Blunt-ended and 5′ recessed ends can be modified with reduced efficiency. T4 PNK ligase is template independent and modifies single-stranded polynucleotides and 5′ overhangs efficiently. The wild-type enzyme also has 3′-phosphatase activity. T4 PNK transfers an organic phosphate from the gamma position on ATP to the 5′-hydroxyl group of DNA and RNA. T4 polynucleotide kinase (T4 PNK) is an enzyme coded for in the genome of the T4 bacteriophage. The following graph shows a comparison of TdT incorporation of several different modified nucleotides. For example, TdT addition of biotin-11-UTP to the 3′ end of complementary DNA probes is an effective way of creating probes for use in nonradioactive electrophoretic mobility shift assays (EMSA) and DNA pull-down assays. TdT can also be used to label the 3′ end of DNA probes with radioactive and nonradioactive tags for a variety of detection and affinity applications. TdT is often used to label DNA probes for RACE (Rapid Amplification of cDNA Ends), TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling) assays and as a method for adding 3′ overhangs to DNA fragments to facilitate cloning. Common sources of DNA templates modified with TdT include unlabeled, single-stranded PCR primers and double-stranded restriction endonuclease fragments with 3′ overhangs (“sticky ends”, 5′ recessed ends). 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. Nucleic acid probes can be labeled with tags or other modifications during synthesis.
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