Lambda exonuclease

Lambda-exonuclease treatment. This procedure is used by Alderon Biosciences [28]. Exonucleases are enzymes that hydrolyse the terminal phosphodiester bonds of DNA strands obtaining mononucleosides monophosphate or biphosphate. In this case a 5 -exo-nuclease that hydrolyses the 5 -end phosphodiester is used. In this way, the strands are destroyed. To achieve the total hydrolysis of the sister strand, the target strand must be protected, for example, by a label at its 5 -end. Therefore, the PCR amplification is carried out using one of the primers biotinylated at the 5 -end and the other one modified at its 5 -end with a phosphate group. The extension of these primers give rise to the 5 -biotinylated target DNA strand and a 5 -phosphate sister DNA strand, which is hydrolysed by 5 -exonuclease. 

R. Kovall, B.W. Matthews, Toroidal Structure of Lambda-Exonuclease , Science, 277, 1824(1997)... 

A. P. Null, J.C. Hannis D.C. Muddiman, Preparation of single-stranded PCR products for ESI-MS using the DNA repair enzyme lambda exonuclease. Analyst, 125... 

A different approach to generate strand-specific probes is offered by lambda exonuclease (lambda exo Table 7.17C). It has 5 - 3 exonuclease activity but only from 5 -phosphoryl termini (5 -hydroxyl termini are resistant) (Little, 1981). One of the two strands can selectively bear a 5 -phosphoryl group by different approaches, such as phosphorylation of one of the two primers prior to PCR or successive restriction, dephosphorylation and restriction steps. In the first approach, the primers are removed after PCR cycling and the strand with the 5 -phosphoryl group digested from the duplex. The phosphorylated or its 5 -P-less equivalent can then be used to generate a strand-specific probe. Synthesis is achieved as described above. 7.6.5. Preparation of nonradioactive probes by PCR... 

Methods to obtain ss DNA labeled at either the 5 - or the 3 -end have been described in Section 7.7. Particularly the methods based on PCR are useful. Submitting one of the primers used for amplification to kinasing with [y- PldNTP renders the strand containing this 5 -phosphate group resistant to lambda exonuclease and only these specific strands will survive digestion. 

The specificity of lambda exonuclease. Interactions with single-stranded DNA. J. Biol Chem., 250, 5438 -5445. 

PREPARATION OF SINGLE-STRANDED DNA FROM PCR AMPLIFIED DNA USING LAMBDA-EXONUCLEASE... 

Com Recursive DNA CoiKtruction 1. T4 polynucleotide kinase (NEB, Ipswich, MA, USA). 2. Thermo-Start DNA polymerase (ABgene). 3. Lambda exonuclease (Epicenter). 

Lambda Exonuclease Digestion of the PCR Product to Regenerate ssDNA... 

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