Direct In Situ Single-Copy (DISC)-PCR

The physical mapping of functional genes and polymorphic markers is obviously important for understanding genome organization in higher organisms. Fluorescence in situ hybridization of labeled DNA to metaphase chromosome spreads has been a very effective means of accomplishing this goal (1 ,2 ), but it is difficult to routinely detect sequences <3–5 kb in length using this technique (3 ). Since the polymerase chain reaction (PCR) (4 ) provides a means to amplify a single copy of DNA to literally hundreds of thousands of copies in a short time, in principle it could be utilized to detect very short sequences directly on metaphase chromosomes. It has been demonstrated that PCR can be performed on the surface of a slide containing cells (5 ) and that the PCR process can incorporate biotin-dUTP into the amplification product (6 ). On-slide PCR with a single primer has been used to detect low-copy sequences on metaphase chromosomes (3 ). Oligonucleotide premed in situ synthesis (PRINS) (7 ), in which an oligonucleotide is annealed to denatured chromosomes and extended by a DNA polymerase, has also proven to be an effective method to detect low-copy sequences.