■ Figure 6-4 (a.) Genomic DNA fragments cut with restriction enzymes Bgl II, BamHI and Hind III and separated by gel electrophoresis. (b.) Autoradiogram of the fragments hybridized to a radioactive or chemiluminescent probe. Control lanes, C, show the restriction pattern of normal DNA. Test lanes, +, show the different restriction patterns that result from the abnormal or translocated DNA.
in the gel. Although short fragments can be denatured directly as described below, larger fragments (>500 bp) are more efficiently denatured if they are depurinated before denaturation (Fig. 6-5). Therefore, for large fragments, the gel is first soaked in HCl solution, a process that removes purine bases from the sugar phosphate backbone. This will "loosen up" the larger fragments for more complete denaturation.
Following depurination, the DNA is denatured by exposing the DNA in the gel to sodium hydroxide. The strong base (NaOH) promotes breakage of the hydrogen bonds holding the DNA strands to one another. The resulting single strands are then available to hydrogen-bond with the single-stranded probe. Further, the single-stranded DNA will bind more tightly than double-stranded DNA to the nitrocellulose membrane upon transfer.
Was this article helpful?