3 -End labeling of DNA

Maxam and Gilbert (1980) have described detailed protocols for the preparation and isolation of dephosphorylated DNA fragments, and reaction schemes for the selective 5 -end and 3 -end labelling of these products either individually or en masse. Pro-... 

Fig. 10. 5 End labeling of DNA and RNA. Labeling is most efficient with single- stranded molecules and at the 5 ends of double-stranded DNA with recessed 3 ends. Blunt-ended DNA and DNA with recessed 5 ends react more slowly. After removal of the S phosphate with alkaline phosphatase, the S OH group is rephosphorylated by the action of polynucleotide kinase and ATP. Alternatively, in the presence of excess ADP, the reverse reaction is encouraged, effectively leading to exchange between then 5 phosphate and the y-phosphate of ATP.

V. Special applications of T4 PNK lacking 3 -phosphatase activity. This mutant enzyme is uniquely suitable for the preparation from 3 -NMP of 3, 5 -biphosphates which can be used for synthesis and labeling of DNA and RNA (38). The 5 -[ P]pCp is a reagent commonly used for the 3 -end labeling of RNA with the T4 RNA ligase (39). The 3 -labeled RNA can then be used for RNA sequencing and hybridization, as well as for the analysis of RNA structure by fingerprinting. 

Terminal deoxynucleotidyl transferase normally adds homopolydeoxynucleotide tails to single-stranded DNA primers in the presence of a deoxynucleoside triphosphate and magnesium. If cobalt is used instead, not only does double-stranded DNA become an acceptable substrate, but ribonucleotides or homopolymer deoxyribo-nucleotide tracts may be added to all forms of duplex DNA at their 3 -ends, regardless of whether these are staggered or even.162 This allows terminal labelling for sequence analysis at the cleavage sites of restriction endonucleases,162- 183 or tail formation for in vitro studies on recombinant DNA.162... 

Detailed procedures for 5 - or 3 -end labelling DNA fragments, following the method of Maxam and Gilbert (1980) are given in Chapter 5. Essentially similar protocols are given by Wu et al. (1976) and Roychoudhury and Wu (1980). 

Under these conditions the octanucleotide linker fragments chromatograph at a similar rate to nucleoside triphosphates. There is usually sufficient of the labelled triphosphate, used for the 3 -end labelling, remaining in the sample to act as a marker for the linker peak which elutes later. The first fractions are enriched in the longer DNA fragments—which tend to clone with lower efficiency—and this crude fractionation decreases the number of random plaques which have to be picked to obtain cloned representatives of all the fragments. 

While the replacement DNA synthesis method for 3 -end labeling requires both the 3 - 5 exonuclease and DNA polymerase functions of DNA polymerases, the activity of the 5 - 3 DNA polymerase alone is sufficient to produce radiolabeled blunt-ended DNA frj ents with substrates containing protruding 5 termini. For example, the recessed 3 terminus shown in Fig. 1C can be extended by DNA polymerase in the presence of dXTP and dCTP to yield the blunt-ended DNA fragment indicated. 

Fig. 7.18. End-labeling can be achieved by different approaches. Kinasing (I) is most effective with 5 -OH overhangs. In the presence of excess ADP, radioactive phosphate can also be exchanged with a 5 phosphate. Whereas kinasing introduces at most one label per DNA end, tailing (II) allows multiple labels to be introduced (Co is necessary with recessed ends). Fill-in of restriction sites (III) also yields 3 -end labeling. RTase or sequenase should then be used. Oligomers are required if the 5 -end is recessed (IV).

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