Preparation of nucleic acid probes

Figure 5.2A ). If the reaction is carried out at a relatively low temperature (about 15° C), the reaction proceeds no further than one complete renewal of the existing nucleotide sequence. Although there is no net DNA synthesis at these temperatures, the synthesis reaction allows the incorporation of labeled nucleotides in place of the previously existing unlabeled ones.

Feinberg and Vogelstein, 1983 ) is based on hybridization of a mixture of all possible hexanucleotides: the starting DNA is denatured and then cooled slowly so that the individual hexanucleotides can bind to suitably complementary sequences within the DNA strands. Synthesis of new complementary DNA strands is primed by the bound hexanucleotides and is catalyzed by the Klenow subunit of DNA polymerase I (which contains the polymerase activity in the absence of associated exonuclease activities). DNA synthesis occurs in the presence of the four dNTPs, at least one of which has a labeled group (see Figure 5.2B ). This method produces labeled DNAs of high specific activity. Because all sequence combinations are represented in the hexanucleotide mixture, binding of primer to template DNA occurs in a random manner, and labeling is uniform across the length of the DNA.

Figure 5.3A ). The same procedure can also be used for labeling double-stranded DNA. In this case, fragments carrying label at one end only can then be generated by cleavage at an internal restriction site, generating two differently sized fragments which can be separated by gel electrophoresis and purified.

Larger DNA fragments can be end-labeled by various alternative methods. Fill-in end-labeling (Figure 5.3B ) is one popular approach, and uses the Klenow subunit of E. coli DNA polymerase. Again, fragments carrying label at one end only can be generated by restriction cleavage and size fractionation. An alternative PCR-based method is primer-mediated 5 end-labeling (see Section 6.1.1 ).

riboprobes ) is most easily achieved by in vitro transcription of insert DNA cloned in a suitable plasmid vector. The vector is designed so that adjacent to the multiple cloning site is a phage promoter sequence, which can be recognized by the corresponding phage RNA polymerase. For example, the plasmid vector pSP64 contains the bacteriophage SP6 promoter sequence immediately adjacent to a multiple cloning site (see Figure 5.4 ). The SP6 RNA polymerase can then be used to initiate transcription from a specific start point in the SP6 promoter sequence, transcribing through any DNA sequence that has been inserted into the multiple cloning site. By using a mix of NTPs, at least one of which is labeled, high specific activity radiolabeled transcripts can be generated (Figure 5.4 ). Bacteriophage T3 and T7 promoter/RNA polymerase systems are also used commonly for generating riboprobes. Labeled sense and antisense riboprobes can be generated from any gene cloned in such vectors (the gene can be cloned in either of the two orientations) and are widely used in tissue in situ hybridization ( Section 5.3.4 ).

Box 5.1 ).

The intensity of an autoradiographic signal is dependent on the intensity of the radiation emitted by the radioisotope, and the time of exposure, which may often be long (one or more days, or even weeks in some applications). ³² P has been used widely in Southern blot hybridization, dot-blot hybridization, colony and plaque hybridization (see below) because it emits high energy b-particles which afford a high degree of sensitivity of detection. It has the disadvantage, however, that it is relatively unstable (see Table 5.1 ). Additionally, its high energy b-particle emission can be a disadvantage under circumstances when fine physical resolution is required to interpret the resulting image unambiguously. For this reason, radionuclides which provide less energetic b-particle radiation have been preferred in certain procedures, for example ³⁵ S-labeled and ³³ P-labeled nucleotides for DNA sequencing and tissue in situ hybridization, and ³ H-labeled nucleotides for chromosome in situ hybridization. ³⁵ S and ³³ P have moderate half-lives while ³ H has a very long half-life. However, the latter isotope is disadvantaged by its comparatively low energy b-particle emission which necessitates very long exposure times.

³² P-labeled and ³³ P-labeled nucleotides used in DNA strand synthesis labeling reactions have the radioisotope at the a-phosphate position, because the b- and g-phosphates from dNTP precursors are not incorporated into the growing DNA chain. Kinase-mediated end-labeling, however, uses [g-³² P]ATP (see Figure 5.3A ). In the case of ³⁵ S-labeled nucleotides which are incorporated during the synthesis of DNA or RNA strands, the NTP or dNTP carries a ³⁵ S isotope in place of the O^- of the a-phosphate group. ³ H-labeled nucleotides carry the radioisotope at several positions. Specific detection of molecules carrying a radioisotope is most often performed by autoradiography (see Box 5.1 ).

Kricka, 1992 ). Two types of non-radioactive labeling are conducted:

• Direct nonisotopic labeling , where a nucleotide which contains the label that will be detected is incorporated. Often such systems involve incorporation of modified nucleotides containing a fluorophore (Figure 5.5A ), a chemical group which can fluoresce when exposed to light of a certain wavelength (fluorescence labeling - see Box 5.2 ).
• Indirect nonisotopic labeling , usually featuring the chemical coupling of a modified reporter molecule to a nucleotide precursor. After incorporation into DNA, the reporter groups can be specifically bound by an affinity molecule, a protein or other ligand which has a very high affinity for the reporter group. Conjugated to the latter is a marker molecule or group which can be detected in a suitable assay (Figure 5.6 ). The reporter molecules on modified nucleotides need to protrude sufficiently far from the nucleic acid backbone to facilitate their detection by the affinity molecule and so a long carbon atom spacer is required to separate the nucleotide from the reporter group.

Two indirect nonisotopic labeling systems are widely used:

• The biotin-streptavidin system utilizes the extremely high affinity of two ligands: biotin (a naturally occurring vitamin) which acts as the reporter, and the bacterial protein streptavidin, which is the affinity molecule. Biotin and streptavidin bind together extemely tightly with an affinity constant of 10^-14 , one of the strongest known in biology. Biotinylated probes can be made easily by including a suitable biotinylated nucleotide in the labeling reaction (see Figure 5.7 ).
• Digoxigenin is a plant steroid (obtained from Dig i- talis plants) to which a specific antibody has been raised. The digoxigenin-specific antibody permits detection of nucleic acid molecules which have incorporated nucleotides containing the digoxigenin reporter group (see Figure 5.7 ).