Polymerase chain reaction probe labeling

A variation on the theme of conventional assay uses both lanthanide-labeled and biotin-labeled single strands to form split probes for sequence of target strands (Figure 12).120 When both of these bind to DNA, the complex binds (via the biotin residue) to a surface functionalized with streptavidin, immobilizing the europium and allowing assay to be carried out. This approach is already very sensitive to DNA sequence, since both sequences must match to permit immobilization of the lanthanide, but can be made even more sensitive by using PCR (the polymerase chain reaction) to enhance the concentration of DNA strands. In this way, initial concentrations corresponding to as few as four million molecules can be detected. This compares very favorably with radioimmunoassay detection limits. 

A CL ISH assay for the detection of human papillomavirus (HPV) DNA was developed, in which the hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes [64], The hybrids were visualized using AP as the enzyme label and a highly sensitive 1,2-dioxetane phosphate as chemiluminescent substrate. This assay was applied to biopsy specimens from different pathologies associated with HPV, which had previously proved positive for HPV DNA by polymerase chain reaction (PCR). The analytical sensitivity was assessed using samples of HeLa and CaSki cell lines, whose content in HPV DNA is known (10-50 copies of HPV 18 DNA in HeLa cells and 400-600 copies... 

FIGURE 2 Expression of exon 5 1- and exon 5 2-containing NOSl mRNAs in human tissues. Reverse transcription was performed using random primers and RNA (2 /ig) derived from leukocyte-enriched platelets (lanes 1 and 2), leukocyte-depleted platelets (lanes 3 and 4), skeletal muscle (lanes 5 and 6), or cerebellum (lanes 7 and 8) with (lanes 1, 3, 5, and 7) or without (lanes 2, 4, 6, and 8) prior treatment with RNase A and T1 followed by polymerase chain reaction (PCR) using Taq DNA polymerase and one twentieth of the reaction mixture and primer pairs specific for either (A) exon 5 2 or (B) exon 5 1. The PCR products were electrophoresed using 3% NuSieve GTG agarose (FMC Bioproducts), transferred to a Duralon-UV membrane (Stratagene), and hybridized with P-labeled probes specific for either (A) exon 5 2 or (B) exon 5T. nt. Nucleotides. 

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