Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—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
  • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

• the present invention relates to a method for detecting variations in the sequences of nucleic acid fragments, particularly in the DNA sequences of genes or gene fragments in patient samples in relation to the wild type genes.
• Methods to scan or screen for mutations may be divided into two groups, i.e. those that identify mutations trough altered properties of heteroduplexes, i.e. base-paired molecules composed of one strand from the normal sequence and a complementary strand derived from the patient sample, and those that observe properties of single stranded molecules or of homoduplexes.
• heteroduplexes i.e. base-paired molecules composed of one strand from the normal sequence and a complementary strand derived from the patient sample
• Examples from the first category of methods are RNAse cleavage of mismatched positions in hybrids between an RNA strand and a complementary DNA strand (Myers R. M. et al. , Science 1985; 230.1242-1246) .
• Such enzyme systems may cleave most or all mismatched positions in DNA strands with a length of at least several hundred bases.
• An exemplary such enzyme system is T4 endonuclease VII (Youil R. et al, Proc. Natl. Acad. Sci. USA 1995; ___. ' 87-91).
• WO 93/20233 discloses a method for identifying a base pair mismatch at a site in a nucleic acid by labelling a single stranded target nucleic acid sequence at two sites on either side of the target site, fixing the doubly labelled nucleic acid to a solid support at one end, hybridizing a corresponding wild type nucleic acid fragment to the target sequence, exposing the nucleic acid hybrid to a mismatch-cleaving enzyme, and after a wash detecting the presence of both labels or one label, that on the end fixed to the solid support or that washed away.
• This method avoids electrophoretic separation, but is disadvantagous in that it inter alia does not permit use for the simultaneous screening of a plurality of different target sequences on a single solid support.
• the object of the present invention is to provide an improved method for identifying sequence discrepancies between nucleic acid sequences, such as normal and patient sample genes or gene fragments, by mismatch-techniques, like those listed above, but which does not use any gel electrophoretic separation, and which may readily be adapted to array formats for multiple screening purposes.
• this object is achieved by providing a single-stranded prototypical normal nucleic acid sequence, or standard (wild type) sequence, immobilized on a solid support, hybridizing a nucleic acid strand derived from a patient sample to the immobilized strand, subjecting the nucleic acid hybridization complex formed to (i) mismatch-induced cleavage or (ii) mismatch- restricted extension reactions, and detecting possible cleavage or extension-termination by optical measurement on the solid support.
• the nucleic acid sequences are preferably DNA sequences, such as genomic DNA sequences.
• sets of the same or different normal nucleic acid strands, especially DNA are immobilized in a linear or, preferably, 2-dimensional planar array to permit either several patient derived samples to be tested in parallel, or, more preferably, several nucleic acid sequences, especially gene fragments, derived from one individual to be tested at the same time.
• either the free ends of the patient DNA strands and/or the normal strands may be labelled, e.g. with a dye, such as with a fluorophore or a chromophore.
• the number of DNA strands bound to their respective positions may be estimated by measuring the local label signal, e.g. fluorescence. Then, cleavage of DNA heteroduplexes at mutation positions is performed using any of the above- mentioned techniques, followed by washes, to remove single strands or base-paired DNA segments, depending on whether the molecules have been cleaved in one or both strands. In general, it is thus desirable to attach the molecules to the support so that the strands that bear detectable functions are not removed under denaturing conditions, unless they have been cleaved. Examples of linkages of the DNA to the support that are suitable include binding by a biotin-avidin/streptavidin interaction or covalent bonds, e.g.
• extension products may be labelled by incorporating detectable functions as modified nucleotides during the extension reaction.
• Measurement of cleavage or extension termination may also be performed by optical "label-free" techniques, such as, for example, mass or refractive index sensing techniques based on evanescent wave sensing, such as surface plasmon resonance (SPR) based methods.
• optical "label-free" techniques such as, for example, mass or refractive index sensing techniques based on evanescent wave sensing, such as surface plasmon resonance (SPR) based methods.
• Differences in the tendency to non-specific cleavage as a function of factors such as hybrid length, base composition or curvature may be weighted in as a background against which to compare the results of the analysis.
• the above method permits a very large number of templates to be simultaneously analyzed in the described manner, and also relatively small contributions of mutant sequences may give rise to a detectably different signal.
• oligonucleotides arrayed on a two- dimensional support may be used to ligate one strand from a specific PCR product by using primers that provide a 5' single stranded extension, as described by Newton C. R., Nucl. Acids Res. 1993; _ ⁇ - 1155-1162.
• This technique would permit the assortment of large sets of different PCR products to the appropriate positions in the array by hybridization to the corresponding immobilized oligonucleotides via a splint.
• the arrays themselves supporting oligonucleotides or longer nucleic acid strands, such as DNA strands, in defined positions and to which the templates may be immobilized may be prepared by any of a number of techniques known in the art.
• One such technique which is described in the International patent application No. PCT/SE95/01420, involves the bundling of structures containing DNA strands, followed by sectioning and deposition on a planar surface.
• the oligonucleotides may be bound to the solid support via a specific binding pair, such as biotin and avidin or streptavidin.
