Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
2001-06-13
2003-12-09
Benzion, Gary (Department: 1645)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S070000, C422S078000, C422S068100, C422S080000, C422S116000, C436S059000, C436S155000, C436S161000
Reexamination Certificate
active
06660229
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods, particularly mass spectrometric methods, for the analysis and sequencing of nucleic acid molecules.
DESCRIPTION OF THE BACKGROUND
Since the recognition of nucleic acid as the carrier of the genetic code, a great deal of interest has centered around determining the sequence of that code in the many forms in which it occurs. Two studies made the process of nucleic acid sequencing, at least with DNA, a common and relatively rapid procedure practiced in most laboratories. The first describes a process whereby terminally labeled DNA molecules are chemically cleaved at single base repetitions (A. M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA 74:560-64, 1977). Each base position in the nucleic acid sequence is then determined from the molecular weights of fragments produced by partial cleavage. Individual reactions were devised to cleave preferentially at guanine, at adenine, at cytosine and thymine, and at cytosine alone. When the products of these four reactions are resolved by molecular weight, using, for example, polyacrylamide gel electrophoresis, DNA sequences can be read from the pattern of fragments on the resolved gel.
In another method DNA is sequenced using a variation of the plus-minus method (Sanger et al. (1977)
Proc. Natl. Acad. Sci. USA
74:5463-67, 1977). This procedure takes advantage of the chain terminating ability of dideoxynucleoside triphosphates (ddNTPs) and the ability of DNA polymerase to incorporate ddNTPs with nearly equal fidelity as the natural substrate of DNA polymerase, deoxynucleoside triphosphates (dNTPs). Briefly, a primer, usually an oligonucleotide, and a template DNA are incubated in the presence of a useful concentration of all four dNTPs plus a limited amount of a single ddNTP. The DNA polymerase occasionally incorporates a dideoxynucleotide that terminates chain extension. Because the dideoxynucleotide has no 3′-hydroxyl, the initiation point for the polymerase enzyme is lost. Polymerization produces a mixture of fragments of varied sizes, all having identical 3′ termini. Fractionation of the mixture by, for example, polyacrylamide gel electrophoresis, produces a pattern that indicates the presence and position of each base in the nucleic acid. Reactions with each of the four ddNTPs permits the nucleic acid sequence to be read from a resolved gel.
These procedures are cumbersome and are limited to sequencing DNA. In addition, with conventional procedures, individual sequences are separated by, for example, electrophoresis using capillary or slab gels, which slow. Mass spectrometry has been adapted and used for sequencing and detection of nucleic acid molecules (see, e.g., U.S. Pat. Nos. 6,194,144; 6,225,450; 5,691,141; 5,547,835; 6,238,871; 5,605,798; 6,043,031; 6,197,498; 6,235,478; 6,221,601; 6,221,605). In particular, Matrix-Assisted Laser Desorption/Ionization (MALDI) and ElectroSpray Ionization (ESI), which allow intact ionization, detection and exact mass determination of large molecules, i.e. well exceeding 300 kDa in mass have been used for sequencing of nucleic acid molecules.
A further refinement in mass spectrometric analysis of high molecular weight molecules was the development of time of flight mass spectrometry (TOF-MS) with matrix-assisted laser desorption ionization (MALDI). This process involves placing the sample into a matrix that contains molecules that assist in the desorption process by absorbing energy at the frequency used to desorb the sample. Time of flight analysis uses the travel time or flight time of the various ionic species as an accurate indicator of molecular mass. Due to its speed and high resolution, time-of-flight mass spectrometry is well-suited to the task of short-range, i.e., less than 30 base sequencing of nucleic acids. Since each of the four naturally occurring nucleotide bases dC, dT, dA and dG, also referred to herein as C, T, A and G, in DNA has a different molecular weight,
M
C
=289.2
M
T
=304.2
M
A
=313.2
M
G
=329.2,
where M
C
, M
T
, M
A
, M
G
are average molecular weights in daltons of the nucleotide bases deoxycytidine, thymidine, deoxyadenosine, and deoxyguanosine, respectively, it is possible to read an entire sequence in a single mass spectrum. If a single spectrum is used to analyze the products of a conventional Sanger sequencing reaction, where chain termination is achieved at every base position by the incorporation of dideoxynucleotides, a base sequence can be determined by calculation of the mass differences between adjacent peaks. In addition, the method can be used to determine the masses, lengths and base compositions of mixtures of oligonucleotides and to detect target oligonucleotides based upon molecular weight.
MALDI-TOF mass spectrometry for sequencing DNA using mass modification (see, e.g., U.S. Pat. Nos. 5,547,835, 6,194,144; 6,225,450; 5,691,141 and 6,238,871) to increase mass resolution is available. The methods employ conventional Sanger sequencing reactions with each of the four dideoxynucleotides. In addition, for example for multiplexing, two of the four natural bases are replaced; dG is substituted with 7-deaza-dG and dA with 7-deaza-dA.
A variety of techniques and combinations thereof have been directed to improving the level of accuracy in determining the nucleotide compositions of mixtures of oligonucleotides using mass spectrometry, and many of these methods employ nucleotide analogs. For example, Muddiman et al. (
Anal. Chem.,
69(8): 1543-1549, 1997) discusses an algorithm for the unique definition of the base composition of PCR-amplified products, especially longer (>100 bp) oligonucleotides. The algorithm places a constraint on the otherwise large number of possible base compositions for long oligonucleotides by taking into account only those masses (measured by electrospray ionization mass spectrometry) that are consistent with that of their denatured complementary strands, assuming Watson-Crick base-pairing. In addition, the algorithm imposes the constraint of known primer compositions, since the primer sequences are known, and this constraint becomes especially significant with shorter PCR products whose mass of “unknown” sequence relative to that of the primer mass is small. Muddiman et al. also discusses invoking additional measurements for defining the base composition with even greater accuracy. These include the possibility of post-modifying the PCR product using e.g., dimethyl sulfate to selectively methylate every “G” in the PCR product, or using a modified base during PCR amplification, conducting mass measurements on the modified oligonucleotides, and comparing the mass measurements with those of the unmodified complementary strands.
Chen et al. (
Anal. Chem.,
71(15): 3118-3125, 1999) reports a method that combines stable isotope
13
C/
15
N labelling of PCR products with analysis of the mass shifts by MALDI-TOF mass spectrometry. The mass shift due to labelling of a single type of nucleotide (i.e, A, T, G or C) reveals the number of that type of nucleotide in a given fragment. While the method is useful in the measurement and comparison of nucleotide compositions of homologous sequences for sequence validation and in scoring polymorphisms, tedious repetitive sequencing reactions (using the four different labelled nucleotides) and mass spectrometric measurements are required.
Hence there is a need in the art for methods that (i) unambiguously assign nucleotides in a sequence, and, (ii) resolve large numbers of oligonucleotides that have the same length, different base compositions, and nearly equal (i.e., less than or equal to about 1 dalton difference) molecular weights. Therefore it is an object herein to provide methods that solve such problems
SUMMARY OF THE INVENTION
Provided herein are methods for sequencing and detecting nucleic acids using techniques, such as mass spectrometry and gel electrophoresis, that are based upon molecular mass. The methods use deoxynucleotide analogs, modified nucleotide terminators and/or mass-labeled
Cantor Charles R.
Siddiqi Fouad A.
Benzion Gary
Chakrabarti Arun K.
Heller Ehrman White & McAuliffe LLP
Seidman Stephanie L.
The Trustees of Boston University
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