Automated band mapping in electrophoretic gel images using background information

Zerr, Troy; Henikoff, Steven

doi:10.1093/nar/gki580

Cited by 65 publications

(48 citation statements)

References 14 publications

(18 reference statements)

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“…The products were separated by electrophoresis on denaturing gels using LI-COR 4200 or 4300 instruments (Lincoln, NE). Gel images were analyzed using GelBuddy (Zerr and Henikoff 2005). Pools containing putative mutant individuals were rescreened to identify the individuals, and then the target was sequenced for confirmation of the lesion and to determine zygosity as previously described (Greene et al 2003;Till et al 2003b).…”

Section: Methodsmentioning

confidence: 99%

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

et al. 2008

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Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, longterm storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for .100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a userfunded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.

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Section: Methodsmentioning

confidence: 99%

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

et al. 2008

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“…2b). Data analysis is aided by the use of GelBuddy, a freely available program for Macintosh and Windows PCs designed for the analysis of TILLING and Ecotilling gel data produced by the LI-COR analyzer 11 . The exact nucleotide change is then determined using standard DNA sequencing methods.…”

Section: Overview Of Tillingmentioning

confidence: 99%

A protocol for TILLING and Ecotilling in plants and animals

et al. 2006

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We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.

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“…Signal is thought to be reduced via nonspecific strand cleavage near the labeled terminus, although different mechanisms have been hypothesized for Endo V [18] and CEL I [19]. Such mutation scanning assays are further hindered by significant background that is characteristic of both end-labeled [2,18,20,21] and ethidium-intercalated [13] detection methods. Typical 5 0 -labeled protocols use only limited enzymatic activity in an attempt to reduce the loss of cleavage fragment signal and optimize the contrast between desired signal and background [19,21].…”

Section: Introductionmentioning

confidence: 99%

Endonucleolytic mutation analysis by internal labeling (EMAIL)

2008

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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.

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Automated band mapping in electrophoretic gel images using background information

Cited by 65 publications

References 14 publications

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

Retention of Induced Mutations in a Drosophila Reverse-Genetic Resource

A protocol for TILLING and Ecotilling in plants and animals

Endonucleolytic mutation analysis by internal labeling (EMAIL)

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