Mass spectrometric methods for sequencing nucleic acids

Details Mass spectrometric methods for sequencing nucleic acids Mass spectrometric methods for sequencing nucleic acids

1997-12-15

2001-07-31

Brusca, John S. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200

Reexamination Certificate

active

06268131

ABSTRACT:

BACKGROUND OF THE INVENTION
Methods of sequencing DNA are typically performed by either the chemical degradation method of Maxam and Gilbert (
Methods in Enzymol.
65:499-560 (1980)) or the enzymatic dideoxynucleotide termination method of Sanger et al. (
Proc. Natl. Acad. Sci. U.S.A.
74:5463-67 (1977)). In the chemical method, base specific modifications result in a base specific cleavage of the radioactive or fluorescently labeled DNA fragment. With the four separate base specific cleavage reactions, four sets of nested fragments are produced which are separated according to length by polyacrylamide gel electrophoresis (PAGE). After autoradiography, the sequence can be read directly since each band (fragment) in the gel originates from a base specific cleavage event. Thus, the fragment lengths in the four “ladders” directly translate into a specific position in the DNA sequence.
In the enzymatic chain termination method, the four base specific sets of DNA fragments are formed by starting with a primer/template system elongating the primer into the unknown DNA sequence area and thereby copying the template and synthesizing a complementary strand using DNA polymerases, such as Klenow fragment of
E. coli
DNA polymerase I, a DNA polymerase from
Thermus aquaticus,
Taq DNA polymerase, or a modified T7 DNA polymerase, Sequenase (e.g., Tabor et al., (1987)
Proc. Natl. Acad. Sci. U.S.A.
84:4767-4771), in the presence of chain-terminating reagents. Here, the chain-terminating event is achieved by incorporating into the four separate reaction mixtures in addition to the four normal deoxynucleoside triphosphates, dATP, dGTP, dTTP and dCTP, only one of the chain-terminating dideoxynucleoside triphosphates, ddATP, ddGTP, ddTTP or ddCTP, respectively, in a limiting small concentration. The four sets of resulting fragments produce, after electrophoresis, four base specific ladders from which the DNA sequence can be determined. Undesirably, most methods for sequencing DNA require the use of polyacrylamide gel electrophoresis (i.e., PAGE) that can result in sequencing artifacts or require detectable labels, such as radioisotopes, enzymes, or fluorescent or chemiluminescent moieties.
Using DNA sequencing methodologies, the entire sequence of the human genome will be determined. The knowledge of the complete sequence of the human genome DNA will certainly help to understand, to diagnose, to prevent and to treat human diseases. To be able to tackle successfully the determination of the approximately 3 billion base pairs of the human genome in a reasonable time frame and in an economical way, rapid, reliable, sensitive and inexpensive methods needed to be and still need to be developed.
Therefore it is an object herein to provide additional methods for sequencing. In particular, it is an object herein to provide mass spectrometric methods of sequencing nucleic acids using RNA polymerase. It is a further object herein to provide methods of sequencing nucleic acids in an array format using RNA polymerase in which nucleic acid probes are immobilized to supports at high densities to facilitate mass spectrometric detection. It is also an object herein to provide methods for identifying transcriptional terminator sequences using mass spectrometric methods.
SUMMARY OF THE INVENTION
Improved methods for sequencing nucleic acids is provided. In particular, a mass spectrometric method for sequencing nucleic acids using RNA polymerases, including DNA-dependent and RNA-dependent RNA polymerases, is provided. The methods utilize a modified Sanger sequencing strategy in which RNA polymerase is used to generate a set of nested RNA transcripts obtained by base-specific chain termination. These are analyzed by mass spectrometry.
In certain embodiments, a double stranded nucleic acid molecule encoding a promoter sequence is isolated from a natural source (e.g., bacteria, viruses, bacteriophages, plants or eukaryotic organisms) or assembled from synthetic sequences and is engineered using recombinant DNA means to contain a single stranded region of at least a 5 nucleotides at the 3′-end of the coding strand. This single stranded region is designed such that it is complementary to a region of the nucleic acid to be sequenced or to a common overlapping sequence (e.g., a restriction endonuclease site). In preferred embodiments, the promoter-containing nucleic acid is covalently coupled via the 3′-end of the noncoding strand or 5′-end of the coding strand to a solid support and, more preferably, is a 5′- or 3′-thiolated DNA linked at high densities to a aminosilane-treated solid support. The linkage may be in the absence or presence of a linker group and is preferably arranged in an array format.
The nucleic acid to be sequenced containing at least a partially single stranded 3′-end is hybridized to the complementary sequences of the promoter-containing DNA. The nucleic acid to be sequenced may be single stranded or double stranded. The hybridization of the two nucleic acid molecules introduces one or more “nicks” in the hybrid at the junction(s) of the adjacent nucleic acid molecules. In certain embodiments, nicks in the coding or non-coding strand, preferably the coding strand, are ligated by the addition of an appropriate nucleic acid ligase prior to initiating transcription (i.e., DNA or RNA ligase).
Transcription is initiated from the promoter by the addition of the appropriate RNA polymerase in the presence of ribonucleoside triphosphates and a selected base-specific chain terminating 3′-deoxyribonucleoside triphosphate. In preferred embodiments, the transcription mixture also contains inosine 5′-triphosphate to reduce the secondary structure of the RNA product and may further contain modified ribonucleoside triphosphates, such as 4-thio uridine 5′triphosphate (UTP), 5-bromo UTP or 5′-iodo CTP to increase the fidelity of termination and turnover of the RNA polymerase enzyme thereby increasing the amount of RNA transcript available for analysis.
The resulting RNA transcripts are analyzed by mass spectrometry. In preferred embodiments, the sample further contains a matrix material and is analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI) and preferably further uses time-of-flight (TOF) analysis. The sequence of the nucleic acid is obtained by aligning the observed mass of the chain-terminated RNA transcripts obtained from sequencing reactions containing each of the four chain-terminating bases.
In other embodiments, the method of sequencing may be used for diagnostic applications to determine the presence of genetic alterations in a known target nucleic acid. For example, a region of the target nucleic acid is amplified and the nucleic acid strand corresponding to the noncoding strand is isolated. The nucleic acid probe containing the promoter may be isolated from a natural source or assembled synthetically by hybridizing two complementary oligonucleotides to form a promoter sequence. A single stranded region of at least 5 nucleotides that is complementary to a region of the nucleic acid to be sequenced or to a common sequence is introduced by recombinant means at the 3′-end of the coding strand. In preferred embodiments, the promoter-containing nucleic acid is covalently coupled via the 3′-end of the noncoding strand or 5′-end of the coding strand to a solid support and, more preferably, is a 5′- or 3′-thiolated DNA linked at high densities to a aminosilane-treated solid support. The linkage may be in the absence or presence of a linker group and is preferably arranged in an array format.
A single stranded 3′ overhang of the nucleic acid to be sequenced, in single stranded or double stranded form, is hybridized to the complementary sequences of the noncoding strand and, in some embodiments, the nick(s) between one or more nucleic acid strands is/are ligated prior to transcription. Transcription is initiated using the appropriate RNA polymerase in the presence of ribonucleoside trip

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