Hybridization single-copy sequences

A crucial aspect of virtually all DNA-DNA hybridization studies is that they involve only so-called single-copy DNA (scDNA) or "unique DNA. Total genomic DNA is made single-stranded by heating and then allowed to reassociate into duplex DNA after cooling. Repeated copies of DNA sequences re-anneal faster than single-copy sequences due to their higher concentration in the solution. Thus, under controlled conditions and time, it is possible to remove excess copies of repeated sequences. 

The second class of rapidly reassociating DNA sequences make up between 20 and 25% of the mouse genome and reassociate at an average rate from a few to 100,000 times faster than would be expected for single copy sequences in a genome the size of a mouse. In RNA/DNA low Cot hybridization reactions, these repeated sequences are the major DNA component in the reaction. 

In situ polymerase chain reaction (PCR) is a very powerful tool, which enhances our ahility to detect minute quantities of a rare, single copy number, target nucleic acid sequences in freshly frozen or paraffin-embedded intact cells or tissue sections (1-10). In 1986, the introduction of PCR methods opened new horizons and revolutionized research in all areas of molecular biology (11,12). Dr. Hasse and his coworkers in 1990 used multiple primers and successfully amplified the target nucleic acid sequences in intact cells by combining a traditional in situ hybridization protocol with a powerful PCR technology (13). 

Ray, R., Komminoth, P., Machado, M., and Wolfe, H. J. (1991) Combined polymerase chain reaction and in situ hybridization for the detection of single copy genes and viral genomic sequences in intact cells. Mod. Pathol. 4,124A. 

Given these constraints that impinge on the reassociation of DNA, experimental designs must be constructed that facilitate (1) the separation of single-copy DNA to be used as tracer DNA from repeated sequences and (2) the hybridization of single-copy tracer DNA with driver DNA from the same and different species. 

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