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

Definitions

• This invention relates to detection of differences between nucleic acids. More particularly, the invention relates to rapid and accurate detection of differences between selected nucleic acids by electrospray mass spectrometry.
• Sequencing a sample in which the locus for a polymorphism is identical for both chromosomes (a homozygote) generally provides a clean signal of the base composition for the polymorphic locus.
• the sequencing signal is complex.
• base-substitution polymorphisms sequencing generates a multiple base signal for the polymorphic position.
• base-deletion or base-insertion polymorphisms sequencing generates multiple base signals starting at the polymorphic locus and propagating throughout the rest of the sequence.
• OLA oligonucleotide ligation assay
• mismatch PCR mismatch PCR
• engineered restriction enzyme site analysis were developed.
• two primers are designed to anneal adjacently on a genomic, amplified template.
• thermocycling in the presence of a thermostable ligase covalently links the primers if they anneal to the template without a gap between them and without a mismatch at the ends to be joined.
• This technique, and other similar techniques is simple and about 5- times faster than sequencing, but it suffers from the drawback of requiring sequence-specific optimization to reduce false positives or false negatives.
• SBH sequence-based hybridization
• the size of the amplified DNA segment is to be limited only by functionality in detecting mass differences between the mass of the amplified DNA segment and the reference mass, the size of the amplified DNA segment is preferably no greater than about 55 base pairs, more preferably no greater than about 50 base pairs, and most preferably no greater than about 45 base pairs.
• a method of detecting heterozygosity at a polymorphic site in the genome of an individual, wherein the polymorphic site comprises a site at which a base substitution or base deletion or insertion occurs in a mutant allele as compared to a wild type allele comprises the steps of:
• a method of detecting a polymorphism at a polymorphic site comprising a base substitution or a base deletion or insertion comprises the steps of:
• FIG. 1 shows a mass-to-charge (m/z) spectrum of a 53-base oligonucleotide (SEQ ID N0:1), wherein the values in brackets denote measured m/z values and the superscripts denote calculated charges.
• FIG. 2 shows a molecular weight transform spectrum obtained from the mass-to-charge spectrum of FIG. 1.
• FIG. 3 shows an m/z spectrum of PCR-amplified DNA.
• FIG. 4 shows a molecular weight transform spectrum obtained from the m/z spectrum of FIG. 3.
• FIG. 5 shows (A) a molecular weight transform spectrum obtained from PCR-amplified DNA wherein the template genomic DNA displayed homozygosity at the polymorphic site, and (B) a molecular weight transform spectrum obtained from PCR-amplified DNA wherein the template genomic DNA displayed heterozygosity at the polymorphic site.
• FIG. 6 shows molecular weight transform spectra of pairwise mixtures of synthetic 53-residue oligonucleotides, wherein the paired oligonucleotides differ only by a single base, as follows: (A) C and A;
• FIG. 7 shows molecular weight transform spectra showing detection of alleles for a C to T base substitution polymorphism according to the present invention: (A) heterozygous mother,- (B) heterozygous father; (C) homozygous daughter 1; (D) homozygous daughter 2; (E) theoretical molecular weights of amplified DNA strands from allele 1; (F) theoretical molecular weights of amplified DNA strands from allele
• FIG. 8 shows molecular weight transform spectra showing detection of alleles for a two-base deletion polymorphism according to the present invention: (A) heterozygous mother; (B) father homozygous for allele
• an amplified segment includes reference to two or more of such amplified segments
• a polymerase includes reference to a mixture of two or more of such polymerases
• reference to “a primer” includes reference to two or more of such primers.
• genomic DNA or similar terms includes reference to transcription products of such genomic DNA. It is well known in the art that RNA can be readily copied as cDNA, which can be amplified by PCR. Thus, amplification of genomic DNA and amplification of cDNA obtained from RNA are considered equivalent.
• Mass spectrometry provides a new method of DNA sequence analysis because differences in sequence composition can be measured from mass differences.
• Mass spectrometry is capable of very rapid sample analysis. For example, mass analysis of sequencing reaction products by electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) achieves mass analysis detection of all the reaction products with a 20-second sampling of the ion beam. Even though most mass spectrometric detectors are capable of determining the mass of components in a sample in less than a second, the rate for sample analysis is limited to about 15 minutes per sample due to other factors, such as signal averaging and sample handling. Matrix-assisted laser desorption ionization time-of- flight mass spectrometry (MALDI-TOF) has been demonstrated as a viable technique for analysis of polymorphisms. L.Y. Ch'ang et al., Detection of delta
• Mass spectrometric analysis has two stages, ionization and ion analysis.
• the ionization process employs either electrospray ionization (ESI) or Matrix-assisted laser desorption ionization (MALDI) .
• the typical analysis hardware for either of these two ionizers can include ion trap (IT) , time-of-flight (TOF) , quadrupole, or Fourier transform ion cyclotron resonance (FTICR) analyzers.
• ESI is typically interfaced with a quadrupole analyzer
• MALDI is generally coupled with a TOF analyzer.
• the accuracy of mass analysis using these methods is approximately as shown in Table l.
• Table 2 shows the absolute value of the mass differences for these six types of substitutions in double-stranded DNA.
• Table 3 shows the absolute value of the mass differences for these six types of substitutions in single-stranded DNA.
• the mass differences for these six types of base substitutions are only 0 or 1 dalton.
• the mass differences range from 9-40 daltons. Therefore, for base-substitution DNA polymorphisms to be analyzed by their mass differences, the samples must be denatured to single strands.
• the separate mass analysis of each of the two strands of double-stranded DNA provides two independent measurements of allelic composition. ESI-quadrupole analysis is ideally suited for analysis of base substitutions because it is inherently single-strand analysis.
• An ESI-MS analyzer is capable of measuring very large ions because, as for all mass analyzers, the property measured is mass to charge ratio (m/z) , and not mass directly.
• Analysis of nucleic acids is generally performed in negative ion mode. As the nucleic acid enters the gas phase, the phosphate backbone oxyanions, all of which are charged in solution, are partially neutralized by proton adduction. The amount of neutralization of oxyanion charge varies between the molecules and thus results in a population of molecules with varying charge states by integral increments.
• mass analysis of a compound by mass spectrometry generates a series of m/z peaks, where the charge state of the peaks increases as the value of measured m/z decreases.
• ESI-MS analysis of a synthetic oligonucleotide containing 53 nucleotide residues generates a series of m/z peaks (FIG. 1) .
• the value inside the brackets is the measured m/z, and the superscript value is the calculated charge.
• M analyte mass
• m/z charge-to-mass ratio
• Z the value of the charge.
• This formula is used to calculate the charge from the measured value of two m/z peaks and their integral relationship of charge.
• the mass of the compound can be calculated from each m/z peak, and thus provide multiple measurements of molecular weight, as shown in Table 4.
• the average of the masses calculated from each m/z peak determines mass accuracy to within 0.01% mass error.
• a computer can automatically transform an m/z spectrum into a molecular weight spectrum according to the Fenn method, J.B. Fenn et al. , Electrospray Ionization for Mass Spectrometry of Large Biomolecules, 246 Science 64-70 (1989) , hereby incorporated by reference, and thus render a molecular weight peak for each related m/z series, as shown in FIG. 2.
• salt e.g. Na' or K +
• This salt adduction generates additional peaks in the ESI-MS spectrum.
• the two peaks to the right of the main peak are salt adducts of Na + and 2 Na 4 .
• salt adduction peaks become the primary species observed. This is undesirable for at least two reasons. First, the intensity of the signal is decreased by distributing the analyte mass over multiple peaks with varying amounts of salt adduction. Second, the mass spectrum of a complex mixture is confused by a much higher degree of m/z peak overlap.
• PCR primers are designed to flank a polymorphic locus such that the nucleotide base pairs involved in the polymorphism are not defined by the primers.
• the distance between the primers is about 1-5 nucleotides.
• amplification In heterozygous individuals, where the sequence composition at the locus is different for each chromosome (two different alleles) , amplification generates two slightly different dsDNA products. Using ESI-quadrupole mass spectrometry, the molecule weight of each of the strands of a dsDNA PCR product can be measured. Each strand can be assigned to an allele type by comparison of the observed molecular weight to the expected weight of the allele types.
• ESI-MS provides several advantages over methods known in the art.
• the ESI-MS technique is highly specific.
• the small size of amplified products enables observation of very narrow windows into the genome. Only the region of DNA between the DNA primers, i.e. the window, represents the genomic DNA.
