Nucleic Acid Hybridization

The key to detection of specific nucleic acid sequences is base pairing between complementary strands of RNA or DNA. At high temperatures (e.g., 90 to 100°C), the complementary strands of DNA separate (denature), yielding single-stranded molecules. If such denatured DNA strands are then incubated under appropriate conditions (e.g., 65°), they will renature to form double-stranded molecules as dictated by complementary base pairing—a process called nucleic acid hybridization. Nucleic acid hybrids can be formed between two strands of DNA, two strands of RNA, or one strand of DNA and one of RNA.

Nucleic acid hybridization provides a means for detecting DNA or RNA sequences that are complementary to any isolated nucleic acid, such as a viral genome or a cloned DNA sequence (Figure 3.28). The cloned DNA is radiolabeled, usually by being synthesized in the presence of radioactive nucleotides. This radioactive DNA is then used as a probe for hybridization to complementary DNA or RNA sequences, which are detected by virtue of the radioactivity of the resulting double-stranded hybrids.

Figure 3.28

Detection of DNA by nucleic acid hybridization. A specific sequence can be detected in total cell DNA by hybridization with a radiolabeled DNA probe. The DNA is denatured by heating to 95°C, yielding single-stranded molecules. The radiolabeled (more...)

Southern blotting (a technique developed by E. M. Southern) is widely used for detection of specific genes in cellular DNA (Figure 3.29). The DNA to be analyzed is digested with a restriction endonuclease, and the digested DNA fragments are separated by gel electrophoresis. The gel is then overlaid with a nitrocellulose filter or nylon membrane, to which the DNA fragments are transferred (blotted) to yield a replica of the gel. The filter is then incubated with a radiolabeled probe, which hybridizes to the DNA fragments that contain the complementary sequence. These fragments are then visualized by exposure of the filter to X-ray film.

Figure 3.29

Southern blotting. Restriction endonuclease fragments of DNA are separated by gel electrophoresis. Specific DNA fragments are then identified by hybridization with an appropriate probe.

Northern blotting is a variation of the Southern blotting technique (hence its name) that is used for detection of RNA instead of DNA. In this method, total cellular RNAs are extracted and fractionated according to size by gel electrophoresis. As in Southern blotting, the RNAs are transferred to a filter and detected by hybridization with a radioactive probe. Northern blotting is frequently used in studies of gene expression—for example, to determine whether specific mRNAs are present in different types of cells.

Nucleic acid hybridization can be used to detect homologous DNA or RNA sequences not only in cell extracts, but also in chromosomes or intact cells—a procedure called in situ hybridization (Figure 3.30). In this case, the hybridization of radioactive or fluorescent probes to specific cells or subcellular structures is analyzed by microscopic examination. For example, labeled probes can be hybridized to intact chromosomes in order to identify the chromosomal regions that contain a gene of interest. In situ hybridization can also be used to detect specific mRNAs in different types of cells within a tissue.

Figure 3.30

Fluorescence in situ hybridization. Hybridization of human chromosomes with chromosome-specific fluorescent probes that label each of the 24 chromosomes a different color. (Courtesy of Thomas Reid and Hesed Padilla-Nash, National Cancer Institute.)

Detection of Small Amounts of DNA or RNA by PCR

Amplification of DNA by the polymerase chain reaction is a much more sensitive technique for detecting cellular DNA or RNA sequences than is Southern or Northern blotting. Approximately 100,000 copies of a DNA or RNA sequence are required for detection by blot hybridization. In contrast, PCR can amplify single copies of DNA (or RNA after reverse transcription) to readily detectable levels.

As discussed earlier, the specificity of amplification in PCR is provided by the use of oligonucleotide primers that hybridize to complementary sequences on the template molecule. Therefore, PCR can selectively amplify a specific DNA molecule from complex mixtures, such as total cell DNA or RNA. Consequently, PCR amplification can be used to detect specific DNA or RNA molecules in very small amounts of starting material, such as extracts of single cells. This extraordinary sensitivity has made PCR an important method for a variety of applications, including the analysis of gene expression in cells available in only limited quantities.

