WO1996029431A2 - Dna diagnostics based on mass spectrometry - Google Patents
Dna diagnostics based on mass spectrometry Download PDFInfo
- Publication number
- WO1996029431A2 WO1996029431A2 PCT/US1996/003651 US9603651W WO9629431A2 WO 1996029431 A2 WO1996029431 A2 WO 1996029431A2 US 9603651 W US9603651 W US 9603651W WO 9629431 A2 WO9629431 A2 WO 9629431A2
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- WIPO (PCT)
- Prior art keywords
- nucleic acid
- mass
- target nucleic
- acid sequence
- dna
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6862—Ligase chain reaction [LCR]
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
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- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
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Definitions
- DNA deoxyribonucleic acid
- chromosomes The Human Genome. T. Strachan. BIOS Scientific Publishers. 1992.
- Each gene codes for a specific protein which after its expression via transcription and translation, fulfills a specific biochemical function within a living cell. Changes in a DNA sequence are known as mutations and can result in proteins with altered or in some cases even lost biochemical activities; this in turn can cause genetic disease. Mutations include nucleotide deletions. insertions or alterations (i.e. point mutations). Point mutations can be either "missense”. resulting in a change in the amino acid sequence of a protein or "nonsense" coding for a stop codon and thereby leading to a truncated protein.
- CF Cystic Fibrosis
- Trisomy 21 Down's Syndrome.
- Trisomy 13 Panau Syndrome
- Trisomy 18 Edward's Syndrome.
- Monosomy X Tuner's Syndrome
- XXY Klienfelter's Syndrome
- certain DNA sequences may predispose an individual to any of a number of diseases such as diabetes. arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g. colorectal. breast. ovarian, lung).
- infectious organisms contain distinct nucleic acid sequences, which are different from the sequences contained in the host cell. Therefore. infectious organisms can also be detected and identified based on their specific DNA sequences.
- nucleic acid sequences Since the sequence of about 16 nucleotides is specific on statistical grounds even for the size of the human genome, relatively short nucleic acid sequences can be used to detect normal and defective genes in higher organisms and to detect infectious
- microorganisms e.g. bacteria, fungi, protists and yeast
- viruses DNA sequences can even serve as a fingerprint for detection of different individuals within the same species.
- nucleic acid sequences can be identified by comparing the mobility of an amplified nucleic acid fragment with a known standard by gel electrophoresis. or by hybridization with a probe, which is complementary to the sequence to be identified. Identification, however, can only be accomplished if the nucleic acid fragment is labeled with a sensitive reporter function (e.g. radioactive (32p. 35s). fluorescent or chemiluminescent). However, radioactive labels can be hazardous and the signals they produce decay over time. Non-isotopic labels (e.g.
- Electrophoresis is particularly error-prone, since the size or the molecular weight of the nucleic acid cannot be directly correlated to the mobility in the gel matrix. It is known that sequence specific effects, secondary structures and interactions with the gel matrix are causing artefacts.
- mass spectrometry provides a means of "weighing" individual molecules by ionizing the molecules in vacuo and making them “fly” by volatilization.
- nucleic acids are very polar biopolymers that are very difficult to volatilize. Consequently, mass spectrometric detection has been limited to low molecular weight synthetic oligonucleotides by determining the mass of the parent molecular ion and through this, confirming the already known oligonucleotide sequence, or alternatively, confirming the known sequence through the generation of secondary ions (fragment ions) via CID in an MS/MS configuration utilizing, in particular, for the ionization and volatilization. the method of fast atomic bombardment (FAB mass spectrometry) or plasma desorption (PD mass spectrometry).
- FAB mass spectrometry fast atomic bombardment
- PD mass spectrometry plasma desorption
- ES mass spectrometry has been introduced by Fenn et al. (J. Phys. Chem. 88, 4451 -59 ( 1984); PCT Application No. WO 90/14148) and current applications are summarized in recent review articles (R.D. Smith et al., Anal. Chem. 62, 882-89 (1990) and B. Ardrey. Electrospray Mass Spectrometry,
- MALDI mass spectrometry in contrast, can be particularly attractive when a time-of-flight (TOF) configuration is used as a mass analyzer.
- TOF time-of-flight
- the MALDI-TOF mass spectrometry has been introduced by Hillenkamp el al. ("Matrix Assisted UV-Laser
- the instant invention provides mass spectrometric processes for detecting a particular nucleic acid sequence in a biological sample.
- the processes can be used, for example, to diagnose (e.g. prenatally or postnatally) a genetic disease or chromosomal abnormality; a predisposition to a disease or condition (e.g. obesity, artherosclerosis, cancer), or infection by a pathogenic organism (e.g. virus, bacteria, parasite or fungus); or to provide information relating to identity, heredity, or compatibility (e.g. HLA phenotyping).
- a nucleic acid molecule containing the nucleic acid sequence to be detected i.e. the target
- Immobilization can be accomplished, for example, based on hybridization between a portion of the target nucleic acid molecule, which is distinct from the target detection site and a capture nucleic acid molecule, which has been previously immobilized to a solid support.
- immobilization can be accomplished by direct bonding of the target nucleic acid molecule and the solid support.
- there is a spacer e.g. a nucleic acid molecule between the target nucleic acid molecule and the support.
- a detector nucleic acid molecule e.g.
- an oligonucleotide or oligonucleotide mimetic which is complementary to the target detection site can then be contacted with the target detection site and formation of a duplex, indicating the presence of the target detection site can be detected by mass spectrometry.
- the target detection site is amplified prior to detection and the nucleic acid molecules are conditioned.
- the target detection sequences are arranged in a format that allows multiple simultaneous detections (multiplexing), as well as parallel processing using oligonucleotide arrays ("DNA chips").
- immobilization of the target nucleic acid molecule is an optional rather than a required step. Instead, once a nucleic acid molecule has been obtain from a biological sample, the target detection sequence is amplified and directly detected by mass spectrometry. In preferred embodiments, the target detection site and/or the detector oligonucleotides are conditioned prior to mass spectrometric detection. In another preferred embodiment, the amplified target detection sites are arranged in a format that allows multiple simultaneous detections (multiplexing), as well as parallel processing using oligonucleotide arrays ("DNA chips").
- nucleic acid molecules which have been replicated from a nucleic acid molecule obtained from a biological sample can be specifically digested using one or more nucleases (using deoxyribonucleases for DNA or ribonucleases for RNA) and the fragments captured on a solid support carrying the corresponding complementary sequences. Hybridization events and the actual molecular weights of the captured target sequences provide information on whether and where mutations in the gene are present.
- the array can be analyzed spot by spot using mass spectrometry.
- DNA can be similarly digested using a cocktail of nucleases including restriction endonucleases.
- the nucleic acid fragments are conditioned prior to mass spectrometric detection.
- At least one primer with 3' terminal base complementarity to an allele is hybridized with a target nucleic acid molecule, which contains the allele.
- An appropriate polymerase and a complete set of nucleoside triphosphates or only one of the nucleoside triphosphates are used in separate reactions to furnish a distinct extension of the primer. Only if the primer is appropriately annealed (i.e. no 3' mismatch) and if the correct (i.e. complementary) nucleotide is added, will the primer be extended. Products can be resolved by molecular weight shifts as determined by mass spectrometry.
- a nucleic acid molecule containing the nucleic acid sequence to be detected (i.e. the target) is initially immobilized to a solid support.
- Immobilization can be accomplished, for example, based on hybridization between a portion of the target nucleic acid molecule, which is distinct from the target detection site and a capture nucleic acid molecule, which has been previously immobilized to a solid support.
- immobilization can be accomplished by direct bonding of the target nucleic acid molecule and the solid support.
- there is a spacer e.g. a nucleic acid molecule between the target nucleic acid molecule and the support.
- a nucleic acid molecule that is complementary to a portion of the target detection site that is immediately 5' of the site of a mutation is then hybridized with the target nucleic acid molecule.
- the addition of a complete set of dideoxynucleosides or 3'-deoxynucleoside triphosphates e.g. pppAdd.
- pppTdd. pppCdd and pppGdd) and a DNA dependent DNA polymerase allows for the addition only of the one dideoxynucleoside or 3'-deoxynucleoside triphosphate that is complementary to X.
- the hybridization product can then be detected by mass spectrometry.
- a target nucleic acid is hybridized with a complementary oligonucleotides that hybridize to the target within a region that includes a mutation M.
- the heterodupiex is then contacted with an agent that can specifically cleave at an unhybridized portion (e.g. a single strand specific endonuclease), so that a mismatch, indicating the presence of a mutation, results in the cleavage of the target nucleic acid.
- the two cleavage products can then be detected by mass spectrometry.
- a target nucleic acid is hybridized with a set of ligation educts and a thermostable DNA ligase, so that the ligase educts become covalently linked to each other, forming a ligation product.
- the ligation product can then be detected by mass spectrometry and compared to a known value. If the reaction is performed in a cyclic manner, the ligation product obtained can be amplified to better facilitate detection of small volumes of the target nucleic acid. Selection between wildtype and mutated primers at the ligation point can result in the detection of a point mutation.
- the processes of the invention provide for increased accuracy and reliability of nucleic acid detection by mass spectrometry. In addition, the processes allow for rigorous controls to prevent false negative or positive results.
- the processes of the invention avoid electrophoretic steps: labeling and subsequent detection of a label. In fact it is estimated that the entire procedure, including nucleic acid isolation, amplification, and mass spec analysis requires only about 2-3 hours time. Therefore the instant disclosed processes of the invention are faster and less expensive to perform than existing DNA detection systems.
- the instant disclosed processes allow the nucleic acid fragments to be identified and detected at the same time by their specific molecular weights (an unambiguous physical standard), the disclosed processes are also much more accurate and reliable than currently available procedures.
- FIGURE 1 A is a diagram showing a process for performing mass
- TDS target detection site
- T target nucleic acid molecule
- C specific capture sequence
- S spacer
- the capture sequence is chosen to specifically hybridize with a complementary sequence on the target nucleic acid molecule (T). known as the target capture site (TCS).
- TCS target capture site
- S spacer
- a detector nucleic acid sequence (D) which is complementary to the TDS is then contacted with the TDS. Hybridization between D and the TDS can be detected by mass spectrometry.
- FIGURE 1B is a diagram showing a process for performing mass spectrometric analysis on at least one target detection site (here TDS 1 and TDS 2) via direct linkage to a solid support.
- the target sequence (T) containing the target detection site (TDS) is a diagram showing a process for performing mass spectrometric analysis on at least one target detection site (here TDS 1 and TDS 2) via direct linkage to a solid support.
- TDS 1 and TDS 2 is immobilized to a solid support via the formation of a reversible or irreversible bond formed between an appropriate functionality (L') on the target nucleic acid molecule (T) and an appropriate functionality (L) on the solid support.
- Detector nucleic acid sequences here Dl and D2
- TDS 1 or TDS 2 Detector nucleic acid sequences
- FIGURE 1 C is a diagram showing a process for detecting a wildtype (D wt ) and/ or a mutant (D mut ) sequence in a target (T) nucleic acid molecule.
- a specific capture sequence (C) is attached to a solid support (SS) via a spacer (S).
- the capture sequence is chosen to specifically interact with a complementary sequence on the target sequence (T), the target capure site (TCS) to be detected through hybridization.
- the target detection site (TDS) includes a mutation.
- X. which changes the molecular weight mutated target detection sites can be distinguished from wildtype by mass spectrometry.
- the detector nucleic acid molecule (D) is designed so that the mutation is in the middle of the molecule and therefore would not lead to a stable hybrid if the wildtype detector oligonucleotide (D wt ) is contacted with the target detector sequence, e.g. as a control.
- the mutation can also be detected if the mutated detector oligonucleotide (D mut ) with the matching base at the mutated position is used for hybridization.
- nucleic acid molecule obtained from a biological sample is heterozygous for the particular sequence (i.e. contain both D wt and D mut ). both D wt and D mut will be bound to the appropriate strand and the mass difference allows both D w and D mut to be detected simultaneously.
- FIGURE 2 is a diagram showing a process in which several mutations are simultaneously detected on one target sequence by employing corresponding detector oligonucleotides.
- the molecular weight differences between the detector oligonucleotides D1 , D2 and D3 must be large enough so that simultaneous detection (multiplexing) is possible. This can be achieved either by the sequence itself (composition or length) or by the introduction of mass-modifying functionalities M 1 - M3 into the detector oligonucleotide.
- FIGURE 3 is a diagram showing still another multiplex detection format. In this embodiment, differentiation is accomplished by employing different specific capture sequences which are position-specifically immobilized on a flat surface (e.g. a 'chip array').
- Detection is achieved by employing appropriately mass differentiated detector oligonucleotides Dl - Dn. which are mass differentiated either by their sequences or by mass modifying functionalities Ml - Mn.
- FIGURE 4 is a diagram showing a format wherein a predesigned target capture site (TCS) is incorporated into the target sequence using PCR amplification. Only one strand is captured, the other is removed (e.g., based on the interaction between biotin and streptavidin coated magnetic beads). If the biotin is attached to primer 1 the other strand can be appropriately marked by a TCS. Detection is as described above through the interaction of a specific detector oligonucleotide D with the corresponding target detection site TDS via mass spectrometry.
- TCS target capture site
- FIGURE 5 is a diagram showing how amplification (here ligase chain reaction (LCR)) products can be prepared and detected by mass spectrometry.
- Mass differentiation can be achieved by the mass modifying functionalities (M1 and M2) attached to primers (P1 and P4 respectively).
- Detection by mass spectrometry can be accomplished directly (i.e. without employing immobilization and target capturing sites (TCS)).
- Multiple LCR reactions can be performed in parallel by providing an ordered array of capturing sequences (C). This format allows separation of the ligation products and spot by spot identification via mass spectrometry or multiplexing if mass differentiation is sufficient.
- FIGURE 6A is a diagram showing mass spectrometric analysis of a nucleic acid molecule, which has been amplified by a transcription amplification procedure.
- An RNA sequence is captured via its TCS sequence, so that wildtype and mutated target detection sites can be detected as above by employing appropriate detector oligonucleotides (D).
- FIGURE 6B is a diagram showing multiplexing to detect two different
- FIGURE 6C is a diagram of a different multiplexing procedure for detection of specific mutations by employing mass modified dideoxynucleoside or 3'-deoxynucleoside triphosphates and an RNA dependent DNA polymerase. Alternatively. DNA dependent
- FIGURE 7A is a diagram showing a process for performing mass
- TDS target detection site
- T target nucleic acid molecule
- pppTdd. pppCdd and pppGdd) and a DNA dependent DNA polymerase allows for the addition only of the one dideoxynucleoside or 3'-deoxynucleoside triphosphate that is complementary to X.
- FIGURE 7B is a diagram showing a process for performing mass spectrometric analysis to determine the presence of a mutation at a potential mutation site (M) within a nucleic acid molecule.
- This format allows for simultaneous analysis of both alleles (A) and (B) of a double stranded target nucleic acid molecule, so that a diagnosis of homozygous normal, homozygous mutant or heterozygous can be provided.
- Allele A and B are each hybridized with complementary oligonucleotides ((C) and (D) respectively), that hybridize to A and B within a region that includes M.
- Each heteroduplex is then contacted with a single strand specific endonuclease. so that a mismatch at M, indicating the presence of a mutation, results in the cleavage of (C) and/or (D), which can then be detected by mass spectrometry.
- FIGURE 8 is a diagram showing how both strands of a target DNA can be prepared for detection using transcription vectors having two different promoters at opposite locations (e.g. the SP6 and the T7 promoter). This format is particularly useful for detecting heterozygous target detection sites (TDS).
- TDS heterozygous target detection sites
- RNAs can be specifically captured and simultaneously detected using appropriately mass-differentiated detector oligonucleotides. This can be accomplished either directly in solution or by parallel processing of many target sequences on an ordered array of specifically immobilized capturing sequences.
- FIGURE 9 is a diagram showing how RNA prepared as described in Figures 6. 7 and 8 can be specifically digested using one or more ribonucleases and the fragments captured on a solid support carrying the corresponding complementary sequences.
- Hybridization events and the actual molecular weights of the captured target sequences provide information on whether and where mutations in the gene are present.
- the array can be analyzed spot by spot using mass spectrometry.
- DNA can be similarly digested using a cocktail of nucleases including restriction endonucleases. Mutations can be detected by different molecular weights of specific, individual fragments compared to the molecular weights of the wildtype fragments.
- FIGURE 10A shows a spectra resulting from the experiment described in the following Example 1.
- Panel i) shows the absorbance of the 26-mer before hybridization.
- Panel ii) shows the filtrate of the centrifugation after hybridization.
- Panel iii) shows the results after the first wash with 50mM ammonium citrate.
- Panel iv) shows the results after the second wash with 50mM ammonium citrate.
- FIGURE 10B shows a spectra resulting from the experiment described in the following Example 1 after three washing/ centrifugation steps.
- FIGURE 10C shows a spectra resulting from the experiment described in the following Example 1 showing the successful desorption of the hybridized 26mer off of beads.
- FIGURE 1 1 shows a spectra resulting from the experiment described in the following Example 1 showing the successful desorption of the hybridized 40mer. The efficiency of detection suggests that fragments much longer than 40mers can also be desorbed.
- FIGURE 12 shows a spectra resulting from the experiment described in the following Example 2 showing the successful desorption and differentiation of an 18-mer and 19-mer by electrospray mass spectrometry. the mixture (top), peaks resulting from 18-mer emphasized (middle) and peaks resulting from 19-mer emphasized (bottom)
- FIGURE 13 is a graphic representation of the process for detecting the Cystic Fibrosis mutation ⁇ F508 as described in Example 3.
- FIGURE 14 is a mass spectrum of the DNA extension product of a ⁇ F508 homozygous normal.
- FIGURE 15 is a mass spectrum of the DNA extension product of a ⁇ F508 heterozygous mutant.
- FIGURE 16 is a mass spectrum of the DNA extension product of a ⁇ F508 homozygous normal.
- FIGURE 17 is a mass spectrum of the DNA extension product of a ⁇ F508 homozygous mutant.
- FIGURE 18 is a mass spectrum of the DNA extension product of a ⁇ F508 heterozygous mutant.
- FIGURE 19 is a graphic representation of various processes for performing apolipoprotein E genotyping.
- FIGURE 20 shows the nucleic acid sequence of normal apolipoprotein E
- FIGURE 21 A shows a composite restriction pattern for various genotypes of apolipoprotein E.
- FIGURE 21 B shows the restriction pattern obtained in a 3.5% MetPhor
- FIGURE 21 C shows the restriction pattern obtained in a 12% polyacrylamide gel for various genotypes of apolipoprotein E.
- FIGURE 22A is a chart showing the molecular weights of the 91. 83. 72, 48 and 35 base pair fragments obtained by restriction enzyme cleavage of the E2. E3 and E4 alleles of apolipoprotein E.
- FIGURE 22B is the mass spectra of the restriction product of a homozygous E4 apolipoprotein E genotype.
- FIGURE 23 A is the mass spectra of the restriction product of a homozygous E3 apolipoprotein E genotype.
- FIGURE 23B is the mass spectra of the restriction product of a E3/E4 apolipoprotein E genotype.
- FIGURE 24 is an autoradiograph of a 7.5% polyacrylamide gel in which 10% (5 ⁇ l) of each PCR was loaded.
- Sample M pBR322 AluI digested: sample 1 : HBV positive in serological analysis; sample 2: also HBV positive; sample 3: without serological analysis but with an increased level of transaminases.
- liver disease sample 4: HBV negative: sample 5: HBV positive by serological analysis: sample 6: HBV negative (-) negative control: (+) positive control). Staining was done with ethidium bromide.
- FIGURE 25A is a mass spectrum of sample 1. which is HBV positive.
- the signal at 20754 Da represents the HBV related PCR product (67 nucleotides. calculated mass: 20735 Da).
- the mass signal at 10390 Da represents the [M+2H] 2+ signal (calculated: 10378 Da).
- FIGURE 25B is a mass spectrum of sample 3. which is HBV negative corresponding to PCR. serological and dot blot based assays. The PCR product is generated only in trace amounts. Nevertheless it is unambiguously detected at 20751 Da (calculated: 20735 Da). The mass signal at 10397 Da represents the [M+2H] 2+ molecule ion (calculated: 10376 Da).
- FIGURE 25C is a mass spectrum of sample 4. which is HBV negative, but CMV positive. As expected, no HIV specific signals could be obtained.
- FIGURE 26 shows a part of the E. coli lacI gene with binding sites of the complementary oligonucleotides used in the ligase chain reaction (LCR).
- LCR ligase chain reaction
- FIGURE 27 is a 7.5% polyacrylamide gel stained with ethidium bromide.
- M chain length standard (pUC 19 DNA. MspI digested).
- Lane 1 LCR with wildtype template.
- Lane 2 LCR with mutant template.
- Lane 3 (control) LCR without template. The ligation product (50 bp) was only generated in the positive reactive containing wildtype template.
- FIGURE 28 is an HPLC chromatogram of two pooled positive LCRs.
- FIGURE 29 shows an HPLC chromatogram the same conditions but mutant template were used.
- the small signal of the ligation product is due to either template-free ligation of the educts or to a ligation at a (G-T. A-C) mismatch.
- the 'false positive' signal is significantly lower than the signal of ligation product with wildtype template depicted in Figure 28.
- the analysis of ligation educts leads to 'double -peaks' because two of the oligonucleotides are 5'- phosphorylated.
- FIGURE 30 In a the complex signal pattern obtained by MALDI-TOF-MS analysis of Pfu DNA-ligase solution is depicted. In b a MALDI -TOF-spectrum of an unpurified LCR is shown. The mass signal 67569 Da probably represents the Pfu DNA ligase.
- FIGURE 31 shows a MALDI-TOF spectrum of two pooled positive LCRs (a).
- the signal at 7523 Da represents unligated oligo A (calculated: 7521 Da) whereas the signal at 15449 Da represents the ligation product (calculated: 15450 Da).
- the signal at 3774 Da is the [M+2H] 2+ signal of oligo A.
- the signals in the mass range lower than 2000 Da are due to the matrix ions.
- the spectrum corresponds to lane 1 in figure 2a and to the chromatogram in figure 2b.
- In b a spectrum of two pooled negative LCRs (mutant template) is shown.
- the signal at 7517 Da represents oligo A (calculated: 7521 Da).
- FIGURE 32 shows a spectrum obtained from two pooled LCRs in which only salmon sperm DNA was used as a negative control, only oligo A could be detected, as expected.
- FIGURE 33 shows a spectrum of two pooled positive LCRs (a).
- the purification was done with a combination of ultrafiltration and streptavidin DynaBeads as described in the text.
- the signal at 15448 Da represents the ligation product (calculated: 15450 Da).
- the signal at 7527 represents oligo A (calculated: 7521 Da).
- the signals at 3761 Da is the [M+2H] 2+ signal of oligo A.
- the signal at 5140 Da is the [M+3H] 2 + signal of the ligation product.
- b a spectrum of two pooled negative LCRs (without template) is shown.
- the signal at 7514 Da represents oligo A (calculated: 7521 Da).
- FIGURE 34 is a schematic presentation of the oligo base extension of the mutation detection primer b using ddTTP (A) or ddCTP (B) in the reaction mix. respectively. The theoretical mass calculation is given in parenthesis. The sequence shown is part of the exon 10 of the CFTR gene that bears the most common cystic fibrosis mutation ⁇ F508 and more rare mutations ⁇ I507 as well as lle506Ser.
- FIGURE 35 is a MALDI-TOF-MS spectra recorded directly from precipitated oligo base extended primers for mutation detection. The spectra on the top of each panel (ddTTP or ddCTP, respectively) show the annealed primer (CF508) without further extension reaction. The template of diagnosis is pointed out below each spectra and the
- FIGURE 36 shows the portion of the sequence of pRFc1 DNA. which was used as template for PCR amplification of unmodified and 7-deazapurine containing 99-mer and 200-mer nucleic acids as well as the sequences of the 19-primers and the two 18-mer reverse primers.
- FIGURE 37 shows the portion of the nucleotide sequence of M13mp18 RFI DNA. which was used for PCR amplification of unmodified and 7-deazapurine containing 103-mer nucleic acids. Also shown are nucleotide sequences of the 17-mer primers used in the PCR.
- FIGURE 38 shows the result of a polyacrylamide gel electrophoresis of PCR products purified and concentrated for MALDI-TOF MS analysis.
- M chain length marker
- lane 1 7-deazapurine containing 99-mer PCR product
- lane 2 unmodified 99-mer
- lane 3 7-deazapurine containing 103-mer
- lane 4 unmodified 103-mer PCR product.
- FIGURE 39 an autoradiogram of polyacrylamide gel electrophoresis of PCR reactions carried out with 5'-[ 32 P]-labeled primers 1 and 4.
- Lanes 1 and 2 unmodified and 7 -deazapurine modified 103-mer PCR product (53321 and 23520 counts)
- lanes 3 and 4 lanes 3 and 4:
- FIGURE 40 a) MALDI-TOF mass spectrum of the unmodified 103-mer PCR products (sum of twelve single shot spectra). The mean value of the masses calculated for the two single strands (31768 u and 31759 u) is 31763 u. Mass resolution: 18. b) MALDI-TOF mass spectrum of 7-deazapurine containing 103-mer PCR product (sum of three single shot spectra). The mean value of the masses calculated for the two single strands (31727 u and 31719 u) is 31723 u. Mass resolution: 67.
- FIGURE 41 a) MALDI-TOF mass spectrum of the unmodified 99-mer PCR product (sum of twenty single shot spectra).
- FIGURE 42 a) MALDI-TOF mass spectrum of the unmodified 200-mer PCR product (sum of 30 single shot spectra). The mean value of the masses calculated for the two single strands (61873 u and 61595 u) is 61734 u. Mass resolution: 28. b) MALDI-TOF mass spectrum of 7-deazapurine containing 200-mer PCR product (sum of 30 single shot spectra). The mean value of the masses calculated for the two single strands (61772 u and 61514 u) is 61643 u. Mass resolution: 39.
- FIGURE 43 a) MALDI-TOF mass spectrum of 7-deazapurine containing 100-mer PCR product with ribomodified primers. The mean value of the masses calculated for the two single strands (30529 u and 31095 u) is 30812 u. b) MALDI-TOF mass spectrum of the PCR-product after hydrolytic primer-cleavage. The mean value of the masses calculated for the two single strands (25104 u and 25229 u) is 25167 u. The mean value of the cleaved primers (5437 u and 5918 u) is 5677 u.
- FIGURE 44 A-D shows the MALDI-TOF mass spectrum of the four sequencing ladders obtained from a 39-mer template (SEQ. ID. No. 13). which was immobilized to streptavidin beads via a 3' biotinylation.
- a 14-mer primer (SEQ. ID. NO. 14) was used in the sequencing.
- FIGURE 45 shows a MALDI-TOF mass spectrum of a solid state sequencing of a 78-mer template (SEQ. ID. No. 15). which was immobilized to streptavidin beads via a 3' biotinylation.
- a 18-mer primer (SEQ ID No. 16) and ddGTP were used in the sequencing.
- FIGURE 46 shows a scheme in which duplex DNA probes with single-stranded overhang capture specific DNA templates and also serve as primers for solid state sequencing.
- FIGURE 47A-D shows MALDI-TOF mass spectra obtained from a 5' fluorescent labeled 23-mer (SEQ. ID. No. 19) annealed to an 3' biotinylated 18-mer (SEQ. ID. No. 20), leaving a 5-base overhang, which captured a 15-mer template (SEQ. ID. No. 21 ).
- FIGURE 48 shows a stacking flurogram of the same products obtained from the reaction described in FIGURE 35, but run on a conventional DNA sequencer.
- the instant invention provides mass spectrometric processes for detecting a particular nucleic acid sequence in a biological sample.
- biological sample refers to any material obtained from any living source (e.g. human, animal, plant, bacteria, fungi, protist. virus).
- the biological sample should contain a nucleic acid molecule.
- appropriate biological samples for use in the instant invention include: solid materials (e.g tissue, cell pellets, biopsies) and biological fluids (e.g. urine, blood, saliva, amniotic fluid, mouth wash).
- Nucleic acid molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art. the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freezethaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials: heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine: and proteinase K extraction can be used to obtain nucleic acid from blood (Rolff. A et al. PCR: Clinical Diagnostics and Research. Springer ( 1994)).
- amplification may be necessary.
- appropriate amplification procedures for use in the invention include: cloning (Sambrook et al..
- a nucleic acid molecule containing a nucleic acid sequence to be detected can be immobilized to a solid support.
- solid supports include beads (e.g. silica gel. controlled pore glass, magnetic. Sephadex/Sepharose. cellulose), flat surfaces or chips (e.g. glass fiber filters, glass surfaces. metal surfaces (steel, gold, silver, aluminum, copper and silicon), capillaries, plastic (e.g. polyethylene, polypropylene, polyamide, polyvinylidenedifluoride membranes or microtiter plates)): or pins or combs made from similar materials comprising beads or flat surfaces or beads placed into pits in flat surfaces such as wafers (e.g. silicon wafers).
- beads e.g. silica gel. controlled pore glass, magnetic. Sephadex/Sepharose. cellulose
- flat surfaces or chips e.g. glass fiber filters, glass surfaces. metal surfaces (steel, gold, silver, aluminum, copper and silicon), capillaries, plastic (e
- Immobilization can be accomplished, for example, based on hybridization between a capture nucleic acid sequence, which has already been immobilized to the support and a complementary nucleic acid sequence, which is also contained within the nucleic acid molecule containing the nucleic acid sequence to be detected (FIGURE 1A). So that hybridization between the complementary nucleic acid molecules is not hindered by the support, the capture nucleic acid can include a spacer region of at least about five nucleotides in length between the solid support and the capture nucleic acid sequence. The duplex formed will be cleaved under the influence of the laser pulse and desorption can be initiated.
