ABSTRACT
Chromosome 21 has often been used as a model system for the development of genome mapping and cloning strategies in humans. In this report methods for systematic chromosome walking, cloning, and mapping are exemplified in the construction of a 1.5-Mb yeast artificial chromosome (YAC) contig encompassing and extending 400 kb beyond each of the genetic loci D21S13 and D21S16. Isolation of insert-terminal sequences from YACs in this contig provides a set of closely spaced physical markers. These have been used to generate a long-range genomic restriction map.
Subject(s)
Chromosome Walking , Chromosomes, Human, Pair 21 , Cloning, Molecular , Genome, Human , Base Sequence , Cell Line , Chromosome Mapping , Chromosomes, Fungal , DNA , DNA Probes , Dinucleoside Phosphates/genetics , Electrophoresis, Gel, Pulsed-Field , Gene Library , Genetic Markers/genetics , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Repetitive Sequences, Nucleic Acid , Restriction MappingABSTRACT
A new method of genetic analysis has been devised. The method, amplification refractory mutation system (ARMS), has been used to genotype the J3.11 MspI restriction fragment length polymorphism (RFLP) closely linked to cystic fibrosis (CF). The DNA sequence for both alleles of this dimorphism has been used to design ARMS primers. These allow genotyping of DNA isolated from blood, Guthrie cards, and buccal cells.
Subject(s)
Cystic Fibrosis/genetics , Alleles , Base Sequence , