Polymerase chain reaction hybridization with labeled probe

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. 

Fig. 1. A low-copy number RFLP cDNA probe used against /fmDIII digests of DNA from 24 barley (Hordeum vulgare, 2C = 10.9 pg 8/) varieties. 10 lg of digested DNA were applied to each lane of a 0.8% agarose gel that was run and transferred to Pall Biodyne B as described in this chapter. The probe was labeled with (hgoxigenin by the polymerase chain reaction (Chapter 10), and then hybridized against the membrane filter, and detected with AMPPD, as desaibed in Chapter 17. The luminograph shown here is a 4-h exposure.

A human gene in a somatic cell hybrid can also be detected and mapped without being expressed at the cellular level, e. g. by restriction enzyme digestion of DNA from the hybrid, followed by Southern blotting and detection of the human gene with a radioactively labeled probe, or by identifying primers which can be used in the Polymerase chain reaction (see) to amplify specifically the human and not the mouse gene. 

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