Probing Questions

Nucleic Acid Labeling Kits Nucleic Acid Labeling Kits (continued) Nucleic Acid Labeling Kits (continued) Genisphere's 3DNA™ Expression Array Detection Kit reagents The ability to label nucleic acids with a variety of tagged molecules has led to astonishing advances in molecular biology. Labeled DNA, RNA, and oligonucleotide probes have made possible techniques like the historic Southern blot, the northern blot, and related dot/slot-blotting procedures, in addition to in situ hybridization studies and colony and plaque lifts. Recently, labeled cDNA probes have become an integral part of the gene expression analysis advances empowered by gene arrays. Beginning with the enzymatic incorporation of radioactivity and eventually employing nonisotopic alternatives, labeling methods and kits have evolved into a host of products and creative technologies that offer some powerful tools to the life scientist. Enzymatic Labeling Techniques Enzymatic labeling techniques are the traditional way to label nucleic acids, although direct labeling techniques are gaining prominence. Nick translation was one of the first techniques molecular biologists used to generate radioactive DNA probes in vitro.1 Prior to the development of nick translation, researchers often relied on in vivo labeling procedures to prepare radiolabeled probes, obtaining specific activities of 106 cpm/µg. In comparison, nick translation generates probes with specific activities near 108 cpm/µg of template DNA. This increase in specific activity enhanced detection sensitivity by 200-fold. To label probes by nick translation, DNase I is used to create free 3' hydroxyl ends (nicks) within an unlabeled DNA template. DNA polymerase I then incorporates radiolabeled deoxynucleotide triphosphates into the nick. This enzyme also shifts, or translates, the nick down the strand in the 3' direction by excising the nucleotide at the 5' end of the nick. As the nick translates down the strand, additional labeled nucleotides are incorporated into it. Andrew P. Feinberg and Bert Vogelstein introduced random prime labeling in 1983, and it has become a common method for labeling DNA.2 This technique relies on a mixture of random primers, usually six to 10 nucleotides long, to prime DNA synthesis in vitro along any double-stranded DNA template. The Klenow fragment of DNA polymerase I is the enzyme used most frequently to synthesize the labeled DNA, but some novel polymerases are also available. Usually, random prime labeling generates radioactive probes with specific activities exceeding 1 x 109 cpm/µg of template DNA. Developed initially using 32P-dATP or 32P-dCTP, random prime labeling can also incorporate nucleotides tagged with 3H, 35S, 33P, and 125I into the newly synthesized probes. Nonradioactive nucleotides such as fluorescein-11-dUTP or biotin-14-dCTP are also popular labels. As an added benefit, the technique successfully labels DNA fragments in low-melt agarose.3 End-labeling reactions designed to attach tagged nucleotides to DNA, RNA, or oligonucleotides form the basis of several commercially available products. Terminal deoxynucleotidyl transferase labels nucleic acids at the 3' end and is included in many products developed primarily for labeling oligonucleotides. Bacteriophage T4 polynucleotide kinase (PNK) catalyzes the transfer of the *-phosphate of [*-32P]ATP to the 5' terminus of DNA, RNA, or oligonucleotides, producing radiolabeled products. Additionally, PNK quantitatively phosphorylates 5' ends using unlabeled ATP. Many cloning vectors contain separate phage promoters for the production of RNA transcripts from cloned DNA templates. The phage polymerases SP6, T7, and T3 are the most widely used for the generation of in vitro transcripts containing radioactive or nonisotopic labels. A few companies tout the benefits of using labeled RNA transcripts in hybridization experiments. These probes offer greater sensitivity than DNA probes because RNA:RNA and RNA:DNA hybrids are more stable than DNA:DNA hybrids. The polymerase chain reaction (PCR) has also found application in the generation of labeled DNA probes. Ambion, KPL, and Roche