Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Labeling the 3 Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase

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Terminal deoxynucleotidyl transferase (TdT, also simply called terminal transferase) is a template-independent polymerase that catalyzes the addition of deoxynucleotides and dideoxynucleotides to the 3-hydroxyl terminus of a DNA molecule. Cobalt (Co2) is a necessary cofactor for the activity of this enzyme. Terminal transferase exhibits a substrate preference for ssDNA but will also add nucleotides to protruding, recessed, and blunt-ended dsDNA fragments, albeit with a lower efficiency. The reaction can be limited to the addition of a single nucleotide when used in the presence of a chain-terminating analog such as a dideoxynucleotide (ddNTP), typically [-32P]ddATP (Yousaf et al. 1984). Alternatively, the enzyme is capable of adding several (2100) nucleotides to 3 ends in a so-called homopolymeric tailing reaction (Deng and Wu 1983) (for more information, see the Additional Protocol Tailing Reaction at the end of this protocol). In addition to adding radioactive nucleotides, terminal transferase can also be used to add nonradioactive labels such as biotin-, DIG-, or fluorescein-labeled nucleotides (Kumar et al. 1988; Igloi and Schiefermayr 1993). Nonradioactive labeling is typically performed under conditions that promote the addition of several nucleotides to increase the sensitivity of the probe. Although the 3-end-labeling reaction is relatively easy to perform (see the Alternative Protocol Synthesizing Nonradiolabeled Probes Using TdT at the end of this protocol), biotin and DIG 3-end-labeling kits are available from several manufacturers (e.g., Thermo Scientific, Roche Applied Science).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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