- the solid support-bound base sequence can be presented through natural oligoribo- or oligodeoxyribonucleotide as well as analogs (e.g. thio-modified phosphodiester or phosphotriester backbone) or employing oligonucleotide mimetics such as PNA analogs (see e.g. Nielsen et al. Science, 254. 1497 (1991)) which render the base sequence less susceptible to enzymatic degradation and hence increases overall stability of the solid support-bound capture base sequence.
- analogs e.g. thio-modified phosphodiester or phosphotriester backbone
- PNA analogs see e.g. Nielsen et al. Science, 254. 1497 (1991)
- a target detection site can be directly linked to a solid support via a reversible or irreversible bond between an appropriate functionality (L') on the target nucleic acid molecule (T) and an appropriate functionality (L) on the capture molecule (FIGURE 1B).
- a reversible linkage can be such that it is cleaved under the conditions of mass spectrometry (i.e., a photocleavable bond such as a charge transfer complex or a labile bond being formed between relatively stable organic radicals).
- the linkage can be formed with L' being a quaternary ammonium group, in which case, preferably, the surface of the solid support carries negative charges which repel the negatively charged nucleic acid backbone and thus facilitate the desorption required for analysis by a mass spectrometer. Desorption can occur either by the heat created by the laser pulse and/or. depending on L.' by specific absorption of laser energy which is in resonance with the L' chromophore.
- the L-L' chemistry can be of a type of disulfide bond (chemically cleavable. for example, by mercaptoethanol or dithioerythrol), a
- L and L,' can also form a charge transfer complex and thereby form the temporary L-L' linkage. Since in many cases the "charge-transfer band" can be determined by UV/vis spectrometry (see e.g. Organic Charge Transfer Complexes by R. Foster. Academic Press, 1969), the laser energy can be tuned to the corresponding energy of the charge-transfer wavelength and. thus, a specific desorption off the solid support can be initiated. Those skilled in the art will recognize that several combinations can serve this purpose and that the donor functionality can be either on the solid support or coupled to the nucleic acid molecule to be detected or vice versa.
- a reversible L-L' linkage can be generated by homolytically forming relatively stable radicals. Under the influence of the laser pulse, desorption (as discussed above) as well as ionization will take place at the radical position.
- desorption as discussed above
- ionization will take place at the radical position.
- other organic radicals can be selected and that, in relation to the dissociation energies needed to homolytically cleave the bond between them, a corresponding laser wavelength can be selected (see e.g. Reactive Molecules by C. Wentrup. John Wiley & Sons. 1984).
- An anchoring function L' can also be incorporated into a target capturing sequence (TCS) by using appropriate primers during an amplification procedure, such as PCR (FIGURE 4).
- PCR FIGGURE 4
- LCR FIGGURE 5
- transcription amplification FIG. 6A
- nucleic acid molecules Prior to mass spectrometric analysis, it may be useful to "condition" nucleic acid molecules, for example to decrease the laser energy required for volatization and/or to minimize fragmentation. Conditioning is preferably performed while a target detection site is immobilized.
- An example of conditioning is modification of the phosphodiester backbone of the nucleic acid molecule (e.g. cation exchange), which can be useful for eliminating peak broadening due to a heterogeneity in the cations bound per nucleotide unit.
- an alkylating agent such as alkyliodide. iodoacetamide. ⁇ -iodoethanol. or 2,3-epoxy-1-propanol.
- the monothio phosphodiester bonds of a nucleic acid molecule can be transformed into a phosphotriester bond.
- phosphodiester bonds may be transformed to uncharged derivatives employing trialkylsilyl chlorides.
- Further conditioning involves incorporating nucleotides which reduce sensitivity for depurination (fragmentation during MS) such as N7- or N9-deazapurine nucleotides, or RNA building blocks or using oligonucleotide triesters or incorporating phosphorothioate functions which are alkylated or employing oligonucleotide mimetics such as PNA.
- Multiplexing can be achieved by several different methodologies. For example, several mutations can be simultaneously detected on one target sequence by employing corresponding detector (probe) molecules (e.g.
- oligonucleotides or oligonucleotide mimetics oligonucleotides or oligonucleotide mimetics.
- the molecular weight differences between the detector oligonucleotides D1, D2 and D3 must be large enough so that simultaneous detection (multiplexing) is possible. This can be achieved either by the sequence itself (composition or length) or by the introduction of mass-modifying
- Mass modifying moieties can be attached, for instance, to either the 5'-end of the oligonucleotide (M 1 ). to the nucleobase (or bases) (M 2 , M 7 ). to the phosphate backbone (M 3 ), and to the 2'-position of the nucleoside (nucleosides) (M 4 , M 6 ) or/and to the terminal 3'-position (M 5 ).
- mass modifying moieties include . for example, a halogen, an azido. or of the type. XR. wherein X is a linking group and R is a mass-modifying
- the mass-modifying functionality can thus be used to introduce defined mass increments into the oligonucleotide molecule.
- the mass-modifying moiety. M can be attached either to the nucleobase. M 2 (in case of the c 7 -deazanucleosides also to C-7. M 7 ). to the triphosphate group at the alpha phosphate. M 3 . or to the 2'-position of the sugar ring of the nucleoside triphosphate. M 4 and M 6 . Furthermore, the mass-modifying functionality can be added so as to affect chain termination, such as by attaching it to the 3'-position of the sugar ring in the nucleoside triphosphate. M 5 .
- chain-elongating nucleoside triphosphates can also be mass-modified in a similar fashion with numerous variations and combinations in functionality and attachment positions.
- the mass-modification. M. can be introduced for X in XR as well as using oligo-/polyethylene glycol derivatives for R.
- the nucleic acid molecule e.g. detector oligonucleotide (D) or the nucleoside triphosphates (FIGURE 6(C)
- the oligo/polyethylene glycols can also be monoalkylated by a lower alkyl such as methyl, ethyl, propyl. isopropyl. t-butyl and the like.
- a selection of linking functionalities. X, are also illustrated.
- Other chemistries can be used in the mass-modified compounds, as for example, those described recently in Oligonucleotides and Analogues. A Practical Approach. F. Eckstein, editor. IRL Press. Oxford. 1991.
- R other than oligo/polyethylene glycols.
- R other than oligo/polyethylene glycols.
- X A simple mass-modification can be achieved by substituting H for halogens like F. Cl, Br and/or I, or pseudohalogens such as SCN, NCS, or by using different alkyl, aryl or aralkyl moieties such as methyl, ethyl, propyl. isopropyl, t-butyl. hexyl, phenyl, substituted phenyl. benzyl, or functional groups such as CH 2 F, CHF 2 , CF 3 , Si(CH 3 ) 3 ,
- the superscript 0-i designates i + 1 mass differentiated nucleotides. primers or tags. In some instances, the superscript 0 can designate an
- unmodified species of a particular reactant and the superscript i can designate the i-th mass-modified species of that reactant. If. for example, more than one species of nucleic acids are to be concurrently detected, then i + 1 different mass-modified detector oligonucleotides (D 0 . D 1 , ...D i ) can be used to distinguish each species of mass modified detector oligonucleotides (D) from the others by mass spectrometry. Different mass-modified detector oligonucleotides can be used to
- all four base permutations at the site of a mutation can be detected by designing and positioning a detector oligonucleotide. so that it serves as a primer for a DNA/RNA polymerase (FIGURE 6C).
- a detector oligonucleotide so that it serves as a primer for a DNA/RNA polymerase (FIGURE 6C).
- mass modifications also can be incorporated during the amplification process.
- FIGURE 3 shows a different multiplex detection format, in which differentiation is accomplished by employing different specific capture sequences which are position-specifically immobilized on a flat surface (e.g. a 'chip array'). If different target sequences T1 - Tn are present, their target capture sites TCS1 - TCSn will specifically interact with complementary immobilized capture sequences C1-Cn. Detection is achieved by employing appropriately mass differentiated detector oligonucleotides D1 - Dn. which are mass differentiated either by their sequences or by mass modifying functionalities M1 - Mn.
- Preferred mass spectrometer formats for use in the invention are matrix assisted laser desorption ionization (MALDI), electrospray (ES), ion cyclotron resonance (ICR) and Fourier Transform.
- MALDI matrix assisted laser desorption ionization
- ES electrospray
- ICR ion cyclotron resonance
- ABI atmospheric pressure ionization interface
- MS/MS Fourier transform and time-of-flight (TOF) configurations as is known in the art of mass spectrometry.
- TOF time-of-flight
- Ion-trap and reflectron configurations can also be employed.
- the mass spectrometric processes described above can be used, for example, to diagnose any of the more than 3000 genetic diseases currently known (e.g hemophilias, thalassemias. Duchenne Muscular Dystrophy (DMD). Huntington's Disease (HD).
- DMD Duchenne Muscular Dystrophy
- HD Huntington's Disease
- CF Cystic Fibrosis
- Example 3 provides a mass spectrometer method for detecting a mutation ( ⁇ F508) of the cystic fibrosis transmembrane conductance regulator gene (CFTR). which differs by only three base pairs (900 daltons) from the wild type of CFTR gene.
- the detection is based on a single-tube, competitive oligonucleotide single base extension (COSBE) reaction using a pair of primers with the 3'-terminal base complementary to either the normal or mutant allele.
- COSBE competitive oligonucleotide single base extension
- products are resolved by molecular weight shifts as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry.
- cystic fibrosis ⁇ F508 polymorphism.
- 28-mer 'normal' (N) and 30-mer 'mutant' (M) primers generate 29- and 31-mers for N and M homozygotes. respectively, and both for heterozygotes. Since primer and product molecular weights are relatively low ( ⁇ 10 kDa) and the mass difference between these are at least that of a single ⁇ 300 Da nucleotide unit, low resolution instrumentation is suitable for such measurements.
- Trisomy 21 Down's Syndrome
- Trisomy 13 Panau Syndrome
- Trisomy 18 Edward's Syndrome
- Monosomy X Teuploidies
- XXY sex chromosome aneuploidies
- DNA sequences may predispose an individual to any of a number of diseases such as diabetes, arteriosclerosis, obesity, various autoimmune diseases and cancer (e.g. colorectal, breast, ovarian, lung);
- Example 4 provides a mass spectometer method for identifying any of the three different isoforms of human apolipoprotein E. which are coded by the E2. E3 and E4 alleles.