Molecular Biochemicals sell PCR-based labeling kits for amplifying radiolabeled, biotinylated, and digoxigenin (DIG)-labeled probes from small amounts of starting material. Can You DIG It? Most of the enzyme-mediated labeling methods were initially developed to incorporate 32P-labeled nucleotides into nucleic acids. While sensitive and useful, these probes suffer from the usual drawbacks associated with the use of radioisotopes. Motivated by the desire to move away from radiation, researchers have developed many nonisotopic labeling alternatives. In 1981, the biotin-avidin/streptavidin system was the first nonradioactive nucleic acid labeling system to be developed, but this system was hampered by background problems.4 In the late 1980s, Boehringer Mannheim Corporation (now Roche Molecular Biochemicals) perfected a labeling system based on digoxigenin, a steroid hapten found in Digitalis plants.5 This system avoids the endogenous background problems encountered with biotin and other methods. Digoxigenin is readily incorporated into nucleic acids using enzymatic labeling methods and is detected with enzyme-labeled anti-DIG antibodies. Roche now has a large collection of nucleic acid labeling products based on digoxigenin. Biotin, however, continues to be a popular nonisotopic alternative that is readily incorporated into nucleic acids enzymatically. For example, KPL's Detector™ nucleic acid probe labeling kits include a biotinylation quantitation standard for determining the relative specific activity of the labeled probe. This enables the user to optimize the amount of probe needed and thus minimizes background problems. New England Biolabs' NEBlot Phototope Kit uses random biotinylated octomers to prime in vitro synthesis from denatured double-stranded DNA and incorporates biotinylated dATP to provide sensitive nucleic acid detection. Regardless of the type of nonisotopic label used, nonradioactive probes have advantages over radioactive ones. While offering comparable sensitivity, nonisotopic probes do not decay and can be stored for at least one year. Frequently, kits for nonisotopic labeling are part of larger detection systems featuring enzyme-labeled streptavidin or anti-DIG antibodies, for example, which are visualized by colorimetric or chemiluminescent substrates.6 Direct Labeling Methods Nonisotopic labeling of nucleic acids does not rely exclusively on enzyme-mediated reactions. A handful of direct labeling kits developed to incorporate nonradioactive tags directly into the nucleic acid starting material are on the market. Most require minimal steps and reagent handling. Unlike random prime labeling and nick translation, most of the direct labeling kits generate full-length, intact, labeled probes. The Label IT® Nucleic Acid Labeling Kits produced by Mirus Corp. and distributed by PanVera Corp. of Madison, Wis., covalently attach rhodamine, fluorescein, biotin, digoxin, Cy3™, Cy5™, or DNP (dinitrophenol) to non-base pairing regions of guanine residues in any nucleic acid. Probes are labeled in a single step with rapid cleanup. The probes generated are appropriate for use in fluorescence in situ hybridization (FISH), hybridization reactions, dot or slot blots, DNA transport studies, and microarray analysis. The VersiTag Labeling Systems from NEN Life Science Products Inc. of Boston label nucleic acids directly with DNP or fluorescein. The system uses a platinum moiety that acts as a linker between the nucleic acid and the hapten. A single kit labels RNA, DNA, and oligonucleotides. Ambion of Austin, Texas, offers the BrightStar™ Psoralen-Biotin kit consisting of the intercalating agent psoralen covalently bound to biotin. The psoralen-biotin reagent intercalates into the nucleic acid and is then covalently bound by exposure to UV light. The kit labels RNA, DNA, PCR products, cDNA, and oligonucleotides in 45 minutes. The Biotin Chem-Link and DIG Chem-Link Labeling kits from Roche Molecular Biochemicals of Indianapolis use the cis-platinum Chem-Link reagent to attach biotin or DIG to the N7 position of guanine and adenine bases. The simple reaction occurs in 30 minutes at 85°C. Recently, the