Multiplex analysis of DNA

A method for analyzing a DNA specimen comprising

• (a) providing a set of at least two vectors, each vector of said set comprising a cloning site and including at least one tag sequence, each tag sequence in each vector differing from each tag sequence of every other vector of said set, and each being incapable of hybridizing to said DNA specimen under stringent conditions,
• (b) providing a first and a second DNA sequence from said DNA specimen, said first DNA sequence being different from said second DNA sequence,
• (c) ligating said first DNA sequence into the cloning site of one of said vectors, and said second DNA sequence into the cloning site of a second of said vectors, thereby producing a plurality of hybrid vectors,
• (d) providing a pool of said hybrid vectors,
• (e) treating separate aliquots of said pool in a plurality of vessels to produce fragments comprising said tag sequence, wherein said fragments in each vessel differ in length from each other and all terminate at a fixed known base or bases, wherein said fixed known base or bases in one of said vessels differ from said fixed known base or bases in another of said vessels,
• (f) separating said fragments comprising tag sequences from each said vessel according to their size,
• (g) hybridizing the separated fragments of step (e) under stringent conditions with a first oligonucleotide probe able to hybridize specifically with one of said tag sequences, and
• (h) detecting the pattern of hybridization wherein said pattern reflects the nucleotide sequence or restriction map of said DNA specimen.

A method for simultaneously analyzing multiple DNA molecules comprising,

• (a) providing a pool of individually different DNA tag sequences, each tag sequence in said pool being foreign to, and incapable of hybridizing under stringent conditions to, any of said DNA molecules to be analyzed;
• (b) from separate aliquots of said pool of tag sequences, generating a plurality of sets of hybrid DNA fragments in separate vessels, said hybrid fragments in each vessel differing in length from one another and terminating in a fixed known base or bases, wherein said fixed known base or bases in one of said vessels differ from said fixed known base or bases in another of said vessels;
• i) each of said fragments comprising at least one of said tag sequences and at least part of one of said DNA molecules to be analyzed, each of said fragments that includes part of any given one said DNA molecules to be analyzed having a tag sequence that is different from each of the tag sequences in fragments that include part of any other one of said DNA molecules to be analyzed,
• ii) each fragment of a first set of said hybrid DNA fragments comprising a first one of said tags, said first set of fragments comprising fragments of different lengths of a first one of said DNA molecules to be analyzed;
• and each fragment of a second set of said sets fragments comprising a second one of said tags, said second set of fragments comprising fragments of different lengths of a second one of said DNA molecules to be analyzed,
• (c) separating said first and said second set of fragments according to their size, yielding size-separated fragments,
• (d) hybridizing said size-separated fragments under stringent conditions with a first oligonucleotide probe able to hybridize specifically with said first one of said tag sequences,
• (e) detecting a first pattern of hybridization with said first probe, wherein said first pattern reflects the nucleotide sequence or restriction map of at least a part of said first DNA molecule to be analyzed,
• (f) hybridizing said size separating fragments under stringent conditions with a second oligonucleotide probe able to hybridize specifically with said second one of said tag sequences, and
• (g) detecting a second pattern of hybridization with said second probe, wherein said second pattern reflects the nucleotide sequence or restriction map of said second DNA molecule to be sequence.