A chromosome 1-specific DNA library from the domestic pig (<i>Sus scrofa domestic</i><i>a</i>)

Abstract

Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS. Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y. DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then religated into pUC18. After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA. The average insert size of the library was 405 bp. Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA. Screening with a (CA)_n probe revealed that roughly 1 % of the clones contained microsatellite sequences. A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1. However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.

Isolation and mapping of porcine chromosome‐specific microsatellites

“…Batches of 1000 flow‐sorted chromosomes were treated with proteinase K as described by D avies et al (1992) and batches of 10 000 chromosomes as described by M iller et al (1992).…”

“…Because of the non‐specific nature of this approach areas of chromosomes poorly covered by polymorphic markers exist. Consequently several groups (M iller et al 1992; G rimm et al 1994) have used specific chromosomes for the construction of chromosome specific libraries. The isolation of microsatellites from targeted chromosomes should be a useful tool to fill any gaps remaining between markers and to construct high density genetic maps for each chromosome.…”

Chromosomes in the flow to simplify genome analysis

“…Comparable libraries were constructed for various animals (Baron et al 1990; Shepel et al 1998) and in wheat (Wang et al 1992). Construction of short-insert libraries became easier after the introduction of methods for representative amplification of chromosomal DNA as only a few hundred or thousand sorted chromosomes (Miller et al 1992; Vooijs et al 1993; Macas et al 1996) or even a single chromosome (Van Devanter et al 1994) was sufficient as starting material. Chromosome specifics of the libraries facilitated gene mapping and targeted the development of DNA markers in human, animals, and plants (Arumuganathan et al 1994; Grady et al 1996; Lan et al 1999; Korstanje et al 2001; Požárková et al 2002).…”

Mapping of microsatellite markers developed from a flow-sorted swine chromosome 6 library

“…1993). Chromosome-enriched libraries have been constructed and characterized for Chr 1, 11, 13, and 18, and a strategy for using pools of several swine chromosomes has also been used to assign loci to the swine map (Miller et al 1992;Davies et al 1994;Dear and Miller 1994;Ellegren and Basu 1995;Riquet et al 1995Riquet et al , 1996. In addition, through the use of coincidence cloning, regionspecific probes have been generated from Chr 6 (Frengen et al 1994).…”