Base-specific cleavage reactions

All the base-specific cleavage reactions described have been used successfully for sequencing DNA. Other novel base-specific modifications have been described e.g. the piperdine-catalysed cleavage at guanine bases after U.V. irradiation in the presence of methylene blue or rose bengal and a specific thymine cleavage by piperidine treatment after reaction of the DNA with osmium tetroxide (Friedmann and Brown, 1978). These latter procedures however, have not, as yet, been widely adopted as sequencing tools. 

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. 

Due to the instabiUty of nucleotides under PCR cycling conditions and issues with incorporation efficiencies, the process features immobiUzation of the PCR product to a solid support, thereby generating single-stranded templates this is then followed by an isothermal primer extension reaction to incorporate the modified nucleotides. In this way, a specific strand of the PCR product can be selected for base-specific cleavage, while the yield of products is not compromised. 

The implication of the hammerhead structures is that, because the base sequences of helices 1 and 111 are not conserved, a hammerhead ribozyme can be constructed from completely unrelated RNAs and perform rapid, site-specific cleavage reactions in trans on virtually any target sequence. This versatility, combined with the structural simplicity and portability, makes the hammerhead ribo-... 

The base-specific chemical cleavage (or Maxam-Gilbert) method starts with a single-stranded DNA that is labeled at one end with radioactive (Double-stranded DNA can be used if only one strand is labeled at only one of its ends.) The DNA strand is then randomly cleaved by reactions that specifically fragment its sugar-phosphate backbone only where certain bases have been chemically removed. There is no unique reaction for each of the four bases. However,... 

The peculiar metal ion specificity of the ATP cleavage reaction may perhaps be explained by reference to some studies on the metal complexes of Schiff bases, which have provided clues to many aspects of biological metal catalysis. It was shown that metal ions will split the carbon-nitrogen double bond in thiophenalde-hyde-ethylenediamine (18, 21) as a consequence of the electronic-drift-to-metal... 

AH fungal RNases (T, T , Ni, Ui, and U2) treated in this section catalyze the reaction shown in Fig. 1. The first step (phosphate transfer) is the cleavage of the phosphodiester bond between the 3 and 5 positions of the ribose moities in the RNA chain with the formation of nucleoside 2, 3 -cyclic phosphates and oligonucleotides with 2, 3 -cyclic phosphate at 3 terminal. The nature of the phosphodiester bonds to be cleaved depends on the base specificity of the enzyme. This phosphoryl transfer step is reversible. In the second step (hydrolysis), these terminal cyclic phosphate groups are hydrolyzed with the formation of corresponding 3 -phosphates. Because the first-step is usually faster than the second step, more or less accumulation of the cyclic phosphate may be observed. 

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