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WO1994008047A1 - Procede d'amplification en chaine par ligase pour la detection de petites mutations - Google Patents

Procede d'amplification en chaine par ligase pour la detection de petites mutations Download PDF

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Publication number
WO1994008047A1
WO1994008047A1 PCT/US1993/008359 US9308359W WO9408047A1 WO 1994008047 A1 WO1994008047 A1 WO 1994008047A1 US 9308359 W US9308359 W US 9308359W WO 9408047 A1 WO9408047 A1 WO 9408047A1
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seq
probes
nucleic acid
probe
kit
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Stanley R. Bouma
Julian Gordon
Wang-Ting Hsieh
Tsung-Hui Jou
Arthur L. Beaudet
Ping Fang
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Abbott Laboratories
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Abbott Laboratories
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    • 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
    • C12Q1/6862—Ligase chain reaction [LCR]

Definitions

  • This invention relates to methods of amplifying target nucleic acids and, particularly, to methods of detecting small mutations using the Ligase Chain Reaction (LCRTM) to selectively amplify the genomic region of interest.
  • LCRTM Ligase Chain Reaction
  • LCRTM target amplification technique
  • two primary (first and second probes) and two secondary (third and fourth) probes are employed in excess.
  • the first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship and so that a ligase can covalently fuse or ligate the two probes into a fused product.
  • a third (secondary) probe can hybridize to the first probe and a fourth (secondary) probe can hybridize to the second probe in a similar abutting fashion.
  • the secondary probes will also hybridize to the target complement in the first instance.
  • the fused strand of primary probes Once the fused strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary fused product.
  • LCRTM LCRTM
  • the fused products are functionally equivalent to either the target or its complement.
  • amplification of the target sequence is achieved. This technique is described more completely in published EP-A-320 308; and in published EP-A-439 182. Genetic mutations can result from deletions, insertions or changes in one or more bases in the genetic code.
  • the cause of a particular illness or disease results from a single or a limited number of mutations or polymorphisms in the genome of the affected individual.
  • determination of the genetic constituency of the individual can be useful in diagnosing the illness or disease.
  • Some diseases or conditions e.g. sickle cell anemia, phenylketonuria, Tay-Sachs disease, medium chain acyl CoA dehydrogenase deficiency, cystic fibrosis
  • Some diseases or conditions e.g. sickle cell anemia, phenylketonuria, Tay-Sachs disease, medium chain acyl CoA dehydrogenase deficiency, cystic fibrosis
  • Others e.g.
  • Duchennes Muscular Dystrophy can manifest themselves by short alterations or deletions in one or a relatively small number of exon locations. It will be recognized that the few examples given above are by no means exhaustive, and that there are hundreds or thousands of other disease related mutations or conditions which can be detected using the methods of the invention. Mutations have been detected in the prior art by tedious and time consuming processes requiring amplification of the relevant genomic region, followed by immobilization of the amplified DNA on a solid membrane and probing the membrane with allele specific probes. This method was improved upon in a procedure known as "reverse dot blot". This procedure is described in Sakai, et al., Proc. Natl. Acad. Sci.
  • ARMS Amplification Refractory Mutation
  • the primer is designed to be perfectly complementary to the normal (or mutant) allele, and to be mismatched at the 3' terminus with respect to the mutant (or normal) allele. Depending on the nature of this mismatch, it alone may be sufficient to cause the primer to be "refractory" to amplification on the mutant (or normal) gene. However, in other cases, particularly when the mismatch results in a pyrimidine/purine mismatch (i.e. G T, T/G, A/C or C/A), the specificity can be improved by introducing an additional mismatch a few bases from the 3' end, thus making the primer even more refractory to amplification in the mutant (or normal) gene.
  • a pyrimidine/purine mismatch i.e. G T, T/G, A/C or C/A
  • K. B. Mullis a co-inventor of the polymerase chain reaction (PCR), has described an improved method in PCR Metlrvods and Applications, 1(1): 1-4 (1991).
  • the improved process comprises preventing the extension reaction from beginning until the temperature is high enough to discourage binding of non-perfectly hybridized primers. This can be accomplished, for example, by adding the polymerase to the other components while the temperature remains high from an initial denaturing step. Mullis indicates that permitting the reaction vessels to cool before adding the polymerase can cause non-specific primer binding and extension, resulting in false positive results. This procedure is referred to as "hot start" PCR.
  • the ligase used by these researchers was T4 DNA ligase, which is not thermostable and not of bacterial origin.
  • the invention provides a method of detecting a small mutation in a target nucleic acid the sequence of which is known, said method including: (a) providing an excess of at least two sets of two probes, the 3' end of a first upstream probe being ligatable to the 5' end of a first downstream probe in the presence of target to form a primary ligation product and the 3' end of a second upstream probe being ligatable to the 5' end of a second downstream probe in the presence of primary ligation product to form a secondary ligation product; (b) reacting under hybridizing conditions said probes and sample suspected to contain said target nucleic acid; (c) ligating the upstream probe to the respective downstream probe using a thermostable ligase; (d) repeatedly denaturing the hybridized, ligated strands, reannealing additional probes and ligating them; and (c) detecting to what extent ligation products have formed; the improvement comprising performing said step b under conditions wherein the concentration of mono
  • the concentration of monovalent cation is between about 100 mM and 200 mM or at least about 180 mM.
  • the monovalent cation is a metal, such as Na + or K + , or NR3H+, where R is independently selected from H or lower alkyl.
  • KC1 is a suitable supply of monovalent cation.
  • the invention provides a method of detecting a small mutation as above, wherein the improvement comprises performing said step b under conditions wherein the initial temperature of mixing ranges from about 50-95 °C, followed by a lower temperature to enhance annealing. Preferred are ranges from about 60-85 °C, and from about 80-85 "C. This method, known as "hot start” may, but need not, be employed in conjunction with the high monovalent cation concentrations noted above.
  • the invention provides a method of detecting a small mutation as above, wherein the improvement comprises providing as at least one of said downstream probes, a mismatched probe having near its 5' end at least one base which is not complementary to the corresponding base in the target.
  • This mismatched base is preferably within 5 bases from the 5' end and is also mismatched in the complementary probe so that the probes do not mismatch one another.
  • This method may, but need not, be employed in conjunction with either or both of the high monovalent cation concentrations noted above and the "hot start" method.
  • the invention provides several distinct probe sets that are useful in LCRTM for detecting cystic fibrosis mutations designated ⁇ F508, G 551 D, Wi282X» G542X. an d the - ⁇ 3J1 polymorphism.
  • Figures 1 to 4 show the alignment of specific oligos with sections of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.
  • Sequence ID Nos. are given in the left-most column (targets are not assigned a Sequence ID No. in these figures) and the numbering is that of Zielenski, et al. Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene , Genomics 10, 214-228 (1991).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • FIG. 1 to 4 show the alignment of specific oligos with sections of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.
  • Sequence ID Nos. are given in the left-most column (targets are not assigned a Sequence ID No. in these figures) and the numbering is that of Zielenski, et al. Genomic DNA sequence of the cystic fibrosis transmembran
  • Figure 1 shows the alignment of oligos 1-17 with the CFTR Gene ⁇ F508 mutation.
  • This mutation involves a three base deletion (either TCT at DNA position 1652-54 or CTT at DNA position 1653-55, both of which leave a ⁇ F 508 mutated sequence of ATTGGT in this region). These deletions cause the loss of a phenylalanine residue which occurs at position 508 of the normal CFTR protein.
  • FIG. 2 shows the alignment of oligos 24-49 with the CFTR Gene G 551 D mutation.
  • This mutation involves a G to A change at DNA position 1784, whereby GGT, which codes for glycine, becomes GAT, which codes for aspartic acid at protein position 551.
  • Figure 3 shows the alignment of oligos 18-23 with the CFTR Gene Wj 282 X mutation. This mutation involves a G to A change at DNA position 3978, whereby TGG, which codes for tryptophan, becomes TGA, a stop codon, at protein position 1282.
  • Figure 4 shows the alignment of oligos 58-67 with the CFTR Gene G 542 X mutation. This mutation involves a G to T change at DNA position 1756, whereby GGA, which codes for glycine, becomes TGA, a stop codon, at protein position 542.
  • Figures 5, 6, 9 (a-f), 12 and 13 are autoradiograms of gels run in examples 7,
  • Figures 7 (a-c), 8, 11 and 14 (a-b) are digitized photos of immunochromatographic strips run in examples 9, 10, 12 and 15, respectively. Detailed descriptions are provided in the respective examples.
  • Figure 10 shows the alignment of oligos 52-57 with the double stranded synthetic target identified as Sequence ID Nos. 50 and 51.
  • Synthetic targets 50 and 51 were synthesized to represent the "antisense” allelic strands of a "normal” sequence containing C at position 23 (No. 50) and a "mutant" strand containing T instead (No. 51).
  • Mutations in DNA can take many forms. As compared to the DNA of a normal individual, mutated DNA may be of three general types. First, it may be missing one or more nucleotides. This is referred to as a "deletion”. Second, it may contain one or more additional nucleotides. This is referred to as an "insertion” or “addition”. Both deletions and insertions can cause "frame shift" translation errors if they occur in a coding region or "exon" of the gene. Such translation errors can cause the synthesis of proteins whose activity is reduced or totally absent. Finally, mutated DNA may contain b the same number of nucleotides, but may have a different nucleotide at one or more crucial position in the gene.
  • mutation encompasses all three types described above.
  • a “small mutation” is a “mutation” as described above provided the number of affected nucleotides is not too large.
  • a mutation is "small” when the number of affected nucleotides is less than about 20% of the average size of the probes used to amplify and detect it.
  • the average size is about 15-25 nucleotides long.
  • mutations of up to about 5 nucleotides are typically covered, although for longer probes, longer mutations would be considered “small”.
  • probe sets can be designed so that the common probe (see infra) hybridizes to the target on one side of the deletion segment; probes for the mutant allele are designed to hybridize to the target at the other end of the deletion segment; and probes for the normal allele are designed to hybridize to the deletion segment at the end adjacent the common probe set .
  • probe sets can be designed so that the common probe (see infra) hybridizes to the target on one side of the insertion segment; probes for the normal allele are designed to hybridize to the target at the other end of the insertion segment; and probes for the mutant allele are designed to hybridize to the inserted segment at the end adjacent the common probe set .
  • a “target sequence” or “target nucleic acid” is a segment of nucleic acid (DNA or RNA). The segment may be from about 10 or 15 to several hundred nucleotides in length.
  • a target segment is usually about 30 to about 60 nucleotides and the sequence is known.
  • a target is "putative" if its presence is expected or anticipated, or if it or a variation of it is expected or anticipated. For example, in multiplex LCRTM of an unknown homozygote to determine which of two mutually exclusive alleles is present, the sequences of both alleles are putative target sequences, even though only one is expected to be present The possible alternatives for each allele are all putative targets.
