WO2002079519A1 - High-fidelity dna sequencing using solid phase capturable dideoxynucleotides and mass spectrometry - Google Patents
High-fidelity dna sequencing using solid phase capturable dideoxynucleotides and mass spectrometry Download PDFInfo
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- WO2002079519A1 WO2002079519A1 PCT/US2002/009752 US0209752W WO02079519A1 WO 2002079519 A1 WO2002079519 A1 WO 2002079519A1 US 0209752 W US0209752 W US 0209752W WO 02079519 A1 WO02079519 A1 WO 02079519A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6872—Methods for sequencing involving mass spectrometry
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Definitions
- DNA deoxyribonucleic acid
- the current state-of-the-art technology for high throughput DNA sequencing is capillary array DNA sequencers using laser- induced fluorescence detection (Smith et al . 1986; Ju et al . 1995, 1996; Kheterpal et al . 1996; Salas-Solan ⁇ ' et al . 1998) . Improvements in the polymerases that : lead to uniform termination efficiency, and the introduction of thermostable polymerases, have also significantly improved the quality of sequencing data (Tabor and Richardson, 1987, 1995) .
- electrophoresis based DNA sequencing methods have difficulty detecting heterozygotes unambiguously and are not 100% accurate on a given base due to compressions in regions rich in nucleotides comprising guanine (G) or cytosine (C) (Bowling et al. 1991; Yamakawa et al . 1997).
- G guanine
- C cytosine
- the first few bases after the priming site are often masked by the high fluorescence signal from excess dye-labeled primers or dye-labeled terminators, and are therefore difficult to identify.
- Mass spectrometry is able to overcome the difficulties (GC compressions and heterozygote detections) typically encountered when using capillary sequencing techniques. However, it is unable to meet the read length and throughput requirements for large scale sequencing projects. In addition, poor resolution prevents the sequence determination of large DNA fragments. At the present time, the read lengths are insufficient for de novo DNA sequencing and the stringent clean sample requirements for using mass spectrometry for DNA sequencing are not entirely met by existing procedures. For this reason, most of the reported mass spectrometry applications have focused on single nucleotide polymorphism (SNP) detection. Several methods have been explored to this end. The most common approach is to extend a primer by a single nucleotide and detect what was added.
- SNP single nucleotide polymorphism
- DNA sequencing results have been reported by several groups using a variety of sample purification procedures. Using cleavable primers, Monforte and Becker (1997) have demonstrated read lengths up to 100 base pairs (bp) . Fu et al .
- biotin and streptavidin are often utilized in biological sample preparation as a way to remove undesired impurities
- the present application discloses the use of biotinylated dideoxynucleotides for a high fidelity DNA sequencing system by mass spectrometry.
- Biotinylated dideoxynucleotides and streptavidin coated magnetic beads can be used to generate high quality sequencing mass spectra of Sanger cycle sequencing DNA fragments on a MALDI-TOF mass spectrometer.
- the method disclosed here provides an efficient way to eliminate false stopped DNA fragments and excess primers and salts in one simple purification step, while still allowing the use of cycle sequencing to generate a high yield of sequencing fragments. Furthermore, it avoids the above-mentioned pitfalls of gel electrophoresis.
- the subject application discloses that mass-tagged dideoxynucleotides which are coupled with biotin or photocleavable biotin can increase the mass separation of the DNA sequencing fragments on the mass spectra, giving better resolution than previously achievable.
- this application discloses a method for creating streptavidin-coated porous channels that can be used in light directed cleavage of the biotin- streptavidin complex. This is important as present commercially available streptavidin coated magnetic beads are inadequate for photocleavage purposes, in that they are opaque to ultraviolet light.
- the system disclosed herein provides a high throughput and high fidelity DNA sequencing system for polymorphism and pharmacogenetics applications. Compared to gel electrophoresis sequencing, this system produces very high resolution of sequencing fragments and extremely fast separation in the time scale of microseconds. The high resolution allows accurate mutation and heterozygosity detection. Also the problematic compressions associated with gel based systems are avoided.
- the method disclosed here allows mass spectrometry based sequencing of much longer read lengths and higher throughput and better mass resolution than previously possible. The method also achieves the stringent sample cleaning required in mass spectrometry, eliminating false stops as well as other unnecessary components.
- SNPs single nucleotide polymorphisms
- This invention is directed to a method for sequencing DNA by detecting the identity of a dideoxynucleotide incorporated to the 3' end of a DNA sequencing fragment using mass spectrometry, which comprises:
- This invention provides a method for sequencing DNA by detecting the identity of a plurality of dideoxynucleotides incorporated to the 3' end of different DNA sequencing fragments using mass spectrometry, which comprises:
- the invention provides a linker for attaching a chemical moiety to a dideoxynucleotide, wherein the linker comprises a derivative of 4-aminomethyl benzoic acid.
- the invention provides a labeled dideoxynucleotide, which comprises a chemical moiety attached via a linker to a 5-position of cytosine or thymine or to a 7 -position of adenine or guanine.
- the invention provides a system for separating a chemical moiety from other components in a sample in solution, which comprises:
- the invention provides a method of increasing mass spectrometry resolution between different DNA sequencing fragments, which comprises attaching different linkers to different dideoxynucleotides used to terminate a DNA sequencing reaction and generate different DNA sequencing fragments, wherein the different linkers ' increase mass separation between the different DNA sequencing fragments, thereby increasing mass spectrometry resolution.
- Figure 1 Schematic of the use of biotinylated dideoxynucleotides and a streptavidin coated solid phase to prepare DNA sequencing samples for mass spectrometric analysis.
- d(A, C, G, T) deoxynucleotide with base adenine (A) , cytosine (C) , guanine (G) , or thymine (T) ;
- dd(A-b, C-b, G-b, T-b) biotinylated dideoxynucleotides .
- Figure 2 DNA sequencing data from solid phase capturable biotinylated dideoxynucleotides. The proper base is identified above each peak. The first peak is at the appropriate position and is used to identify the 13bp primer plus the first base, adenine. The mass difference between a peak and the previous peak is indicated above the base . The region between 6500 and 12000 (m/z) is magnified for clarity. Data obtained using biotinylated dideoxynucleotides dcLATP-11-biotin, ddGTP-11-biotin, ddCTP-11-biotin and ddTTP-11- biotin.
- Figure 3 Sequencing data collected using biotinylated terminators to produce sequencing fragments that are then analyzed on a mass spectrometer. All four bases can be clearly distinguished using biotinylated terminators ddATP- 11-biotin, ddGTP-11-biotih, ddCTP-11-biotin and ddTTP-16-biotin.
- Figure 4 Structure of four mass tagged biotinylated ddNTPs. Any of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) can be used with any of the illustrated linkers .
- Figure 5 Synthesis scheme for mass tag linkers.
- the linkers are labeled to correspond to the specific ddNTP with which they are shown coupled in Figures 4, 6, 8, 9 and 10.
- any of the three linkers can be used with any ddNTP.
- Figure 6 The synthesis of ddATP-Linker-II-ll-Biotin.
- FIG. 7 DNA sequencing products are purified by a streptavidin coated porous silica surface. Only the biotinylated fragments are captured. These fragments are then cleaved by ultraviolet irradiation (hv) to release the captured fragments, leaving the biotin moiety still bound to the streptavidin.
- hv ultraviolet irradiation
- Figure 8 Mechanism for the cleavage of photocleavable linkers.
- Figure 9 The structures of ddNTPs linked to photocleavable (PC) biotin " . Any of the four ddNTPs
- ddATP ddCTP, ddGTP, ddTTP
- ddTTP ddATP, ddCTP, ddGTP, ddTTP
- Figure 11 Schematic for capturing a DNA fragment terminated with a ddNTP on a surface and then for freeing the ddNTP and DNA fragment .
- the dideoxynucleotide (ddNTP) which is on one end of the DNA fragment (not shown) , is attached via a linker to a chemical moiety "X" which interacts with a compound "Y" on the surface to capture the ddNTP and DNA fragment .
- the ddNTP and DNA fragment can be freed from the surface either by disrupting the interaction between chemical moiety : X and compound Y (lower panel) or by cleaving a cleavable linker (upper panel) .
- Figure 12 Schematic of a high throughput channel based streptavidin purification system. Sample solutions can be pushed back and forth between the two plates through glass capillaries and the streptavidin coated channels in the chip. The whole chip can be irradiated to cleave the samples after immobilization.
- Figure 13 The synthesis of streptavidin coated porous surface.
- nucleotide bases are used as follows: adenine (A), cytosine (C) , guanine (G) , thymine (T) , and uracil (U) .
- This invention is directed to a method for sequencing DNA by detecting the identity of a dideoxynucleotide incorporated to the 3' end of a DNA sequencing fragment using mass spectrometry, which comprises :
- This invention provides a method for sequencing DNA by detecting the identity of a plurality of dideoxynucleotides incorporated to the 3' end of different DNA sequencing fragments using mass spectrometry, which comprises: (a) attaching a chemical moiety via a linker to a plurality of different dideoxynucleotides to produce labeled dideoxynucleotides; (b) terminating a DNA sequencing reaction with the labeled dideoxynucleotides to generate labeled DNA sequencing fragments, wherein the DNA sequencing fragments have a 3' end and the chemical moiety is attached via the linker to the 3' end of the DNA sequencing fragments ; (c) capturing the labeled DNA sequencing fragments on a surface coated with a compound that specifically interacts with the chemical moiety attached via the linker to the DNA sequencing fragments, thereby capturing the DNA sequencing fragments;
- the chemical moiety is attached via a different 'linker to different dideoxynucleotides.
- the different linkers increase mass separation between different labeled DNA sequencing fragments and thereby increase mass spectrometry resolution.
- the dideoxynucleotide is selected from the group consisting of 2 ' , 3 ' -dideoxyadenosine 5 ' -triphosphate (ddATP) , 2 ', 3 ' -dideoxyguanosine 5'- triphosphate (ddGTP) , 2 ' , 3 ' -dideoxycytidine 5'- triphosphate (ddCTP) , and 2 ', 3 ' -dideoxythymidine 5'- triphosphate (ddTTP) .
- ddATP 2 ' , 3 -dideoxyadenosine 5 ' -triphosphate
- ddGTP 2 ', 3 ' -dideoxyguanosine 5'- triphosphate
- ddCTP 2 ' , 3 ' -dideoxycytidine 5'- triphosphate
- ddTTP 2 ', 3 ' -dideoxythym
- the interaction between the chemical moiety attached via the linker to the DNA sequencing fragment and the compound on the surface comprises a biotin-streptavidin interaction, a phenylboronic acid-salicylhydroxamic acid interaction, or an antigen-antibody interaction.
- the step of freeing the DNA sequencing fragment from the surface comprises disrupting the interaction between the chemical moiety attached via the linker to the DNA sequencing fragment and the compound on the surface.
- the interaction is disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
- the interaction is disrupted by ultraviolet light.
- the interaction is disrupted by ammonium hydroxide, formamide, or a change in pH (-log H + concentration) .
- the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings.
- the dideoxynucleotide comprises a cytosine or a thymine with a 5 -position, or an adenine or a guanine with a 7-position, and the linker is attached to the 5- position of cytosine or thymine or to the 7-position of adenine or guanine .
- the step of freeing the DNA sequencing fragment from the surface comprises cleaving the linker.
- the linker is cleaved by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical ' chemical means, heat, and light.
- the linker is cleaved by ultraviolet light.
- the linker is cleaved by ammonium hydroxide, formamide, or a change in pH (-log H + concentration) .
- the linker comprises a derivative of 4-aminomethyl benzoic acid. In one embodiment, the linker comprises one or more fluorine atoms .
- the linker is selected from the group consisting of:
- a plurality of different labeled dideoxynucleotides is used to generate a plurality of different labeled DNA sequencing fragments.
- a plurality of different linkers is used to increase mass separation between different labeled DNA sequencing fragments and thereby increase mass spectrometry resolution.
- the chemical moiety comprises biotin
- the labeled dideoxynucleotide is a biotinylated dideoxynucleotide
- the labeled DNA sequencing fragment is a biotinylated DNA sequencing fragment
- the surface is a streptavidin-coated solid surface.
- the biotinylated dideoxynucleotide is selected from the group consisting of ddATP-11-biotin, ddCTP-11-biotin, ddGTP-11-biotin, and ddTTP-16-biotin.
- the biotinylated dideoxynucleotide selected from the group consisting of:
- ddNTPl ddNTP2 , ddNTP3 , and ddNTP4 represent four different dideoxynucleotides.
- biotinylated dideoxynucleotide is selected from the group consisting of:
- ddNTPl ddNTP2 , ddNTP3 , and ddNTP4 represent four different dideoxynucleotides .
- biotinylated dideoxynucleotide is selected from the group consisting of:
- the streptavidin-coated solid surface is a streptavidih-coated magnetic bead or a streptavidin-coated silica glass.
- steps (b) to (e) are performed in a single container or in a plurality of connected containers.
- the mass spectrometry is matrix- assisted laser desorption/ionization time-of-flight mass spectrometry.
- the invention provides for the use of any of the methods described herein for detection of single nucleotide polymorphisms, genetic mutation analysis, serial analysis of gene expression, gene expression analysis, identification in forensics, genetic disease association studies, genomic sequencing, translational analysis, or transcriptional analysis.
- the invention provides a linker for attaching a chemical moiety to a dideoxynucleotide, wherein the linker comprises a derivative of 4-aminomethyl benzoic acid.
- the dideoxynucleotide is selected from the group consisting of 2 ' , 3 ' -dideoxyadenosine 5 ' -triphosphate (ddATP) , 2 ', 3 ' -dideoxyguanosine 5'- triphosphate (ddGTP) , 2 ', 3 ' -dideoxycytidine 5'- triphosphate (ddCTP) , and 2 ', 3 ' -dideoxythymidine 5'- triphosphate (ddTTP) .
- ddATP 2 ' ' -dideoxyadenosine 5 ' -triphosphate
- ddGTP 2 ', 3 ' -dideoxyguanosine 5'- triphosphate
- ddCTP 2 ', 3 ' -dideoxycytidine 5'- triphosphate
- ddTTP 2 ', 3 ' -dideoxythymidine 5'
- the linker comprises one or more fluorine atoms.
- the linker is selected from the group consisting of:
- the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings .
- the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light. In one embodiment, the linker is cleavable by ultraviolet light. In different embodiments, the linker is cleavable by ammonium hydroxide, formamide, or a change in pH (-log H + concentration) .
- the chemical moiety comprises biotin, streptavidin, phenylboronic acid, salicylhydroxamic acid, an antibody, or an antigen.
- the dideoxynucleotide comprises a cytosine or a thymine with a 5 -position, or an adenine or a guanine with a 7-position, and the linker is attached to the 5-position of cytosine or thymine or to the 7-position of adenine or guanine.
- the invention provides for the use of any of the linkers described herein in DNA sequencing using mass spectrometry, wherein the linker increases mass separation between different dideoxynucleotides and increases mass spectrometry resolution.
- the invention provides a labeled dideoxynucleotide, which comprises a chemical moiety attached via a linker to a 5-position of- -cytosine or thymine or to a 7-position of adenine or guanine.
- the dideoxynucleotide is selected from the group consisting of 2 ', 3 ' -dideoxyadenosine 5 ' -triphosphate (ddATP) , 2 ', 3 ' -dideoxyguanosine 5'- triphosphate (ddGTP) , 2 ' , 3 ' -dideoxycytidine 5 ' - triphosphate (ddCTP) , and 2 ', 3 ' -dideoxythymidine 5'- triphosphate (ddTTP) .
- ddATP 2 ', 3 ' -dideoxyadenosine 5 ' -triphosphate
- ddGTP 2 ', 3 ' -dideoxyguanosine 5'- triphosphate
- ddCTP 2 ' ' -dideoxycytidine 5 ' - triphosphate
- ddTTP 2 ', 3 ' -dideoxythy
- the linker can comprise a chain structure, or a structure comprising one or more rings, or a structure comprising a chain and one or more rings.
- the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a -2,7- chemical means, a physical chemical means, heat, and light.
- the linker is cleavable by ultraviolet light.
- the linker is cleavable by ammonium hydroxide, formamide, or a change in pH (-log H + concentration) .
- the ' chemical moiety comprises biotin, streptavidin, phenylboronic acid, salicylhydroxamic acid, an antibody, or an antigen.
- the labeled dideoxynucleotide is selected from the group consisting of :
- ddNTPl ddNTP2 , ddNTP3 , and ddNTP4 represent four different dideoxynucleotides .
- the labeled dideoxynucleotide is selected from the group consisting of:
- the labeled dideoxynucleotide is selected from the group consisting of:
- ddNTPl ddNTP2 , ddNTP3 , and ddNTP4 represent four different dideoxynucleotides.
- the labeled dideoxynucleotide is selected from the group consisting of:
- the invention provides the use of any of the labeled dideoxynucleotide described herein in DNA sequencing using mass spectrometry, wherein the linker increases mass separation between different labeled dideoxynucleotides and increases mass spectrometry resolution.
- the labeled dideoxynucleotide has a molecular weight selected from the group consisting of 844, 977, 1,017, and 1,051. In one embodiment, the labeled dideoxynucleotide has a molecular weight selected from the group consisting of 1,049, 1,182, 1,222, and 1,257.
- the mass spectrometry is matrix- assisted laser desorption/ionization time-of-flight mass spectrometry.
- the invention provides a system for separating a chemical moiety from other components in a sample in solution, which comprises ' :
- the interaction between the chemical moiety and the compound coating the surface is a biotin-streptavidin interaction, a phenylboronic acid-salicylhydroxamic acid interaction, or an antigen-antibody interaction.
- the chemical moiety is a biotinylated moiety and the channel is a streptavidin-coated silica glass channel.
- the biotinylated moiety is a biotinylated DNA sequencing fragment .
- the chemical moiety can be freed from the surface by disrupting the interaction between the chemical moiety and the compound coating the surface.
- the interaction can be disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
- the interaction can be disrupted by ammonium hydroxide, formamide, or a change in pH (-log H + concentration) .
- the chemical moiety is attached via a linker to another chemical compound.
- the other chemical compound is a DNA sequencing fragment.
- the linker is cleavable by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
- the channel is transparent to ultraviolet light and the linker is cleavable by ultraviolet light. Cleaving the linker frees the DNA sequencing fragment or other chemical compound from the chemical moiety which remains captured on the surface .
- the invention provides a multi-channel system which comprises a plurality of any of the single channel systems disclosed herein.
- the channels are in a chip.
- the multi-channel system comprises 96 channels in a chip.
- the invention provides for the use of any of the systems described herein for separating one or more DNA sequencing fragments, wherein each fragment is terminated with a dideoxynucleotide attached via a linker to the chemical moiety.
- the invention provides a method of increasing mass spectrometry resolution between different DNA sequencing fragments, which comprises attaching different linkers to different dideoxynucleotides used to terminate a DNA sequencing reaction and generate different DNA sequencing fragments, wherein the different linkers increase mass separation between the different DNA sequencing fragments, thereby increasing mass spectrometry resolution.
- one or more of the different linkers comprises one or more fluorine atoms.
- one or more of the different linkers is selected from the group consisting of:
- Matrix-assisted laser desorption/ionization time-of- flight mass spectrometry has recently been explored widely for DNA sequencing.
- the Sanger dideoxy procedure (Sanger et al . 1977) is used to generate the DNA sequencing fragments and no labels are required.
- the mass resolution in theory can be as good as one dalton.
- mass spectrometry produces very high resolution of the sequencing fragments and extremely • fast separation in the time scale of microseconds. The high resolution allows accurate mutation and heterozygosity detection.
- Another advantage of sequencing with mass spectrometry is that the compressions associated with gel based systems are completely eliminated.
- the samples must be free from alkaline and alkaline-earth salts. Samples must be desalted and free from contaminants before the MS analysis.
- affinity systems other than biotin-streptavidin can be used.
- affinity systems include but are not limited to phenylboronic acid-salicylhydroxamic acid (Bergseid et al . 2000) and antigen-antibody systems.
- DNA template deoxynucleotides - (dNTPs) (A, C, G, T) and biotinylated dideoxynucleotides (ddNTP-biotin) (A-b, C-b, G-b, T-b) , primer, and DNA polymerase are combined in one tube. After polymerase extension and termination reactions, a series of DNA sequencing fragments with different lengths are generated. The sequencing reaction mixture is then incubated for a few minutes with a streptavidin coated solid phase. Only the DNA sequencing fragments that are terminated with biotinylated dideoxynucleotide at the 3' end are captured on the solid phase.
- False stops occur in sequencing when a deoxynucleotide rather than a dideoxynucleotide terminates a sequencing fragment.
- a deoxynucleotide terminated false stop has a mass difference of 16 daltons with its dideoxy counterpart. This mass difference is identical to the difference between adenine and guanine.
- false stops can be wrongly interpreted or interfere with existing peaks decreasing accuracy. Salts can ruin spectra by broadening the observed peaks beyond recognition. The method disclosed here eliminates all these problems.
- ddTTP-16-biotin is used since it is commercially available (Enzo, Boston) and has a large mass difference in comparison to ddCTP-11-biotin (see Table 1) . It is paired with ddCTP-11-biotin, ddATP-11-biotin, and ddGTP-11-biotin to allow unambiguous assignment of the mass spectra sequencing ladder (see Figure 3) .
- Sample preparation is performed in one tube by executing the sequencing reactions with biotinylated ddNTPs, regular dNTPs, DNA polymerase, and reaction buffer. The sample is then placed in a thermocycler for 30 cycles to create extension fragments. Streptavidin beads are then added to the sample and incubated to allow the biotin-streptavidin complex to form. The beads are collected by placing the reaction tube in a magnet and thoroughly washing them with an ammonium acetate solution to remove all impurities such as false stops, primers, and salts. Dilute ammonium hydroxide solution is then used to dissociate the biotin streptavidin complex at 60 °C (Jurinke et . al . , 1997) .
- the current application discloses systematic modification of the biotinylated dideoxynucleotides by incorporating mass linkers assembled using 4-aminomethyl benzole acid derivatives to increase the mass separation of the individual bases.
- the mass linkers can be modified by incorporating one or two fluorine atoms to further space out the mass differences between the nucleotides.
- the structures of four biotinylated ddNTPs are shown in Figure 4. ddCTP-11-biotin is commercially available (New England Nuclear, Boston) .
- ddTTP-Linker I-11-Biotin ddTTP-Linker I-11-Biotin
- ddATP-Linker II-11-Biotin ddGTP-Linker III-11-Biotin are synthesized as shown, for example, for ddATP-Linker II-11-Biotin in Figure 6.
- the linkers are attached to the 5-position on the pyrimidine bases (C and T) , and to the 7-position on the purines (A and G) for subsequent conjugation with biotin.
