Hybrid selection

James, P., Halladay, J., and Craig, E. A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425-1436. 

Joung, J. K., Ramm, E. I., and Pabo, C. 0. (2000). A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. Proc. Natl. Acad. Sci. USA 97, 7382-7387. 

PAL cDNA clones have been isolated from a number of species (Table 1). The first PAL cDNA clone was isolated from French bean suspension culture cells treated with fungal elicitor (Edwards et al., 1985). The clones were preselected by differential hybridisation to RNA from non-treated and elicitor-treated suspension culture cells. Further identification involved in vitro translation of hybrid-selected RNA and immunoprecipi-tation (Edwards et al., 1985), using an antibody raised against the purified bean PAL enzyme (Bolwell etal., 1985). This work identified pPAL5 as a PAL cDNA clone. 

Under what conditions could the high hybridization selectivity be fully utilized for direct (reversible) sensing ... 

Colbere-Garapin F, Horodniceanu F, Kourilsky P, Garapin A (1981), A new dominant hybrid selective marker for higher eucaryotic cells, J. Mol. Biol. 150 1-14. 

Orum, H., Nielsen, P. E., Jorgensen, M., Larsson, C., Stanley, C., and Koch, T. (1995). Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection. BioTechniques 19, 472-480. 

The effect of pH on the hybridization selectivity of the DNA-conjugated TA-polyallylamine-immobilized substrate was studied. At pH 8.0 and 10, the... 

Table 1 Comparison of hybridization selectivity and efficiency of DNA conjugated TA-PAAm at various pH...

The hybridization selectivity and efficiency of DNA-conjugated PDPH-PAA were investigated with 1 pg mL 1 of injected sample DNA. The response to fully matched DNA (89 RU) was equivalent to 0.92 pmol cm 2, which indicates a hybridization efficiency of 33%, based on the amount of probe DNA on the surface (2.8 pmol cm 2). This hybridization efficiency was lower than that of DNA-conjugated TA-polyallylamine however, for DNA-conjugated TA- polyallylamine and PDPH-PAA, the maximum response when 1 pgmL-1 of unmatched DNA sample was injected decreased from 94 RU to 21 RU. These results suggest that less nonspecific DNA adsorption occurs with DNA-conjugated PDPH-PAA due to the electrostatic repulsion between the car-... 

The effect of various target DNA for codon 72 concentrations (20, 40, 70, 100 nM) on hybridization selectivity was investigated. The relation between fluorescence intensity and target DNA concentration is shown in Fig. 13. For tG-DNA (the fully matched sequence), the fluorescence intensity clearly increased with an increase in the target DNA concentration. On the other hand, the fluorescence intensities in the presence of tC, tT, and tA-DNA (SNP sequences) and unmatched DNA were essentially the same at all target DNA concentrations. This result indicates that this DNA-conjugated polymer can... 

Nylon, particularly the positively charged membranes, has largely replaced nitrocellulose or diazotized paper for hybrid selection. DNA, e.g., from linearized plasmid, can be applied in 0.4 N NaOH to denature the DNA and to promote a strong fixation. The nylon membrane (2-cm squares) is soaked for 5 min in water and then 30 min in 0.4 N NaOH. Concurrently, DNA is denatured for 10 min in 0.4 N NaOH. DNA is then applied repeatedly with drying between applications. The membrane is then washed twice with 1 M NH4OAC and twice with 1 X SSG. The membrane is blotted and dried (may contain > 100 p,g/cm ). An alternative method is presented in Section 8.3.1. Disks of 0.5 cm are then punched out with a sterile one-hole paper punch. It is also possible to use Southern blots, or fragments thereof, for hybrid selection. 

The major problem in hybrid selection is degradation of mRNA by contaminants of formamide. Formamide is required to lower the T, to prevent RNA degradation due to high temperatures and should be deionized (stir 10 ml of formamide with 1 g of AG-501SA from Bio-Rad for 1 h at room temperature, filter and store at -70°C). 

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