  • multiplex LCRTM is the performance of LCRTM on a plurality of targets using at least one set of four probes for each putative target sequence (subject to the situation described below with regard to “common” probes).
  • N-plex LCRTM refers to LCRTM performed to amplify or detect one or more of N target sequences. Multiplex LCRTM is described in detail in the aforementioned U.S. application No.07/860,702, filed March 31, 1992.
  • multiplex LCRTM is not essential to the present invention
  • duplex LCRTM is useful with the present invention.
  • This special case of multiplex which also can be viewed as "competitive" LCRTM, occurs when information is desired simultaneously about both the mutant and the normal allele for a single mutation, for example, when heterozygote carrier status must be determined.
  • probes designed to be specific for the normal allele essentially "compete" with probes designed to be specific for the mutant allele, since their target is the same except for the mutation.
  • Certain advantages can be gained in this process because competition appears to enhance specificity, the prefectly matched probe being ligated more successfully than the imperfectly matched probe.
  • Example 13 illustrates this principle and the need to optimize the balance of probe concentrations.
  • Multiplex LCRTM is especially useful when more than one mutation locus must be examined to diagnose a particular disease or condition.
  • LCRTM Ligase Chain Reaction
  • the first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the 3' hydroxyl end of an "upstream” probe abuts the 5' phosphate end of a "downstream” probe, and so that a ligase can covalently ligate the two probes into a fused primary ligation product.
  • LCRTM employs upstream and downstream secondary probes.
  • a third probe downstream secondary
  • a fourth probe upstream secondary
  • the second probe portion of the primary ligation product i.e. the portion formed principally by the downstream primary probe
  • the secondary probes can also hybridize to the target complement in the first instance.
  • the primary and secondary ligation products are separated from their respective complementary strands, they can hybridize with the secondary and primary probes, respectively. It is important to realize that the ligation products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization, ligation and separation, amplification of the target sequence is achieved.
  • the step of "ligating" as recited above encompasses several known methods of joining two probes together.
  • the preferred method is by the use of a thermostable ligase, especially a thermostable bacterial ligase, such as that of the Thermus genus.
  • Ligases are discussed in EP-A-320 308 and in EP-A-373 962.
  • Joining also encompasses the possible intermediate step of "correcting" a modified end as taught in EP-A-439 182. Correction includes the filling of a gap with an extending reagent (such as polymerase) as well as the cleaving of a blocking group (such as with endonuclease IV).
  • a reaction solution is typically prepared by collecting the sample from a patient and disrupting the cells to release the DNA.
  • Detergents may be used, but other known methods of sample preparation are also included. Specific extraction buffer compositions are available from the literature, and from the examples. The DNA is rendered single-stranded by altering the stringency conditions, usually by heating. It has unexpectedly been found that the use of high concentrations of monovalent cations enhances the present invention. While typical reaction buffers of the prior art have included the monovlaent cations NaCl or KC1 at about 20 - 30 mM, the present invention employs up to 10 fold higher concentrations: for example, from about 100 to about 200 mM.
  • the monovalent cation is a metal ion such as K + or Na + , or is an ammonium ion of the form NHR3+, where R is independently selected from H or lower alkyl.
  • “Lower alkyl” in this application refers to monovalent straight or branched aliphatic radicals which may be derived from alkanes by the removal of one hydrogen, and have the general formula CnH2n+i; wherein n is from 1 to about 6. Examples of lower alkyl include CH3-, CH3CH2-, CH3CH(CH3)-, and CH3(CH2)4---
  • specificity can be improved by initiating the amplification reaction at a relatively high temperature; i.e. a temperature above about 50" C.
  • the reaction is initiated when ligase is added to a mixture of denatured probes and target. When this mixing takes place at temperatures from 50-95° C, the specificity is improved.
  • the reaction is started at 60-85°, more preferably 80-85" C.
  • the reaction may also be started by supplying a necessary cofactor or other required component while the reaction mixture is at an elevated temperature. To facilitate detection at least one probe of each probe set should bear a detectable label.
  • the detectable labels from each of the probe sets must all be differentiable, one from the other, either by signal differentiation or by spatial differentiation.
  • signal differentiation refers to the ability to distinguish targets in essentially the same location (i.e homogeneous assay) by virtue of differences in the signal (e.g. different fluorescent emission wavelengths or different colors).
  • spatial differentiation refers to the ability to distinguish targets based on the position or location of the signal. Spatial differentiation is also known as separation and may be accomplished by size, molecular weight, charge density, or magnetic or specific binding properties, and the like. Dot-blots, electrophoretic gels and immunochromatography are examples of spatial differentiation.
  • Isotopic labels can be detected by gel electrophoresis and autoradiography as in example 5.
  • Hapten labels can be detected by solid phase separation and development, such as in the immunochromatography detection of example 6.
  • Multiplex LCRTM generally requires probes equalling four times the number of putative sequences. However, by careful probe design, the general rule of four probes for each putative target is modified in the present case, where the mutations are "small" and well defined. Two probes from one set (say, arbitrarily, the right two from the normal allele) will also serve as the two (right) probes for the mutant allele and only six probes will be required at each mutation to derive information about both alleles. It will be noted that the common probes may be on the same side of the point of ligation with reference to a fixed point (i.e. they are an upstream probe and its complementary downstream probe).
  • left-side probes can serve as the common probes for CF mutations ⁇ F508 (oligos 1 and 2, Table 1 and fig. 1), G55 1 D (oligos 24 and 25, Table 1 and fig. 2), and W 1 2 82 X (oligos 18 and 19, Table 1 and fig. 3).
  • right-side probes can serve as common probes, e.g. for CF mutation G5 4 2X (oligos 62 and 63, Table 1 and fig. 4).
  • "Right" and "left" side probes in this context refer to the complementary probes which are distal and proximal, respectively, of the point of ligation with reference to the origin of replication.
  • the common probes need not be those on the same side of the point of ligation (i.e. they need not be an upstream probe and its complementary downstream probe). Common probes can span the ligation junction, i.e. they may both be upstream or downstream probes on opposite strands. This is demonstrated by probes 14 and 15 ( Figure 1) for CF mutation ⁇ F508 (Oligo 14 is a primary downstream probe on the right side and oligo 15 is a secondary downstream probe on the left side); and by probes 67 and 68 ( Figure 4) for CF mutation G542X (Oligo 68 is a primary downstream probe on the right side and oligo 67 is a secondary downstream probe on the left side). In cases where common probes are used, it is possible to determine both alleles using only six distinct probes.
  • probes are designed so that a terminal nucleotide causes perfect complementarity with one allele (say, normal) and a mismatch viz-a-viz the mutant allele. In some cases, adequate specificity can be achieved by this mismatch alone, particularly when the mismatch results in a purine/purine or a pyrimidine/pyrimidine pairing.
  • LCRTM LCRTM
  • the specificity of LCRTM is improved when one or more additional mismatches are deliberately introduced into the oligonucleotide probes, particularly near the 5' end of the downstream probes. "Near" in this context, means within about 5 bases from the 5' end. If the deliberate mismatch is too far from the end, it would not be expected to improve specificity.
  • the "tail” can be made complementary to a capture probe which might be immobilized on a membrane or other solid phase.
  • the "tail” might be made complementary to a labeled detector probe, thus providing a way to identify the presence or not of the amplification product.
  • these tails are not essential to the invention and are provided merely to facilitate a more convenient method of detection.
  • EDTA a metal chelator, ethylenediamine tetraacetic acid EPPS a buffer comprising N-(2-hydroxyethyl)piperazine-N'-(3- propanesulfonic acid) oligos oligonucleotides, generally oligo-2-deoxyribonucleotides.
  • TRIS a buffer comprising t (hydroxymethyl)aminomethane TRITON a detergent, O-(polyoxyethylene)-4-octylphenol Mutations are described herein, in a manner generally accepted in the art.
  • ⁇ F508 specifies a three base deletion at DNA position 1653 which causes the loss of the phenylalanine which occurs at position 508 of the normal CFTR protein
  • G551D specifies a G to A change at DNA position 1784, causing replacement of the normally-occurring glycine at position 551 with aspartic acid
  • G542X and W1282X specify truncation of the CFTR protein after positions 541 or 1281, respectively, because these so-called nonsense mutations (G to T at DNA position 1756 for G542X, and G to A at DNA position 3978 for W1282X) cause the gene to code for no amino acid at positions 551 or 1282, rather than the normally occurring glycine or tryptophan.
  • Oligonucleotides were synthesized following standard protocols using automated synthesizers and nucleoside cyanoethylphosphoramidites (Applied Biosystems, Foster City, CA). For immunochromatographic detection, the probes were labeled at either the 3'- or 5'-end with a hapten as specified in Table 1. The labeling procedure followed standard solid-phase DNA synthesis methods, and used either commercially available haptens (Fluorescein-ONTM and Biotin-ONTM, Clontech, Palo Alto, CA) or a dansyl phosphoramidite synthesized at Abbott Laboratories.
  • the sequences were selected based on the mutation to be detected and the method of LCRTM. They were derived from the published sequence of the Cystic Fibrosis (CF) Transmembrane Conductance Regulator gene (J. Zielenski, R. Rozmahel, D. Bozon, B-S. Kerem, Z. Grzelczak, J. Riordan, J. Rommens, and L-C Tsui: Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (1991) Genomics 10, 214-228). The specific sequences used are listed in Table 1. The manner in which oligos are listed in Table 1.
  • fig. 1-17 align with the CFTR, in particular with the ⁇ F508 mutation is shown in fig. 1.
  • the manner in which oligos 24-33 align with the CFTR gene, in particular with the G551D mutation, is shown in fig. 2.
  • the manner in which oligos 18-23 align with the CFTR gene, in particular with the W1282X mutation, is shown in fig. 3.
  • DNA samples were obtained from the blood of affected cystic fibrosis (CF) patients, asymptomatic carriers, or normal human volunteers. The whole blood was obtained by venipuncture, and the DNA extracted using a DNA extractor (Applied Biosystems, Foster City, CA). The LCRTM process itself occurred in either a
  • TempcyclerTM Coy, Ann Arbor, MI
  • a model 480 Thermal Cycler Perkin-Elmer, Norwalk, CT
  • Example 3 Blunt LCRTM with rapid cycling Blunt LCRTM similar to example 2 was also performed in a total volume of 15 ⁇ L with the following final concentrations: 20 mM tra(hydroxymethyl)aminomethane (TRIS) at pH 7.6, 25 mM potassium acetate, 10 mM magnesium acetate, 10 mM dithiothreitol, 0.6 mM NAD, 0J % Triton X-100, and other concentrations as specified in subsequent examples. DNA samples were obtained as described in example 2. Altematively, synthetic 51 -base targets with sequence identical to the wild-type or mutant sequence were used. The LCRTM process itself occurred in a FTS-1 Thermal SequencerTM (Corbett Research, Mortlake, NSW, Australia) using a temperature profile specified in subsequent examples.
  • FTS-1 Thermal SequencerTM Corbett Research, Mortlake, NSW, Australia
  • the LCRTM was performed using the so-called “single-gap” or “double-gap” strategy as described by Backman, et al. in European Patent Application 0439 182 (1991). Reactions were performed in a total volume of 50 ⁇ L with the following final .
  • i, o _ concentrations 50 mM EPPS pH 7.8, 20 mM potassium (added as KOH to adjust the pH of the buffer and as KC1 to achieve 20 mM K + ), 30 mM MgCl2 100 ⁇ M NAD, 1 ⁇ M nucleoside triphosphate (dATP, dCTP, dGTP, or TTP as necessary to fill the particular gap), 0.5 unit/50 ⁇ L reaction DNA polymerase (Amplitaq®, Perkin- Elmer/Cetus, Emeryville, CA), and other concentrations as specified in subsequent examples. DNA samples were obtained as described in example 2. The LCRTM process itself occurred in either a TempcyclerTM (Coy, Ann Arbor, MI) or a model 480 Thermal Cycler (Perkin-Elmer, Norwalk, CT) using temperature profiles specified in subsequent examples.
  • TempcyclerTM Coy, Ann Arbor, MI
  • a model 480 Thermal Cycler Perkin-Elmer, Norwalk, CT
  • LCRTM For electrophoretic detection, one of the LCRTM probes was labeled at its 5'- end with 32p following standard procedures (Sambrook, et al.: Molecular cloning: a laboratory manual (1989) Cold Spring Harbor Laboratory Press). Following LCRTM, 10 ⁇ L portions of each reaction mixture were lyophilized and resuspended in 3 ⁇ L of 95% formamide, 20 mM EDTA with 0.05% bromophenol blue and 0.05% xylene cyanol, and loaded onto a 12% polyacrylamide gel containing 7 M urea. Following electrophoresis, the gels were exposed for autoradiography to Hyperf ⁇ lm® (Amersham, Arlington Heights, IL).
  • Antisera to fluorescein and biotin were raised in rabbits against fluorescein-BSA or biotin-BSA. Antiserum against dansyl was a mouse monoclonal obtained from the University of Pennsylvania (S-T. Fan and F. Karush: Molecular Immunology (1984) 21, 1023-1029). These antisera were purified by passage through protein A Sepharose® or protein G Sepharose® (Pharmacia, Piscataway, NJ) and diluted in 0J M TRIS pH 7.8, 0.9% NaCl, 0.1% BSA, 1% sucrose, and a trace of phenol red. Portions (0.2 ⁇ L) of these diluted antisera were spotted onto 4 x 40 mm strips of nitrocellulose (AE 98, 5 ⁇ m, Schleicher and Schuell, Dassel, Germany).
  • Colloidal selenium was prepared following the procedure of D. A. Yost et al. (U. S. Patent 4,954,452). The colloid was diluted in water to achieve an optical density of 16 at 545 nm. To 1 mL of this suspension was added 1 ⁇ L of a i- biotin at 1 mg/mL and 60 ⁇ L of BSA at 100 mg/mL. This suspension was mixed on a vortex mixer for 1 minute, and was ready for use.
  • the LCRTM reaction mixtures were analyzed by Abbott TestPack PlusTM immunochromatography essentially following the protocol of European Patent Application EP-A-357-011. Briefly, 15 ⁇ L of the anti-biotin colloid (selenium or blue latex) was diluted with 14 ⁇ l buffer (OJ M TRIS pH 7.8, 0.9% NaCl, 3% alkali- treated casein), and mixed with 1 ⁇ L LCRTM product A nitrocellulose strip containing ⁇ nri-fluorescein or ⁇ wri-dansyl, or both, was introduced to the colloid suspension, and chromatography was allowed to proceed for 5 minutes. The strip was dried. The presence of a colored spot at the locus of antibody application indicated the presence of a specific LCRTM product. In some instances, the intensity of color was measured by digitizing an image of the strip and assigning a value based on the gray level determined in the digitizing process.
  • Example 7 CF ⁇ F508 mutation, gap-LCRTM: electrophoretic detection
  • Extracted DNA from patients either homozygous for the CF ⁇ F508 mutation ( ⁇ F508/ ⁇ F508 DNA, fig. 5), from heterozygous carriers (NL/ ⁇ F508 DNA, fig. 5), or from normal human controls (NL/NL, fig. 5) was amplified by single-gap and double- gap LCRTM using TTP as the fill base as described in example 4.
  • oligos 1-6 Table 1 were present at 20 nM each, and oligo 1 was labeled at the 5 '-end with 32p.
  • DNA ligase and DNA polymerase were added to the mixture at 80°C.
  • the LCRTM then proceeded for 45 cycles of 85°C for 30 seconds and 45°C for 45 seconds in a Perkin-Elmer thermal cycler.
  • the reaction products were analyzed by electrophoresis as described in example 5.
  • Normal DNA yielded only the 49 bp product of the ligation of oligos 1-4 (NL NL; lane 2, fig. 5).
  • Homozygous CF ⁇ F508 DNA yielded only the 43 bp product of the ligation of oligos 1, 2, 5, and 6 ( ⁇ F508/ ⁇ F508; lane 3, fig. 5).
  • DNA from heterozygous carriers amplified both products (NIV ⁇ F508; lane 4, fig. 5). Amplification in the absence of target DNA produced insufficient product to detect (None; lane 1, fig. 5).
  • oligos 1, 3, 4, 5, 6, and 7 were present at 20 nM each, and oligo 1 was labeled at the 5'-end with 32p.
  • the same cycling protocol was used as for double-gap LCRTM, with the exception that the annealing temperature was 46°C.
  • Normal DNA yielded only the 49 bp product of the ligation of oligos 1, 3, 4, and 7 (NL/NL; lane 6, fig. 5).
  • Homozygous CF ⁇ F508 DNA yielded only the 43 bp product of the ligation of oligos 1, 5, 6, and 7 ( ⁇ F508/ ⁇ F508; lane 7, fig. 5).
  • Extracted DNA from patients either homozygous or heterozygous for the CF ⁇ F508 mutation was amplified by blunt LCRTM as described in example 2.
  • Oligos 3, 8, 9, and 10 (Table 1) were used at 10 nM each, and oligos 5 and 11 were present at 15 nM.
  • Oligo 8 was labeled at the 5 '-end with 32p.
  • the reaction mixture, without enzyme, was incubated at 98°C for 3 minutes, then cooled to 80°C.
  • DNA ligase was added to the mixture at 80°C.
  • the LCRTM then proceeded for 42 cycles of 85°C for 30 seconds and 49°C for 45 seconds in a Perkin-Elmer thermal cycler.
  • the reaction products were analyzed by electrophoresis as described in example 5.
  • Both the 48 bp product of the ligation of oligos 3, 8, 9, and 10 and the 42 bp product of oligos 5, 8, 9, and 11 were amplified from normal DNA (NL/NL), homozygous DNA ( ⁇ F508/ ⁇ F508). and heterozygous DNA (NL ⁇ F508). and even in the absence of any DNA target (None) when the potassium (KCl) concentration was 20 mM or 100 mM (lanes 1-8, fig. 6). When the KCl concentration was 180 mM, then the 48 bp product was produced from normal DNA (lane 10, fig. 6) and the 42 bp product from homozygous DNA (lane 12, fig. 6).
  • Example 9 Effect of salt concentration on the CF ⁇ F508 mutation, blunt-LCRTM: immunochromatographic detection DNA was extracted and amplified as described in example 7, except that the buffer used was that used for gap LCRTM (example 4). Oligos 12 and 13 were present at 27 nM and oligos 14-17 at 13.6 nM. The reaction mixture, without enzyme, was incubated at 100°C for 3 minutes, then cooled to 85°C. DNA ligase and 0.5 unit DNA polymerase were added to the mixture at 85°C (subsequent studies showed that the DNA polymerase was not necessary — only the detergent, which in this case was
  • the LCRTM then proceeded for 42 cycles of 85°C for 1 minute and 55°C for 1 minute in a Perkin-Elmer thermal cycler.
  • the reaction products were analyzed by immunochromatography as described in example 6.
  • concentration of KCl was 80 mM or 100 mM
  • both the ligation product of oligos 12-15 containing dansyl at one end and biotin at the other, and the ligation product of oligos 13-17 containing fluorescein at one end and biotin at the other were amplified from homozygous ( ⁇ F508), heterozygous (het), and normal (W) DNA (figs. 7a and 7b).
  • Synthetic 51-base targets homologous for the normal CFTR gene (oligo 58, "N” in fig. 8) or for the G542X mutation (oligo 59, "M” in fig. 8) were amplified by blunt LCRTM with rapid cycling as described in example 3. Oligos 60-63 were present at 22 nM, 55 nM, or 110 nM each, and the synthetic targets were present at 1 x 10 -- copies each.
  • the KCl concentration was 75 mM, 100 mM, or 125 mM, as specified in fig. 8 and table 2.
  • the reaction mixture, with enzyme was assembled at 0°C and transfered to the Corbett thermal sequencer.
  • the LCRTM proceeded for 30, 35, or 40 cycles of 85°C for 5 seconds and 45°C for 5 seconds. A conplete cycle took approximately 55 seconds.
  • the reaction products were analyzed by immunochromatography and quantitated by digital imaging as described in example 6.
  • Extracted DNA from patients heterozygous for the CF G551D mutation (HET DNA, figs. 9) or from normal human controls (N DNA, figs. 9) was amplified by blunt LCRTM as described in example 2. Oligos 24, 25, 26, 27, and 30-38 were present at 10 nM each and oligos 28, 29, and 40-49 at 8 nM each. Oligo 24 was labeled at the 5'-end with 32p. T e reaction mixture, without enzyme, was incubated at 98°C for 3 minutes, then cooled to 80°C. DNA ligase was added to the mixture at 80°C. The LCRTM then proceeded for 30 cycles of 85°C for 30 seconds and 49°C for 45 seconds in a Perkin-Elmer thermal cycler. The reaction products were analyzed by electrophoresis as described in example 5.
  • the LCRTM was performed with "normal" oligos 24-27 (oligo N, fig. 9, producing a 44 bp product) or "mutant" oligos 24, 25, 28, and 29 (oligo M, fig. 9, producing a 39 bp product).
  • Amplified product was produced by both sets of oligos from any of the human DNA targets (lanes 3-6, fig. 9a) although little, if any, amplification was seen in the absence of target DNA (lanes 1-2, fig. 9a).