- the molecular weights of DNA fragments obtained after restriction with appropriate restriction endonucleases can be used to detect the presence of a mutation.
- the diagnosis for a genetic disease, chromosomal aneuploidy or genetic predisposition can be preformed either pre- or post-natally.
- Viruses, bacteria, fungi and other infectious organisms contain distinct nucleic acid sequences, which are different from the sequences contained in the host cell. Detecting or quantitating nucleic acid sequences that are specific to the infectious organism is important for diagnosing or monitoring infection. Examples of disease causing viruses that infect humans and animals and which may be detected by the disclosed processes include:
- Retroviridae e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV. See Ratner, L. et al., Nature. Vol. 313. Pp. 227-284 ( 1985): Wain Hobson. S. et al, Cell. Vol. 40: Pp. 9-17 (1985)): HIV-2 (See Guyader et al., Nature. Vol. 328, Pp. 662-669 (1987); European Patent Publication No. 0 269 520:
- HIV-LP International Publication No. WO 94/00562 entitled "A Novel Human Immunodeficiency Virus”: Picornaviridae (e.g.. polio viruses, hepatitis A virus. (Gust. I.D.. et al.. Intervirology. Vol. 20. Pp. 1 -7 (1983): entero viruses, human coxsackie viruses, rhinoviruses. echoviruses): Calcivihdae (e.g.. strains that cause gastroenteritis); Togaviridae (e.g... equine encephalitis viruses, rubella viruses);
- Picornaviridae e.g.. polio viruses, hepatitis A virus. (Gust. I.D... et al.. Intervirology. Vol. 20. Pp. 1 -7 (1983): entero viruses, human coxsackie viruses, rhinoviruses. echoviruses): Calcivihdae
- Flaviridae e.g., dengue viruses, encephalitis viruses, yellow fever viruses
- Coronaviridae e.g., coronaviruses
- Rhabdoviridae e.g., vesicular stomatitis viruses, rabies viruses
- Filoviridae e.g., ebola viruses
- Paramyxoviridae e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
- Orthomyxoviridae e.g., influenza viruses
- Bungaviridae e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses
- Arena viridae hemorrhagic fever viruses
- Reovihdae e.g.. reoviruses. orbiviurses and rotaviruses
- Birnaviridae Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses):
- Papovaviridae papilloma viruses, polyoma viruses
- Adenoviridae most adenoviruses
- infectious bacteria examples include: Helicobacter pylons, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis. M. avium. M. intracellulare. M. kansaii. M. gordonae). Staphylococcus aureus. Neisseria gonorrhoeae. Neisseria memngitidis. Listeria monocytogenes. Streptococcus pyogenes (Group A
- Streptococcus Streptococcus). Streptococcus agalactiae (Group B Streptococcus). Streptococcus (viridans group). Streptococcus faecalis. Streptococcus bovis. Streptococcus (anaerobic sps.).
- Streptococcus pneumoniae pathogenic Campylobacter sp.. Enterococcus sp.. Haemophilus influenzae. Bacillus antracis. corynebacterium diphtheriae. corynebacterium sp.,
- Erysipelothrix rhusiopathiae Clostridium perfringers, Clostridium tetani. Enterobacter aerogenes. Klebsiella pneumoniae. Pasturella multocida. Bacteroides sp.. Fusobacterium nucleatum. Streptobacillus moniliformis. Treponema pallidium. Treponema per pneumonia.
- Leptospira. and Actinomyces israelli examples include: Cryptococcus neoformans. Histoplasma capsulatum. Coccidioides immilis, Blastomyces dermatitidis.Chlamydia trachomatis.
- Candida albicans Other infectious organisms (i.e.. protists) include: Plasmodium falciparum and Toxoplasma gondii.
- Example 5 provides a nested PCR and mass spectrometer based method that was used to detect hepatitis B virus (HBV) DNA in blood samples.
- HBV hepatitis B virus
- other blood-borne viruses e.g.. HIV-1. HIV-2. hepatitis C virus (HCV).
- HCV hepatitis A virus
- HAV hepatitis viruses
- other hepatitis viruses e.g., non-A-non-B hepatitis, hepatitis G. hepatits E
- cytomegalovirus. and herpes simplex virus (HSV) can be detected each alone or in combination based on the methods described herein.
- nucleic acid sequences can be used to detect normal and defective genes in higher organisms and to detect infectious microorganisms (e.g. bacteria, fungi, protists and yeast) and viruses. DNA sequences can even serve as a fingerprint for detection of different individuals within the same species. (Thompson. J.S. and M.W. Thompson, eds., Genetics in Medicine, W.B. Saunders Co.. Philadelphia. PA (1986).
- FIG. 1C One process for detecting a wildtype (D wt ) and/ or a mutant (D mut ) sequence in a target (T) nucleic acid molecule is shown in Figure 1C.
- a specific capture sequence (C) is attached to a solid support (ss) via a spacer (S).
- the capture sequence is chosen to specifically interact with a complementary sequence on the target sequence (T). the target capture site (TCS) to be detected through hybridization.
- TDS target detection site
- X which increases or decreases the molecular weight
- mutated TDS can be distinguished from wildtype by mass spectrometry. For example, in the case of an adenine base (dA) insertion, the difference in molecular weights between D wt and D mut would be about 314 daltons.
- the detector nucleic acid (D) is designed such that the mutation would be in the middle of the molecule and the flanking regions are short enough so that a stable hybrid would not be formed if the wildtype detector oligonucleotide (D wt ) is contacted with the mutated target detector sequence as a control.
- the mutation can also be detected if the mutated detector oligonucleotide (D mut ) with the matching base at the mutated position is used for hybridization. If a nucleic acid obtained from a biological sample is heterozygous for the particular sequence (i.e. contain both D wt and D mut ).
- both D wt and D mut will be bound to the appropriate strand and the mass difference allows both D wt and D mut to be detected simultaneously.
- the process of this invention makes use of the known sequence information of the target sequence and known mutation sites. Although new mutations can also be detected. For example, as shown in FIGURE 8. transcription of a nucleic acid molecule obtained from a biological sample can be specifically digested using one or more nucleases and the fragments captured on a solid support carrying the corresponding complementary nucleic acid sequences. Detection of hybridization and the molecular weights of the captured target sequences provide information on whether and where in a gene a mutation is present.
- DNA can be cleaved by one or more specific endonucleases to form a mixture of fragments. Comparison of the molecular weights between wildtype and mutant fragment mixtures results in mutation detection.
- Tetrahedron, 37, 362 (1981)) was performed to synthesize a 3'-T5-50mer oligonucleotide sequence in which 50 nucleotides are complementary to a "hypothetical" 50mer sequence.
- T5 serves as a spacer.
- Deprotection with saturated ammonia in methanol at room temperature for 2 hours furnished according to the determination of the DMT group CPG which contained about 10 umol 55mer/g CPG.
- This 55mer served as a template for hybridizations with a 26mer (with 5'-DMT group) and a 40mer (without DMT group).
- the reaction volume is 100 ul and contains about 1nmol CPG bound 55mer as template, an equimolar amount of oligonucleotide in solution (26mer or 40mer) in 20mM Tris-HCl. pH 7.5. 10 mM MgCI 2 and 25mM NaCl. The mixture was heated for 10' at 65°C and cooled to 37°C during 30' (annealing). The oligonucleotide which has not been hybridized to the polymer-bound template were removed by centrifugation and three subsequent washing/centrifugation steps with 100 ul each of ice-cold 50mM
- DNA fragments at a concentration of 50 pmole/ul in 2-propanol/10mM ammoniumcarbonate ( 1/9. v/v) were analyzed simultaneously by an electrospray mass spectrometer.
- FIGURE 12 The successful desorption and differentiation of an 18-mer and 19-mer by electrospray mass spectrometry is shown in FIGURE 12.
- PCR Amplification and Strand Immobilization were carried out with exon 10 specific primers using standard PCR conditions (30 cycles: 1'@95°C, 1'@55°C. 2'@72°C); the reverse primer was 5' labelled with biotin and column purified
- PCR products were purified by column separation (Qiagen Quickspin) and immobilized on streptavidin coated magnetic beads (Dynabeads. Dynal. Norway) according to their standard protocol; DNA was denatured using 0.1M NaOH and washed with 0.1M NaOH. 1 ⁇ B+W buffer and TE buffer to remove the non-biotinylated sense strand. COSBE Conditions. The beads containing ligated antisense strand were resuspended in 18 ⁇ l of Reaction mix 1 (2 ⁇ l 10X Taq buffer. 1 ⁇ L (1 unit) Taq Polymerase. 2 ⁇ L of 2 mM dGTP.
- Mass Spectrometry After washing, beads were resuspended in 1 ⁇ L 18 Mohm/cm H 2 O. 300 nL each of matrix (Wu et al., 1993) solution (0.7 M 3-hydroxypicolinic acid. 0.7 M dibasic ammonium citrate in 1 : 1 H 2 O:CH 3 CN) and resuspended beads (Tang et al. (1995) Rapid Commun Mass Spectrom 8:727-130) were mixed on a sample target and allowed to air dry.
- matrix Wang et al. (1995) Rapid Commun Mass Spectrom 8:727-130
- Figures 14 - 18 show the representative mass spectra of COSBE reaction products. Better results were obtained when PCR products were purified before the biotinylated anti-sense strand was bound.
- Example 4 Differentiation of Human Apolipoprotein E Isoforms hy Mass Spectrometry
- Apolipoprotein E (Apo E). a protein component of lipoproteins. plays an essential role in lipid metabolism. For example, it is involved with cholesterol transport. metabolism of lipoprotein particles, immunoregulation and activation of a number of lipolytic enzymes.
- a sample of DNA encoding apolipoprotein E can be obtained from a subject, amplified (e.g. via PCR); and the PCR product can be digested using an appropriate enzyme (e.g. CfoI). The restriction digest obtained can then be analyzed by a variety of means.
- the three isotypes of apolipoprotein E (E2, E3 and E4 have different nucleic acid sequences and therefore also have distinguishable molecular weight values.
- Example 5 Detection of hepatitis B virus in serum samples.
- Phenol/choloform extraction of viral DNA and the final ethanol precipitation was done according to standard protocols.
- Each reaction was performed either with 1 ⁇ l of the first reaction or with a 1 : 10 dilution of the first PCR as template, respectively. 100 pmol of each primer. 2.5 u
- Pfu(exo-) DNA polymerase (Stratagene. Heidelberg, Germany), a final concentration of 200 ⁇ M of each dNTPs and 5 ⁇ l 10x Pfu buffer (200 mM Tris-HCl. pH 8.75. 100 mM KCl. 100 mM (NH 4 ) 2 SO 4 , 20 mM MgSO 4 . 1% Triton X-100. 1mg/ml BSA. (Stratagene. Heidelberg.
- thermocycler (OmniGene. MWG-Biotech. Ebersberg. Germany) using the following program: 92°C for 1 minute. 60°C for 1 minute and 72°C for 1 minute with 20 cycles.
- PCR For the recording of each spectrum, one PCR, 50 ⁇ l, (performed as described above) was used. Purification was done according to the following procedure: Ultrafiltration was done using Ultrafree-MC filtration units (Millipore. Eschborn, Germany) according to the protocol of the provider with centrifugation at 8000 rpm for 20 minutes. 25 ⁇ l (10 ⁇ g/ ⁇ l) streptavidin Dynabeads (Dynal, Hamburg. Germany) were prepared according to the instructions of the manufacturer and resuspended in 25 ⁇ l of B/W buffer (10 mM Tris-HCl. pH7.5, 1mM EDTA. 2 M NaCl).
- B/W buffer 10 mM Tris-HCl. pH7.5, 1mM EDTA. 2 M NaCl
- This suspension was added to the PCR samples still in the filtration unit and the mixture was incubated with gentle shaking for 15 minutes at ambient temperature.
- the suspension was transferred in a 1.5 ml Eppendorf tube and the supernatant was removed with the aid of a Magnetic Particle Collector. MPC. (Dynal. Hamburg, Germany).
- the beads were washed twice with 50 ⁇ l of 0.7 M ammonium citrate solution. pH 8.0 (the supernatant was removed each time using the MPC). Cleavage from the beads can be accomplished by using formamide at 90°C.
- the supernatant was dried in a speedvac for about an hour and resuspended in 4 ⁇ l of ultrapure water (MilliQ UF plus Millipore.
- a nested PCR system was used for the detection of HBV DNA in blood samples employing oligonucleotides complementary to the c region of the HBV genome (primer 1 : beginning at map position 1763, primer 2 beginning at map position 2032 of the complementary strand) encoding the HBV core antigen (HBVcAg).
- DNA was isolated from patients serum according to standard protocols.
- a first PCR was performed with the DNA from these preparations using a first set of primers. If HBV DNA was present in the sample a DNA fragment of 269 bp was generated.
- a further advantage is that the amount of fragments generated in the second PCR is high enough to ensure an unproblematic detection although purification losses can not be avoided.
- the samples were purified using ultrafiltration to remove the primers prior to immobilization on streptavidin Dynabeads. This purification was done because the shorter primer fragments were immobilized in higher yield on the beads due to steric reasons.
- the immobilization was done directly on the ultrafiltration membrane to avoid substance losses due to unspecific absorption on the membrane.
- the beads were washed with ammonium citrate to perform cation exchange (Pieles. U. et al., (1993) Nucleic Acids Res 21 :3191-3196).
- the immobilized DNA was cleaved from the beads using 25% ammonia which allows cleavage of DNA from the beads in a very short time, but does not result in an introduction of sodium cations.
- each sample of the first PCR was used undiluted as template and in a 1 :10 dilution, respectively.
- Sample 1 was collected from a patient with chronic active HBV infection who was positive in HBs- and HBe-antigen tests but negative in a dot blot analysis.
- Sample 2 was a serum sample from a patient with an active HBV infection and a massive viremia who was HBV positive in a dot blot analysis.
- Sample 3 was a denatured serum sample therefore no serologicial analysis could be performed but an increased level of transaminases indicating liver disease was detected.
- Figure 24 shows the results of a PAGE analysis of the nested PCR reaction.
- a PCR product is clearly revealed in samples 1, 2, 3, 5 and 6.
- sample 4 no PCR product was generated, it is indeed HBV negative, according to the serological analysis.
- Negative and positive controls are indicated by + and -. respectively.
- Amplification artifacts are visible in lanes 2, 5, 6 and + if non-diluted template was used. These artifacts were not generated if the template was used in a 1 : 10 dilution.
- sample 3. PCR product was only detectable if the template was not diluted.
- the results of PAGE analysis are in agreement with the data obtained by serological analysis except for sample 3 as discussed above.
- Figure 25 A shows a mass spectrum of a nested PCR product from sample number 1 generated and purified as described above.
- the signal at 20754 Da represents the single stranded PCR product (calculated: 20735 Da, as the average mass of both strands of the PCR product cleaved from the beads).
- the mass difference of calculated and obtained mass is 19 Da (0.09%).
- sample number 1 generated a high amount of PCR product, resulting in an unambiguous detection.
- Fig. 25B shows a spectrum obtained from sample number 3. As depicted in Fig. 24. the amount of PCR product generated in this section is significantly lower than that from sample number 1. Nevertheless, the PCR product is clearly revealed with a mass of 20751 Da (calculated 20735). The mass difference is 16 Da (0.08%).
- the spectrum depicted in Fig. 25C was obtained from sample number 4 which is HBV negative (as is also shown in Fig 24). As expected no signals corresponding to the PCR product could be detected. All samples shown in Fig. 25 were analyzed with MALDI-TOF MS. whereby PCR product was detected in all HBV positive samples, but not in the HBV negative samples. These results were reproduced in several independent experiments.
- biotinylated one and all other oligonucleotides were synthesized in a 0.2 ⁇ mol scale on a MilliGen 7500 DNA Synthesizer (Millipore. Bedford. MA. USA) using the ⁇ -cyanoethylphosphoamidite method (Sinha. N.D. et al.. (1984) Nucleic Acids Res.. Vol. 12, Pp. 4539-4577).
- the oligodeoxynucleotides were RP-HPLC-purified and deprotected according to standard protocols.
- the biotinylated oligodeoxynucleotide was purchased (HPLC-purified) from Biometra. Gottingen. Germany).
- the LCR was performed with Pfu DNA ligase and a ligase chain reaction kit (Stratagene. Heidelberg. Germany) containing two different pBluescript KII phagemids. One carrying the wildtype form of the E.coli lacI gene and the other one a mutant of this gene with a single point mutation at bp 191 of the lacI gene.
- Ion exchange HPLC (IE HPLC) was performed on the SMART-system
- DNA was pipetted onto the sample holder, then immediately mixed with 0.5 ⁇ l matrix solution (0.7 M 3-hydroxypicolinic acid in 50% acetonitrile. 70 mM ammonium citrate). This mixture was dried at ambient temperature and introduced into the mass spectrometer. All spectra were taken in positive ion mode using a Finnigan MAT Vision 2000 (Finnigan MAT. Bremen. Germany), equipped with a reflectron (5 keV ion source. 20 keV
- the E. coli lacI gene served as a simple model system to investigate the suitability of MALDI-TOF-MS as detection method for products generated in ligase chain reactions.
- This template system consists of an E.coli lacI wildtype gene in a pBluescript KII phagemid and an E. coli lacI gene carrying a single point mutation at bp 191 (C to T transition) in the same phagemid.
- Four different oligonucleotides were used, which were ligated only if the E. coli lacI wildtype gene was present (Figure 26). LCR conditions were optimized using Pfu DNA ligase to obtain at least 1 pmol ligation product in each positive reaction.
- Figure 27 shows a PAGE of a positive LCR with wildtype template (lane 1 ). a negative LCR with mutant template ( 1 and 2) and a negative control which contains enzyme. oligonucleotides and no template.
- the gel electrophoresis clearly shows that the ligation product (50bp) was produced only in the reaction with wildtype template whereas neither the template carrying the point mutation nor the control reaction with salmon sperm DNA generated amplification products.
- Figure 28 HPLC was used to analyze two pooled LCRs with wildtype template performed under the same conditions. The ligation product was clearly revealed.
- Figure 29 shows the results of a HPLC in which two pooled negative LCRs with mutant template were analyzed. These chromatograms confirm the data shown in Figure 27 and the results taken together clearly demonstrate, that the system generates ligation products in a significant amount only if the wildtype template is provided. Appropriate control runs were performed to determine retention times of the different compounds involved in the LCR experiments. These include the four
- oligonucleotides (A, B, C. and D).
- a synthetic ds 50-mer (with the same sequence as the ligation product), the wildtype template DNA.
- streptavidin-coated magnetic beads were used. As was shown in a recent paper, the direct desorption of DNA immobilized by Watson-Crick base pairing to a complementary DNA fragment covalently bound to the beads is possible and the non-biotinylated strand will be desorbed exclusively (Tang. K et al., (1995) Nucleic Acids Res. 23:3126-3131). This approach in using immobilized ds DNA ensures that only the non-biotinylated strand will be desorbed. If non-immobilized ds DNA is analyzed both strands are desorbed (Tang, K. et. al., (1994) Rapid Comm. Mass Spectrom.
- Figure 31 A shows a MALDI-TOF mass spectrum obtained from two pooled LCRs (performed as described above) purified on streptavidin DynaBeads and desorbed directly from the beads showed that the purification method used was efficient (compared with Figure 30).
- a signal which represents the unligated oligo A and a signal which corresponds to the ligation product could be detected.
- the agreement between the calculated and the experimentally found mass values is remarkable and allows an unambiguous peak assignment and accurate detection of the ligation product.
- no ligation product but only oligo A could be detected in the spectrum obtained from two pooled LCRs with mutated template (Figure 31B).
- Figure 32 shows a spectrum obtained from two pooled LCRs in which only salmon sperm DNA was used as a negative control, only oligo A could be detected, as expected.
- the lower intensity of the ligation product signal could be due to different desorption/ionization efficiencies between 24- and a 50-mer. Since the T m value of a duplex with 50 compared to 24 base pairs is significantly higher, more 24-mer could be desorbed. A reduction in signal intensity can also result from a higher degree of fragmentation in case of the longer oligonucleotides.
- Figure 33 shows spectra obtained from two pooled positive (33A) and negative (33B) LCRs. respectively.
- the positive reaction was performed with a chemically synthesized, single strand 50mer as template with a sequence equivalent to the ligation product of oligo C and D.
- Oligo C was 5'-biotinylated. Therefore the template was not detected.
- only the ligation product of Oligo A and B (calculated mass 15450 Da) could be desorbed from the immobilized and ligated oligo C and D.
- This newly generated DNA fragment is represented by the mass signal of 15448 Da in Figure 33A.
- Figure 32A shows that this method of sample preparation produces signals with improved resolution and intensity.
- Example 7 Mutation detection by solid phase oligo base extension of a primer and analysis by MALDI-TOF mass spectrometry Summary
- the solid-phase oligo base extension method detects point mutations and small deletions as well as small insertions in amplified DNA.
- the method is based on the extension of a detection primer that anneals adjacent to a variable nucleotide position on an affinity-captured amplified template, using a DNA polymerase. a mixture of three dNTPs. and the missing one didesoxy nucleotide.
- the resulting products are evaluate and resolved by MALDI-TOF mass spectrometry without further labeling procedures.
- the aim of the following experiment was to determine mutant and wildtype alleles in a fast and reliable manner. Description of the experiment
- the method used a single detection primer followed by a oligonucleotide extension step to give products differing in length by some bases specific for mutant or wildtype alleles which can be easily resolved by MALDI-TOF mass spectrometry.
- the method is described by using an example the exon 10 of the CFTR-gene. Exon 10 of this gene bears the most common mutation in many ethnic groups ( ⁇ F508) that leads in the homozygous state to the clinical phenotype of cystic fibrosis.
- Genomic DNA were obtained from healthy individuals, individuals homozygous or heterozygous for the ⁇ F508 mutation, and one individual heterozygous for the 1506S mutation. The wildtype and mutant alleles were confirmed by standard Sanger sequencing.
- the primers for PCR amplification were CFE ⁇ 10-F (5- GCAAGTGAATCCTGAGCGTG-3' (SEQ ID No. 13) located in intron 9 and biotinylated) and CFE ⁇ 10-R (5 -GTGTGAAGGGCGTG-3'. (SEQ ID No. 14) located in intron 10).
- Primers were used in a concentration of 8 pmol.
- Taq-polymerase including 10x buffer were purchased from Boehringer-Mannheim and dTNPs were obtained from Pharmacia. The total reaction volume was 50 ⁇ l. Cycling conditions for PCR were initially 5 min. at 95°C.
- Amplification products were purified by using Qiagen's PCR purification kit (No. 28106) according to manufacturer's instructions. The elution of the purified products from the column was done in 50 ⁇ l TE-buffer ( 10mM Tris. 1 mM EDTA. pH 7.5).
- 5'CTATATTCATCATAGGAAACACCA-3' (SEQ ID No. 15) was performed in 50 ⁇ l annealing buffer (20 mM Tris. 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO, 1 % Triton X-100, pH 8, 75) at 50°C for 10 min. The wells were washed three times with 200 ⁇ l washing buffer and oncein 200 ⁇ l TE buffer. The extension reaction was performed by using some components of the DNA sequencing kit from USB (No. 70770) and dNTPs or ddNTPs from Pharmacia. The total reaction volume was 45 ⁇ l. consisting of 21 ⁇ l water.
- the extended primer was denatured in 50 ⁇ l 10%-DMSO (dimethylsufoxide) in water at 80°C for 10 min. For precipitation. 10 ⁇ l NH4-Acetat (pH 6.5). 0.5 ⁇ l glycogen (10 mg/ml water. Sigma No. Gl 765), and 100 ⁇ l absolute ethanol were added to the supernatant and incubated for 1 hour at room temperature. After centrifugation at 13.000 g for 10 min the pellet was washed in 70% ethanol and resuspended in 1 ⁇ l 18 Mohm/cm H 2 O water.
- Sample preparation and analysis on MALDI-TOF mass spectrometry Sample preparation was performed by mixing 0.3 ⁇ l of each of matrix solution (0.7 M 3-hydroxypicolinic acid. 0.07 M dibasic ammonium citrate in 1 :1 H 2 O:CH 3 CN) and of resuspended DNA/glycogen pellet on a sample target and allowed to air dry. Up to 20 samples were spotted on a probe target disk for introduction into the source region of an unmodified Thermo Bioanalysis (formerly Finnigan) Visions 2000 MALDI-TOF operated in reflectron mode with 5 and 20 kV on the target and conversion dynode. respectively.
- matrix solution 0.7 M 3-hydroxypicolinic acid. 0.07 M dibasic ammonium citrate in 1 :1 H 2 O:CH 3 CN
- resuspended DNA/glycogen pellet Up to 20 samples were spotted on a probe target disk for introduction into the source region of an unmodified Thermo Bioanalysis (formerly Finnigan) Visions
- M r (calc) Theoretical average molecular mass (M r (calc)) were calculated from atomic compositions: reported experimental Mr (M r (exp)) values are those of the singly-protonated form, determined using external calibration.
- the aim of the experiment was to develop a fast and reliable method independent of exact stringencies for mutation detection that leads to high quality and high throughput in the diagnosis of genetic diseases. Therefore a special kind of DNA sequencing (oligo base extension of one mutation detection primer) was combined with the evaluation of the resulting mini-sequencing products by matrix-assisted laser desorption ionization
- MALDI mass spectrometry
- TOF time-of-flight
- the schematic presentation as given in Figure 34 shows the expected short sequencing products with the theoretically calculated molecular mass of the wildtype and various mutations of exon 10 of the CFTR-gene.
- the short sequencing products were produced using either ddTTP ( Figure 34A) or ddCTP ( Figure 34B) to introduce a definitive sequence related stop in the nascent DNA strand.
- the MALDI-TOF-MS spectra of healthy, mutation heterozygous, and mutation homozygous individuals are presented in Figure 34. All samples were confirmed by standard Sanger sequencing which showed no discrepancy in comparison to the mass spec analysis. The accuracy of the experimental measurements of the various molecular masses was within a range of minus 21.8 and plus 87.1 dalton (Da) to the range expected.
- the method described is highly suitable for the detection of single point mutations or microlesions of DNA. Careful choice of the mutation detection primers will open the window of multiplexing and lead to a high throughput including high quality in genetic diagnosis without any need for exact stringencies necessary in comparable allele-specific procedures. Because of the uniqueness of the genetic information, the oligo base extension of mutation detection primer is applicable in each disease gene or polymorphic region in the genome like variable number of tandem repeats (VNTR) or other single nucleotide polymorphisms (e.g.. apolipoprotein E gene).
- VNTR variable number of tandem repeats
- apolipoprotein E gene single nucleotide polymorphisms
- Example 8 Detection of Polymerase Chain Reaction Products Containing 7- Deazapurine Moieties with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry
- oligodeoxynucleotide primers were either synthesized according to standard phosphoamidite chemistry (Sinha. N.D., et al., (1983) Tetrahedron Let. Vol. 24. Pp. 5843-5846: Sinha. N.D., et al., (1984) Nucleic Acids Res., Vol. 12, Pp. 4539-4557) on a MilliGen 7500 DNA synthesizer (Millipore. Bedford. MA, USA) in 200 nmol scales or purchased from MWG-Biotech (Ebersberg, Germany, primer 3) and Biometra (Goettingen. Germany, primers 6-7).
- the 103-mer DNA strands (modified and unmodified) were amplified from M13mp18 RFI DNA (100 ng. Pharmacia. Freiburg. Germany) in 100 ⁇ L reaction volume using primers 4 and 5 all other concentrations were unchanged.
- the reaction was performed using the cycle: denaturation at 95°C for 1 min.. annealing at 40°C for 1 min. and extension at 72°C for 1 min. After 30 cycles for the unmodified and 40 cycles for the modified 103-mer respectively, the samples were incubated for additional 10 min. at 72°C.
- Primers 1 and 4 were 5'-[ 32 -P]-labeled employing T4-polynucleotidkinase (Epicentre Technologies) and ( ⁇ - 32 P)-ATP. (BLU/NGG/502A, Dupont. Germany) according to the protocols of the manufacturer. The reactions were performed substituting 10% of primer 1 and 4 in PCR with the labeled primers under otherwise unchanged reactionconditions. The amplified DN As were separated by gel electrophoresis on a 10%
- the amplified DNA was purified using Ultrafree-MC filter units (30.000 NMWL), it was then redissolved in 100 ⁇ l of 0.2 mol/L NaOH and heated at 95°C for 25 minutes. The solution was then acidified with HCl (1 mol/L) and further purified for MALDI-TOF analysis employing Ultrafree-MC filter units ( 10.000 NMWL) as described below.
- MALDI-TOF MS Aliquots of 0.5 ⁇ L of analyte solution and 0.5 ⁇ L of matrix solution (0.7 mol/L 3-HPA and 0.07 mol/L ammonium citrate in acetonitrile/water (1 :1. v/v)) were mixed on a flat metallic sample support. After drying at ambient temperature the sample was introduced into the mass spectrometer for analysis.
- the MALDI-TOF mass spectrometer used was a Finnigan MAT Vision 2000 (Finnigan MAT. Bremen. Germany). Spectra were recorded in the positive ion reflector mode with a 5 keV ion source and 20 keV
- the instrument was equipped with a nitrogen laser (337 nm wavelength).
- the vacuum of the system was 3-4 ⁇ 10 -8 hPa in the analyzer region and 1-4 ⁇ 10 -7 hPa in the source region.
- Spectra of modified and unmodified DNA samples were obtained with the same relative laser power: external calibration was performed with a mixture of synthetic oligodeoxynucleotides (7-to50-mer).
- RNA polymerases such as the SP6 or the T7 RNA polymerase. must be used
- the modified 103-mer nucleic acids.
- fragmentation causes a broad (M+H) + signal.
- the maximum of the peak is shifted to lower masses so that the assigned mass represents a mean value of (M+H) + signal and signals of fragmented ions, rather than the (M+H) ⁇ signal itself.
- the modified 103-mer still contains about 20% A and G from the oligonucleotide primers, it shows less fragmentation which is featured by much more narrow and symmetric signals.
- peak tailing on the lower mass side due to depurination. is substantially reduced.
- the difference between measured and calculated mass is strongly reduced although it is still below the expected mass.
- the 7-deazapurine containing nucleic acids seem to give higher sensitivity despite the fact that they still contain about 20% unmodified purine nucleotides.
- the unmodified 99-mer required 20 laser shots in contrast to 12 for the modified one and the 103-mer required 12 shots for the unmodified sample as opposed to three for the 7-deazapurine nucleoside-containing PCR product.
- Figures 10a and 10b display the spectra of the PCR product before and after primer cleavage.
- Figure 10b shows that the hydrolysis was successful: Both hydrolyzed PCR product as well as the two released primers could be detected together with a small signal from residual uncleaved 100-mer. This procedure is especially useful for the MALDI-TOF analysis of very short PCR-products since the share of unmodified purines originating from the primer increases with decreasing length of the amplified sequence.
- the remarkable properties of 7-deazapurine modified nucleic acids can be explained by either more effective desorption and/or ionization. increased ion stability and/or a lower denaturation energy of the double stranded purine modified nucleic acid.
- the exchange of the N-7 for a methine group results in the loss of one acceptor for a hydrogen bond which influences the ability of the nucleic acid to form secondary structures due to non-Watson-Crick base pairing (Seela. F. and A. Kehne ( 1987) Biochemistry, 26, 2232-2238.). which should be a reason for better desorption during the MALDI process.
- Depurination may either be accompanied by loss of charge which decreases the total yield of charged species or it may produce charged fragmentation products which decreases the intensity of the non fragmented molecular ion signal.
- Oligonucleotides were purchased from Operon Technologies (Alameda. CA) in an unpurified form. Sequencing reactions were performed on a solid surface using reagents from the sequencing kit for Sequenase Version 2.0 (Amersham. Arlington Heights. Illinois).
- template strand DNA 1 1683 was 3'-biotinylated by terminal deoxynucleotidyl transferase.
- a 30 ⁇ l reaction containing 60 pmol of DNA 11683, 1.3 nmol of biotin 14-dATP (GIBCO BRL. Grand Island, NY), 30 units of terminal transferase (Amersham, Arlington Heights. Illinois), and 1x reaction buffer
- the 0.1 mg beads from previous step were resuspended in a 10 ⁇ l volume containing 2 ⁇ l of 5x Sequenase buffer (200 mM Tris-HCl. pH 7.5. 100 mM MgCl2. and 250 mM NaCl) from the Sequenase kit and 5 pmol of corresponding primer PNA16/DNA.
- the annealing mixture was heated to 70°C and allowed to cool slowly to room temperature over a 20-30 min time period. Then 1 ⁇ l 0.1 M dithiothreitol solution.
- 1 ⁇ l Mn buffer (0.15 M sodium isocitrate and 0.1 M McC12). and 2 ⁇ l of diluted Sequenase (3.25 units) were added.
- the reaction mixture was divided into four aliquots of 3 ⁇ l each and mixed with termination mixes (each consists of 3 ⁇ l of the appropriate termination mix: 32 ⁇ M c7dATP. 32 ⁇ M dCTP. 32 ⁇ M c7dGTP. 32 ⁇ M dTTP and 3.2 ⁇ M of one of the four ddTNPs. in 50 mM NaCl).
- the reaction mixtures were incubated at 37°C for 2 min. After the completion of extension. the beads were precipitated and the supernatant was removed. The beads were washed twice and resuspended in TE and kept at 4°C.
- CM1B3B was immobilized on Dynabeads M280 with streptavidin (Dynal.
- CM1B3B by incubating 60 pmol of CM1B3B with 0.3 magnetic beads in 30 ⁇ l 1M NaCl and TE (1 x binding and washing buffer) at room temperature for 30 min.
- the beads were washed twice with TE and redissolved in 30 ⁇ l TE, 10 or 20 ⁇ l aliquot (containing 0.1 or 0.2 mg of beads respectively) was used for sequencing reactions.
- the duplex was formed by annealing corresponding aliquot of beads from previous step with 10 pmol of DF1 1a5F (or 20 pmol of DF1 1a5F for 0.2 mg of beads) in a 9 ⁇ l volume containing 2 ⁇ l of 5x Sequenase buffer (200 mM Tris-HCl, pH 7.5. 100 mM MgCl1 , and 250 mM NaCl) from the Sequenase kit.
- the annealing mixture was heated to 65 °C and allowed to cool slowly to 37°C over a 20-30 min time period.
- the duplex primer was then mixed with 10 pmol of TSlo (20 pmol of TS10 for 0.2 mg of beads) in 1 ⁇ l volume, and the resulting mixture was further incubated at 37°C for 5 min. room temperature for 5-10 min. Then 1 ⁇ l 0.1 M dithiothreitol solution. 1 ⁇ l Mn buffer (0.15 M sodium isocitrate and 0.1 M MnCl 2 ). and 2 ⁇ l of diluted Sequenase (3.25 units) were added. The reaction mixture was divided into four aliquots of 3 ⁇ l each and mixed with termination mixes (each consists of 4 ⁇ l of the appropriate termination mix: 16 ⁇ M dATP. 16 ⁇ M dCTP.
- HPA hydrohydropicolinic acid
- ammonium citrate 10: 1 mole ratio in 50% acetonitrile
- a primer is directly annealed to the template and then extended and terminated in a Sanger dideoxy sequencing.
- a biotinylated primer is used and the sequencing ladders are captured by streptavidin-coated magnetic beads. After washing, the products are eluted from the beads using EDTA and formamide.
- our previous findings indicated that only the annealed strand of a duplex is desorbed and the immobilized strand remains on the beads. Therefore, it is advantageous to immobilize the template and anneal the primer.
- the beads with the immobilized template and annealed sequencing ladder can be loaded directly onto the mass spectrometer target and mix with matrix. In MALDI.
- a 39-mer template (SEQ. ID. No. 23) was first biotinylated at the 3' end by adding biotin-14-dATP with terminal transferase. More than one biotin-14-dATP molecule could be added by the enzyme. However, since the template was immobilized and remained on the beads during MALDI. the number of biotin-14-dATP would not affect the mass spectra.
- a 14-mer primer (SEQ. ID. No. 29) was used for the solid-state sequencing.
- the sequencing reaction produced a relatively homogenous ladder, and the full-length sequence was determined easily.
- a possible explanation is that a small portion of the template formed some kind of secondary structure, such as a loop, which hindered sequenase extension. Mis-incorporation is of minor importance, since the intensity of these peaks were much lower than that of the sequencing ladders.
- 7-deaza purines were used in the sequencing reaction, which could stabilize the N-glycosidic bond and prevent depurination. minor base losses were still observed since the primer was not substituted by 7-deazapurines.
- the full length ladder, with a ddA at the 3' end. appeared in the A reaction with an apparent mass of 1 1899.8. However, a more intense peak of 122 appeared in all four reactions and is likely due to an addition of an extra nucleotide by the Sequenase enzyme.
- a 78-mer template containing a CTG repeat (SEQ. ID. No. 25) was 3'-biotinylated by adding biotin-14-dATP with terminal transferase.
- An 18-mer primer (SEQ. ID. No. 26) was annealed right outside the CTG repeat so that the repeat could be sequenced immediately after primer extension.
- the four reactions were washed and analyzed by MALDI-TOFMS as usual.
- An example of the G-reaction is shown in Figure 35 and the expected sequencing ladder is shown in Table III with theoretical mass values for each ladder component. All sequencing peaks were well resolved except the last component (theoretical value 20577.4) was indistinguishable from the background.
- Duplex DNA probes with single-stranded overhang have been demonstrated to be able to capture specific DNA templates and also serve as primers for solid-state sequencing.
- the scheme is shown in Figure 46. Stacking interactions between a duplex probe and a single-stranded template allow only 5-base overhand to be sufficient for capturing. Based on this format, a 5' fluorescent-labeled 23-mer (5'-GAT GAT CCG ACG CAT CAC AGC TC) (SEQ. ID. No. 29) was annealed to a 3'-biotinylated 18-mer (5'-GTG ATG CGT CGG ATC ATC) (SEQ. ID. No. 30). leaving a 5-base overhang.
- a 15-mer template (5'-TCG GTT CCA AGA GCT) (SEQ ID. No. 31) was captured by the duplex and sequencing reactions were performed by extension of the 5-base overhang.
- MALDI-TOF mass spectra of the reactions are shown in Figure 47A-D. All sequencing peaks were resolved although at relatively low intensities. The last peak in each reaction is due to unspecific addition of one nucleotide to the full length extension product by the Sequenase enzyme.
- the same products were run on a conventional DNA sequencer and a stacking fluorogram of the results is shown in Figure 48. As can be seen from the Figure, the mass spectra had the same pattern as the fluorogram with sequencing peaks at much lower intensity compared to the 23-mer primer.
- Sample distribution can be made more homogenous and signal intensity could potentially be increased by implementing the picoliter vial technique.
- the samples can be loaded on small pits with square openings of 100 um size.
- the beads used in the solid-state sequencing is less than 10 um in diameter, so they should fit well in the microliter vials.
- Microcrystals of matrix and DNA containing "sweet spots" will be confined in the vial. Since the laser spot size is about 100 ⁇ m in diameter, it will cover the entire opening of the vial. Therefore, searching for sweet spots will be unnecessary and high repetition-rate laser (e.g. > 10Hz) can be used for acquiring spectra.
- the resolution of the run-through peak in Figure 33 is greater than 200 (FWHM) which is enough for sequence determination in this case. This resolution is also the highest reported for MALDI desorbed DNA ions above the 70-mer range. Use of the delayed extraction technique may further enhance resolution.
Abstract
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EP96910464A EP0815261B3 (en) | 1995-03-17 | 1996-03-18 | Dna diagnostics based on mass spectrometry |
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DE69626196T DE69626196T3 (en) | 1995-03-17 | 1996-03-18 | DNA DIAGNOSTICS USING MASS SPECTROMETRY |
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DK96910464T DK0815261T3 (en) | 1995-03-17 | 1996-03-18 | DNA diagnostics based on mass spectrometry |
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