Bacterial artificial chromosome libraries for mouse sequencing and functional analysis.
Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.
Osoegawa
K
K
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263 USA.
Tateno
M
M
Woon
P Y
PY
Frengen
E
E
Mammoser
A G
AG
Catanese
J J
JJ
Hayashizaki
Y
Y
de Jong
P J
PJ
D000818:Animals; D017101:Bacteriophage P1 / Q000235:genetics / Q000378:metabolism; D001923:Brain Chemistry / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D005260:Female; D005819:Genetic Markers / Q000235:genetics; D015698:Genomic Library; D007668:Kidney / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013154:Spleen / Q000737:chemistry
D005819:Genetic Markers; D004247:DNA