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Project Documentation & Protocols: Maize Gene Discovery Project: RescueMu Library Plates: Overview

Contents: Index | Progress | Ordering | Screening | Sequences | Phenotypes | Protocols | FAQs

Library plates are your opportunity to acquire genomic clones of a maize gene and to identify insertional mutations in genes of interest. Library plates represent the diversity of germinal plus somatic RescueMu insertion sites in a field of corn plants immortalized in a suite of small genomic libraries of maize DNA. These plasmid library plates can be screened by PCR or by transforming into E. coli cells followed by colony hybridization to identify clones of interest. Because typical inserts are 4- 8 kb, many intact genes are represented in these plasmid libraries. More importantly, a subset of RescueMu insertions are germinal and thus are an important resource for identifying mutations in "your favorite gene."

The Maize Gene Discovery Project is sequencing to the right and left of these plasmids to generate maize genomic DNA that is highly enriched for "gene-like" sequences. RescueMu and other Mu elements insert preferentially into genes, hence libraries of the insertion sites are an efficient route of gene discovery. We expect that many genes expressed at low levels or only during a transient developmental stage or physiological condition will not be identified by EST sequencing. RescueMu plasmid sequencing is our strategy to identify many of these otherwise elusive genes. Alignment of genomic sequence information with available ESTs will allow identification of introns and provide the opportunity for ZmDB to extend annotation into promoter, 5' UTR and 3' UTR features. Because RescueMu elements tie together functional genomics through the analysis of mutants and genomic sequencing for gene identification, they are the key technology in our project. Production scale genomic sequencing started on February 15, 2001, on Grid G (for more about gene tagging using RescueMu, click here.). We are sequencing plasmids from the 46 rows of this plant grid, plus two columns, to verify recovery of germinal insertions in both a row and a column library. Presently, RescueMu sequencing is 1/3 effort and EST sequencing is 2/3 effort. Later this year, we will probably increase genomic sequencing effort to speed discovery of genes that are unlikely to be found by EST sequencing.

On the other pages in this section of ZmDB you can consult instructions for the library plates, for PCR screening (including tabular information on positive controls verified to work with a particular plate), for charting progress in the plasmid sequencing project from particular library plates, a tabular sample of initial RescueMu gene "hits" from Grid G, links from the library plates to the phenotype database for each grid of plates that generated a library plate, and an order form to request library plates. As of March 2001, the majority of phenotype information is from grids A-E, but we are starting library plate distribution with Grid G, followed by H - L then F. Phenotype information for these grids will accumulate during 2001.

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