DIG CHEM-Link reagent was used to directly label mRNA probes intended for hybridization to nucleic acid cDNA arrays.7 Finally, Amersham Pharmacia Biotech of Uppsala, Sweden, sells the AlkPhos Direct Labelling and Detection System for direct labeling of DNA, RNA, and oligonucleotides with alkaline phosphatase. Similarly, the company's ECL Direct Nucleic Acid Labeling and Detection System directly labels DNA or RNA probes with horseradish peroxidase (HRP). The procedure entails a 20-minute reaction, and detection is achieved by the HRP-catalyzed breakdown of luminol. Erase, Erase, Erase With the development of its Strip-EZ™ StripAble™ probe synthesis technology, Ambion provides researchers with an easy and efficient way to extend the life of valuable DNA and RNA blots. Invented for synthesizing easily stripped RNA, DNA, and PCR probes labeled primarily with radioisotopes, the Strip-EZ kits incorporate specially modified nucleotides into new probes. Stable during the normal steps of blot hybridization, washing, and detection, the StripAble probes are easily removed from the blot by a 10-minute incubation in Probe Degradation Buffer. This buffer cleaves the full-length probes into short oligonucleotides at the site of each modified nucleotide. These short oligonucleotides then melt away from the blot during the mild 68°C washing step in Degradation Buffer. Because of the mild conditions used, very little of the bound target material is lost from the membrane. Consequently, these blots may be used repeatedly. Ambion's newest Strip-EZ kit, the Strip-EZ RT StripAble cDNA Probe Synthesis and Removal Kit, makes strippable probes for hybridization to expensive gene arrays. Preparing either radioactive or nonradioactive probes, the Strip-EZ RT Kit allows a single gene array to be probed up to 10 times. Roche Molecular Biochemicals' PCR DIG Probe Synthesis Kit contains an alkali-labile DIG-11-dUTP formulation. Incorporated into probes amplified by PCR, the DIG label is easily removed after detection, allowing subsequent rehybridization with multiple DIG-labeled probes. Specialty Kits While most nucleic acid labeling kits synthesize probes for standard applications like northern and Southern blotting, a few kits prepare probes for specific applications. For example, Nick Translation Kits from Vysis Inc. of Downers Grove, Ill., label DNA with fluorescent tags destined for use in FISH and genomic microarray experiments. GenHunter Corporation of Nashville, Tenn., a company specializing in products for differential display, offers the ReversePrime™ cDNA Labeling Kit for preparing labeled cDNA probes for use in "reverse northern" blots. Its HotPrime™ DNA Labeling Kit is optimized for labeling differential display fragments but can label other DNA probes efficiently as well. Lastly, Geno Technology of St. Louis offers the Message Hunter™ System, an mRNA labeling kit to synthesize 32P-labeled cDNA for use in characterizing expression levels of multiple genes on prepared blots. S Michael D. Brush can be contacted at mdbrush@compuserve.com. References 1. P.W. Rigby et al., "Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I," Journal of Molecular Biology, 113:237-51, 1977. 2. A.P. Feinberg, B. Vogelstein, "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity," Analytical Biochemistry, 132:6-13, 1983. 3. A.P. Feinberg, B. Vogelstein, "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity," Addendum, Analytical Biochemistry, 137:266-7, 1984. 4. P.R. Langer et al., "Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes," Proceedings of the National Academy of Sciences, 78:6633-7, 1981. 5. R. Martin et al., "A highly sensitive, nonradioactive DNA labeling and detection system," BioTechniques, 9:762-8, 1990. 6. D.A. Wilkinson, "A shot in the dark: products for the chemiluminescent detection of DNA," The Scientist, 12[10]:21, May 11, 1998. 7. T. Hoevel et al., "Cisplatin-Digoxigenin mRNA labeling for nonradioactive detection of mRNA hybridized onto nucleic acid cDNA arrays," BioTechniques, 27:1064-7, 1999.

Probing Questions

Enzymatic Labeling Techniques

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