Direct cloning of a long restriction fragment aided with a jumping clone.
Long-range physical mapping with rare-cutting restriction enzymes (rare cutters) is an important step for structural analysis of complex genomes. Combination of two types of DNA clones bearing the rare-cutter sites, linking clones and jumping clones (Fig. 1a), facilitates the physical mapping [Poustka et al., Nature 325 (1987) 353-355]. A step followed by the physical mapping is the cloning of the large (rare-cutter-generated) restriction fragment of interest. For facilitating this step, we devised a method to directly clone a long restriction fragment without constructing the whole genomic DNA library using the jumping clone as starting material. The short DNA segments of a jumping clone, which are derived from the 5' and 3' terminal regions of the large restriction fragment, are inserted into the yeast artificial chromosome plasmid (pYAC) vector, and then converted into single strands with T7 gene 6-encoded 5'----3' exonuclease. The total genomic DNA digested with the restriction enzyme is also treated with the exonuclease to convert the terminal regions of the restriction fragments into single strands. In the resulting products, only the fragment corresponding to the jumping clone can form hybrids with the just-mentioned, single-stranded DNAs, which are connected to the pYAC, and only this fragment is cloned in yeast. We describe the protocol of this method with Escherichia coli DNA as a model experiment. Judging from the cloning efficiency, this method could be applied to cloning single-copy regions of the human genome, provided a jumping clone is available. The instability of inserts in the pYAC vector is also discussed.
Matsuoka
T
T
Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan.
Kato
H
H
Hashimoto
K
K
Kurosawa
Y
Y
D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015183:Restriction Mapping; D013604:T-Phages / Q000201:enzymology
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D005090:Exodeoxyribonucleases; C046264:gene b exonuclease