End labelling DNA fragments

The most widely used procedure for cleaving the end-labelled DNA fragments are the base-specific chemical degradation reac-... 

The initial requirement is for a singly end-labelled DNA fragment or a uniformly labelled single-strand. If the fragment molecule is single-stranded e.g. a chemically synthesized primer, all that... 

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). 

The abbreviated protocols above indicate how four base-specific cleavage reactions are coordinated to sequence one end-labelled DNA fragment. They are given only as a chronological guide, and initially the more detailed descriptions in Procedures H-K should be followed. DMS is dimethylsulphate, HZ is hydrazine, and pip-for is piperidinium formate. 

In order to facilitate analysis of FeBABE produced fragments, the prey protein or biomolecule is labeled at one end with a tag that can be detected after electrophoresis, usually in a transfer blot. The tag can be a fusion tag, such as 6X His, or any other group that can be targeted with an antibody and detected. Alternatively, radiolabels and fluorescent labels have been used with prey molecules, including the use of end-labeled DNA to study where DNA binding proteins dock onto the oligonucleotide sequence. 

In this method the single-stranded DNA fragment to be sequenced is end-labelled by treatment with alkaline phosphatase to remove the 5 phosphate, followed by reaction with 32P-labelled ATP in the presence of polynucleotide kinase, which attaches 32P to the 5 terminal. The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base as follows. 

Wijsman JH, Jonker RR, Keijzer R, van de Velde CJ, Cornelisse CJ, van Dierendonck JH. A new method to detect apoptosis in paraffin sections In situ end-labeling of fragmented DNA. J Histochem Cytochem 1993 41 7-12. 

Tateyama H, Tada T, Hattori H, Murase T, Li WX, Eimoto T. Effects of prefixation and fixation times on apoptosis detection by in situ end-labeling of fragmented DNA. Arch Pathol Lab Med 1998 122 252-255. 

Fig. 3.10. Autoradiograph of a sequencing gel prepared using the Maat and Smith procedure. The sequence shown is that derived from a 440 nucleotide-long fragment from a Hinfl digest of a 5 -end labelled HirtdUl fragment of adenovirus type 5 DNA. Samples from each base-specific reaction mixture were loaded every 2 hours (runs I, II, III, and IV). Electrophoresis was carried out at a constant current of 30 mA. Nucleotide sequence analysis of the complementary DNA strand revealed one mistake in the sequence as written. At position 2870 (in run III) two C s should be read instead of one. The zone of compression responsible for this error is not very apparent and emphasizes the importance of sequencing both DNA strands.

Fig. 5.3. Two routes for preparing singly-labelled DNA fragments. In scheme (a) the duplex fragment, labelled at both ends is cleaved with a secondary restriction enzyme at R to yield two singly-labelled duplex fragments. In scheme (b) the doubly-labelled fragment is denatured and the separated strands resolved by polyacrylamide gel electrophoresis.

Contamination of end-labelled fragment with other end-labelled DNAs... 

G6. Gorczyca, W., Tuziak, T., Kram, A., Melamed, M. R., and Darzynkiewicz, Z., Detection of apoptosis-associated DNA strand breaks in fine-needle aspiration biopsies by in situ end labelling of fragmented DNA. Cytometry 15, 169-175 (1994). 

Figure 5-15. Restriction mapping. Restriction mapping is best carried out using end-labelled DNA, which has been tagged either radioactively or with some suitable label (digoxygenin, fluorescent group etc.). Partial cleavage with different restriction enzymes produces fragments whose...

Figure 5-20. DNA sequencing by the Maxam-Gilbert method. End-labelled DNA is treated in four separate aliquots with chemical reagents which cleave specifically after A, G, C+Tand C. The fragments generated by partial chemical...

Protein-DNA interactions can be characterized experimentally by the DNA footprinting technique and the electrophoretic mobility shift assay (EMSA). (a) In vitro DNAse I footprinting (b) EMSA. Reactions contained an end-labeled radioactive fragment (a) or oligonucleotide (b) incubated with the zinc-finger DNA binding domain of the... 

FIGURE 2 Footp rinting results of RNA polymerase binding to the lac promoter (see Fig. 26-5). In this experiment, the 5 end of the nontemplate strand was radioactively labeled. Lane C is a control in which the labeled DNA fragments were cleaved with a chemical reagent that produces a more uniform banding pattern. 

Figure 6. Schematic illustrating DNA footprinting methodology. DNA cleaved by a sequence-neutral cleaving agent yields an even distribution of cuts on end labeled and then denatured fragments (top). When protein is bound to a specific site on the DNA, cleavage at that site is inhibited, and the cleavage pattern on end-labeled DNA shows a blank spot, or footprint (bottom).

The fragments are fractionated and the sizes of the labeled pieces are measured, usually in four parallel electrophoretic lanes. The pattern of bands seen is often called a ladder. To read base sequence form 5 -end beginning with the bottom-most band, proceed upward remembering that the two ladders, A + G and C + T contain all bands that arise from partial cleavage of the 5 -end labeled DNA. If a band appears in the A + G ladder, it derives from cleavage at a purine. This purine is a G if it falls under G ladder, otherwise it is an A. Likewise the C + T ladder indicates a pyrimidine, and if it is also under C ladder, that pyrimidine is a C, if not it is a T. 

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