Endonucleolytic mutation analysis by internal labeling (EMAIL)

Cross, Michael J., Waters, Daniel L. E., Lee, L. Slade and Henry, Robert J. (2008) Endonucleolytic mutation analysis by internal labeling (EMAIL). Electrophoresis, 29 6: 1291-1301.


Author Cross, Michael J.
Waters, Daniel L. E.
Lee, L. Slade
Henry, Robert J.
Title Endonucleolytic mutation analysis by internal labeling (EMAIL)
Journal name Electrophoresis   Check publisher's open access policy
ISSN 0173-0835
1522-2683
Publication date 2008-03
Sub-type Article (original research)
DOI 10.1002/elps.200700452
Volume 29
Issue 6
Start page 1291
End page 1301
Total pages 11
Place of publication Weinheim, Germany
Publisher Wiley
Language eng
Formatted abstract Mismatch-specific endonucleases are efficient tools for the targeted scanning of populations for subtle DNA variations. Conventional protocols involve 5′-labeled amplicon substrates and the detection of digestion products by LIF electrophoresis. A shortcoming of such protocols, however, is the limited 5′-signal strength. Normally the sensitivity of fluorescent DNA analyzers is superior to that of intercalating dye/agarose systems, however, pooling capacities of the former and latter approaches to mismatch scanning are somewhat similar. Detection is further limited by significant background. We investigated the activity of CEL nucleases using amplicon substrates labeled both internally and at each 5′-terminus. The amplicons were generated from exon 8 of the rice starch synthase IIa encoding gene. Signal of both 5′-labels was significantly reduced by enzyme activity, while that of the internal label was largely unaffected. In addition, background resulting from internal labeling was a significant improvement on that associated with 5′-labeling. Sizing of the multilabeled substrates suggests that 5′-modification enhances exonucleolytic activity, resulting in the removal of the dye-labeled terminal nucleotides. We have developed an alternative approach to mismatch detection, in which amplicon labeling is achieved via the incorporation of fluorescently labeled deoxynucleotides, which we have named Endonucleolytic Mutation Analysis by Internal Labeling (EMAIL). The strength of the EMAIL assay was demonstrated by the reclassification of a rice line as being heterozygous for the starch gene. This cultivar was assigned as being homozygous by a previous resequencing study. EMAIL shows potential for the clear identification of multiple mutations amongst allelic pools.
Keyword Cel I Endonuclease
Internal Labeling
Mismatch Detection
Strand-specific Nucleases
Surveyor(tm) Nuclease
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Alliance for Agriculture and Food Innovation
ERA 2012 Admin Only
 
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