International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

I14 Radiation Hybrid Mapping of Mouse Expressed Sequence Tags (ESTs)

A. R. Haynes1, C. Davison1, C. Heuston1, P. J. Trickett1, A. Southwell1, S. Greenaway1, M. A.Strivens1, T. Matise2, H. Doi3, J. Kitchen3, M. S. H. Ko4, S. D. M. Brown1 and P. Denny1. 1MRC UK Mouse Genome Centre and Mammalian Genetics Unit, Harwell, OX11 ORD, UK; 2The Rockefeller University, 1230 York Ave, Box 192, New York, NY, USA.; 3ERATO Doi Bioasymmetry Project, Wayne State University, Detroit, MI 48202, USA.; 4Laboratory of Genetics, National Institute on Aging, NIH, Baltimore, MD 21224, USA

There are ~6700 mapped genes in the Mouse Genome Database (July 1999; http://www.informatics.jax.org/). This means that the map density is too sparse for the routine use of the positional candidate approach. Several groups, including our own, are mapping ESTs as a way to increase the density of transcript maps and hence increase the likelihood of finding candidates for any novel mutation.

Radiation hybrid (RH) mapping has several advantages over traditional meiotic mapping:

  1. markers can be typed in a simple plus/minus gel assay
  2. markers need only exhibit mouse/hamster variance.
  3. high resolution can be achieved by typing a moderate number (94) of hybrid lines (<150kb in some genome regions using the T31 (Research Genetics) mouse/hamster panel - C. Hayes, personal communication).

Our focus is the mapping of ESTs that appear to lack human homologs. It is assumed that these ESTs will have human equivalents, but due to bias in the choice of tissues used in the construction of cDNA libraries, they have not been sequenced. In this way, we will add value to the existing human transcript maps. Mouse ESTs derived from early embryonic stage cDNA are particularly enriched in such novel sequences. We aim to map about 10,000 ESTs over the next 2-3 years and are currently adding ~60 ESTs each week to the map.

Sequence from 3' expressed sequence tags (3' ESTs) is used to design primers which are then optimised using a hot-start PCR protocol. We now use a 384 well based system, and a Biomek 2000 robot to set up PCRs, with the reaction products being resolved on a modified agarose gel. Gel image data are to be handled by EMPATH (EST Mapping Project AT Harwell) which is a software package with image analysis code for semi-automated scoring of the presence of PCR products. It is designed to record, analyse and manage data from the EST mapping project, and is based around a commercial database. The system interface is via any web browser and allows monitoring of the mapping process from primer creation to incorporation into the Whitehead/MIT framework maps.

 


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