Genetic mapping is provided by combinations of two related general procedures. In the first procedure, mapping is provided by identifying genetic regions from which DNA fragments derived from two individuals can combine to form extensive hybrids free of base mismatches. DNA is processed by a method that allows perfectly-matched hybrid DNA molecules formed between DNAs from the two individuals, to be separated from imperfectly-paired DNA hybrids or hybrids in which both strands are from the same individual's DNA. The perfectly-matched hybrid DNAs can then be labeled and the labeled DNA used as probes to identify loci of identity-by-descent between the two individuals. In the second procedure, nicks are introduced specifically into DNA hybrids formed between non-identical alleles from a region of heterozygosity in an individual diploid genome. The nicked DNA molecules are then specifically labeled to provide probes for identifying regions of heterozygosity in the genome of an individual.

A novel process is disclosed utilizing a subtraction hybridization technique for producing DNA hybridization probes of high specific activity or cDNA subtraction libraries, useful in connection with the cloning of differentially expressed genes. A preparation of single stranded cDNA derived from transcript mRNA of a target cell source is subjected to subtraction hybridization using excess single stranded "driver" nucleic acid from a reference cell source so that all the cDNA having a nucleotide sequence complementary to transcript mRNA of the reference cell source is subtracted by annealing with the "driver" nucleic acid to form duplex molecules. The strands of these duplex molecules are then chemically cross-linked by treatment with an aziridinylbenzoquinone interstrand cross-linking agent, and the remaining unsubtracted single stranded unique cDNA derived solely from the target cell source is processed in the presence of the chemically cross-linked duplex molecules to provide labelled probe material or a subtraction cDNA library, using random priming and a DNA polymerase lacking exonuclease activity and inactive with respect to the cross-linked duplex molecules.

The invention relates to methods for rapidly determining the sequence and/or length a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguish from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution. Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders. Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.

Disclosed is a method for detecting one or more mismatches in a test nucleic acid which preferentially hybridizes to a reference nucleic acid. The method involves a) providing the test nucleic acid in its single-stranded form; b) providing the reference nucleic acid immobilized on a solid support, the reference nucleic acid being in its single-stranded form and the solid support being compatible with a slot in an electrophoretic gel; c) contacting the single-stranded test nucleic acid with the single-stranded immobilized reference nucleic acid under conditions allowing heteroduplex formation, whereby the heteroduplex is immobilized on the solid support; d) contacting the immobilized heteroduplex with a resolvase (for example, T4 endonuclease VII) capable of recognizing at least one single base pair mismatch in the heteroduplex under conditions which permit the resolvase to cleave the heteroduplex; e) inserting the solid support into the slot of the electrophoretic gel under conditions sufficient to release all or a cleaved portion of the immobilized heteroduplex into the slot; and f) analyzing the released product by gel electrophoresis, the presence of a cleavage product being an indication of a mismatch in the test nucleic acid.

Methodology is provided for developing probes for identifying sequence differences between two related DNA populations, sets of DNA fragments or collections of restriction-endonuclease-cleaved DNA or CODA. The method employs an initial stage to obtain a representation of both DNA populations, namely using the PCR to produce relatively short fragments, referred to as amplicons. Tester amplicons containing target DNA, sequences of interest, are ligated to adaptors and mixed with excess driver amplicons under melting and annealing conditions, followed by PCR amplification. The process may be repeated so as to greatly enrich the target DNA. Optionally, the target DNA may then be cloned and the DNA used as probes.