Antibodies as Probes for Proteins

Studies of gene expression and function require the detection not only of DNA and RNA, but also of specific proteins. For these studies, antibodies take the place of nucleic acid probes as reagents that can selectively react with unique protein molecules. Antibodies are proteins produced by cells of the immune system (B lymphocytes) that react against molecules (antigens) that the host organism recognizes as foreign substances—for example, the protein coat of a virus. The immune systems of vertebrates are capable of producing millions of different antibodies, each of which specifically recognizes a unique antigen, which may be a protein, a carbohydrate, or a nonbiological molecule. An individual lymphocyte produces only a single type of antibody, but the antibody genes of different lymphocytes vary as a result of programmed gene rearrangements during development of the immune system (see Chapter 5). This variation gives rise to an array of lymphocytes with distinct antibody genes, programmed to respond to different antigens.

Antibodies can be generated by inoculation of an animal with any foreign protein. For example, antibodies against human proteins are frequently raised in rabbits. The sera of such immunized animals contain a mixture of antibodies (produced by different lymphocytes) that react against multiple sites on the immunizing antigen. However, single species of antibodies (monoclonal antibodies) can also be produced by the culturing of clonal lines of B lymphocytes from immunized animals (usually mice). Because each lymphocyte is programmed to produce only a single antibody, a clonal line of lymphocytes produces a monoclonal antibody that recognizes only a single antigenic determinant, thereby providing a highly specific immunological reagent.

Although antibodies can be raised against proteins purified from cells, other materials may also be used for immunization. For example, animals may be immunized with intact cells to raise antibodies against unknown proteins expressed by a specific cell type (e.g., a cancer cell). Such antibodies may then be used to identify proteins specifically expressed by the cell type used for immunization. In addition, antibodies are frequently raised against proteins expressed in bacteria as recombinant clones. In this way, molecular cloning allows the production of antibodies against proteins that may be difficult to isolate from eukaryotic cells. Moreover, antibodies can be raised against synthetic peptides that consist of only 10 to 15 amino acids, rather than against intact proteins. Therefore, once the sequence of a gene is known, antibodies against peptides synthesized from part of its predicted protein sequence can be produced. Because antibodies against these synthetic peptides frequently react with the intact protein as well, it is possible to produce antibodies against a protein starting with only the sequence of a cloned gene.

Antibodies can be used in a variety of ways to detect proteins in cell extracts. Two common methods are immunoblotting (also called Western blotting) and immunoprecipitation. Western blotting (Figure 3.31) is another variation of Southern blotting. Proteins in cell extracts are first separated according to size by gel electrophoresis. Because proteins have different shapes and charges, however, this process requires a modification of the methods used for electrophoresis of nucleic acids. Proteins are separated by a method known as SDS-polyacrylamide gel electrophoresis (SDS-PAGE), in which they are dissolved in a solution containing the negatively charged detergent sodium dodecyl sulfate (SDS). Each protein binds many detergent molecules, which denature the protein and give the protein an overall negative charge. Under these conditions, all proteins migrate toward the positive electrode—their rates of migration determined (like those of nucleic acids) only by size. Following electrophoresis, the proteins are transferred to a filter, which is then allowed to react with antibodies against the protein of interest. The antibody bound to the filter can be detected by various methods, thereby identifying the protein against which the antibody is targeted.

Figure 3.31

Western blotting. Proteins are separated according to size by SDS-polyacrylamide gel electrophoresis and transferred from the gel to a filter. The filter is incubated with an antibody directed against a protein of interest. The antibody bound to the filter (more...)

In immunoprecipitation, antibodies are used to isolate the proteins against which they are directed (Figure 3.32). Typically, cells are incubated with radioactive amino acids to label their proteins. Such a radiolabeled cell extract is then incubated with an antibody, which binds to its antigenic target protein. The resulting antigen-antibody complexes are isolated and subjected to electrophoresis, allowing detection of the radioactive antigen by autoradiography.

Figure 3.32

Immunoprecipitation. Radiolabeled proteins are incubated with an antibody, which forms complexes with the protein against which it is directed (the antigen). These antigen-antibody complexes are collected on beads that bind the antibody. The beads are (more...)

