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  • 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
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  • 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
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • C—CHEMISTRY; METALLURGY
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  • 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
  • C12Q1/6869—Methods for sequencing

Definitions

• This invention pertains to high throughput screening of nucleic acid samples in order to identify the presence of one or more genetic alterations of interest in those samples.
• This invention also pertains to the identification of specific target nucleic acid sequences associated with genetic disorders.
• the methods of the present invention can be used to identify genetic polymorphisms, to determine the molecular basis for genetic diseases, and to provide carrier and prenatal diagnosis for genetic counseling.
• the invention pertains to specific high-resolution identification of disease-causing microorganisms.
• the first group designed to scan for mutations within a gene, includes single-strand conformational polymorphism (SSCP) (7), denaturing gradient-gel electrophoresis (DGGE) (8), heteroduplex analysis (HET) (9) , chemical cleavage analysis (CCM) (10), ribonuclease cleavage (RNAase) (11), and direct sequencing of the target (12).
• SSCP single-strand conformational polymorphism
• DGGE denaturing gradient-gel electrophoresis
• HET heteroduplex analysis
• CCM chemical cleavage analysis
• RNAase ribonuclease cleavage
• direct sequencing of the target (12.
• the present invention encompasses high-throughput methods for detecting and identifying sequences or genetic alterations (defined as nucleotide additions, deletions, or substitutions) in a large number of nucleic acid samples, which is achieved by: immobilizing a plurality of the nucleic acid samples on a support; providing a multiplicity of purine and pyrimidine containing polymers; hybridizing the immobilized samples with the multiplicity of purine and pyrimidine containing polymers at substantially the same time; identifying the hybridized purine and pyrimidine containing polymers wherein the identification of the hybridized purine and pyrimidine containing polymers identifies the nucleic acid sequence or one or more genetic alterations.
• the hybridized purine and pyrimidine polymers can be identified by any method well-known in the art, such as, for example, sequencing, direct labeling, indirect labeling, and labeling with a unique length marker.
• the present invention also encompasses certain embodiments wherein the sample is not immobilized, and is reacted with the purine and pyrimidine containing polymers in solution (i.e., rather than on a support) .
• Both the target nucleic acid sequence and/or the hybridized purine and pyrimidine containing polymers may be amplified to facilitate detection and identification.
• amplification methods include polymerase chain reaction (PCR) , ligase chain reaction (LCR) , gap-LCR, ligation amplification reaction (LAR) , oligonucleotide ligation assay (OLA) , amplification refractory mutation system (ARMS) , competitive oligonucleotide priming (COP) , allele specific PCR, Q-beta replicase amplification, nucleic acid sequence based amplification (NASBA) and branched chain amplification.
• Hybridizations can be carried out under conditions that minimize the differences in melting temperature of hybrids formed between different purine and pyrimidine containing polymers and the target nucleic acid sequence.
• Figure 1 shows autoradiographic results obtained from hybridizing multiple identical filters containing human genomic DNA with 32 P-labelled ASOs specific for different alleles of the cystic fibrosis transmembrane regulator (CFTR) gene.
• the ASOs used in each hybridization are identified on the left of each filter.
• Lane 1 in each case contains DNA carrying the mutant sequence complementary to each ASO; lanes 2-6 contain wild-type "normal" sequences.
• Figure 2A-2D show autoradiographic results obtained from hybridizing four identical filters containing human genomic DNA with 32 P-labelled ASOs specific for different alleles of the cystic fibrosis transmembrane regulator (CFTR) gene.
• the ASOs used in each hybridization are identified on the left of each filter.
• the lanes marked A contain positive control DNA samples.
• Rows B-E contain patient samples analyzed in duplicate, with the exception of 8C (amplification failure on duplicate sample), and D7, D8 and E7 (positive controls) .
• FIGS 3A and 3B show the identification of specific mutations in pool-positive samples identified in Figure 1.
• the top row of each filter contains positive control samples for ASOs in pool 1 and pool 2 as indicated.
• Row B contains pool-1 or pool-2 positive patient samples.
• Pool 1 lanes 1 and 2 contain sample 4, lanes D and E from Figure 1.
• Pool 2 lanes 1 and 2 contains sample 3, lanes D and E from Figure 2.
• Lanes 3 and 4 contain sample 5, lanes D and E from Figure 2.
• Figure 4 shows a schematic representation of the methods of the present invention.
• Figure 5 depicts general schemes for ligation based techniques for hybridizing purine and pyrimidine polymers to immobilized samples and identifying the hybridized products.
• Figure 6 depicts a scheme for identifying ligation products through chemical cleavage sequencing of the products, i.e., Maxam-Gilbert sequencing.
• Figure 7 depicts a scheme for identifying ligation products using a conventional Sanger type sequencing reaction.
• Figure 8 depicts another method for identifying ligation products by Sanger sequencing.
• Figure 9 depicts Sanger sequencing of ligation products that are amplified using a universal priming sequence.
• Figure 11 shows simultaneous detection of 106 different mutations within 33 target regions from seven different genes in a single hybridization assay.
• Rows 1-7 Detection of 66 CFTR mutations present in cystic fibrosis (CF) .
• Rows 8-9 Negative control samples for cystic fibrosis (CF-) mutations.
• Rows 10-11 Detection of 14 ⁇ - globin mutations in ⁇ -thalassemia ( ⁇ Thal.) and 2 ⁇ -globin mutations in sickle cell anemia (SCA) .
• Row 12 Detection of 3 HEXA mutations present in Tay Sachs (TSD) .
• Row 13 Detection of 8 GCR mutations present in Gaucher's disease (GCR) .
• GCR Gaucher's disease
• Row 14 Detection of 4 ASPA mutations present in Canavan Disease (CD) .
• Row 15 Detection of 5 BRCA 1 mutations present in breast cancer (BRC) .
• Row 16 Detection of 4 FACC mutations present in Fanconi anemia
• Figure 12 shows the band patterns generated by chemical cleavage of eluted ASOs and reveals the identity of the mutation.
• C C cleavage reaction
• G G cleavage reaction.
• Figure 13 shows the band patterns generated by an enzymatic sequencing procedure reveal the identity of the mutation.
• the ASO(s) eluted from mutation-positive samples were used to prime cycle sequencing reactions in a complex mixture of templates. Each template contained a region complementary to a specific ASO (the priming site) , a common "stuffer region" (Region B) and a downstream mutation-specific identifier sequence (Region A) .
