Repeated sequences hybridization

Nakajima T, Ota N, Kodama T, Emi M. Isolation and radiation hybrid mapping of a highly polymorphic CA repeat sequence at the SREBP cleavage-activating protein (SCAP) locus. J Hum Genet 1999 44 350-351. 

Polyclonal antibodies can react with many epitopes, whereas MAbs are restricted to one epitope on proteins that do not have repeating sequences.24 By definition, polyclonal immunoassays are generally much more sensitive but less specific than monoclonal assays. Bispecific or hybrid antibodies can be used to increase the affinity. Bispecific antibodies are formed by the fusion of two previously established hybridomas to produce antibodies displaying the binding characteristics of both of the antibodies in one molecule.25... 

Given these constraints that impinge on the reassociation of DNA, experimental designs must be constructed that facilitate (1) the separation of single-copy DNA to be used as tracer DNA from repeated sequences and (2) the hybridization of single-copy tracer DNA with driver DNA from the same and different species. 

Fig. 2. Map of the Haloferax volcanii genome (top) the chromosome (bottom) the plasmids. Cosmids are represented by arrows, site-rich regions (oases) by heavy line, and markers placed by hybridization with unique probes are shown inside the circle (all from ref. [15]). Outside the circle are the results of probing with repeated sequences (ISH51, D), with labelled tRNA, 7S and ribosomal RNAs and (outermost) auxotrophic markers mapped by complementation. From ref. [86].

Formamide hybridization mix For repeat sequence probes (e.g., centromeric probes), 2X SSC, 500 ug/mL SSDNA, 10% dextran sulphate, 70% formamide. 

Controls. Test all your probes on normal chromosomes prepared from lymphocytes. This is essential no matter what your final application is. In addition, for interphase cytogenetics, use sections from normal tissue. Pay particular attention to specificity of hybridization. Some probes such as chromosome-specific repeat-sequence probes can bind to several chromosomes if the hybridization conditions are not correct. Use commercial probes to test your reagents. 

Suppression hybridization is used when repeated DNA is present in both target and probe sequences. Since these repeat sequences are present in a relatively much higher concentration and since they have a lower complexity, they would give a much stronger signal than less abundant sequences. These nonspecific hybridization signals can be suppressed by hybridizing first with unlabeled repetitive DNA probes. Such probes are now commercially available for different species (e.g., from BRL). 

These sequences are junk DNA in the same way we save the junk in our attic with the anticipation that we may need it someday. They are certainly not garbage DNA if this were true, these DNA would have been eliminated during the evolutionary process, the same way we throw away our garbage. Even before the human genome project, there were solid indications for the existence of the so-called repeat sequences in higher organisms, including humans, from the DNA hybridization experiments by Britten and Kohne (1968). 

Figure 6.11, Amo radio grams of doned, repeated sequences pCaod 5 (a LINE 1 probe) and Blur 8 (an Alu I probe) hybridized on nitrocellulose transfers from a gel electrophoresis on 0,6% agarose gel of Kpn I digests of unfractionated human DNA (lanes T) and its major eomponcms 1.698 (lanes 1), 1.700 (lanes 2). 1,704 (lanes 3), and 1.708 (lanes 4) loaded in equal amounts (I pg), (Modified from Soriano et al., 198.3).

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