A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica).
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 re-ligated 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.
Miller
J R
JR
AFRC Institute of Animal Physiology and Genetics Research, Cambridge Research Station, UK.
Dixon
S C
SC
Miller
N G
NG
Tucker
E M
EM
Hindkjaer
J
J
Thomsen
P D
PD
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D017403:In Situ Hybridization; D007621:Karyotyping; D008214:Lymphocytes / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013552:Swine / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D004247:DNA