Molecular structure of human chromosomes / edited by Jorge J. Yunis.

Date:
1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Molecular structure of human chromosomes / edited by Jorge J. Yunis. Source: Wellcome Collection.

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    3. Gene Localization by RNA:DNA Hybridization 65 know, the chromosomal DNA remains immobiUzed on the slide in the single- stranded state. Apparently the majority of DNA is blocked from complete RNA hybridization by chromosomal protein. The whole procedure of DNA denatura- tion should be reinvestigated for several reasons. Formamide should be used first because it denatures DNA without using acid or base. Seventy percent forma¬ mide in 2X SSC at 60°—70° would provide denaturation conditions that are above the Tu. of most DNA sequences. D. Time, Concentration, and Temperature Before hybridization is begun, it is necessary to make a few calculations and do preliminary experiments. Operationally, most people have used a concentra¬ tion between 0.1 and 1 pig per ml with mRNA, 5 S, 18 S, and 28 S rRNA (vertebrate chromosomes) and with tRNA {Drosophila). The highly repeated satellite DNA, 1.705 gm/cm^ of Drosophila (Peacock et al, 1973; Peacock and Steffensen, 1975) will reach saturation within a few hours with ^H-cRNA at 0.01 jUg/ml with either salivary or mitotic chromosomes. These highly repeated sequences of 1.705 gm/cm^ DNA are repeating units of 5-nucleotides, AAGAG (Endow et al, 1975). This rapid annealing is characteristic of constitutive heterochromatin. The rate is explained by the fact that short repeats have a high probability of finding complementary strands. When the kinetics of in situ hybridization are compared to DNA immobilized on filters, the reaction rates are very similar (Szabo et al, 1975). In both cases, the DNA is immobilized, so one can estimate from filter data about how fast or how slowly a particular RNA will anneal DNA on slides. For example, the messenger RNA for silk fibroin at 1-2 Aig/ml was sufficient to reach saturation on filters at hybridization for 24-48 hours (Suzuki et al, 1972). Again, the reader is reminded that this hybridization was done with formamide so that traces of ribonuclease would not degrade the messenger RNA during a long incubation period. Recently it has been possible to quantitate the kinetics of in situ hybridiza¬ tion. Szabo et al. (1975) extended the work of Birnstiel et al (1971) in the use of what is called a C^t plot, an abbreviation with Cy in moles/nucleotides/liter and t in sec. Given a concentration of RNA, the time to reach saturation at the 95% level can be calculated. E. Conditions of Hybridization A number of workers have hybridized in varying amounts of SSC, usually between 2X SSC and 6X SSC. The salt concentration has an effect on the rate, temperature and Гщ of hybridization. These methods are essentially those derived from procedures using nitrocellulose filters (Gillespie and Spiegelman,