- 7-I-ddA is coupled with linker II in the presence of tetrakis (triphenylphosphine) palladium(O) to produce 7 -Linker II-ddA, which is phosphorylated with P0C1 3 in butylammonium pyrophosphate (Burgess and Cook, 2000) .
- 7 -Linker II-ddATP is produced, which then couples with sulfo-NHS-LC-Biotin (Pierce, Rockford IL) to yield the desired ddATP-Linker 11-11- Biotin.
- ddTTP-Linker I-11-Biotin, and ddGTP-Linker III-11-Biotin can be synthesized.
- this application discloses the use of ddNTPs containing a photocleavable biotin (PC-biotin) .
- PC-biotin photocleavable biotin
- a schematic of capture and cleavage of the photocleavable linker on the streptavidin coated porous surface is shown in Figure 7.
- the reaction mixture consists of excess primers, enzymes, salts, false stops, and the desired sequencing fragments.
- This reaction mixture is passed over a streptavidin-coated surface and allowed to incubate.
- the biotinylated sequencing fragments are captured by the streptavidin surface, while everything else in the mixture is washed away.
- the fragments are released into solution by cleaving the photocleavable linker with ultraviolet (UV) light, while the biotin remains attached to the streptavidin that is covalently bound to the surface.
- UV ultraviolet
- the pure DNA fragments can then be crystallized in matrix solution and analyzed by mass > spectrometry. It is advantageous to cleave the biotin moiety since it contains sulfur which has several relatively abundant isotopes.
- the rest of the DNA fragments and linkers contain only carbon, nitrogen, hydrogen, oxygen, fluorine and phosphorous, whose dominant isotopes are found with a relative abundance of 99% to 100%. This allows high resolution mass spectra to be obtained.
- the photocleavage mechanism (Olejnik et al .
- ddCTP- PC-Biotin Four new biotinylated ddNTPs disclosed here, ddCTP- PC-Biotin, ddTTP-Linker I-PC-Biotin, ddATP-Linker II- PC-Biotin and ddGTP-Linker III-PC-Biotin are shown in Figure 9. These compounds are synthesized by a similar chemistry as shown for the synthesis of ddATP-Linker II-11-Biotin in Figure 6. The only difference is that in the final coupling step NHS-PC- LC-Biotin (Pierce, Rockford IL) is used, as shown in Figure 10.
- the photoclea-rable linkers disclosed here allow the use of solid phase capturable terminators and mass spectrometry to be turned into a high throughput sequencing technique .
- the ddNTP is attached via a linker to a chemical moiety ("X" in Figure 11) .
- the dideoxynucleotide and DNA fragment are captured on the surface through interaction between chemical moiety "X” and a compound on or attached to the surface ( "Y” in Figure 11) .
- the present application discloses two methods for freeing the captured dideoxynucleotide and DNA fragment. In the situation illustrated in the lower part of Figure 11, the dideoxynucleotide and DNA fragment are freed from the surface by disrupting or breaking the interaction between chemical moiety "X" and compound "Y” . In the upper part of Figure 11, the dideoxynucleotide is attached to chemical moiety "X" via a cleavable linker which can be. cleaved to free the dideoxynucleotide and DNA fragment .
- X and compounds can be used for the "X" - "Y" affinity system, which include but are not limited to, biotin-streptavidin, phenylboronic acid- salicylhydroxamic acid (Bergseid et al . 2000), and antigen-antibody systems.
- the cleavable linker can be cleaved and the "X" - "Y" interaction can be disrupted by a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
- a means selected from the group consisting of one or more of a physical means, a chemical means, a physical chemical means, heat, and light.
- ultraviolet light can be used to cleave the cleavable linker.
- Chemical means include, but are not limited to, ammonium hydroxide (Jurinke et . al . , 1997), formamide, or a change in pH (-log H + concentration) of the solution.
- V High density streptavidin-coated, porous silica channel system.
- Streptavidin coated magnetic beads are not ideal for using the photocleavable biotin capture and release process for DNA sequencing fragments, since they are not transparent to UV light. Therefore, the photocleavage reaction is not efficient.
- a high-density surface coated with streptavidin is essential. It is known that the commercially available 96-well streptavidin coated plates cannot provide a sufficient surface area for efficient capture of the biotinylated DNA fragments. Disclosed in this application is a new porous silica channel system designed to overcome this limitation.
- porous channels are coated with a high density of streptavidin.
- Ninety-six (96) porous silica glass channels can be etched into a silica chip ( Figure 12) .
- the surfaces of the channels are modified to contain streptavidin as shown in Figure 13.
- the channel is first treated with 0.5 M NaOH, washed with water, and then briefly pre-etched with dilute hydrogen fluoride. Upon cleaning with water, the capillary channel is coated with high density 3- aminopropyltrimethoxysilane in aqueous ethanol (Woolley et al . 1994) .
- each end of a channel is connected to a single well.
- the end of a channel could be connected to a plurality of wells.
- Pressure is applied to drive the samples through a glass capillary into the channels on the chip. Inside the channels the biotin is captured by the covalently bound streptavidin. After passing through the channel, the sample enters into a clean plate in the other end of the chip. Pressure applied in reverse drives the sample through the channel multiple times arid ensures a highly efficient solid phase capture. Water is similarly added to drive out the reaction mixture and thoroughly wash the captured fragments.
- the chip After washing, the chip is irradiated with ultraviolet light to cleave the photosensitive linker and release the DNA fragments. The fragment solution is then driven out of the channel and into a collection plate. After matrix solution is added, the samples are spotted on a chip and allowed to crystallize for detection by MALDI-TOF mass spectrometry.
- The- purification cassette is cleaned by chemically cleaving the biotin- streptavidin linkage, and is then washed and reused.
- a synthetic DNA template can be synthesized which mimics a portion of the human immunodeficiency virus type 1 protease gene.
- the sequence of the template (SEQ ID NO: 3) and that of the sequencing primer (SEQ ID NO: 4) are shown below (Schmit et al . 1996) :
- the tumor suppressor gene p53 can also be used as a model system.
- the p53 gene is one of the most frequently mutated genes in human cancer (O'Connor et al . 1997) . Since most of the p53 mutation hot spots are clustered within exons 5-8, this region of the p53 gene is selected as a sequencing target.
- a synthetic sequencing template containing a portion of the sequences from exon 7 and exon 8 of the p53 gene and an appropriate primer can be prepared:
- This template (SEQ ID NO: 5) was chosen to explore the use of the mass spectrometry sequencing procedure disclosed herein for the .detection of clustered hot spot single base mutations.
- the potentially mutated bases are underlined (A, G, C and T) in the synthetic template shown above.
- DNA templates generated by polymerase chain reaction can also be used to further validate the high fidelity MALDI- TOF mass spectrometry sequencing technology.
- the sequencing templates are generated by PCR using flanking primers in the intron region located at each p53 exon boundary from a pool of genomic DNA
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Abstract
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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CA002442862A CA2442862A1 (en) | 2001-03-30 | 2002-03-29 | High-fidelity dna sequencing using solid phase capturable dideoxynucleotides and mass spectrometry |
EP02728606A EP1383923A4 (en) | 2001-03-30 | 2002-03-29 | High-fidelity dna sequencing using solid phase capturable dideoxynucleotides and mass spectrometry |
JP2002577927A JP2004533608A (en) | 2001-03-30 | 2002-03-29 | High fidelity DNA sequencing using solid phase captureable dideoxynucleotides and mass spectrometry |
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US09/823,181 US20030027140A1 (en) | 2001-03-30 | 2001-03-30 | High-fidelity DNA sequencing using solid phase capturable dideoxynucleotides and mass spectrometry |
US09/823,181 | 2001-03-30 |
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EP (1) | EP1383923A4 (en) |
JP (1) | JP2004533608A (en) |
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