Solid phase sequencing of biopolymers
This invention relates to methods for detecting and sequencing target nucleic acid sequences, to mass modified nucleic acid probes and arrays of probes useful in these methods, and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Probes may be affixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.
1. A method for detecting whether a target nucleic acid having a known sequence is present in a sample comprising nucleic acid fragments, comprising:
(a) under hybridization conditions, contacting the nucleic acid fragments of the sample with an array of nucleic acid probes, wherein each probe in the array of nucleic acid probes comprises a double-stranded region, a single-stranded region and a variable sequence within the single-stranded region, and wherein the single-stranded region of the probes comprises a sequence complementary to a sequence of the target nucleic acid;
(b) releasing the nucleic acid fragments hybridized to the probes, thereby producing released fragments;
(c) determining molecular weights of the released fragments by mass spectrometry;
(d) determining whether the sequence of the target nucleic acid is present in the released fragments from the molecular weights of the released fragments; and
(e) detecting whether the target nucleic acid is present in the sample based on whether the target nucleic acid sequence is present in the released fragments.
2. The method of claim 1 , wherein the array is attached to a solid support.
3. The method of claim 2 , wherein the probes are attached to the solid support via a cleavable attachment.
4. The method of claim 3 , wherein the cleavable attachment is cleavable by an enzyme, a chemical agent or electromagnetic radiation.
5. The method of claim 2 , wherein the solid support comprises a matrix chemical that facilitates volatilization of nucleic acids for mass spectrometry.
6. The method of claim 5 , wherein the volatilization of nucleic acid is done using fast atom bombardment, plasma desorption, matrix-assisted laser desorption/ionization, electrospray ionization, photochemical release, electrical release, droplet release, resonance ionization or a combination thereof.
7. The method of claim 2 , wherein the solid support is selected from the group consisting of plates, beads, microbeads, whiskers, combs, hybridization chips, membranes, single crystals, ceramics and self-assembling monolayers.
8. The method of claim 1 , wherein the sample is obtained from an environmental source.
9. The method of claim 1 , wherein the probes are from about 8 to about 100 nucleotides in length.
10. The method of claim 1 , wherein the length of the single-stranded regions of the probes is about 4 to about 20 nucleotides, about 5 to about 12 nucleotides or about 6 to about 10 nucleotides.
11. The method of claim 1 , wherein the sample is subjected to a purifying step before step (a).
12. The method of claim 11 , wherein the purifying step comprises removing toxins or infectious substances from the sample if said toxins or infectious substances are present in the sample.
13. The method of claim 11 , wherein the purifying step comprises removing substances that interfere with step (a).
14. The method of claim 1 , wherein the array includes mass modified probes.
15. The method of claim 1 , wherein the nucleic acid fragments are produced by enzymatic or physical cleavage.