Telomerase, a ribonucleoprotein enzyme, is expected to be a new marker for cancer diagnosis. Thedevelopment of the telomeric repeat amplification protocol (TRAP) and its modified versions have facilitatedthe detection of telomerase activity in small tissue and tumour samples. But most of these techniques requirecomplex post-PCR procedures. As for the two real-time quantitative methods (SYBR Green and Amplifluormethods) reported so far, both use fluorogenic probes without specificity. To overcome these problems wedeveloped a new real-time method for the detection of telomerase activity. In this method a duplex scorpionprimer and reverse primers with hairpin-like structures were used. The use of duplex scorpion primers showsa series of advantages: the target-specific probe sequence provides higher specificity than the SYBR Greenand Amplifluor methods; the unimolecular probing mechanism allow the assay to be conducted under fastcycling conditions and a single operation can be completed in 1.5h; the closed-tube system reduces the riskof carryover contamination and supports high throughput. This method may be a useful tool to rapidly,specifically and precisely quantify telomerase activity.

The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservationbut their function is unknown. We have determined that these fingers mediate homodimerization andbinding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammaliancells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cellsresults in altered expression of HOX genes that are targets for regulation by MLL. These alterations aresuppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHDfingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.

A modified molecular beacon that possesses a stem-hairpinstructure as seen in conventional molecular beacons and canbe cleaved during PCR is designed, and it can specificallyrecognize the presence of the target and was obviously moresensitive than conventional molecular beacons.

A novel real-time quantitative method for measuring telomerase activity is described in which the duplex scorpionprimer is used to provide an intramolecular probing mechanism for specific detection of telomerase activity. Usingthis method, linearity from 10 to 104 cells expressing telomerase activity could be obtained (R2=0.994). Therequirement of post-PCR steps is thus obviated.

A novel method for the detection of specific nucleic acids in homogenous solution was developed. The method is based onthe use of duplex probes in which fluorescent donor and quencher labeled on either oligonucleotide are held in close proximity, so that fluorescence is quenched. Amplification of the target sequence results in the cleavage of the probe and the resultingfluorescence can be detected. The fluorescent data analysis demonstrated that the duplex probes can specifically recognize thepresence of target, and a significantly higher lever of relative fluorescent signal than TaqMan probes is obtainable. Combinedwith real-time PCR instruments, the assay can be used to quantify the input target molecules. As few as five copies of initialtarget molecules can be detected, and a large dynamic linear ranger (five orders of magnitude) is obtained.