Once nucleic acids have been affixed to a membrane, specific sequences can be detected by hybridization with a labeled, denatured, single-stranded probe that binds to homologous RNA or DNA. These probes might be composed of either RNA or DNA, and labeling methods might be radioactive or nonra-dioactive.
11.1. NICK TRANSLATION The method of nick translation relies on Escherichia coli DNA polymerase I—a polymerizing enzyme that also possesses a 5'^3' exonuclease activity that degrades double-stranded DNA and RNA : DNA hybrids (26). First, discontinuities ("nicks") are generated in the phosphodiester backbone of the double-stranded DNA by brief treatment with pancreatic DNase I, producing free 3'-hydroxyl termini along the strand of DNA. DNA polymerase I then extends the 3'-OH termini in the presence of the four dNTPs, utilizing its exonuclease activity to hydrolyze nucleotides in the 5'^3' direction (see Fig. 7). The use of radioactive nucleoside triphosphates in the reaction with DNA polymerase I produces uniformly labeled DNA. Disadvantages of nick translation include the strict requirements in the protocol to time and temperature limitations and the large amount of template DNA (0.5 |g) required per reaction. It is also important to note that small DNA fragments (<200 bp) are not suitable for nick translation. Nick translation kits are commercially available and provide a reliable source for the buffers and enzymes to carry out the reaction. Most protocols recommend separating radiolabeled DNA from unincorporated dNTPs by centrifugation through a small column of Sephadex G-50 (6,28).
11.2. RANDOM PRIMER EXTENSION An alternative method for generating labeled DNA involves the utilization of oligonucleotide primers of random sequence (29). The double-stranded DNA is denatured and random nanomers or hexamers
are annealed to the template DNA strands (see Fig 8). The primers are extended by the large fragment of DNA polymerase (Klenow fragment) or T7 DNA polymerase in the presence of radiolabeled dNTPs. Random-primed probes can be labeled to a higher specific activity than nick-translated probes, although they are typically shorter (approx 500 nucleotides). The reaction can also be carried out at room temperature with small quantities of template DNA (25-50 ng) and is not significantly affected by longer incubation times (overnight). This method could also be used to label small fragments (200 bp). Random primer extension kits are available commercially and provide the user with enzyme, cold dNTPs, and reaction buffers. Because the majority of radioactive dNTP is incorporated into DNA, purification of the probe is not usually necessary. However, if purification is needed, centrifugation through Sephadex G-50 is sufficient.
11.3. GENERATION OF STRAND-SPECIFIC PROBES The generation of radiolabeled probes from double-stranded DNA works well when the target sequences are present in sufficient quantity and the hybridization between the labeled DNA probe and target sequences is strong. When hybridization between probe and target is weak, hybrids form between the complementary DNA sequences of the probe, resulting in segregation of the probe and decreased detection of target sequences. Single-stranded probes are composed of only one of two strands of a nucleic acid sequence, thus allowing detection of target sequences without unwanted reannealing of probe. These probes are particularly useful when analyzing target sequences that are only partially homologous to the probe (such as the detection of homologous genes in multiple species).
Radiolabeled cDNA probes are generated by primer extension of single-stranded DNA derived from a recombinant bac-teriophage Ml3 and can yield probes of extremely high specific activity [1 x 109 counts per million (CPM)/|g] (29). Primers are commonly chosen that anneal to the single-stranded viral DNA in a region upstream from the site of insertion. Extension of the annealed primers is typically accomplished with the Klenow fragment of DNA polymerase I
in the presence of three nonradioactive dNTPs and one a-32P-labeled dNTP. The major drawback of this method is the required separation of labeled probe from the template and smaller DNA fragments. Labeled probes can be separated by polyacrylamide gel electrophoresis or by alkaline chromatography through Sepharose CL-4B (Pharmacia Biotech) (28).
The ability to generate single-stranded RNA probes ("ribo-probes") has been made possible by the development of plas-mid vectors, which contain multiple cloning sites downstream from strong bacteriophage promoters (SP6, T7, and T3). These promoters are recognized by bacteriophage-specific, DNA-dependent RNA polymerases, which fail to recognize bacterial, plasmid, or eukaryotic promoters that are present within the construct (30). As a result, labeled RNA can be synthesized directly from these linearized plasmids at an extremely high efficiency and specific activity when radioactive NTPS are included in the reaction (see Fig 9). The greater stability of RNA hybrids makes RNA probes superior to double-stranded and single-stranded DNA probes in Southern and Northern analysis. In addition, the production of radiolabeled RNA is more efficient than the generation of single-stranded cDNA probes, because unwanted template DNA can be eliminated by simply treating the samples with RNase-free DNase I. When these RNA probes are to be applied to Northern blots, it is important to generate the probe in the "antisense" direction, so that the probe is complementary and not identical to the mRNA of interest.
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