• the primers can be provided with biotin handles in connection with their preparation, and then the biotin-labelled oligonucleotides can be attached to a streptavidin-coated support.
• the oligonucleotides can also be bound by a linker arm, such as a covalently bonded hydrocarbon chain, e.g. a C ⁇ o-20 chain.
• the oligonucleotides can be bound directly to the solid support, such as by epoxide/amine coupling chemistry.
• the solid support can be a plate or chip of glass, silicon or other material.
• the solid support can also be coated, such as with gold or silver. Coating may facilitate attachment of the oligonucleotides to the solid support.
• Measurement of the fluorescence from fluorophore- labelled duplexes may be performed by methods known in the art, such as by a two-dimensional diode array or a CCD (charge-coupled device) camera, for example.
• CCD charge
• the patient samples may be simply labelled by selecting a 5' fluorophore-labelled primer in the amplification reaction used for amplifying the DNA strand or strands of interest. It is also possible to label the standard sequence on the solid support in the 3 ' position by tailing with terminating fluorophore-labelled nucleotides (Prober J. M. et al. , Science 1987; 238: 336- 341) , and using the enzyme terminal deoxynucleotidyl transferase (Maniatis T. et al. , Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Press, 1982) .
• the hybridization of patient strands to the arrayed templates is simplified if these are first rendered single- stranded, e.g. by digestion with lambda exonuclease (Higuchi R. G. et al. , Nucl. Acids Res. 1989; 12(14): 1989, and Nikiforov T. T. et al., PCR Meth. Applic. 1994; 3_: 285- 291) .
• the fluorophore label which is present at the 5' end of the amplified strands, conveniently protects this strand against degradation.
• the method of the invention may be mentioned the circumstance that a normal sequence may be selected as a standard against which to compare the patient sample, which means that both homozygous and heterozygous mutations may be monitored.
• the technique may have obvious advantages for scanning for clinically important mutations, ultimately in all estimated human 65,000 genes.
• the technique could also be useful as a forensic tool, rapidly identifying differences between DNA samples or for the typing of genes such as transplantation genes.
• the method of the invention would provide access to a much larger set of genetic markers than RPLFs or microsatellites, since any point mutation occurring in segments of DNA could be scored.
• the method of the invention would be highly useful to identify the location of homozygous genomic regions in individuals affected by recessive disorders by being homozygous by decent, i.e. having inherited the same mutated gene through parental lineages.
• PCR product from an individual having the normal form of the amplified globin gene is generated in a reaction where one of the primers has a 5' extension- sequence, interrupted by non-nucleotide residues, such as for instance hexaethylene glycol residues (HEG) (other oligonucleotide sequence modifications serving a similar purpose have been described by Newton et al., Nucl. Acids Res. 1993; 21:1155-1162).
• HOG hexaethylene glycol residues
• the 5* extension will remain single stranded and is used to hybridize and ligate the PCR product to a solid support which has previously been modified by the coupling of a suitable oligonucleotide with a free 3 'end, and to which a complementary oligonucleotide has been hybridized such that this hybridized oligonucleotide can also hybridize to the 5" extension of the PCR product, and permit the PCR product to be ligated to the oligonucleotide on the support.
• the free 3' end of the amplified strand remaining on the support is modified with a dideoxynucleotide with an added fluorophore, using the enzyme terminal deoxynucleotide transferase.
• a corresponding PCR product is derived from a patient in order to investigate if the globin gene in this patient differs from the normal sequence.
• a 5' phosphorylated primer lacking any non- nucleotidic sequences is used instead of the HEG-modified primer used for the amplification of the normal gene.
• the opposite primer is modified through the addition of a biotin 5' reside which will protect this strand from digestion by the 5' exonuclease ⁇ -exonuclease (another 5' modification, serving a similar purpose has been described by Nikiforov et al. , PCR Meth. Appl. 1994; -1:285-291) .
• An excess of the single strands from the amplified patient sample is then hybridized to the single strands bound to the support. After the fluorescence from the immobilized molecules has been recorded, the duplex molecules are exposed to reagents that cleave mismatched positions in duplex DNA, such as T4 endonuclease VII, as taught by Youil et al. , Proc.
• the above described example of carrying out the invention may be modified in two ways to increase the probability and speed of detecting any mutations.
• the supports may be designed so that not only the 5 ' end of the standard sequence but also the 3 ' end of the patient sample are stably attached to the support, e.g.
• the 5 ' end of the patient sample may be modified with a fluorophore, for instance by attaching a fluorophore instead of the biotin group in the example above.
• cleavage of either or both strands of the immobilized heteroduplex may be detected, increasing the probability of detecting mutations.
• the other suggested modification of the above protocol is by performing the analysis for a large number of samples in parallel. Thus, at defined locations on a 2-dimensional array the corresponding genes or gene fragments from many patients may be hybridized. Alternatively, and more importantly, many different gene sequences in one individual may be compared to standard variants of the corresponding sequences, immobilized in discrete locations.

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• 1995
  • 1995-01-30 SE SE9500341A patent/SE9500341D0/xx unknown
• 1996
  • 1996-01-30 WO PCT/SE1996/000095 patent/WO1996023903A1/en not_active Application Discontinuation
  • 1996-01-30 EP EP96902038A patent/EP0871766A1/de not_active Withdrawn
  • 1996-01-30 JP JP8523465A patent/JPH10513055A/ja active Pending
  • 1996-01-30 CA CA002211666A patent/CA2211666A1/en not_active Abandoned

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