Chemical Cleavage Method

Specific site(s) of cleavage Reactions/reagents used Ladder 

A+G Depurination by protonation of purines with HCOOH followed by piperidine cleavage of the chain at G and A A + G 

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In another method for sequencing, the DNA is labeled at internal base positions by enzymes which repHcate DNA strands in the presence of specific chain terrninators, thus producing an array of labeled fragments which reflect the sequence of the DNA. The rest of the procedure is the same as described for the chemical cleavage method (22). 

Maxam, A. M. Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980, 65, 499-560. 

Alternatively, cleavage can be done chemically, but here specificity of cleavage at a single defined position is complicated, since most chemical cleavage methods react with single amino acids (Table 5.13). For instance, chemical cleavage at the C-terminal side of methionine residue can be effected using CNBr, but the use of this method is limited, since most proteins are likely to contain internal methionine residues. 

The application of this highly selective chemical cleavage method to bovine pancreatic ribonuclease will be described in Section V. 

Two basic protocols for DNA sequencing are commonly adapted in laboratories the chemical cleavage method (Maxam and Gilbert, 1977) and the enzymatic chain termination or dideoxy method (Sanger et al, 1977). Of the two approaches, the Sanger dideoxy... 

Abstract Site-selective peptide/protein degradation through chemical cleavage methods is an important modification of biologically relevant macromolecules which complements enzymatic hydrolysis. In this review, recent progress in chemical, site-selective peptide btuid cleavage is overviewed, with an emphasis oti postulated mechanisms and their implications on reactivity, selectivity, and substrate scope. 

In this chapter, we survey recent (during the past 10 years) progress in site-selective peptide/protein degradation through chemical cleavage methods that complement enzymatic hydrolysis. 

Fig. 3. Nucleic acid sequencing. Scheme showing the chemical cleavage method of Maxam and Gilbert applied to the determination of the nucleotide sequence in a hypothetical length of DNA. a = original DNA, i.e. the uncleaved but P-labeled DNA being sequenced, b > location of each nucleotide in the DNA being sequenced, numbering from the 5 -end.

For a number of years, the Chemical Cleavage Method was more extensively used than Sanger s Chain Termination Method, because it did not require such specialized reagents, e.g. specific oligonucleotide primers and appropriate DNA polymerases. The latter method, however, is now the one of choice for most workers, largely because the specialized reagents are now readily available and the procedure has been automated. 

Just after 1975, several methods for DNA sequencing were developed. Two of them, the chemical cleavage method of Allan Maxam and Walter Gilbert, and the chain terminator method of Frederick Sanger, are today used. The chemical cleavage method is explained below. 

The Chemical Cleavage Method- Rather than just discussing this method in a textbook style let s try and actually visualize how we sequence a given piece of DNA. Let us take any arbitrary 10 nucleotide DNA sequence and see how it is manipulated amd what the results are. Following is the sequence that we randomly select. 

The first step in the chemical cleavage method is to radioactively label one end of the single stranded DNA. In most cases the DNA fragment will have a phosphate at the 5 -end. To remove this the DNA is treated with alkaline phosphatase which will carry out the... 

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