Cloneless genomic DNA analysis: an efficient and simple methods for de novo genomic sequencing projects and gap filling.
The utility of using genomic DNA directly in agarose, i.e. cloneless libraries, in place of large clone libraries, radiation hybrid panels, or chromosome dissection was demonstrated. The advantage of the cloneless library approach is that, in principle, a targeted genomic resource can be developed rapidly for any genomic region using any genomic DNA sample. Here, a human chromosome 20 Not I fragment library was generated by slicing a pulsed field gel lane containing fractionating Not I cleaved DNA from a monosomic hybrid cell line into 2 mm pieces. A reliable PCR method using agarose embedded DNA was developed. InterAlu PCR generated unique patterns of products from adjacent slices (e.g. fractions). Further, the specificity of the interAlu products was demonstrated by FISH analysis and in other hybridization experiments to arrayed interAlu products. STS content mapping was used to order the fractions and also demonstrate the unique content of the library fractions.
Nguyen
Giang
G
Molecular Biotechnology Research Laboratory, Department of Biomedical Engineering, 36 Cummington Street, Boston, MA 02215, USA.
Bukanov
Nikolayev
N
Oshimura
M
M
Smith
Cassandra L
CL
D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*