  • the LCRTM was performed with the oligo sets as specified in Table 3, below. With these oligos, amplification occurred predominantly when the probes matched the target exactly at the point of ligation, i.e. "normal” oligos amplified control and heterozygous G551D DNA (lanes 3 and 5, figs. 9b, 9c, 9d, 9e, and 9f), and "mutant" oligos amplified only the heterozygous G551D DNA (lanes 4 and 6, figs. 9b, 9c, 9d, 9e, and 9f)- Little amplification occurred in the absence of human DNA target (lanes 1 and 2, figs. 9b, 9c, 9d, 9e, and 9f). This pattern persisted, whether the additional mismatch was 1, 2, 3, or 4 bases removed from the point of ligation (see CF G551D oligo alignments, fig.2).
  • the CF J3. 11 mutation is a polymorphism loosely linked to cystic fibrosis, but removed from the CFTR gene by several hundred bp. It is a point mutation; I. Bartels, et al, Am. J. Human Genetics, 38: 280-7 (1986).
  • Synthetic 50-base targets homologous for allele 1 or allele 2 of the CF J3.
  • 11 polymorphism (oligos 50 and 51, respectively) were amplified by gap LCRTM as described in example 4, using dGTP and dATP as the fill bases. Potassium as KCl was present at 100 mM. Oligos 52-57, where used, were present at 13.6 nM.
  • reaction mixture without enzyme, was incubated at 100°C for 3 minutes, then cooled to 85°C.
  • DNA ligase was added to the mixture at 85°C.
  • the LCRTM then proceeded for 42 cycles of 85°C for 30 seconds and 45°C for 30 seconds in a Perkin-Elmer thermal cycler.
  • the reaction products were analyzed by immunochromatography on strips spotted with ⁇ nft ' -dansyl antibody as described in example 6.
  • oligos 52-55 For analysis with homologous oligos, the LCRTM was performed with oligos 52-55. These oligos have perfect sequence homology with target 50, but have a mismatch with target 51 at the point of ligation. Nevertheless, these oligos amplified both targets (strips 1 and 2, fig. 11).
  • a third analysis used oligos 54-57, in which oligos 56 and 57 had 1 mismatch with respect target 50 and 2 mismatches with target 51, but no mismatches with respect to each other. These oligos amplified target 50 (strip 5) where probe and target share complete homology at the point of ligation, but target 51, which has a mismatch at the point of ligation, amplified very little (strip 6, fig. 11).
  • Example 13 Effect of probe balance on CF W1282 mutation Extracted DNA from patients homozygous for the CF W1282X mutation
  • the LCRTM then proceeded for 42 cycles of 85°C for 30 seconds and 50°C for 45 seconds in a Perkin-Elmer thermal cycler.
  • the reaction products were analyzed by electrophoresis as described in example 5.
  • oligos were classified as "normal” if they were completely homologous with the normal CFTR gene sequence (oligos 20 and 21, "N” in fig. 12), "mutant” if they were completely homologous with the sequence of the CF W1282X mutation (oligos 22 and 23, "M” in fig. 12), or "common” if they were completely homologous to either sequence (oligos 18 and 19, "C” in fig. 12).
  • Amplified product was produced in insufficient quantity to detect above background from control reactions containing no human DNA (lanes 1, 5, 9, and 13; fig. 12).
  • the combination of normal (N) and common (C) oligos produced a 45 bp product from normal DNA (NL NL, lanes 2, 6, 10, and 14; fig. 12) and from heterozygous DNA (NL/Wi282X > lanes 4, 8, 12, and 16; fig.
  • the amount of amplified product produced dependsed very critically on the relative concentration of the oligos present.
  • the spot from the 45 bp "normal" product was much more intense that that from the 40 bp "mutant" product (lanes 1-4, fig. 12).
  • the concentration of mutant oligos increased to 16, 17, and 18 nM
  • the spot from the 40 bp product increased in intensity
  • the spot from the 45 bp product decreased in intensity (lanes 5-8, 9-12, and 13-16; fig. 12). It is important to note that very small changes in oligo concentration (as little as 5%) effected a major change in the relative amounts of the different amplified materials produced.
  • DNA was extracted and amplified as described in example 11 using oligos 24- 29 at the concentrations stated in example 11. Oligo 24 was labeled at the 5 '-end with 32p.
  • LCRTM with "hot start” the reaction mixture, without enzyme, was incubated at 98°C for 3 minutes, then cooled to 40" C, 50°C, 60°C, or 80°C; at which temperature DNA ligase was added.
  • LCRTM without "hot start” the reaction mixture, without enzyme, was incubated at 98°C for 3 minutes, then cooled to room temperature. DNA ligase was added to the mixture at room temperature. Under both conditions, the LCRTM then proceeded for 34 cycles of 85°C for 30 seconds and 49°C for 45 seconds in a Perkin-Elmer thermal cycler. The reaction products were analyzed by electrophoresis as described in example 5.
  • amplified product was produced in the presence of normal DNA (N, fig. 13) or heterozygous DNA (HET, fig. 13) with either normal oligos (oligos 24-27, oligo N of fig. 13, producing a 44 bp product; lanes 3, 5, 9, and 11) or mutant oligos (oligos 24, 25, 28, and 29; oligo M of fig. 13, producing a 39 bp product, lanes 4, 6, 10, and 12). Mutant oligos also produced an amplified product in the absence of human DNA at these temperatures (lanes 2 and 8, fig. 13).
  • mutant oUgos amplified only the heterozygous CF G551D DNA (HET, lanes 18, 24, and 30, fig. 13), and neither the normal human DNA (lanes 16, 22, and 28) nor the control without human DNA (lanes
  • oligos 66-69 were present at the concentrations stated in table 4.
  • the LCRTM proceeded for the number of cycles stated in table 4, each cycle comprising 85°C for 5 seconds and the lower temperature stated in table 4 for 5 seconds.
  • the reaction products were analyzed by immunochromatography and quantitated by digital imaging as described in example 6.
  • oligos 66-69 were present at 92 nM each.
  • reaction mixtures were prepared separately, with 8 ⁇ L of DNA ligase (1700 units) being introduced to the reaction capillary first, followed by an air space of 2.5 ⁇ L, and finally 10 ⁇ L of reaction mix containing oligos and target.
  • the capillaries were placed in the Corbett thermal sequencer, heated to 85°C for 30 seconds, and the liquids mixed by compressing the contents of the capillary. This was easily accomplished by briefly pushing down on the plunger of the capillary syringe.
  • the LCRTM proceeded for 40 cycles of 85 ⁇ C for 5 seconds and 57°C for 5 seconds.
  • the reaction products were analyzed by immunochromatography as described in example 6.
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Abstract

La présente invention se rapporte à des procédés de détection de petites mutations dans l'acide nucléique cible à l'aide de l'Amplification en Chaîne par Ligase (LCRTM). Les petites mutations détectables comprennent des délétions de base unique, des insertions et des modifications, ainsi que des mutations multiples (des délétions, des insertions et des modifications) où la taille de la mutation est inférieure à environ 15 % de la longueur de sonde moyenne. Les procédés présentent des teneurs élevées en cations monovalents, notamment Na+, K+ et NH¿4?+, et/ou des températures de mélange initiales élevées. Les procédés incluent également des conceptions de sondes présentant des mauvais appariements délibérés par rapport à la cible, les mauvais appariements étant situés près des extrémités 5'. On décrit également plusieurs ensembles de séquences de sondes destinées à la détection de mutations associées à la mucoviscidose.
PCT/US1993/008359 1992-09-25 1993-09-07 Procede d'amplification en chaine par ligase pour la detection de petites mutations Ceased WO1994008047A1 (fr)

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WO1995027078A1 (fr) * 1994-04-04 1995-10-12 Ciba Corning Diagnostics Corp. Dosages d'hybridation-ligature destines a la detection de sequences specifiques d'acides nucleiques
WO1996040996A1 (fr) * 1995-06-07 1996-12-19 Abbott Laboratories Procede de detection de sequences d'acide nucleique ayant recours a une amplification competitive
US5888731A (en) * 1995-08-30 1999-03-30 Visible Genetics Inc. Method for identification of mutations using ligation of multiple oligonucleotide probes
WO2000015837A3 (fr) * 1998-09-15 2000-05-25 Deutsches Krebsforsch Procede de controle qualite lors de la constitution de trames oligomeriques
US6946296B2 (en) 1995-11-30 2005-09-20 Maxygen, Inc. Methods and compositions for polypeptide engineering
US8283121B2 (en) 1996-05-29 2012-10-09 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US8288521B2 (en) 1996-02-09 2012-10-16 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
EP2473634B1 (fr) * 2009-09-03 2019-05-01 Seegene, Inc. Sonde td et ses utilisations

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GENOMICS, Vol. 4, issued 1989, WU et al., "The Ligation Amplification Reaction (LAR)--Amplification of Specific DNA Sequences Using Sequential Rounds of Template-Dependent Ligation", pages 560-569. *
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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995027078A1 (fr) * 1994-04-04 1995-10-12 Ciba Corning Diagnostics Corp. Dosages d'hybridation-ligature destines a la detection de sequences specifiques d'acides nucleiques
WO1996040996A1 (fr) * 1995-06-07 1996-12-19 Abbott Laboratories Procede de detection de sequences d'acide nucleique ayant recours a une amplification competitive
US5955598A (en) * 1995-06-07 1999-09-21 Abbott Laboratories Primer compositions and kits for detecting hepatitis B virus
US5888731A (en) * 1995-08-30 1999-03-30 Visible Genetics Inc. Method for identification of mutations using ligation of multiple oligonucleotide probes
US6025139A (en) * 1995-08-30 2000-02-15 Visible Genetics Inc. Method for identification of mutations using ligation of multiple oligonucleotide probes
US6946296B2 (en) 1995-11-30 2005-09-20 Maxygen, Inc. Methods and compositions for polypeptide engineering
US8624016B2 (en) 1996-02-09 2014-01-07 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US8288521B2 (en) 1996-02-09 2012-10-16 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US8703928B2 (en) 1996-02-09 2014-04-22 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US9206477B2 (en) 1996-02-09 2015-12-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US9234241B2 (en) 1996-02-09 2016-01-12 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US8283121B2 (en) 1996-05-29 2012-10-09 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US8597890B2 (en) 1996-05-29 2013-12-03 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US8597891B2 (en) 1996-05-29 2013-12-03 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US8642269B2 (en) 1996-05-29 2014-02-04 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled polymerase chain reactions
US8802373B2 (en) 1996-05-29 2014-08-12 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US6582917B1 (en) 1998-09-15 2003-06-24 Deutsches Krebsforschungszentrum Method for controlling quality in the construction of oligomer grids
WO2000015837A3 (fr) * 1998-09-15 2000-05-25 Deutsches Krebsforsch Procede de controle qualite lors de la constitution de trames oligomeriques
EP2473634B1 (fr) * 2009-09-03 2019-05-01 Seegene, Inc. Sonde td et ses utilisations

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