As discussed in Chapter 1, antibodies can also be used to visualize proteins within cells, as well as in cell lysates. For example, cells can be stained with antibodies labeled with fluorescent dyes, and the subcellular localization of the antigenic proteins can be visualized by fluorescence microscopy (see Figure 1.28). Antibodies can also be labeled with tags that are visible in the electron microscope, such as heavy metals, allowing visualization of antigenic proteins at the ultrastructural level.

Probes for Screening Recombinant DNA Libraries

The same basic methods for detecting nucleic acids and proteins in cell extracts are used for identifying molecular clones that contain specific cellular DNA inserts. For example, nucleic acid hybridization can be used to identify genomic or cDNA clones that contain DNA sequences for which a probe is available. Cloned cDNAs in expression vectors can also be identified by the use of antibodies against their encoded proteins.

The first step in isolation of either genomic or cDNA clones is frequently the preparation of recombinant DNA libraries—collections of clones that contain all the genomic or mRNA sequences of a particular cell type (Figure 3.33). A genomic library of human DNA, for example, might be prepared by the cloning of random DNA fragments with average sizes of about 15 kb in a λ vector. Since the size of the human genome is about 3 × 10⁶ kb, the DNA equivalent of one genome would be represented by 200,000 such λ clones. Because of statistical fluctuations in sampling, however, many genes will not be represented in a library of 200,000 recombinants, while other genes will be represented by multiple clones. Therefore, larger libraries, consisting of approximately 1 million recombinant phages are usually prepared to ensure a high likelihood that any gene of interest is represented in the collection.

Figure 3.33

Screening a recombinant library by hybridization. Fragments of cell DNA are cloned in a bacteriophage λ vector and packaged into phage particles, yielding a collection of recombinant phage carrying different cell inserts. The phages are used to (more...)

Any gene for which a probe is available can readily be isolated from such a recombinant library. The recombinant phages are plated on E. coli, and each phage replicates to produce a plaque on the lawn of bacteria. The plaques are then blotted onto filters in a process similar to the transfer of DNA from a gel to a filter during Southern blotting, and the filters are hybridized with a radiolabeled probe to identify the phage plaques that contain the gene of interest. The appropriate plaque can then be isolated from the original plate in order to propagate the recombinant phage that carries the desired cell insert. Similar procedures can be used to screen bacterial colonies carrying plasmid DNA clones, so specific clones can be isolated by hybridization from either phage or plasmid libraries.

A variety of probes can be used for screening recombinant libraries. For example, a cDNA clone can be used as a probe to isolate the corresponding genomic clone, or a gene cloned from one species (e.g., mouse) can be used to isolate a related gene from a different species (e.g., human). In addition to isolated DNA fragments, synthetic oligonucleotides can be used as probes, enabling the isolation of genes on the basis of partial amino acid sequences of their encoded proteins. For example, oligonucleotides consisting of 15 to 20 bases can be synthesized on the basis of the partial amino acid sequence of a protein of interest. These oligonucleotides can then be used as probes to isolate cDNA clones, which (as already discussed) are much easier to sequence and to characterize than is the protein itself. It is thus possible to proceed experimentally from a partial peptide sequence of a protein to the isolation of a cloned gene.

An alternative approach to isolating a gene on the basis of its protein product is the use of antibodies as probes to screen expression libraries. In this case a cDNA library is generated in an expression vector that drives protein synthesis in E. coli. Phage plaques or bacterial colonies are then transferred to a filter as already described, but the filter is then reacted with an antibody (as in Western blotting) to identify clones that are producing the protein of interest.

These procedures for identifying molecular clones and detecting genes and gene products in cells illustrate the flexibility of recombinant DNA technology. Starting with any cloned gene, it is possible not only to determine the nucleotide sequence of that gene and use it as a probe for studies of gene organization and transcription, but also to express and raise antisera against its encoded protein. Conversely, genes can be cloned on the basis of limited characterization of a protein of interest, using either oligonucleotide or antibody probes. Thus, recombinant DNA has allowed experimental analyses to proceed either from DNA to protein or from protein to DNA, providing great versatility to the strategies currently employed for studies of eukaryotic genes and their encoded proteins.