Construction of two near-kilobase resolution restriction maps of the 5' regulatory region of the human apolipoprotein B gene by quantitative DNA fiber mapping (QDFM).
Quantitative DNA fiber mapping (QDFM) is a high-resolution technique for physical mapping of DNA. The method is based on hybridization of fluorescently labeled DNA probes to individual DNA molecules stretched on a chemically modified glass surface. We now demonstrate and validate a rapid QDFM-based approach for the mapping of multiple restriction sites and precise localization of restriction fragments in large genomic clones. Restriction fragments of a 70-kb P1 clone (P1-70) containing the 5' region of the human apolipo-protein B gene (APOB) were subcloned and mapped along straightened P1-70 DNA molecules. Multicolor fluorescence in situ hybridization (FISH) and digital image analysis allowed us to rapidly position 29 restriction fragments, ranging in size from 0.5 kb to 8 kb, and to map 43 restriction sites. The restriction map obtained by QDFM was in excellent agreement with information obtained by RecA-assisted restriction endonuclease (RARE) cleavage, long-range PCR, and DNA sequence analyses of the P1-70 clone. These data demonstrate that QDFM is a rapid, reliable method for detailed restriction site-mapping of large DNA clones.
Duell
T
T
Life Sciences Division, Lawrence Berkeley National Laboratory, CA 94720, USA.
Nielsen
L B
LB
Jones
A
A
Young
S G
SG
Weier
H U
HU
D001055:Apolipoproteins B / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D004586:Electrophoresis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011938:Rec A Recombinases; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015183:Restriction Mapping / Q000379:methods*; D017422:Sequence Analysis, DNA
D001055:Apolipoproteins B; D015345:Oligonucleotide Probes; D011938:Rec A Recombinases; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII