interfere with successful use of a supported oligonucleotide as a primer in the PCR.
  • the total spacer arms conform to the general formula (N)x-(L 1 )y-S-S-(L 2 ) z e.g. in the manner illustrated below, using arbitrary, imaginary sequences:
  • N represents a nucleotide in the spacer arm upstream of the end of the "c-target" sequence
  • L 1 and L 2 represent atoms in a covalent chain from the end nucleotide to the support, on each side of the two sulphur atoms of the -S-S-linkage;
  • x, y and z are 0 or Integers and the sum of x, y and z is 3 to 25, preferably 4 to 25, especially 6 to 20.
  • the invention is usable merely for isolating a polynucleotide without necessarily hybridising or annealing it. In such circumstances a spacer arm will frequently offer no advantage.
  • oligo primers oligonucleotide primers
  • the forward primer because it primes 5' to 3' synthesis of complement to the coding strand of the polynucleotide.
  • the other is the reverse primer which primes 5' to 3' synthesis of complement to the non-coding strand. If neither strand codes, the designations are arbitrary.
  • the PCR normally comprises broadly three steps per cycle:
  • annealing forward and reverse oligonucleotide primers to the strands of a polynucleotide to be amplified, these strands having been first separated, annealing being carried out typically at a temperature of 30 to 60°C;
  • the cycle is then repeated, using the products of each separation step (3) as the templates for the ensuing annealing step (1) of the next cycle.
  • Products are conveniently detected by labelling the nucleotides used for the chain extension and determining the label attached to the products. Labelling can be done radioactively e.g. with 32 P or by biotinylation, for example, and the labelled products immobilised and separated from the reaction medium.
  • the PCR can be carried out in solution and the products detected by carrying the PCR to the end of a separation step (3), and probing the strands with a probe which is attached to a support or to which a support is later attached. The amount of label attached to the support is then determined.
  • PCR products can be subjected to a sandwich assay in which a two-site probe is used, site A being capable of binding specifically to the PCR product which it is hoped to detect and site B being capable of hybridising with an immobilised polynucleotide of the invention.
  • Another method of detecting PCR products on a solid support depends on measuring the thickness of the layer deposited on the solid phase. This can be done by measuring changes of optical path through the layered film: see literature of
  • Products attached to the solid phase through the linkage of the invention can be released therefrom by any of the known methods of breaking -S-S- bonds, such as by prolonged action of a high concentration of dithiothreitol or 2-mercaptoethanol. Note, however, that the relatively low concentrations of these reagents often present in conventional PCR buffer systems will not normally break the S-S bonds.
  • polynucleotides can be linked to the same integral unit (particle, sheet, bead, tube etc.) of the support material, provided that there is no interference between polynucleotides which would hinder their end use, e.g. in an assay.
  • Example 1 illustrates the hybridisation capacity of an oligonucleotide bonded to a support via an -S-S- linkage, in accordance with the invention.
  • the oligonucleotide was used with and without a 4-nucleotide spacer between the c-target sequence and the 5'-end.
  • the oligonucleotide synthesised for coupling to the beads is that of the forward primer complementary to a sequence of the human papilloma virus type 16 successfully applied in the PCR for amplification of a 120 base-pair fragment of HPV16 DNA:
  • It is a methyl phosphoramidite of formula CF 3 CO NH(CH 2 ) 6 OP(OCH 3 )N[CH(CH 3 ) 2 ] 2 and is used in the beta-cyanoethyl phosphoamidite synthesis to introduce an aminohexyl termination (the terminal CF 3 CO- group being cleaved off at the end of the synthesis).
  • hybridisation probes prepared were either a sequence complementary to D99 i.e:
  • the ollgos were synthesised on a Biotech Instruments BT8500 automatic machine, using the well established cyanoethyl phosphoamidite coupling chemistry.
  • the last base to be added at the 5' end of the oligo was a fully dlmethoxytrltyl protected, thiol phosphoamidite, introduced via line Q on the BT8500.
  • the oligo was cleaved from the controlled pore glass solid phase in the normal way with aqueous ammonia, using a 'trityl ON' cleavage method. The free oligo was then dried at 50°C under vacuum. It is stable for long periods at -20°C. COUPLING OF THIOL-TERMINATED OLIGONUCLEOTIDE TO BEADS VIA DITHIO LINKAGES,
  • oligo 0.04 ⁇ mole of the oligo, (10 OD 260 units) was dissolved in 100 ⁇ l of 0.1M triethanolamine acetate (TEAA) buffer pH 7.5. 15 ⁇ l of 1M silver nitrate solution was added, vortexed, and incubated at room temperature for 30 mins. This cleaved the dlmethoxytrltyl protective group off the thiol, with approx. 70% efficiency. 20 ⁇ l of 1.0M dlthiothreitol (DTT) was added, vortexed and reacted for 5 mins. The reaction mixture was centrifuged at 3000 rpm for 5 mins and the supernatant removed.
  • TEAA triethanolamine acetate
  • the supernatant (minus beads) should be brown at this stage; if not, iodine solution should be added in 20 ⁇ l aliquots until it turns brown.
  • the solution and beads were then vortexed and reacted for 30 min. to couple the beads to the oligo. After coupling, the beads were centrifuged and washed three times with 0.05M TEAA buffer to remove iodine and were then ready for use.
  • 70% of the activated oligo has been shown to bind to the beads, i.e. a total of 0.02 ⁇ moles per lmg or 10 mg of beads. Since the protective group is cleaved with 70% yield and 70% of the activated oligo binds to the beads, the yield is 70% - 49% with respect to the original oligo.
  • the amino-terminated D99 (4dTTP spaced) oligo (2) prepared above was coupled to Dynabead M450 beads (Dynal UK Ltd) having -OH active groups according to the method described by V. Lund et. al ., 10c. (0.04 ⁇ mole DNA to 10 mg of beads).
  • Beads were activated by incubation for 30 mins. in distilled water, followed by raising the pH to 11.5 with 2M sodium hydroxide and incubation for 50 mins. 30mg of cyanogen bromide was added and the pH re-adjusted to 11.5 with 2M sodium hydroxide. The beads were incubated on a roller for 10 mins followed by 5 washes with distilled water and resuspended in lOmM sodium bicarbonate buffer, pH 8.4
  • oligo 0.08 ⁇ mole of oligo was added to 1ml of 1-methyl imidazole buffer pH 7 (Sigma) containing 0.1M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma), 0.25M ethylenediamine (Sigma) and incubated at 50oC for 3 hrs. 60 ⁇ l of 3M sodium acetate, pH6.5 were added and 1 ml ice cold ethanol, mixed and incubated at -80oC for 2 hrs. The precipitate (comprising the amineactivated oligo), after centrifugation, was dried in a vacuum dessicator and reconstituted in 600 ⁇ l 10 mM sodium bicarbonate buffer, pH8.0.
  • Oligo was coupled to M450 beads via carbodiimide according to the above Lund et al. reference. Thus, 0.08 ⁇ mole of activated oligo was added to 10 mg beads in the presence 0.04M sodium
  • Anti-D99 oligo was 3' end labelled with 32 P-dideoxyATP using a kit supplied by Amersham International pic and separated on a G-50 "Sepharose" column. Labelled anti-D99 was added to unlabelled anti-D99 to give a 4-5% label concentration.
  • Beads with or without oligo (0.02 ⁇ mole per mg. or 10mg) attached by the methods described above were incubated with a molar excess (0.03 ⁇ mole) of anti-D99 (per mg. or 10 mg of beads) (4-5% 3'-end labelled with 32 P) in 2 ⁇ saline sodium citrate (SSC) buffer at 40oC for 2.5 hrs (1ml total volume). The supernatants were removed and discarded and the beads washed 4 times in 2 ⁇ SSC at 40°C, to remove thoroughly excess label led probe.
  • SSC saline sodium citrate
  • the hybridisation capacity of the oligos attached to the beads was then measured by dissociating the labelled probe DNA strand from the bead-bonded oligo strand and measuring the amount of label released into solution.
  • the beads were then sequentially Incubated in 0.5ml of 2 ⁇ SSC for 5 mins at 10°C temperature increments up to 90oC, supernatants were removed at the end of each cycle and the number of radioactivity counts per minute (cpm) assessed by Cerenkov-counting.
  • a final further incubation of 90°C in the presence of 2% mercaptoethanol in 2 ⁇ SSC was performed and the cpm in the supernatant and remaining on the beads determined.
  • Figure 1 relates to 10 mg beads dithlo-coupled to 0.2 ⁇ mole of oligo D99.
  • the filled squares show the cpm values obtained using the dithio-coupled beads hybridised to labelled complementary oligonucleotide, while the open triangles show the cpm values for the same beads incubated under the hybridisation conditions with the control non-complementary oligonucleotide.
  • the open circles depict the amount of label in the supernatant using unactivated thiol beads (i.e. in the absence of D99) incubated under hybridisation conditions with the complementary (anti-D99) or non-complementary probes (the results were the same whichever probes were used).
  • the right hand side shows the cpm remaining on the beads after the 90°C wash in the presence.
  • 2-mercaptoethanol breaks the -S-S- bond, so these results are a measure of non-specific binding. Similar results (not shown) were obtained in the absence of mercaptoethanol (mean of two experiments).
  • Figure 2 shows the results obtained from the same experiments performed using 10 mg of beads with or without 0.2 ⁇ mole of oligo
  • D99/non-complementary oligo gave a radioactivity count on the beads between that for di thio-linked D99/non-complementary ol igo (fi l l ed square) and the unacti vated/compl ementary ol i go (oeen circle).
  • the results for the dithio-linked beads of the invention were more erratic than in Figure 1, doubtless due to the low ratio of oligo to beads in the coupling reaction.
  • Figure 4 shows the results obtained as in Figure 3 using beads linked to D99 via carbodiimide chemistry.
  • This Example illustrates the effect on hybridisation of including an 8dT spacer arm in the immobilised oligo.
  • the results shown in Figure 5 were obtained.
  • the filled circles represent results from the 8dT spaced immobilised D99, while the filled squares are those for the unspaced immobilised D99.
  • Beads which were not coupled to the D99 gave counts of virtually zero. It is seen that the counts were somewhat higher from the nucleotide spacer arm-linked beads, indicating improved hybridisation.
  • This Example illustrates the effect on the hybridisation of including a long (15 dT) spacer arm in the immobilised oligo.
  • Example 2 Following the procedure of Example 1, using 10 mg of the beads and, in one run, pre-washing the oli go-coupled beads for 5 minutes at 80°C instead of at room temperature, and using a thiol-terminated oligo with a 15 dT instead of a 4 dT spacer, the data shown in the Table below were generated. There is a reduction in the amount of specific hybridisation compared with Example 1 in which the spacer was shorter. Possibly such a long spacer creates steric hindrance to hybridising anti-D99.
  • This Example illustrates immobilisation of DNA to a non-thiolated support (control pore glass) by constructing a dithio linkage.
  • the D99 oligos coupled to the activated CPG were then hybridised to labelled anti-D99, as in previous Examples.
  • the same hybridisation was performed on 0.2 ⁇ mole of D99 coupled to lmg of magnetic beads as in Example 1.
  • the hybridisation capacity of this DNA is considered as good as (if not a little better) than that of the magnetic beads.
  • Example 2 demonstrates that double-stranded DNA simulating a PCR product, immobilised onto magnetic particles (Biomag) via the dithlo-linkage described in Example 1, can be labelled by a 3' end-labelling reaction.
  • the magnetic beads carrying this duplex of immobilised double-stranded DNA were used as template in the 3' end-labelling reaction employing 32 P-dCTP as the labelling nucleotide, and T ag polymerase or Klenow fragment as the enzymes in Tag polymerase buffer.
  • cytidine (dC) bases are added to complement the guanosine (dG) tail
  • the reaction mixture was heated to 90°C, and whilst still at this temperature, the beads were removed by centrifugation.
  • the beads were washed with 2 ⁇ SSC at 90°C and the radioactivity (cpm) remaining on them was then determined by Cerenkov-counting.
  • Table 1 were compared to those obtained using D99-beads without enzyme present, or using magnetic beads alone (i.e. no immobilised D99).
  • DNA (oligo D99) dithio-linked to magnetic beads can function as the forward primer in the polymerase chain reaction for the amplification of target DNA (the E6 region of human papilloma virus type 16 (HPV16) genomic DNA), resulting in immobilisation of the PCR products as double stranded or (by heating the product to 90oC immediately before separating solid from liquid phases) single stranded DNA.
  • target DNA the E6 region of human papilloma virus type 16 (HPV16) genomic DNA
  • 3 mg magnetic beads coupled to oligo D99 were added to a total 100 ⁇ l PCR reaction mixture composed of recommended PCR buffer (Perkin-Elmer Cetus) in the presence of Caski DNA (a cell line infected with HPV16 genomic DNA) free (i.e. not immobilised) reverse primer (referred to as non-complementary primer in Example 1) or both free non-complementary primer and oligo D99.
  • recommended PCR buffer Perkin-Elmer Cetus
  • Reactants were preheated to 98°C for 7 minutes 5 units of Tag polymerase were added and a PCR was carried out for 40 temperature cycles composed of 96°C for 30 seconds (denaturation), 40°C for 30 seconds (annealing of primer to target) and 72°C for 1 minute (DNA synthesis by primer extension) performed on a Wolfson Programmable Thermal Cycler. Following cycling, supernatants were removed and the beads resuspended in 2xSSC heated to 90°C. The magnetic beads were removed by centrlfugatlon and washed once with 2xSSC buffer at room temperature. This results in the dithio-linked oligo D99 and its extension product remaining on the magnetic beads as single-stranded DNA.
  • Hybridisation of pobe and assessment of hybridised probe were performed as described in Example 1, except that the amount of radioactivity remaining on the beads was determined.
  • Table 2 also includes the data obtained using magnetic beads alone (i.e. no dithio-linked oligo D99) in the presence or absence of Tag polymerase and in reaction mixtures containing free (i.e. unlinked) reverse primer and forward primer.
  • F refers to forward oligo pirimer (i.e. 1 399) and R refers to reverse primer.
  • Tag Tag polymerase.
  • the reaction scheme below shows a simplified version of a PCR carried out in the manner of Example 7, with a forward primer (D99) immobilised, a reverse primer in solution, and a single molecule of double-stranded HPV16 DNA.
  • AAAGATTCCA TAATATAAGG GGCGGTGACC GGTCGATG 38

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On peut lier des nucléotides provenant d'un résidu 5'-phosphate à un support au moyen d'une liaison dithio (-S-S-). Des polynucléotides ainsi immobilisés peuvent être utilisés dans une réaction en chaîne de polymérase (RCP) nécessitant de façon typique une température de 90°C. La liaison -S-S- peut être produite par réaction d'un support à base de thiol avec un polynucléotide à terminaison thiol en position 5'. Pour faciliter l'hybridation du polynucléotide immobilisé, la séquence à hybrider devrait être espacée de façon adequate du support. L'invention s'applique à tous les cas où un polynucléotide tel que l'ADN doit être séparé en phase solide à partir d'une phase liquide, surtout dans des méthodes de dosage hétérogène ainsi que pour l'isolement des produits de ladite réaction en chaîne de polymérase.
PCT/GB1990/001040 1989-07-07 1990-07-05 Polynucleotides immobilises Ceased WO1991000868A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB8915607.9 1989-07-07
GB898915607A GB8915607D0 (en) 1989-07-07 1989-07-07 Immobilised polynucleotides
GB8921327.6 1989-09-21
GB898921327A GB8921327D0 (en) 1989-09-21 1989-09-21 Immobilised polynucleotides(2)
GB9008274.4 1990-04-11
GB909008274A GB9008274D0 (en) 1990-04-11 1990-04-11 Immobilised polynucleotides(3)

Publications (1)

Publication Number Publication Date
WO1991000868A1 true WO1991000868A1 (fr) 1991-01-24

Family

ID=27264570

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1990/001040 Ceased WO1991000868A1 (fr) 1989-07-07 1990-07-05 Polynucleotides immobilises

Country Status (2)

Country Link
IE (1) IE902467A1 (fr)
WO (1) WO1991000868A1 (fr)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004199A3 (fr) * 1991-08-20 1993-04-15 Scient Generics Ltd Procedes de detection et de quantification d'acides nucleiques, et de production d'acides nucleiques marques, immobilises
FR2707296A1 (fr) * 1993-07-09 1995-01-13 Genset Sa Procédé de synthèse d'acides nucléiques sur support solide et composés utiles notamment comme support solide dans ledit procédé.
US5412087A (en) * 1992-04-24 1995-05-02 Affymax Technologies N.V. Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces
US5501947A (en) * 1990-07-19 1996-03-26 Royal Free Hospital School Of Medicine PCR diagnosis of human papilloma virus type 16
FR2726286A1 (fr) * 1994-10-28 1996-05-03 Genset Sa Procede d'amplification d'acides nucleiques en phase solide et trousse de reactifs utile pour la mise en oeuvre de ce procede
US6207385B1 (en) 1996-11-19 2001-03-27 Amdex A/S Use of nucleic acids bound to carrier macromolecules
WO2001025247A1 (fr) * 1999-10-05 2001-04-12 Quiatech Ab Composes de protection des hydroxyles et procedes d'utilisation
US6277975B1 (en) 1992-10-23 2001-08-21 Genetics Institute, Inc. Fusions of P-selectin ligand protein and polynucleotides encoding same
EP0910670A4 (fr) * 1997-03-05 2001-11-21 Orchid Biosciences Inc Fixation covalente de molecules d'acide nucleique sur des phases solides au moyen de ponts disulfures
US6849462B1 (en) 1991-11-22 2005-02-01 Affymetrix, Inc. Combinatorial strategies for polymer synthesis
US6919211B1 (en) 1989-06-07 2005-07-19 Affymetrix, Inc. Polypeptide arrays
US6955915B2 (en) 1989-06-07 2005-10-18 Affymetrix, Inc. Apparatus comprising polymers
US7087732B2 (en) 1989-06-07 2006-08-08 Affymetrix, Inc. Nucleotides and analogs having photoremovable protecting groups
US7329496B2 (en) 1990-12-06 2008-02-12 Affymetrix, Inc. Sequencing of surface immobilized polymers utilizing microflourescence detection
US7442499B2 (en) 1994-06-17 2008-10-28 The Board Of Trustees Of The Leland Stanford Junior University Substrates comprising polynucleotide microarrays
US7625697B2 (en) 1994-06-17 2009-12-01 The Board Of Trustees Of The Leland Stanford Junior University Methods for constructing subarrays and subarrays made thereby
US7691330B1 (en) 1991-11-22 2010-04-06 Affymetrix, Inc. Combinatorial strategies for polymer synthesis
US7869958B2 (en) 2004-08-09 2011-01-11 Research Development Foundation Structure-based modulators of B1 G-protein coupled receptors
US10093967B2 (en) 2014-08-12 2018-10-09 The Regents Of The University Of Michigan Detection of nucleic acids
US10948450B2 (en) 2018-07-23 2021-03-16 Genmark Diagnostics, Inc. Electrochemical measurements of components in coatings
US12460249B2 (en) 2017-03-08 2025-11-04 The Regents Of The University Of Michigan Protein analyte detection by analizing time-dependent signals from transient binding events of labeled low-affinity probes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0192197A2 (fr) * 1985-02-22 1986-08-27 Molecular Diagnostics, Inc. Immobilisation rapide et spécifique d'acides nucléiques sur un support solide
WO1988001302A1 (fr) * 1986-08-11 1988-02-25 Siska Diagnostics, Inc. Procedes et compositions d'analyse a l'aide de sondes d'acide nucleique
EP0269445A2 (fr) * 1986-11-26 1988-06-01 Cetus Oncology Corporation Procédé de détection des virus HTLVI et HTLVII par hybridation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0192197A2 (fr) * 1985-02-22 1986-08-27 Molecular Diagnostics, Inc. Immobilisation rapide et spécifique d'acides nucléiques sur un support solide
WO1988001302A1 (fr) * 1986-08-11 1988-02-25 Siska Diagnostics, Inc. Procedes et compositions d'analyse a l'aide de sondes d'acide nucleique
EP0269445A2 (fr) * 1986-11-26 1988-06-01 Cetus Oncology Corporation Procédé de détection des virus HTLVI et HTLVII par hybridation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Nucleic Acids Research, Volume 15, No. 13, 10 July 1987, R. ZUCKERMANN et al.: "Efficient Methods for Attachment of Thiol Specific Probes to the 3'-ends of Synthetic Oligodeoxyribonucleotides", page 5316 *

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6919211B1 (en) 1989-06-07 2005-07-19 Affymetrix, Inc. Polypeptide arrays
US7087732B2 (en) 1989-06-07 2006-08-08 Affymetrix, Inc. Nucleotides and analogs having photoremovable protecting groups
US6955915B2 (en) 1989-06-07 2005-10-18 Affymetrix, Inc. Apparatus comprising polymers
US5501947A (en) * 1990-07-19 1996-03-26 Royal Free Hospital School Of Medicine PCR diagnosis of human papilloma virus type 16
US7459275B2 (en) 1990-12-06 2008-12-02 Affymetrix, Inc. Sequencing of surface immobilized polymers utilizing microfluorescence detection
US7329496B2 (en) 1990-12-06 2008-02-12 Affymetrix, Inc. Sequencing of surface immobilized polymers utilizing microflourescence detection
WO1993004199A3 (fr) * 1991-08-20 1993-04-15 Scient Generics Ltd Procedes de detection et de quantification d'acides nucleiques, et de production d'acides nucleiques marques, immobilises
US7736906B2 (en) 1991-11-22 2010-06-15 Affymetrix, Inc. Combinatorial strategies for polymer synthesis
US6849462B1 (en) 1991-11-22 2005-02-01 Affymetrix, Inc. Combinatorial strategies for polymer synthesis
US7691330B1 (en) 1991-11-22 2010-04-06 Affymetrix, Inc. Combinatorial strategies for polymer synthesis
US6864101B1 (en) 1991-11-22 2005-03-08 Affymetrix, Inc. Combinatorial strategies for polymer synthesis
US5412087A (en) * 1992-04-24 1995-05-02 Affymax Technologies N.V. Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces
US7927835B2 (en) 1992-10-23 2011-04-19 Genetics Institute, Llc Nucleic acids encoding P-selectin ligand fusion proteins
US7563760B2 (en) 1992-10-23 2009-07-21 Genetics Institute, Llc P-selectin ligand protein
US8232252B2 (en) 1992-10-23 2012-07-31 Genetics Institute, Llc P-selectin ligand protein
US6277975B1 (en) 1992-10-23 2001-08-21 Genetics Institute, Inc. Fusions of P-selectin ligand protein and polynucleotides encoding same
FR2707296A1 (fr) * 1993-07-09 1995-01-13 Genset Sa Procédé de synthèse d'acides nucléiques sur support solide et composés utiles notamment comme support solide dans ledit procédé.
WO1995001987A1 (fr) * 1993-07-09 1995-01-19 Genset Procede de synthese d'acides nucleiques sur support solide et comoposes utiles notamment comme support solide dans ledit procede
US7625697B2 (en) 1994-06-17 2009-12-01 The Board Of Trustees Of The Leland Stanford Junior University Methods for constructing subarrays and subarrays made thereby
US7442499B2 (en) 1994-06-17 2008-10-28 The Board Of Trustees Of The Leland Stanford Junior University Substrates comprising polynucleotide microarrays
WO1996013609A1 (fr) * 1994-10-28 1996-05-09 Genset Procede d'amplification d'acides nucleiques en phase solide et trousse de reactifs utile pour la mise en ×uvre de ce procede
FR2726286A1 (fr) * 1994-10-28 1996-05-03 Genset Sa Procede d'amplification d'acides nucleiques en phase solide et trousse de reactifs utile pour la mise en oeuvre de ce procede
US6274351B1 (en) 1994-10-28 2001-08-14 Genset Solid support for solid phase amplification and sequencing and method for preparing the same nucleic acid
US6277604B1 (en) 1994-10-28 2001-08-21 Genset Methods for solid-phase nucleic acid amplification and sequencing
US7279311B2 (en) 1996-11-19 2007-10-09 Oakville Trading Hong Kong Limited Use of nucleic acids bound to carrier macromolecules
US6207385B1 (en) 1996-11-19 2001-03-27 Amdex A/S Use of nucleic acids bound to carrier macromolecules
EP0910670A4 (fr) * 1997-03-05 2001-11-21 Orchid Biosciences Inc Fixation covalente de molecules d'acide nucleique sur des phases solides au moyen de ponts disulfures
US7279563B2 (en) 1999-10-05 2007-10-09 Helicos Biosciences Corporation Compounds for protecting hydroxyls and methods for their use
US6639088B2 (en) 1999-10-05 2003-10-28 Quiatech Ab Compounds for protecting hydroxyls and methods for their use
WO2001025247A1 (fr) * 1999-10-05 2001-04-12 Quiatech Ab Composes de protection des hydroxyles et procedes d'utilisation
US7869958B2 (en) 2004-08-09 2011-01-11 Research Development Foundation Structure-based modulators of B1 G-protein coupled receptors
US10093967B2 (en) 2014-08-12 2018-10-09 The Regents Of The University Of Michigan Detection of nucleic acids
EP3705609A1 (fr) 2014-08-12 2020-09-09 The Regents of The University of Michigan Détection d'acides nucléiques
EP4105338A1 (fr) 2014-08-12 2022-12-21 The Regents of The University of Michigan Détection d'acides nucléiques
US12460249B2 (en) 2017-03-08 2025-11-04 The Regents Of The University Of Michigan Protein analyte detection by analizing time-dependent signals from transient binding events of labeled low-affinity probes
US10948450B2 (en) 2018-07-23 2021-03-16 Genmark Diagnostics, Inc. Electrochemical measurements of components in coatings
US11796500B2 (en) 2018-07-23 2023-10-24 Roche Molecular Systems, Inc Electrochemical measurements of components in coatings

Also Published As

Publication number Publication date
IE902467A1 (en) 1991-02-13

Similar Documents

Publication Publication Date Title
GB2233654A (en) Immobilised polynucleotides
WO1991000868A1 (fr) Polynucleotides immobilises
AU702125B2 (en) Immunoassay and kit with two reagents that are cross-linked if they adhere to an analyte
CA2266749C (fr) Utilisation d'oligonucleotides modulaires comme sondes ou amorces dans des dosages a base d'acide nucleique
WO1991008307A1 (fr) Capture amelioree d'acide nucleique cible faisant appel a des oligonucleotides attaches de façon covalente a des polymeres
JP2006254924A (ja) 溶液相サンドイッチハイブリダイゼーションアッセイに用いるためのｈｉｖプローブ
JP2004511220A (ja) 型特異的ハイブリッド捕捉法による核酸の検出方法
IE880697L (en) Method of assaying of nucleic acids, a reagent combination and¹kit therefor
JPS60237361A (ja) 目標遺伝物質の検出方法
EP2358897B1 (fr) Procédés et compositions de détection à base de particules de molécules cibles utilisant des molécules de liaison
WO1995014106A2 (fr) Detection cyclique par scission de sondes de sequences d'acides nucleiques
EP1615948A2 (fr) Amplification immunologique
EP0672187A1 (fr) Dosages pcr permettant de determiner la presence et la concentration d'une cible
CA2450139A1 (fr) Techniques de detection multiplexees
WO1993013120A1 (fr) Sondes d'amplification de vhb utlisees dans des methodes d'hybridation en sandwich a phase en solution
EP0286642A1 (fr) Procede d'analyse de polynucleotides base sur le deplacement des brins, et complexe reactif
IE911534A1 (en) Protein-nucleic acid probes and immunoassays using same
JP5341291B2 (ja) ディップスティック検定における改良型結合相互作用
JP2644419B2 (ja) 核酸の固定化
JP2004000154A (ja) 核酸の配列特異的検出
JPWO2021113290A5 (fr)
CA2119701A1 (fr) Methode d'essai pour l'acide nucleique
WO1989009281A1 (fr) Procede d'amplification et de detection d'acide nucleique dans un liquide test
CN101233410B (zh) 用于标记或处理包含目的生物分子特别是核酸的生物样品的方法
WO1999058716A1 (fr) Procede servant a quantifier une sequence specifique de genes et reactif de quantification

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB IT LU NL SE