Base-specific cleavage

COMPARATIVE SEQUENCE ANALYSIS USING BASE-SPECIFIC CLEAVAGE AND MALDI-TOF MS... 

Hartmer R, Storm N, Boecker S, Rodi CP, Hillenkamp F, Jurinke C, van den Boom D. RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis. Nucleic Acids Res 2003 31 e47. 

Bocker S. SNP and mutation discovery using base-specific cleavage and MALDI-TOF mass spectrometry. Bioinformatics 2003 19(suppl l) i44—i53. 

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. 

In the Maxam and Gilbert method (M2) the starting point is a defined DNA restriction fragment. The DNA strand to be sequenced must be radioactively labeled at one end with a 32P-labeled phosphate group. This DNA can be single stranded or of duplex form. The base-specific cleavages depend upon the following points ... 

The underlying principle of this method, namely base-specific cleavage, is now used more widely for the sequence analysis of target regions. Further applications... 

Whilst several methods have been developed to achieve the base-specific cleavage of amplification products, the main difference between them relates to the type of analyte, with base-specific cleavage occurring either on DNA or RNA. 

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. 

Drosten et al. [92] in conjunction with base-specific cleavage, 16 DEN4 strains all tied with a relative coincidence of 1.0. Serotypes 1,2, and 3 all tied with much lower scores of 0.30. 

Honisch C, Chen Y, Mortimer C, Arnold C, Schmidt O, van den Boom D, Cantor CR, Shah HN, Gharbia SE. Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing. Proc Natl Acad Sci USA. 

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