7:  7.5 Analyzing Specific Nucleic Acids in Complex Mixtures

Southern Blotting Detects Specific DNA Fragments

The technique of Southern blotting, named after its originator Edwin Southern, can identify specific restriction fragments in a complex mixture of restriction fragments. The DNA to be analyzed, such as the total DNA of an organism, is digested to completion with a restriction enzyme. For an organism with a complex genome, this digestion may generate millions of specific restriction fragments. The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size. However, many different fragments are of exactly the same length, and these do not separate from one another.

Even though all the fragments are not resolved by gel electrophoresis, an individual fragment that is complementary to a specific DNA clone can be detected. The restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting (Figure 7-32). This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter, much like the replica filter produced from plaques of a λ library. (The blot is used because probes do not readily diffuse into the original gel.) The filter then is incubated under hybridization conditions with a specific radiolabeled DNA probe, which usually is generated from a cloned restriction fragment. The DNA restriction fragment that is complementary to the probe hybridizes, and its location on the filter can be revealed by autoradiography.

Southern blotting permits a comparison between the restriction map of DNA isolated directly from an organism and the restriction map of cloned DNA. This comparison is necessary to be certain that no rearrangements have occurred during the cloning procedure such as might happen if two restriction fragments that do not normally lie next to each other were inadvertently ligated together before ligation into a cloning vector. Southern blotting also is used to map restriction sites in genomic DNA next to the sequence of a cloned DNA fragment. This provides a rapid method of comparing the restriction maps of different individual organisms in the region surrounding a cloned fragment. Deletion and insertion mutations are readily detected, as well as sequence differences in specific restriction sites.

Northern Blotting Detects Specific RNAs

Northern blotting, humorously named because it is patterned after Southern blotting, is used to detect a particular RNA in a mixture of RNAs. An RNA sample, often the total cellular RNA, is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation. The individual RNAs then are separated according to size by gel electrophoresis and transferred to a nitrocellulose filter to which the extended denatured RNAs adhere. The filter then is exposed to a labeled DNA probe and subjected to autoradiography. Because the amount of a specific RNA in a sample can be estimated from a Northern blot, the procedure is widely used to compare the amounts of a particular mRNA in cells under different conditions (Figure 7-33).

Specific RNAs Can Be Quantitated and Mapped on DNA by Nuclease Protection

Another important method for detecting and quantitating specific RNA molecules employs endonucleases that digest single-stranded but not double-stranded nucleic acids. The method was originally designed using endonuclease S1, an enzyme from the mold Aspergillus oryzae that digests singlestranded RNA and DNA but not double-stranded molecules. A labeled DNA strand, or probe, complementary to an RNA of interest is prepared from a cloned DNA. A source of RNA, such as the total polyadenylated RNA isolated from a particular tissue or type of cultured cell, is incubated with a high concentration of the labeled DNA probe under conditions in which all the RNA complementary to the probe hybridizes to it (Figure 7-34a). The preparation then is treated with endonuclease S1, which digests all the unhybridized RNA and probe molecules, leaving only the double-stranded region in the RNA-DNA hybrids, which is protected from nuclease digestion. Treatment of the digested preparation with alcohol precipitates the probe – target RNA hybrid, which then is subjected to gel electrophoresis followed by autoradiography or phosphorimager analysis of the gel to detect the protected probe. The amount of radioactivity in the resulting band is a measure of the amount of RNA complementary to the probe in the initial sample of RNA. Nuclease protection also can be performed with a complementary labeled RNA and ribonuclease A, a single-strand-specific pancreatic ribonuclease.

The DNA region that encodes a particular RNA can be mapped with the nuclease-protection technique by use of restriction-fragment probes in which one end is complementary to only a portion of the RNA of interest. In this case, the RNA-DNA hybrid protected from S1 digestion is shorter than the RNA being probed; its length corresponds to that of the DNA region extending from one end of the coding region to a restriction site within it (Figure 7-34b). Comparison of the protected doubled-stranded fragments obtained with two or more such “partial” probes can map the RNA sequence relative to restriction sites in the complementary DNA (Figure 7-34c).

Transcription Start Sites Can Be Mapped by S1 Protection and Primer Extension

As discussed in Chapter 10, some of the DNA regulatory elements that control transcription of genes into mRNA are located near the transcription start site. Mapping the start site for synthesis of a particular mRNA often helps in identifying the DNA regulatory sequences that control its transcription. Two methods are used to map the 5′ end of a particular mRNA on a complementary DNA: S1 protection and primer extension. The first step in both methods is to identify the general region of a DNA that includes the start site of interest (Figure 7-35a); this can be done by Northern blot analysis or nuclease protection using various cloned restriction fragments as probes.

In the S1-protection method, the identified DNA region is treated with appropriate restriction enzymes to produce a single-stranded DNA fragment that will hybridize with the 5′ portion of the mRNA. This fragment is radiolabeled at the 5′ end, hybridized with the mRNA, and then trimmed with S1 endonuclease (Figure 7-35b). From the length of the labeled probe segment protected from digestion, the position of the start site in the original DNA can be located.

The primer-extension method uses a synthetic oligonucleotide that is complementary to an approximately 20- nucleotide stretch of the mRNA located 50 – 200 nucleotides from its 5′ end. This synthetic oligonucleotide is end-labeled at the 5′ end and then used to prime DNA synthesis by reverse transcriptase with the mRNA as the template (Figure 7-35c). The position of the start site can be mapped from the length of the resulting extension product.

SUMMARY

• Cloned DNA fragments that have been isolated and labeled (usually radioactively) are widely used as probes to identify and quantitate a complementary DNA or RNA in a complex mixture. When added to a mixture of polynucleotide chains, a probe base-pairs only with complementary chains.

• Southern and Northern blots detect specific DNAs or RNAs, respectively, that have been separated by gel electrophoresis. Polynucleotides in the gel are transferred and attached to a filter or membrane replica of the gel (see Figure 7-32). The membrane is then incubated with a probe that hybridizes to the complementary polynucleotide of interest. After excess probe is washed away, the position and amount of labeled probe bound to the filter is determined by autoradiography or phosphorimager analysis.

• The amount of a specific RNA in a mixture can be detected by nuclease-protection analysis. Hybridization of an RNA to a labeled, single-stranded probe protects it from digestion by a nuclease that digests single-stranded but not double-stranded nucleic acids (see Figure 7-34a). The nuclease-resistant probe, hybridized to the target RNA, is recovered, subjected to gel electrophoresis, and detected by autoradiography or phosphorimager analysis. A variant of nuclease-protection analysis can map an RNA relative to restriction sites in complementary DNA (see Figure 7-34b, c).

• A target mRNA can also be quantitated and its transcription start site mapped by nuclease-protection analysis with a 5′-end-labeled DNA probe that hybridizes with the 5′ end of the mRNA (see Figure 7-35a, b). In the alternative primer-extension method, a short 5′-end-labeled DNA probe that hybridizes to the target mRNA acts as a primer for extension by reverse transcriptase to the 5′ end of the mRNA template strand (see Figure 7-35a, c).