Positional sequencing by hybridization

A method for creating an array of probes fixed to a solid support comprising the steps of:

• a) synthesizing a first set of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus;
• b) fixing the first set to the solid support;
• c) synthesizing a second set of nucleic acids each comprising a sequence complimentary to the constant region of the first set;
• and
• d) hybridizing the nucleic acids of the first set with the second set to create the array.

A method for creating an array of probes comprising the steps of:

• a) synthesizing an array of single-stranded nucleic acids each containing a constant sequence at the 3′-terminus, another constant sequence at the 5′-terminus, and a random internal sequence of length R flanked by the cleavage sites of a restriction enzyme;
• b) synthesizing an array of primers each compliementary to a portion of the constant sequence of the 3′-terminus, hybridizing the two arrays together to form hybrids;
• c) extending the sequence of each primer by polymerization using a sequence of the nucleic acid as a template;
• and
• d) cleaving the extended hybrids with the restriction enzyme to form an array of probes with a double-stranded portion at one terminus, a single-stranded portion containing the random sequence at the opposite terminus.

A method for creating an array of probes comprising the steps of:

• a) synthesizing an array of single-stranded nucleic acids each containing a constant sequence at the 3′- terminus, another constant sequence at the 5′-terminus, and a random internal sequence of length R flanked by the cleavage sites of a restriction enzyme;
• b) synthesizing an array of primers with a sequence complimentary to the constant sequence at the 3′-terminus;
• c) hybridizing the two arrays together to form hybrids;
• d) enzymatically extending the primers using the nucleic acids as templates to form full-length hybrids;
• e) cloning the full-length hybrids into vectors;
• f) amplifying the cloned sequences by multiple polymerase chain reactions;
• and
• g) cleaving the amplified sequences with the restriction enzyme to form the array of probes with a double-stranded portion at one terminus and a single-stranded portion containing the random sequence at the opposite terminus.

A method for replicating an array of single-stranded probes on a solid support comprising the steps of:

• a) synthesizing an array of nucleic acids each comprising a constant sequence of length C at a 3′-terminus and a random sequence of length R at a 5′-terminus;
• b) fixing the array to a first solid support;
• c) synthesizing a set of nucleic acids each comprising a sequence complimentary to the constant sequence;
• d) hybridizing the nucleic acids of the set with the array;
• e) enzymatically extending the nucleic acids of the set using the random sequences of the array as templates;
• f) denaturing the set of extended nucleic acids;
• and
• g) fixing the denatured nucleic acids of the set to a second solid support to create the replicated array of single-stranded probes.

A method for creating a probe comprising the steps of:

• a) synthesizing a plurality of first nucleic acids and a plurality of second nucleic acids comprising a random terminal sequence and a sequence complimentary to a sequence of the first nucleic acids;
• b) hybridizing the first nucleic acids with the second to form partial duplexes;
• c) hybridizing a target nucleic acid to the partial duplexes;
• d) ligating the hybridized target to the first nucleic acid of the partial duplexes;
• e) isolating the second nucleic acid from the ligated duplexes;
• and
• f) synthesizing a plurality of third nucleic acids each complimentary to the constant sequence of the second nucleic acid and hybridizing the third nucleic acids with the isolated second nucleic acids to create a probe.

A method for creating a probe comprising the steps of:

• a) synthesizing a plurality nucleic acids and a plurality of second nucleic acids each comprising a random terminal sequence and a sequence complimentary to the sequence of the first nucleic acids;
• b) hybridizing the first nucleic acids with the second nucleic acids to form partial duplexes having a double-stranded portion and a single-stranded portion with the random sequence within the single-stranded portion;
• c) hybridizing a target nucleic acid to the partial duplexes;
• d) ligating the hybridized target to the first nucleic acid of the partial duplex;
• e) hybridizing the ligated target with a set of oligonucleotides comprising random sequences;
• f) ligating the hybridized oligonucleotide to the second nucleic acid;
• g) isolating the oligonucleotide ligated second nucleic acid;
• and
• h) synthesizing another plurality of first nucleic acids and hybridizing the first nucleic acids with the isolated second nucleic acid to create the probe.

A method for creating a probe comprising the steps of:

• a) synthesizing a plurality of first nucleic acids and a plurality of second nucleic acids comprising a random terminal sequence and a sequence complimentary to a sequence of the first nucleic acid;
• b) hybridizing the first nucleic acids to the second nucleic acids to form partial duplexes having a double-stranded portion and a single-stranded portion with the random nucleotide sequence within the single-stranded portion;
• c) hybridizing a target nucleic acid to the partial duplexes;
• d) ligating the hybridized target to the first nucleic acid of the partial duplex;
• e) enzymatically extending the second nucleic acid using the target as a template;
• f) isolating the extended second nucleic acid;
• and
• g) synthesizing another first nucleic acid and hybridizing the first nucleic acid with the isolated and extended second nucleic acid to create a probe.