chapter  21
6 Pages

Detection of a Single-Copy Sequence on Human Metaphase by Cyclic PRINS

Primed in situ labeling (PRINS) is not merely an alternative to conventional fluorescent in situ hybridization (FISH) but a complementary approach. By combining features of FISH with polymerase chain reaction, it provides high sensitivity and specificity for in situ target sequence detection. In the original protocol described by Koch et al.1 an unlabeled oligonucleotide primer is annealed to its complementary target sequence and extended by DNA polymerase in presence of labeled nucleotides. The extended fluorescent labeled target is then visualized by fluorescent microscopy. In contrast to standard “tube PCR,” however, the mean length of extension generated by PRINS is only approximately 40 nucleotides2 and the amount of incorporated label is relatively low. Consequently, PRINS was mainly applied for targets containing repetitive sequences and the identification of single-copy sequences was achieved only in very few cases during the first decade after its invention.3-5 Recently, however, several improvements of the PRINS technique have been described that allowed successful localization of singlecopy genes such as DMD, SRY, and SOX3.6-9 Moreover, PRINS has been successfully applied in the screening for chromosomal microdeletions in PraderWilli/Angelman and DiGeorge/velo-cardiofacial syndrome.10 Interestingly, various reports emphasize different modification steps of the original PRINS protocol. Hindkjaer et al.4 used a primer cocktail of 18 primers to detect a sequence of the p53 gene on chromosome 17pl3. The disadvantage of this approach is the

same as in other multiplex PCRs; primer selection and construction is as difficult since all must have the same annealing temperature. Troyer et al.5 used a cyclic PRINS approach for target amplification. However, technical limitations restrict this approach since most commercially available cover slips and glue do not withstand several cycles of annealing, extending, and denaturation. Kandandale8-9 and Tharapel et al.10 improved the sensitivity mainly by applying the Tyramide Signal Amplification System− (NEN Life Science Products, Boston, MA) in the immunochemistry step of the PRINS protocol. We have introduced a new chamber system for in situ applications of PRINS on slides6 which allows overcoming of some of the main disadvantages of the original PRINS protocol: (1) reaction conditions remain constant during many cycles, guaranteeing reliable results, (2) the open chamber allows for hot start and nested in situ PCR which is a unique improvement in the development of PRINS, and (3) since the reaction mix can be removed after cyclic amplification, aliquots can be run on a gel to check for successful amplification of an internal control. Our system can be used for the detection of high and low copy repetitive sequences, and it worked well for a 273-bp sequence of the single-copy DMD gene.