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Labeling Methods Overview

Nick Translational Labeling

The nick translation method1 is based on the ability of DNase I to introduce randomly distributed nicks into DNA at low enzyme concentrations in the presence of Mg2+. E. coli DNA polymerase I synthesizes DNA complementary to the intact strand in a 5´ 3´ direction using the 3´-OH termini of the nick as a primer2. The 5´ 3´ exonucleolytic activity of DNA Polymerase I simultaneously removes nucleotides in the direction of synthesis3. The polymerase activity sequentially replaces the removed nucleotides with isotope-labeled or hapten-labeled deoxyribonucleoside triphosphates1. At low temperature (+15°C), the unlabeled DNA in the reaction is thus replaced by newly synthesized labeled DNA.

References

  1. Rigby, P.W.J. et al. (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237251.
  2. Kelly, R.B. et al. (1970) Enzymatic Synthesis of Deoxyribonucleic Acid. J. Biol. Chem. 245, 3945.
  3. Klett, R.P. et al. (1968) Exonuclease VI, a new nuclease activity associated with E. coli DNA polymerase. Proc. Natl. Acad. Sci. USA 60, 943950.

Additional application information can be found in the Related Downloads.

Random Primed DNA Labeling

The method of "random primed" DNA labeling developed by Feinberg and Vogelstein1,2 is based on the hybridization of a mixture of all possible hexanucleotides to the DNA to be labeled. All sequence combinations are represented in the hexanucleotide primer mixture, which leads to binding of primer to the template DNA in a statistic manner. Thus an equal degree of labeling along the entire length of the template DNA is guaranteed. The complementary strand is synthesized from the 3´ OH termini of the random hexanucleotide primer using Klenow enzyme, labeling grade. Modified deoxyribonucleoside triphosphates ([32P]-, [35S]-, [3H]-, [125I]-, digoxigeninor biotinlabeled) present in the reaction are incorporated into the newly synthesized complementary DNA strand.

References

  1. Feinberg, AP. & Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specificactivity. Anal. Biochem., 132 (1), 613.
  2. Feinberg, AP. & Vogelstein, B. (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem., 137 (1), 266.

PCR Labeling of DNA Probes

The polymerase chain reaction (PCR) allows the amplification of minute amounts of DNA to levels above 1 μg. The only prerequisite is that some sequence information of the target sequence is known for synthesizing the appropriate primers.

The nonradioactive DIG system uses digoxigenin, a steroid hapten, to label DNA, RNA, or oligonucleotides for hybridization, and subsequent color- or luminescent detection. The digoxigenin is coupled to dUTP via an alkali-labile ester bond. The labeled dUTP can be easily incorporated by enzymatic nucleic-acid synthesis using DNA polymerases.

The combination of nonradioactive labeling with PCR is a powerful tool for the analysis of PCR products, and also for the preparation of labeled probes from small amounts of a respective target sequence.

Oligonucleotide 3´ / 5´ End Labeling

3´end labeling

Oligonucleotides are enzymatically labeled at their 3´-end with terminal transferase either by incorporation of a single digoxigenin-labeled dideoxyuridine-triphosphate (DIG-ddUTP) or by the addition of a longer nucleotide tail1.

Terminal Transferase catalyzes the template independent addition of deoxyand dideoxynucleoside triphosphates to the 3´OH ends of double and single-stranded DNA fragments and oligonucleotides. Terminal Transferase incorporates digoxigenin-, biotin-, and fluorochrome-labeled deoxy- and dideoxynucleotides as well as radioactive labeled deoxy- and dideoxynucleotides.

5´end labeling

Oligonucleotides are reacted with a phosphoramidite in a final step according to the classical solid phase phosphoramidite synthesis method. By this process a 5´-terminal aminofunction is created. Treatment with ammonia releases the oligonucleotide from the support and cleaves the protecting groups. In the subsequent step the digoxigenin moiety is introduced at the 5´-position.  

References

  1. Schmitz, G. G.; Walter, T.; Seibl, R.; Kessler, C. (1991) Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 192, 222–231.

Additional application information can be found in the Related Downloads.

Oligonucleotide Tailing

Oligonucleotides are enzymatically labeled at their 3´ -end with terminal transferase by the addition of a longer nucleotide tail1. For the generation of tailed oligonucleotide probes, a mixture of the deoxynucleotides-triphosphate dATP and DIG-dUTP is used in a template independent reaction (DIG Oligonucleotide Tailing Kit, 2nd Generation).

Terminal transferase catalyzes the template independent addition of deoxy- and dideoxynucleoside triphosphates to the 3´OH ends of double- and single-stranded DNA fragments and oligonucleotides. Terminal transferase incorporates digoxigenin-, biotin-, and fluorochrome-labeled deoxy- and dideoxynucleotides as well as radioactively labeled deoxy- and dideoxynucleotides.

labeling-methods-overview-fig1

Figure 1. Nonradioactive oligonucleotide tailing and detection.

References

  1. Schmitz, G. G.; Walter, T.; Seibl, R.; Kessler, C. (1991) Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal. Biochem. 192, 222–231.

Additional application information can be found in the Related Downloads.

Transcriptional Labeling of RNA Probes

The DNA to be transcribed is cloned into the polylinker site of appropriate transcription vectors (e.g., pSPT 18 or 19), which contain promoters for SP6 and T7 RNA polymerases. Adjacent template DNA is linearized at a suitable site. The RNA polymerases are used to produce "run off" transcripts. DIG-UTP is incorporated into the transcript. Every 20-25th nucleotide of the newly synthesized RNA is a DIG-UTP. Since the nucleotide concentration does not become limiting in the standard transcription reaction, this reaction can generate large amounts of labeled RNA.

labeling-methods-overview-fig2

Figure 2. DNA insertion into the polylinker site of the transcription vectors pSPT18 or pSPT19.

DNA is inserted into the polylinker site of the transcription vectors pSPT18 or pSPT19 (see Figure 2). These two vectors differ only in the orientation of their polylinker regions. The promoters for SP6 and T7 RNA polymerases are located on either side of the polylinker. SP6 and T7 RNA polymerases specifically transcribe DNA sequences downstream of the SP6 or T7 promoters respectively1,2. Cloned inserts within the polylinker region are transcribed from either promoter. The first DNA strand may be transcribed with SP6 RNA polymerase and the opposite strand using T7 RNA polymerase. It is also possible to transcribe the first and opposite strands by inserting the same DNA into both pSPT18 and pSPT19 in opposite orientations and transcribing with only one of the RNA polymerases. SP6 and T7 RNA polymerase use the cloned DNA as template and synthesize complementary RNA in the presence of Mg2+ and ribonucleoside triphosphates. Spermidine stimulates enzyme activity. Specifically labeled transcripts are obtained when using radioactively (e.g., 32P,3H, 35S) or non radioactively (e.g., digoxigenin or biotin) labeled ribonucleotide triphosphates.

References

  1. Dunn, J. J. & Studier, F. W. (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477535.
  2. Kassavetis, G. A. et al. (1982) Bacteriophage SP6specific RNA polymerase. II. Mapping of SP6 DNA and selective in vitro transcription. J. Biol. Chem. 257, 57795788.

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