• Regular C and G sequencing Lanes C and G for each sample
• the fingerprint generated from the mutation-specific identifier sequence unequivocally identified the specific ASO, and therefore the mutation present in the target DNA.
• CF cystic fibrosis
• BT ⁇ -thalassemia
• BRC Breast
• Figure 14 shows the simultaneous detection of 106 different mutations in over 500 different patient samples in a single ASO hybridization assay.
• An "allele-specific oligonucleotide” as defined herein is an oligonucleotide having a sequence that is identical or almost identical to a known nucleic acid portion. Often, an ASO contains a small change relative to the prevalent "wild type" sequence. This change may comprise addition, deletion, or substitution of one or more nucleotides. ASOs can be designed to identify any addition, deletion, or substitution, as long as the nucleic acid sequence is known.
• a "variant" sequence as used herein encompasses a nucleic acid sequence that differs from a known sequence by the addition, deletion, or substitution of one or more nucleotides.
• Amplification of a nucleic acid sequence as used herein denotes the use of polymerase chain reaction (PCR) (Saiki et al. , Science 239:487, 1988), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189, 1991) (LCR), gap-LCR (Abravaya et al. , Nuc. Acids Res. 23:675, 1995), ligation amplification reaction (Wu et al. , Genomics 4:560, 1989); ASPCR (Wu et al. , Proc. Natl. Acad. Sci. USA 86:2757, 1989), ARMS (Newton et al. , Nucl. Acids Res. 17:2503, 1989), or other methods to increase the concentration of a particular nucleic acid sequence.
• PCR polymerase chain reaction
• LCR ligase chain reaction
• gap-LCR Abravaya et al. , Nu
• “Chemical sequencing” of nucleic acids denotes methods such as that of Maxim and Gilbert (Maxim- Gilbert sequencing, Maxam and Gilbert, 1977, Proc. Natl. Acad. Sci. USA 74:560), in which nucleic acids are randomly cleaved using individual base-specific reactions.
• Enzymatic sequencing of nucleic acid denotes methods such as that of Sanger (Sanger et al. , 1977, Proc. Natl. Acad. Sci. USA, 74:5463), in which a single-stranded DNA is copied and randomly terminated using DNA polymerase. 6.
• bound and hybridized are used interchangeably to denote the formation of nucleic acid:purine and pyrimidine containing polymer duplexes.
• affinity purified denotes purification using hybridization.
• High-throughput denotes the ability to simultaneously process and screen a large number of nucleic acid samples (e.g., in excess of 50 or 100 nucleic acid samples) and a large number of target sequences within those samples.
• PNA polypeptide containing polymers
• DNA, RNA and other polymers containing purines and pyrimidines that are capable of Watson-Crick base pairing, and which do not necessarily carry a sugar phosphate backbone, such as PNA. See, J. Am. Chem. Soc, 114:1895-97 (1992) .
• the present invention provides refinements of a modified allele-specific oligonucleotide approach for the simultaneous analysis of large numbers of patient samples for multiple CF mutations (25) .
• Invention methods further provide a Multiplex Allele-Specific Diagnostic Assay (MASDA) , which has the capacity to cost effectively analyze large numbers of samples (>500) for a large number of mutations (>100) in a single assay.
• MASDA uses oligonucleotide hybridization to interrogate DNA sequences.
• the target DNA is immobilized to the solid support, and interrogated in a combinatorial fashion with a pool of ASOs (i.e. a single mixture of mutation-specific oligonucleotides) in solution.
• ASOs i.e. a single mixture of mutation-specific oligonucleotides
• the ASO(s) corresponding to the specific mutation(s) present in a given sample is hybrid- selected from the pool by the target DNA.
• sequence-specific band patterns associated with the bound ASOs are generated by chemical or enzymatic sequencing, and the mutation or mutations present in the sample are easily identified.
• CFTR 26
• ⁇ -globin (1) HEXA
• GCR 28
• ASPA 29
• BRCAl (3) ASPA
• FACC 30
• the present invention encompasses a high- throughput method for screening nucleic acid samples for target sequences or sequence alterations and, more particularly, for specific DNA sequences in DNA isolated from a patient.
• the method is applicable when one or more genes or genetic loci are targets of interest. It will also be appreciated that this method allows for rapid and economical screening of a large number of nucleic acid samples for target sequences of interest.
• the specific nucleic acid sequence comprises a portion of a nucleic acid, a particular gene, or a genetic locus in a genomic DNA known to be involved in a pathological condition or syndrome.
• Non-limiting examples include cystic fibrosis, sickle-cell anemia, ⁇ -thalassemia, and Gaucher's disease.
• the specific nucleic acid sequence comprises part of a particular gene or genetic locus that may not be known to be linked to a particular disease, but in which polymorphism is known or suspected.
• the specific nucleic acid sequence comprises part of a foreign genetic sequence e.g. the genome of an invading microorganism.
• a foreign genetic sequence e.g. the genome of an invading microorganism.
• Non-limiting examples include bacteria and their phages, viruses, fungi, protozoa, and the like.
• the present methods are particularly applicable when it is desired to distinguish between different variants or strains of a microorganism in order to choose appropriate therapeutic interventions.
• the target nucleic acid represents a sample of nucleic acid isolated from a patient.
• This nucleic acid may be obtained from any cell source or body fluid.
• Non-limiting examples of cell sources available in clinical practice include blood cells, buccal cells, cervicovaginal cells, epithelial cells from urine, fetal cells, or any cells present in tissue obtained by biopsy.
• Body fluids can include blood, urine, cerebrospinal fluid, semen, and tissue exudates at the site of infection or inflammation.
• Nucleic acids can be extracted from the cell source or body fluid using any of the numerous methods that are standard in the art. It will be understood that the particular method used to extract the nucleic acid will depend on the nature of the source.
• the minimum amount of DNA, for example, that can be extracted for use in a preferred form of the present invention is about 5 pg (corresponding to about 1 cell equivalent of a genome size of 4 x IO 9 base pairs) .
• the target nucleic acid may be employed in the present invention without further manipulation.
• one or more specific regions present in the target nucleic acid may be amplified by PCR.
• the amplified regions are specified by the choice of particular flanking sequences for use as primers.
• Amplification at this step provides the advantage of increasing the concentration of specific nucleic acid sequences within the target sequence population.
• the length of nucleic acid sequence that can be amplified ranges from 80 bp to up to 30 kbp (Saiki et al. , 1988, Science, 239:487) .
• the target nucleic acid is bound to a solid phase or semi-solid phase matrix.
• matrices suitable for use in the present invention include nitrocellulose or nylon filters, glass beads, magnetic beads coated with agents for affinity capture, treated or untreated microtiter plates, polymer gels, agarose and the like. It will be understood by a skilled practitioner that the method by which the target nucleic acid is bound to the matrix will depend on the particular matrix used. For example, binding to nitrocellulose can be achieved by simple adsorption of nucleic acid to the filter, followed by baking the filter at 75-80°C under vacuum for 15 min-2h.
• charged nylon membranes can be used that do not require any further treatment of the bound nucleic acid.
• Beads and microtiter plates that are coated with avidin can be used to bind target nucleic acid that has had biotin attached (via e.g. the use of biotin-conjugated PCR primers) .
• antibodies can be used to attach target nucleic acid to any of the above solid supports by coating the surfaces with the antibodies and incorporating an antibody-specific hapten into the target nucleic acid.
• polymers While immobilization of the target nucleic acid is generally preferred, in some embodiments it may be desirable to hybridize the polymers to the target in solution, i.e., without having bound the target to a support.
• polymers may be hybridized to the target in solution, and then ligated in solution as part of a technique according to the invention for high throughput screening. Ligation techniques are discussed further below.
• the untreated or amplified target nucleic acid preferably bound to a solid phase or semi-solid phase matrix
• a mixture of purine and pyrimidine containing polymers hereinafter also referred to as "polymer” or “polymers”.
• These polymers are preferably allele-specific oligonucleotides (ASOs) .
• 10-200 ASOs can be pooled for a single hybridization, preferably 50-100, and most preferably 50.
• the length of individual ASOs may be 16-25 nucleotides, preferably 17 nucleotides in length.
• the purine and pyrimidine containing polymers may be synthesized chemically by methods that are standard in the art, e.g., using commercially available automated synthesizers. These polymers may then be radioactively labelled (e.g. end-labelled with 32 P using polynucleotide kinase) or conjugated to other commonly used "tags" or reporter molecules. For example, fluorochromes (such as FITC or rhodamine) , enzymes (such as alkaline phosphatase) , biotin, or other well-known labelling compounds may be attached directly or indirectly. Furthermore, using standard methods, a large number of randomly permuted polymers can be synthesized in a single reaction. As detailed below, the present invention does not require that individual hybridizing sequences be determined prior to the hybridization. Rather, the sequence of bound polymers can be determined in a later step.
• fluorochromes such as FITC or rhodamine
• enzymes such as alkaline
• the hybridization reaction can be performed under conditions in which polymers such as those containing different sequences hybridize to their complementary DNA with equivalent strength. This is achieved by: 1) employing polymers of equivalent length; and 2) including in the hybridization mixture appropriate concentrations of one or more agents that eliminate the disparity in melting temperatures among polymers of identical length but different guanosine+cytosine (G+C) compositions. Agents that may be used for this purpose include without limitation quaternary ammonium compounds such as tetramethylammonium chloride (TMAC) .
• TMAC tetramethylammonium chloride
• TMAC acts through a non-specific salt effect to reducing hydrogen-bonding energies between G-C base pairs. At the same time, it binds specifically to A-T pairs and increases the thermal stability of these bonds. These opposing influences have the effect of reducing the difference in bonding energy between the triple-hydrogen bonded G-C based pair and the double-bonded A-T pair.
• TMAC acts through a non-specific salt effect to reducing hydrogen-bonding energies between G-C base pairs. At the same time, it binds specifically to A-T pairs and increases the thermal stability of these bonds.
• agent that exhibits these properties can be used, if desired, in practicing the present invention.
• agents can be easily identified by determining melting curves for different test oligonucleotides in the presence and absence of increasing concentrations of the agent.
• a solid matrix such as a nylon filter
• the target nucleic acid and polymers can be incubated for sufficient time and under appropriate conditions to achieve maximal specific hybridization and minimal non-specific, i.e. background, hybridization.
• the conditions to be considered include the concentration of each polymer, the temperature of hybridization, the salt concentration, and the presence or absence of unrelated nucleic acid.
• the polymers can be separated into at least two groupings, each grouping containing a sufficient number of polymers to allow for hybridization. For example, it may be preferred to divide the total number of polymers of a pool to be hybridized to the nucleic acid samples into groupings of about 50 polymers.
• Each group of polymers can be hybridized to the nucleic acid immobilized on the support in a sequential manner, but the polymers comprising each group can be hybridized to the nucleic acid at substantially the same time. Additionally, immobilized nucleic acid samples may be hybridized to at least one pool of polymers, the identity of the hybridizing polymers determined, and then the nucleic acid samples hybridized again with the same or different polymer pools.
• the concentration of each purine and pyrimidine containing polymer generally ranges from 0.025 to 0.2 pmol per ml of hybridization solution.
• the optimal concentration for each polymer can be determined by test hybridizations in which the signal-to-noise ratio (i.e. specific vs. non-specific binding) of each polymer is measured at increasing concentrations of labeled polymer.
• oligonucleotides containing the uniabeled non-variant (i.e. wild-type) sequence may be included in the reaction mixture at a concentration equivalent to 1-100 times the concentration of the labelled polymer.
• the temperature for hybridization can be optimized to be as high as possible for the length of the polymers being used. This can be determined empirically, using the melting curve determination procedure described above. It will be understood by skilled practitioners that determination of optimal time, temperature, polymer concentration and salt concentration should be done in concert.
• hybridized polymers identified by the invention be those that are perfectly hybridized. Methods described above minimize imperfect hybridization. Such methods, however, are not always necessary. In the ligation procedure described in the Examples, imperfect hybrids may form, but perfect hybrids are selectively identified.
• unbound polymers are, if necessary, removed such as by washing the matrix-bound nucleic acid in a solution containing TMAC or similar compounds, under conditions that preserve perfectly matched nucleic acid:polymer hybrids. Washing conditions such as temperature, nature and concentration of salts, and time of washing, are determined empirically as described above. At this stage, the presence of bound polymers may be determined. Different methods for detection will depend upon the label or tag incorporated into the polymers. For example, radioactively labelled or chemiluminescent ASOs that have bound to the target nucleic acids can be detected by exposure of the filter to X-ray film.
• polymers containing a fluorescent label can be detected by excitation with a laser or lamp- based system at the specific absorption wavelength of the fluorescent reporter.
• polymers can each carry, in addition to the probe sequence, a molecular weight modifying entity (MWME) that is unique for each member of the polymer pool.
• MWME molecular weight modifying entity
• the MWME does not participate in the hybridization reaction but allows direct identification of the separated polymer by determination of the relative molecular weight by any number of methods. Other methods for detection and identification are described below.
• the bound polymers are separated from the matrix-bound target nucleic acid. Separation may be accomplished by any means known in the art that destabilizes nucleic acid to polymer hybrids, i.e. lowering salt concentration, raising temperature, exposure to formamide, alkali, etc.
• the bound polymers may be separated by incubating the target nucleic acid-polymer complexes in water, and heating the reaction above the melting temperature of the nucleic acid:polymer hybrids. This obviates the need for further treatment or purification of the separated polymers.
• the hybridized polymers with or without separation from the target nucleic acid, can be identified by a number of different methods that will be readily appreciated by those of skill in the art.
• sequencing the polymers it is possible to correspondingly identify target sequences or genetic alterations in the nucleic acid samples.
• the polymers can also be identified by directly labeling them with a unique reporter that provides a detectable signal.
• Polymers that are directly labeled can be detected using radioactivity, fluorescence, colorimetry, x-ray diffraction or absorption, magnetism, enzymatic activity, chemiluminescence, and electrochemiluminescence, and the like.
• Suitable labels include fluorophores, chromophores, radioactive atoms (such as 32 P and 125 I) , electron dense reagents, and enzymes that produce detectable products. See L. Kricka, Nonisotopic DNA Probe Techniques, Chapters 1 and 2, Academic Press, 1992 (hereinafter "Kricka”).
• indirect labeling may also be used.
• Many binding pairs are known in the art for indirect labeling, including, for example, biotin - avidin, biotin - streptavidin, hapten - antihapten antibody, sugar - lectin, and the like.
• one member of a binding pair can be attached to the polymer and the other member of the binding pair directly labeled as described above.
• the polymers that are bound to target nucleic acid sequences may be identified by incubation with the labeled member and subsequent detection of the binding pair-label complex. See, Bioconjugate Chemistry, 1:165-187 (1990); Kricka, Chapters 1 and 2.
• the polymers can still further be identified by using unique length markers. That is, by providing polymers having components that contribute a predetermined and unique molecular weight to each individual polymer, in addition to the portions that participate in hydrogen bonding interactions with target nucleic acids, it is possible to identify an individual polymer by molecular weight. See, e.g., Nucleic Acids Res., 22:4527-4534 (1994) .
• hybridized polymers can be identified by use of hybridization arrays.
• purine and pyrimidine containing polymers of predetermined seguence are immobilized at discrete locations on a solid or semi-solid support.
• the sequence of each immobilized polymer comprising the array is complementary to the sequence of a member of the polymer pool.
• Members of the polymer pool that hybridize with target nucleic acids can be identified after separation from target nucleic acids by rehybridization with immobilized polymers forming the array.
• the identity of the polymer is determined by the location of hybridization on the array. See, U.S. Patent No. 5,202,231 and WO 8910977.
• Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
• the hybridized polymer is directly subjected to sequencing, using a chemical method standard in the art (e.g. Maxam- Gilbert sequencing, Maxam and Gilbert, 1977, Proc. Natl. Acad. Sci., USA, 74:560).
• This method does not require that polymers be separated from the target nucleic acid prior to sequencing, and, further, is particularly applicable when randomly permuted mixtures of polymers are used.
• the hybridized polymers are identified by enzymatic sequencing (Sanger et al. , 1977, Proc. Natl. Acad. Sci., USA, 74:5463) .
• oligonucleotides are synthesized that contain sequences complementary to the polymers and additional pre-determined co-linear sequences that act as sequence "tags" (see Example 4 below) . Separation of the polymers from the target nucleic acid is performed in the presence of a mixture of these complementary, "tagged" oligonucleotides.
• the polymers hybridize to their complementary sequences and act as primers for the sequencing reaction. Determination of the resulting primed sequence "tag” then identifies the polymer(s) present in the reaction.
• the hybridized polymers are incubated with complementary oligonucleotides that may contain universal primer sequences and/or a sequencing primer sequence with or without an additional "tag" sequence (see Example 4 below) .
• complementary oligonucleotides that may contain universal primer sequences and/or a sequencing primer sequence with or without an additional "tag” sequence (see Example 4 below) .
• initial hybridization of a polymer to its complementary oligonucleotide allows the polymer to serve as the initial primer in a single extension reaction.
• the extension product is then used directly as template in a cycle sequencing reaction. Cycle sequencing of the extension products results in amplification of the sequencing products.
• the sequencing primer is oriented so that sequencing proceeds through the polymer itself, or, alternatively, through the "tag" sequence.
• the extension product includes a universal primer sequence and a sequencing primer sequence.
• This extension product is then added to a linear amplification reaction in the presence of universal primer.
• the oligonucleotides containing complementary sequences to bound polymers are therefore selectively amplified.
• these amplified sequences are subjected to Sanger sequencing, using the built-in sequencing primer sequence.
• the sequencing primer is placed immediately upstream of a "tag" sequence as above. Thus, determination of the "tag" sequence will identify the colinear polymer sequence.
• the present invention accommodates the simultaneous screening of a large number of potential polymers in a single reaction.
• the actual number of polymers that are pooled for simultaneous hybridization is determined according to the diagnostic need. For example, in cystic fibrosis (CF) , one particular mutation ( ⁇ 508) accounts for more than 70% of CF cases.
• CF cystic fibrosis
• a preliminary hybridization with a labelled or tagged ⁇ 508-specific polymer according to the present methods, followed by detection of the bound polymer, will identify and eliminate ⁇ 508 alleles.
• a second (“phase two") hybridization a large number of polymers encoding other, less frequent, CF alleles are utilized, followed by separation and sequencing as described above.
• pools of polymers are determined only by the number of independent hybridizations that would be needed in a phase two analysis on a pool positive sample.
• the present invention accommodates the simultaneous screening of large numbers of nucleic acid samples from different sources, including different mammals, with a large number of polymers that are complementary to mutations in more than one potential disease-causing gene.
• the present invention provides for simultaneous screening for a large number of potential foreign nucleic acids. Furthermore, particular strains, variants, mutants, and the like of one or more microorganisms can also be distinguished by employing appropriate polymers in the screening.
• the methods of the present invention also make it possible to define potentially novel mutant alleles carried in the nucleic acid of a patient or an invading microorganism, by the use of randomly permuted polymers in phase one or phase two screening. In this embodiment, separation of the bound polymers, followed by sequencing, reveals the precise mutant sequence.
• kits for carryinj out high-throughput screening of nucleic acid samples will include, in packaged combination, at least the following components: a support, a multiplicity of purine and pyrimidine containing polymers, appropriate labeling components, and enzymes and reagents required for polymer sequence determination.
• Tables 1-8 The specific mutations examined within each disease gene are shown in Tables 1-8. These tables also include the size (bp) of regions amplified, and the primers used for each amplification.
• multiplex PCR was performed to reduce the number of PCR reactions needed (Table 1, column 5) . A total of 9 reactions facilitated amplification of 33 different loci. All single or multiplex PCR reactions were performed in a disease-specific manner. In other words, DNA samples were amplified for the relevant disease gene only, and not for all loci examined in the assay. Examples of the various disease-specific amplification products are shown in Figure 10. In order to include 66 CF mutations within the study discussed herein, 17 different regions within the CFTR gene were amplified using two multiplex PCR reactions ( Figure 10 lanes 1 and 2) . Amplicon sizes ranged from 130 - 510 bp.
• a single amplification product of 1600 bp was sufficient to include 14 ⁇ -thalassemia and two sickle cell anemia associated mutations within the ⁇ -globin gene (Figure 10 lane 3) .
• a 2-plex amplification reaction was designed to examine 3 mutations ( Figure 10 lane 4) .
• 3-plex amplification reactions were performed ( Figure 10 lane 6) .
• a separate 4-plex amplification was performed for five Breast Cancer Susceptibility-related mutations ( Figure 10 lane 7) and a single 3-plex amplification for Fanconi Anemia-associated mutations ( Figure 10 lane 8) .
• the specific mutation present within any pool- positive sample was identified by eluting the hybridized oligonucleotide from the sample DNA and directly interrogating the oligonucleotide sequence.
• the eluted oligonucleotides were attached to a solid support, G and C base specific chemical modification reactions were performed, and the reaction products separated by polyacrylamide gel electrophoresis (32) .
• Figure 12 represents an example of the CG limited sequencing fingerprints produced from some of the oligonucleotides eluted from the pool-positive control samples in Figure 3 (CF30, CF31, TSI, TS2, TS3, BT2, BT3*, BT6 and BT7) .
• Each eluted oligonucleotide shown in Figure 12 generated a characteristic fingerprint which unambiguously identified the specific mutation present in the pool-positive sample DNA. Unique band patterns were generated for each of 106 ASOs. Sequence analysis of all
• BT3* generated a unique fingerprint made up of two superimposed oligonucleotide-specific band patterns. This demonstrated that a compound heterozygote genotype was readily identified using this technique.
• an enzymatic protocol for eluted oligonucleotide identification was developed. This procedure involved using the eluted mutation specific oligonucleotide as a primer in a cycle sequencing reaction.
• the eluted oligonucleotide was added to a cycle sequencing reaction mix containing a mixture of synthetic (77-mer) templates.
• Each synthetic template contained a different priming sequence complementary to one of the mutation- specific oligonucleotides present in the pooled hybridization reaction, and a downstream specific identifier sequence to generate unique, mutation-specific fingerprints identifying the eluted ASO.
• Figure 13 is an example of the C and G band patterns generated from cycle sequencing reactions utilizing oligonucleotides eluted from positive (mutant genotype CF17, CF20, BT5 and BRC5) and negative (normal genotype CF neg., BT neg., and BRC neg.) control samples.
• Each reaction performed with oligonucleotides eluted from positive control samples ( Figure 13 lanes 1-4) generated a common band pattern contained within all synthetic templates (Region B) followed by a mutation-specific fingerprint (Region A) .
• the pattern observed in the mutation-specific fingerprints allowed unequivocal identification of the corresponding ASO primer, and consequently the specific mutation present in the patient sample.
• No band patterns were observed when cycle sequencing reactions were performed with eluates from negative control samples ( Figure 13 lanes 5-7).
• Figure 14 represents the hybridization results generated from analyzing >500 different DNA samples for the presence of 106 different mutations, in a single hybridization assay. All samples known to carry one of the 106 different mutations were correctly identified as pool-positive in the multiplex hybridization ( Figure 14) . The specific mutations were correctly identified within each pool-positive sample by performing the chemical modification and cleavage procedure. It is significant to note that no increase in non-specific background was observed when sample throughput was increased to more than 500 samples in a single hybridization assay.
• Buccal cells were collected on a sterile cytology brush (Scientific Products) or female dacron swab
• DNA was prepared as follows, immediately or after storage at room temperature or at 4°C. The brush or swab was immersed in 600 ⁇ l of 50mM
• the solution containing DNA was then neutralized with 60 ⁇ l of IM Tris, pH 8.0, and vortexed again (Marchall et al. , J.Med.Genet. 27:658, 1990) .
• the DNA was stored at 4°C.
• oligonucleotides representing 40 bp of endogenous gene sequence including the mutation were synthesized, cloned into pGEM ® -3Zf(+) vectors (Promega
• mutations were selected from 33 regions in seven different genes.
• the genes included the cystic fibrosis transmembrane conductance regulator gene (CFTR) , the ⁇ - globin gene, the Tay-Sachs hexosaminidase gene (HEXA) , the Gaucher gene (GCR) , the Canavan aspartoacylase gene (ASPA) , the breast cancer susceptibility gene (BRCAl) and the Fanconi Anemia Complementation Group C gene (FACC) .
• CTR cystic fibrosis transmembrane conductance regulator gene
• HEXA Tay-Sachs hexosaminidase gene
• GCR Gaucher gene
• ASPA Canavan aspartoacylase gene
• BRCAl the breast cancer susceptibility gene
• FACC Fanconi Anemia Complementation Group C gene
• PCR amplifications were performed using 1-2 ⁇ g of genomic DNA or 10 ng of plasmid DNA in 100 ml of reaction buffer containing lOmM TrisHCl pH 8.3, 50mM KCl, 1.5mM MgCl 2 , 200 mM dNTPs and 0.05-0.1 units / ⁇ l Taq polymerase (Perkin-Elmer, Norwalk, CT) .
• concentration of primers ranged from 0.2 - 1.6 mM.
• DNA amplifications were performed using a Perkin- Elmer 9600 Thermal Cycler (Perkin-Elmer, Norwalk, CT) .
• Perkin-Elmer 9600 Thermal Cycler Perkin-Elmer, Norwalk, CT
• the amplifications were carried out for 28 cycles with ramping (94°C/10 sec. hold with 48 sec. ramp, 60°C/10 sec. hold with 36 sec. ramp, 72°C/10 sec. hold with 38 sec. ramp) with a final 74°C hold for 5 minutes before cooling.
• ramping 94°C/10 sec. hold with 48 sec. ramp, 60°C/10 sec. hold with 36 sec. ramp, 72°C/10 sec. hold with 38 sec. ramp
• a final 74°C hold for 5 minutes before cooling.
• the amplification program consisted of 28 cycles with a 55°C anneal (94°C/10 sec. hold, 55°C/10 sec. hold, 74°C/10 sec. hold) with a final 74°C hold for 5 minutes before cooling.
• Amplification products were analyzed by 2% agarose gel electrophoresis followed by ethidium bromide staining and visualization on a UV transilluminator (Fotodyne, New Berlin, WI)
• Mutations from 7 different genes were selected as candidates for a complex mutation detection assay.
• the 106 mutations examined included point mutations, deletions and insertions. Details of the selected mutations and gene amplifications are listed in Tables 1-8.
• Allele-specific oligonucleotides were 17- ers synthesized and HPLC-purified by Operon Technologies (Alameda, CA) . All oligonucleotides were quantitated by spectrophotometry and tested in independent hybridizations before being pooled. Specified amounts of individual ASOs were combined into a pool of 106 ASOs so that the pool would contain the required amount of each specific ASO determined to be optimal for the pool hybridization.
• ASOs representing known cystic fibrosis (CF) mutations are set forth below.
• Amplified products were denatured using 1. ON NaOH, 2.0M NaCl, 25mM EDTA pH 8.0, containing bromophenol blue (30 ml of 0.1% bromophenol blue/ 10 ml denaturant) for 5 minutes at room temperature.
• Denatured products were blotted onto Biotrans membrane (ICN Biomedicals Inc., Aurora, OH) using a 96-well format dot blot apparatus (Life Technologies, Gaithersburg, MD) .
• Membranes were neutralized in 2x SSC (0.15M NaCl, 0.015M trisodium citrate) for 5 minutes at room temperature and baked in a vacuum oven at 80°C for 15 minutes. Immediately before use, the membranes were rinsed in distilled water, and placed in hybridization solution.
• Polynucleotide kinase (New England Biolabs, Beverly, MA) .
• the labeling reactions were incubated at 37°C for 1 hour.
• the efficiency of the kinase reactions were monitored by chromatography on cellulose polyethyleneimine (PEI) plates (J.T. Baker Inc., Phillipsburg, NJ) using 0.75M NaH 2 P0 4 pH 3.5 buffer, followed by exposure of the plates to Kodak X- Omat X-Ray film (Eastman Kodak Company, Rochester, NY) at room temperature for 5 minutes.
• PEI cellulose polyethyleneimine
• Hybridizations were carried out in plastic bags containing the filters prepared as in Example 1 above, to which pooled radiolabelled ASOs were added in a TMAC hybridization buffer (3.0M TMAC, 0.6% SDS, lmM EDTA, lOmM sodium phosphate pH 6.8, 5X Denhardt's Solution, and 40 ⁇ g/ml yeast RNA) .
• TMAC hybridization buffer 3.0M TMAC, 0.6% SDS, lmM EDTA, lOmM sodium phosphate pH 6.8, 5X Denhardt's Solution, and 40 ⁇ g/ml yeast RNA
• the 96-well array of spotted genomic samples was marked with a grid so that positives identified in the hybridization could be easily located for the subsequent elution and ASO sequencing.
• Signal intensities generated from the different mutation-positive samples were optimized by adjusting the concentrations of each mutation-specific oligonucleotide within the hybridization.
• the final concentration of each labeled mutant ASO in the pool hybridization ranged from 0.008-1.8 pMol/ml, with the concentration of cold normal ASOs ranging from 0- 200 fold excess of the corresponding mutant ASO.
• Hybridizations were allowed to proceed overnight at 52°C, with agitation.
• the blots were wrapped in plastic wrap and exposed to Kodak X-Omat X-Ray film (Eastman Kodak Company, Rochester, NY) at -80°C for 15 minutes to 1 hour.
• Kodak X-Omat X-Ray film Eastman Kodak Company, Rochester, NY
• sequence of the polymers may be determined directly using chemical sequencing.
• polymer ⁇ may be used in conjunction with complementary oligonucleotides that contain other sequences in addition to sequences complementary to the polymers.
• the polymers serve as primers to form extension products that contain the additional sequences, and the extension products are subjected to DNA sequencing.
• the ASO hybridized to each mutation-positive sample was identified by eluting and sequencing the ASO.
• sequencing "C” and "G” bases only was sufficient to unambiguously identify the ASO sequence and therefore allowed unequivocal identification of the corresponding mutation in the DNA sample.
• the region of membrane containing each mutation- positive sample identified in the pool hybridization was excised and the disc of Biotrans membrane placed in 100 ml distilled water and heated at 95°C for 10 minutes to elute the bound ASO. After cooling to room temperature, the membrane disc was discarded, and the eluted ASO was subjected to chemical sequencing.
• Solid-phase chemical cleavage of the ASOs attached to a solid support was performed according to Rosenthal et al. (32) with minor changes. This method permitted simultaneous sequencing of all bound ASOs in a single reaction vessel.
• a small, labeled piece (6 mm x 3 mm) of CCS paper (32) was immersed in each tube containing eluted ASO, and incubated at 65°C for 1 hour. All pieces of paper were then combined into a single 50 ml tube containing 25 ml of distilled water. The papers were then washed at room temperature 3 times (30 sec. /wash) with distilled water (25 ml/wash) followed by 3 washes (30 sec. /wash) with 96% ethanol (25 ml/wash) . Papers with attached ASOs could be batch washed without cross- contamination.
• the eluted ASOs were recovered from the top portion of the Microcon-3 TM concentrator by 3 serial rinses with 20 ⁇ l distilled water/rinse, and the fractions pooled. The samples were lyophilized in a UniVapoTM concentrator
• sequencing reactions included a pool of oligonucleotide templates with each template (77-mer) consisting of a 3 ' region (17bp) as the primer binding site uniquely complementary to a specific ASO, and a second unique region (17bp) consisting of an "ASO-specific identifier sequence" . Sequencing products were only observed when an eluted ASO was bound to the complementary region of a unique template, acted as a primer and permitted cycle sequencing to reveal the identity of the downstream "ASO-specific identifier sequence" .
• the cycle sequencing reactions contained a pool of ASO-specific templates (5 f oles/template) , 0.5 ⁇ l of Thermosequenase buffer concentrate (Amersham Life Science, Cleveland, OH), 0.125 ⁇ l of Thermosequenase (32U/ ⁇ l) , and either l G' termination mix (15mM dATP, 15mM dCTP, 15mM dTTP, 15mM 7-deaza-dGTP and 4mM ddGTP) or 'C termination mix (15mM dATP, 15mM dGTP, 15mM dTTP, 15mM 7-deaza-dGTP and 4mM ddCTP) in a reaction volume of 8 ⁇ l.
• Cycle sequencing was performed between 95°C for 30 seconds and 70°C for 1 minute for 30 cycles, followed by a 2 minute incubation at 70°C. Sequencing products were resolved on a 15% acrylamide/7M urea gel before being exposed to Kodak X-Omat
• an ASO is incubated with the complementary oligonucleotide in a Sanger sequencing reaction, and the sequence is determined directly.
• Version 2 Cycle sequencing of eluted ASO
• an ASO serves as a primer for a single extension reaction.
• the extension product is then subjected to cycle sequencing, using the universal primer to prime the sequencing reaction (see Example 5 below.) .
• an ASO serves as a primer for a single extension reaction.
• the extension product is then amplified using the universal primer sequence and the ASO as amplification primers.
• the amplification products are subjected to Sanger sequencing using as a primer an oligonucleotide corresponding to the sequencing target (see Example 6 below. ) .
• Example 5 Cycle Sequencing of ASOs
• the complementary oligonucleotide (Version 2 in Example 4 above) contains a universal primer sequence at its 5' end, separated by 25-30 bases from the complement to R334 at its 3 ' end.
• the extension reaction contains the following components:
• the reaction is allowed to proceed at room temperature for 30 minutes.
• ddNTPs dideoxynucleotide analogues
• a separated mutation-specific oligonucleotide, designated R334W and having the sequence 5 ' -TTCCAGAGGATGATTCC-3 ' is added to a reaction mix containing reaction components for extension as in Example 5, Step A.
• the complementary oligonucleotide (Version 3 in Example 4 above) contains a universal primer sequence at its 5' end, a "tag” sequence, "sequencing target” sequence, followed by the complement to R334W at its 3 ' end.
• an aliquot of the reaction is added to an amplification mixture containing the following components: 3 ⁇ l extension products
• the reaction is then subjected to 35 cycles of amplification, using a GeneAmp PCR System 9600 Thermocycler. 2 ⁇ l of the amplification products are then removed and subjected to Sanger sequencing, using the Sanger sequencing primer.
• RNA may also be used as a target nucleic acid.
• Cells are collected by centrifugation, and the supernatant is removed.
• the cell pellet is suspended in cold lysis buffer (140 mM NaCl, 1.5 mM MgCl 2 , 1.0 mM Tris- Cl, pH 8.5, 0.5% NP-40, and Rnasin® (Promega, Inc.)) .
• Cellular debris is pelleted by centrifugation for 5 minutes at 4°C at 5000 x g. The supernatant is transferred to a fresh tube and the EDTA concentration brought to 10 mM.
• Proteins are removed by extraction with phenol-chloroform saturated with aqueous 10 mM Tris, pH 8.5. The aqueous phase is precipitated with sodium acetate at pH 5.2 and 2.5 volumes of ice cold ethanol overnight at 10°C RNA is collected by centrifugation at 10,000 x g at 4°C for 30 minutes.
• RNA may be used directly in the manner of the present invention, or converted to amplified DNA via a reverse transcription PCR protocol.
• 1 ⁇ g of RNA is mixed with 100 pmol of appropriate primers, lmM dNTPs, lU/ ⁇ l RNasin® in 20 ⁇ l PCR buffer (50 mM KCl, 20 mM Tris, pH 8.4, 2.5 mM MgCl 2 ) and 200 U of reverse transcriptase.
• the mixture is incubated at 23°C for
• Protocols similar to those described in Example 1, may be used to amplify the resultant cDNA.
• Oligonucleotides are hybridized to immobilized nucleic acid targets in a similar manner as described in Example; 2 above, except that each ASO in the pool is labeled with a unique fluorescent probe instead of 3 P.
• ASOs designated ⁇ F508M, G542XM, G55IDM and R553XM are labeled with Texas Red, tetramethylrhodamine, fluorescein, and Cy3, respectively.
• bound ASOs can be detected as having been bound prior to separation.
• ASO binding is detected by fluorescence of the conjugated label either visually or by any number of automated methods. After separation, the ASO can be positively identified by measuring emission wavelength in response to fluor excitation.
• Oligonucleotides are hybridized to immobilized nucleic acid targets in a similar manner as described in Example 2 above, except that each ASO in the pool is additionally labeled with a unique molecular weight modifying entity.
• the four ASOs described in Example 8A are each derivatized with a 5 ' oligomeric hexaethyleneoxide (HEO) tail of differing length.
• ASOs designated ⁇ F508M, G542XM, G551DM and R553XM can be labeled with lengths of 5, 10, 15 and 20 HEO units, respectively.
• the tails are added using standard DNA synthesis protocols such as those described in Nucleic Acid Res, 22: 4527.
• the HEO tail does not participate in hydrogen bonding but does give a unique molecular weight to each ASO.
• the ASO can be identified without further modification by distinguishing the separated ASOs by molecular weight, using any number of commonly recognized methods, such as gel or capillary electrophoresis.
• An additional method of utilizing molecular weight identification of the hybridizing polymer is to add an additional number of nucleotides to the polymer enzymatically after separation from target nucleic acid.
• the separated polymer after hybridization to the immobilized nucleic acid target, is collected into a tube containing oligonucleotides, each of which is complementary to one member of the polymer pool used to probe the target nucleic acid.
• the oligonucleotide also contains an additional sequence, the length of which is unique for that oligonucleotide.
• the polymer When the polymer and oligonucleotide hybridize, the polymer can subsequently be used as a primer to enzymatically extend the polymer to the full length of the complementary oligonucleotide.
• a direct or indirect label as described above, may be incorporated.
• the extended oligonucleotide can be identified by determining the relative molecular weight of the labeled product by any number of established methods, such as gel or capillary electrophoresis.
• Ligation based techniques are known for identifying polymers probes that have perfectly hybridized to a sample. Ligation is often used in such techniques to distinguish perfect from imperfect hybridization at the junction of adjacent polymer probes. This is particularly useful for determining genetic alterations. (Landegren et al., 1988, Science 241:1078) .
• LCR is one technique that results in amplification of the ligated products and can be used to aid in obtaining sufficient copies of the product to determine the presence of the target sequence.
• Gap-LCR is a modification of LCR that reduces the background generated by target-independent ligation. Other ligation related amplification techniques are listed above.
• Figure 5 schematically depicts some examples of ligation techniques that can be used in the present invention.
• polymers form ligation probes that, e.g. , flank the site of a genetic alteration.
• One of the polymers (or probes) of each pair has a capture molecule attached.
• the other has a reporter molecule attached.
• any non-hybridized polymers may be washed away. Washing away of unhybridized polymers is not always necessary, although it is strongly preferred, particularly where a large number of polymers is reacted with the samples (an excess of unhybridized polymers can interfere with identification of ligated polymers by e.g. sequencing) . Nor does the sample need to be immobilized since it.
• the ligated products can then be amplified to allow easier identification. For example, LCR thermocycling accomplishes this. Alternately, it is possible to amplify the ligated products using other techniques, such as PCR. It is also possible to amplify the amount of sample DNA prior to the hybridization step so that, if a sufficient quantity of polymer probes is used, the ligated products will not need to be amplified.
• the hybridized and ligated polymers are then captured on a solid support.
• the presence of the reporter molecule is then determined. If the reporter is present, then ligation has occurred, and the presence of the target molecule determined.
• Models Various schemes to identify the target sequence that the ligated polymers are specific for will be apparent to one skilled in the art. Four such schemes are depicted in Figures 6-9, indicated as "Models" 1-4.
• Model 1 Figure 6
• Genetic alterations, target nucleic acid sequences, or randomly permuted alterations that resulted in ligation of the polymers are identified through chemical cleavage sequencing of the products, i.e., Maxam-Gilbert type sequencing.
• Model 2 Figure 7
• the ligation products are sequenced in a conventional Sanger type sequencing reaction. Specifically, after the ligation products are captured on a solid phase, a heterogeneous population of sequences is added that includes a sequence that hybridizes to the ligation product.
• ligation products are directly sequenced by conventional Sanger sequencing using heterogeneous primers that are complementary to sequences in the "B" portion of the ligation product depicted. Alternately, sequencing is done using a primer complementary to a common 3 ' tether added to the "B" probes.
• the ligation product is used as a template for a linear amplification using a universal priming sequence. The reaction can be performed with the product either attached to a solid phase or in solution. The products are conventionally sequenced by Sanger sequencing using a primer complementary to the tether sequence.
• the amplification refractory mutation system is a known PCR type system for determining mutations and can be used to practice the present invention.
• polymers are synthesized that are complementary to a sequence that may contain a mutation, and that act as primers when hybridized to the target DNA.
• Primers that are complementary to wild type sequence(s) are unlabelled.
• Primers that are complementary to a mutant sequence(s) are labelled.
• a second polymer is synthesized that is designed to act as a primer allowing PCR amplification when used in combination with the first set of primers.
• the second polymer is attached to one member of a binding pair, e.g., biotin, that allows capture of amplified PCR products on a solid phase.
• the polymer/primers are hybridized to the sample, and PCR thermocycling is then carried out.
• the amplified products are then exposed to a solid phase having a binding partner (e.g. avidin) attached to its surface. Presence of a signal on the amplified products bound to the support surface indicates that a mutant sequence was present in the sample.
• the bound products can then be identified using methods described above, e.g. Sanger sequencing.
• Invention methodologies provided herein retain the capacity for large sample throughput while reducing the number of hybridizations involved in performing multiple mutation analysis. If the number of probes for any diagnostic test is extremely large, the large number of independent hybridizations to be performed on pool positive samples reduces the cost effectiveness of the invention methodology. Using the MASDA approach of the instant invention, the disadvantages of both individual sample hybridizations and independent probe hybridizations are avoided. By eluting and interrogating the sequence of the mutation-specifie oligonucleotides hybridized to a DNA sample, MASDA eliminates the need for secondary independent hybridizations.
• MASDA is an extremely flexible, modular platform. This is very- important in a field such as genetic diagnostics, where the number of relevant genes, and mutations identified in each gene, change rapidly. Having oligonucleotide probes in solution, it is possible to mix and match probes on demand, therefore allowing clinical laboratories to cost- effectively customize diagnostic assays. There is also flexibility in sample preparation, and target detection.
• the sample nucleic acid can be either DNA or RNA.
• PCR was utilized as the amplification procedure.
• the present invention MASDA
• MASDA is compatible with any amplification technology, and does not require any processing of amplification products prior to mutation detection and identification. This becomes a very- important issue when large numbers of samples need to be analyzed in a single assay. Since the sample nucleic acid does not need to be fragmented, long PCR products can be analyzed, as well as the multiplex amplicons demonstrated in this study.
• the preesnt invention MASDA will facilitate the development of oligonucleotide libraries representative of previously identified expressed sequence tags or bi- allelic markers identified within the human genome.
• Phelps R..,, Haugen-Strano, A., Katcher, H. , Yaku o, K. , Gholami, Z. Shaffer, D. , Stone, S., Bayer, S., Wray, C, BBooqgddee ,, RR..,, Dayananth, P., Ward, J. , Tonin, P., Narod, S., Bristow, P. K. , Norris, F. H. , Helvering, L. , Morrison, P., Rosteck, P., Lai, M. , Barrett, J. C, Lewis, C, Neuhausen, S., Cannon-Albright, L., Goldgar, D.

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