Selection of hybrids by affinity capture (SHAC): a method for the generation of cDNAs enriched in sequences from a specific chromosome region.
We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hybrids by affinity capture (SHAC). This procedure can be described in two stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplification with a universal degenerate primer. This material is referred to as the "target" DNA. In the second stage, a mouse liver cDNA library with unique linker-adapter ends, referred to as the "source" cDNA, was hybridized to the biotin-labeled target DNA prepared during the first stage. The resulting DNA duplexes were captured by streptavidin-coated magnetic beads. The cDNAs were released from their biotin-labeled target homologs by alkaline denaturation and recovered by PCR amplification. These cDNAs were referred to as the SHACcDNAs. Specificity of the SHACcDNA to chromosome 11 was verified by FISH analysis. To examine representation of the SHACcDNA, we confirmed the presence of seven genes or single-copy DNA segments known to be localized on mouse chromosome 11, using a dot blot assay. In addition, a second round of SHAC was performed to achieve even higher specificity for the resulting chromosome 11 SHACcDNA. The SHAC technology should facilitate construction of cytogenetically defined cDNA libraries and should assist in the fields of gene discovery and genome mapping.
Chen-Liu
L W
LW
Applied Genetics Laboratories, Inc., Melbourne, Florida 32901, USA.
Huang
B C
BC
Scalzi
J M
JM
Hall
B K
BK
Sims
K R
KR
Davis
L M
LM
Siebert
P D
PD
Hozier
J C
JC
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D017931:DNA Primers; D018076:DNA, Complementary; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008969:Molecular Sequence Data
D017931:DNA Primers; D018076:DNA, Complementary