Analysis of region-specific library constructed by sequence-independent amplification of microdissected fragments surrounding weaver (wv) gene on mouse chromosome 16.
The C3-C4 region of mouse chromosome 16 was microdissected and amplified directly by sequence-independent amplification (SIA). The SIA product was proved to originate from the microdissected region by fluorescence in situ hybridization (FISH) and was cloned into the PCR II vector (mean insert size 506 bp). Colony hybridization showed that about 59% of the clones contained either unique or low copy number sequences. Southern blot analysis of 100 unique clones demonstrated that 50 clones hybridized with single (33 clones) or multiple (17 clones) bands on blots of DNA from a hamster-mouse hybrid cell line that contains mouse chromosome 16, 13 clones hybridized with mouse but not with the hamster-mouse hybrid DNA, 19 clones contained repetitive sequences, and the remaining 18 clones failed to yield bands. One third of the 100 unique clones hybridized to human genomic DNA. Thirty-three clones were sequenced. None of them was found in GenBank. Our results demonstrate that this relatively simple method of microdissection and cloning can produce a library of good quality.
Wei
J
J
Department of Pathology, Indiana University School of Medicine, Indianapolis 46202-5251.
Dlouhy
S R
SR
Zhu
J
J
Ghetti
B
B
Hodes
M E
ME
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D005819:Genetic Markers; D015698:Genomic Library; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D017931:DNA Primers; D005819:Genetic Markers