Filters

Filters

The sea urchin genome research resource includes an extensive set of high-density arrayed cDNA and genomic libraries. These are carried permanently in 384-well plates and are reproduced for screening robotically, by spotting the clones in a format such that a 22 cm² membrane carries 18,432 clones in fixed locations with respect to both 384-well plate identity and X Y well location (Maier et al, 1994). Thus a whole cDNA library or large-fragment genomic library can be represented on five or six such filters. Each clone is spotted in duplicate to provide indicators for false positives. Each filter can
be re used for screening many times, and after hybridization with a radioactive probe analysis of the filters can be carried out by digital methods. The large robot required for arraying and spotting clone libraries is housed at Caltech and thus we have continuous access to it.

The advantage of array technologies for the following proposals are crucial:

1. Instant availability of clones without purification by successive rescreening, i.e., when a high density screen reveals a desired clone as a pair of spots hybridizing with a given probe, it is at once available, pure, in a given position in a 384-well plate in the freezer.

2. Fast and easy mapping of extensive regions of the genome by use of overlaps in screens of BAC and PAC libraries. These libraries contain large genomic fragments. For example, in our S. purpuratus PAC library the average insert size is 80 kb; and in our BAC library it is 140 kb. The determination of the gene order in the Hox gene cluster of S. purpuratus performed by Martinez, P., Rast, J. P., Arenas-Mena, C.. and Davidson, E. H. (Organization of an echinoderm Hox gene cluster. Proc. Natl. Acad. Sci. USA 96, 1469-1474, 1999.) illustrates how such libraries can be exploited for genomic mapping.

3. Molecular techniques including subtractive hybridization methods and macro-array screening.

TABLE 1. The complement of arrayed libraries prepared as part of the Sea Urchin Genome Project. The progress in preparation of each library is indicated.

Source	Made	Arrayed	Spotted
72 hr Sp cDNA	X	X	X
Larval Sp cDNA	X	X	X
PMC Sp cDNA	X	X	X
Ovary Sp cDNA	X	X	X
Testis Sp cDNA	X	X	X
Am cDNA	X	X	X
Blastula-Gastrula Ap cDNA	X	X	X
Sp BAC genomic	X	X	X
Sp small BAC	X	X	X
Sp BAC FR2	X	X	X
Lv BAC	X	X	X
Et small BAC	X	X	X
Lv small BAC	X	X	X
Pl small BAC	X	X	X
Am small BAC	X	X	X
Ap BAC	X	X	X
Pf BAC	X	X	X
Sp BAC(Mbo1)	X	X	X
Am large BAC	X	X	X

Abbreviations:

Sp: Strongylocentrotus purpuratus
Sf: Strongylocentrotus franciscanus
Lv: Lytechinus variegatus
Ap: Arbacia punctulata
Et: Eucidaris tribuloides
Am: Asterina miniata
Pf: Ptychodera flava
Pl: Paracentrotus lividis
PMC: primary mesenchyme cell