• the primers define the polymerization of their opposite strand base pairs.
• the ESI-MS technique does not require empirical optimization of analysis conditions to elucidate polymorphy. Once the PCR amplification has been optimized for specificity and yield, the preparation of samples for mass analysis is the same for any amplified product. As a result, successful ESI-MS analysis appears to be dependent primarily on sample cleanup and to be independent of nucleotide composition. Electrophoretic techniques such as SSCP, however, require extensive exploration of running conditions to detect specific polymorphisms. Mismatch PCR and polymorphism selective restriction site engineering both are site-specific, thus must be re-optimized for each locus analyzed.
• Enhanced resolution of a T-to-A base-substitution polymorphism can be achieved on a quadrupole detector by substituting deoxyuridine for deoxythymidine in the PCR reaction protocol. This modification results in a 23-dalton separation between the alleles, thus enabling a quadrupole detector to readily distinguish the alleles of all types of base substitution polymorphisms in a 53-nucleotide substrate.
• ESI-MS is complementary to current technology by enabling confirmation of putative polymorphic loci and then rapid screening of sample sets.
• the ESI-MS technique improves reliability of diagnosis because it directly measures the mass of the alleles involved in a polymorphism, not their relative mobilities.
• ESI-MS analysis appears well suited for detecting base- substitution polymorphisms or base deletion/insertion polymorphisms, which are difficult to detect by current methods.
• Many disease genes result from nonsense, missense, and frame-shifting mutations. These mutations, which are primarily caused by base substitutions, or one- or two-base deletions or insertions, are readily detectable by ESI-MS analysis.
• ESI-MS can be used in the fields of disease gene detection, genotyping, tissue typing, and DNA forensics.
• ESI-MS analysis of a set of base substitution polymorphisms can be use to uniquely identify an individual, and thus may be a useful forensic tool.
• ESI-MS can also be used for tissue typing transplant or graft matching or for identifying pathogens, such as bacteria and viruses. This technique is also amenable to cancer dianostics by detecting the presence of a somatically mutated allele in either tissue biopsies or in blood.
• ESI-MS analysis can be used as a technique to confirm the content of polymorphisms by detecting the expected alleles and then to rapidly screen for the presence of the alleles in genomes.
• Genomic templates were prepared by phenol/chloroform extraction of blood or of biopsy material according to methods well known in the art, e.g. J. Sambrook et al. , Molecular Cloning: A Laboratory Manual (2d ed., 1989), hereby incorporated by reference. Oligonucleotides were either purchased from Genset or National Biosciences, or were synthesized according to methods well known in the art, e.g. S.A. Narang et al. , 68 Meth. Enzymol. 90 (1979); E.L. Brown et al. , 68 Meth. Enzymol. 109
• Taq polymerase was purchased from either Perkin-Elmer or as "PCR SUPERMIX" from Gibco/BRL. PCR was carried out in either a Model 9600 or a Model 2400 Perkin-Elmer thermocycler according to methods well known in the art, e.g. U.S. Patent No. 4,683,195; U.S. Patent No. 4,683,202; U.S. Patent No. 4,800,159; U.S. Patent No. 4,965,188; PCR Technology: Principles and Applications for DNA Amplification (H. Erlich ed., Stockton Press,
• PCR Protocols A guide to Methods and Applications (Innis et al . eds, Academic Press, San Diego, Calif., 1990), hereby incorporated by reference.
• PCR amplification was generally optimized at 40 cycles as follows: annealing for 30 seconds at
• the primers were SEQ ID NO:2 and SEQ ID NO:3.
• PCR product purification was performed by either reverse phase HPLC, molecular weight cut-off spin filtration, or ethanol precipitation.
• the PCR products were isolated by reverse phase HPLC, lyophilized to dryness, resuspended in deionized water, and then ethanol precipitated. The supernate was then removed from the precipitate by aspiration, and the precipitate was lyophilized once again. The dry precipitate was then dissolved in 80% methanol, 10 mM triethylamine (TEA) , and subjected to quadrupole mass analysis according to methods well known in the art.
• TAA triethylamine
• Reverse phase HPLC was performed on a Waters HPLC with buffer A: 100 mM TEA-bicarbonate, pH 7, and buffer B: 100 mM TEA-bicarbonate, pH 7, and 50% methanol. Gradient elution was with 0 to 100% buffer B in 60 minutes, and the column was a 4 mm x 300 mm PRP-3 from Hamilton. Molecular weight cut-off spin filters were MICROCON 3 and MICROCON 10 from Amicon. Ethanol precipitation was performed at a final concentration of 70% ethanol and 0.7 M ammonium acetate at -20°C for greater than 1 hour.
• Samples for ESI-MS analysis were solvated in 80% methanol, 10 mM TEA, and then were injected into a Sciex QE mass spectrometer through a charged capillary. Ions were measured as a mass-to-charge ratio spectrum and then transformed into molecular weight spectra by the Fenn method, supra .
• FIGS. 3 and 4 show, respectively, the m/z spectrum and the molecular weight transform spectrum of PCR products amplified according to this example. Four major peaks were observed (FIG. 4) , which are further characterized in Table 5.
• peaks I and II correspond to the sense and antisense strands of the predicted amplification product.
• peaks III and IV correspond to the 3 ' -mono-adenylation products of peaks I and II.
• Taq polymerase is known to add such adenylate residues to amplification products in PCR.
• Table 5 also shows that comparison of the masses measured from the four amplification products, peaks I-IV, to the expected masses for such products demonstrates a mass- determining accuracy to within 0.01% mass error.
• Example 2 In this example, the procedure of Example 1 was followed except that genomic templates were from an individual homozygous at a polymorphic site and from another individual heterozygous at the polymorphic site.
• SEQ ID N0:1 illustrates a portion of the sequence of the sense strand of one allele of the polymorphic site
• SEQ ID NO:7 illustrates a corresponding portion of the sequence of the sense strand sequence of the other allele at the polymorphic site.
• the two alleles differ only by a single base substitution.
• the mass analysis resulted in one peak for each of the four expected strands (FIG. 5A) .
• the mass analysis generated doublet peaks for each strand (FIG. 5B) .
• the molecular weight observed for each peak in the doublet corresponds to the expected molecular weight from each allele.
• the spectrum from the homozygote shows a peak for the expected adenylated antisense strand, but the spectrum from the heterozygote does not appear to have an expected doublet at this position.
• the three other doublet peaks in the spectrum from the heterozygote each provide independent measurements of heterozygosity, thus the inherent redundancy of the technique enhances its robustness.
• Example 3 In this example, oligonucleotides were synthesized to model all possible base substitution polymorphisms at the locus examined in Example 2. These four oligonucleotides were identical except for having a different base at the polymorphic site. The four oligonucleotides are SEQ ID N0:1, SEQ ID NO: 7,
• Example 4 In this example, the procedure of Example 3 is followed except that SEQ ID NO:10 is substituted for SEQ ID NO:7.
• the 23 dalton difference in molecular weight between dA and dU at the polymorphic locus results in the mixture of SEQ ID NO:8 and SEQ ID NO:10 being easily resolved.
• ESI-MS genotyping in a four-member family for a C to T base substitution polymorphism was performed at a silent polymorphism locus in a candidate gene for Benign Neonatal Familial Convulsions (BNFC) .
• the procedure was as in Example 1 except that the primers were SEQ ID NO:11 and SEQ ID NO:12.
• the molecular weight spectra demonstrate detection of alleles, namely two alleles in the heterozygotic mother (A) and father (B) , and only one allele in the homozygotic daughters (C and D) .
• ESI-MS analysis and assignment were performed without knowledge of which samples were homozygotic or heterozygotic. The assignment was confirmed by checking the sequence results from an automated sequencer that had previously been used to diagnose allele inheritance in the family. In comparison to the sequencing results, ESI-MS analysis demonstrates at least two advantages.
• ESI-MS generates data on the composition of each strand, sense and antisense, in the analysis of one sample, whereas automated sequencing requires two separate sequencing reactions, i.e. with a forward primer and a reverse primer, to achieve data on each strand.
• Example 6 In this example, ESI-MS technique was applied to disease inheritance detection. Analysis was performed on a family group with a defective allele for the Attenuated Polyposis Coli (APC) gene. The defective allele comprises a two-base deletion, which results in a 618 dalton difference between the PCR products of the alleles.
• FIGS. 8A and 8D show the presence of peaks that are not present in the spectra FIGS. 8B and 8C, indicating the presence of the mutant allele.

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