TdT has poor activity towards double-stranded DNA with blunt ends or 5′ overhangs. TdT has the highest activity towards the 3′ end of single-stranded DNA but can also modify the 3′ overhang of double-stranded DNA with lower efficiency. TdT is template independent and not significantly affected by DNA sequence, but DNA structure is important. TdT typically adds numerous deoxynucleotides to the 3′ terminus of a DNA strand, but reaction conditions can be optimized such that only 1–3 incorporation events occur. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase enzyme expressed in certain populations of lymphoid cells.
However, assays requiring protein interactions (i.e., gel shift and pull-down assays) require end-labeling to allow protein binding. Typically, nucleic acids hybridization reactions (i.e., northern blotting) benefit from the high specific activity gained through random incorporation of label into a probe. The choice of method needed is determined in part by the degree of labeling required and whether the modification will cause steric hindrance that prevents the desired interactions. There are enzymatic and chemical methods for creating probes labeled at either the 5′ or 3′ ends of the oligonucleotide as well as randomly incorporated throughout the sequence.
In contrast, chemical methods are amenable to larger scale reactions.
For small-scale probe generation needs, enzymatic methods are an economical method for labeling probes. Numerous reagents are available for quick and efficient benchtop oligonucleotide labeling, and they are most useful for making small amounts of probe or when many different probes with the same label are required (i.e., for mutational analysis). Because of this, many researchers may choose in-house methods or labeling kits for probe generation. Additionally, the minimum order quantity for modified oligonucleotides is typically much higher than unmodified versions and may be excessive compared to the amount required for the intended application. However, purchasing custom oligonucleotide probes (especially RNA) can be quite expensive depending on the modification and whether costly purification services are required. As in the case with the kinase labeling of DNA ( Chapter 39), the DNA fragment is cleaved with a second restriction enzyme, to generate fragments of unequal size that are labeled only in one end.The fragments are subsequently separated on the basis of their different molecular weights and isolated ( chapter 7 and chapter 10).Nucleic acid probes can be labeled with tags or other modifications during synthesis. The DNA fragment to be labeled is incubated in the presence of one or more deoxyribonucleotide triphosphates (one of which is labeled with 32P in the a-phosphate group) and the Klenow fragment of DNA polymerase. The principle of the method is shown in Fig.
The method described here is for generating 3′ end-labeled DNA fragments that are suited for sequencing by the method of Maxam and Gilbert ( Chapter 51), but can in principle also be used when radioactively labeled DNA is required for other purposes (e.g., Southern hybridization). Radiolabeling of DNA fragments is most efficient with DNA fragments that contain recessed 3′ ends ( see Fig. The fragment still has the polymerase and 3′–5′ exonuclease activity, but lacks the 5′–3′ exonuclease activity of the holoenzyme ( 1). The Klenow fragment of DNA polymerase I is a proteolytic fragment obtained by the treatment of DNA polymerase I with subtilisin.
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