Multiple-complete-digest restriction fragment mapping: generating sequence-ready maps for large-scale DNA sequencing.
Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of x15-30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a "shotgun" method.
Wong
G K
GK
The Human Genome Center, Department of Medicine, University of Washington, Seattle, WA 98195, USA.
Yu
J
J
Thayer
E C
EC
Olson
M V
MV
D001482:Base Composition; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D018244:Chromosomes, Artificial, Yeast; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D006801:Humans; D015203:Reproducibility of Results; D015183:Restriction Mapping / Q000379:methods*
D004247:DNA; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; C048503:ATGCAT-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific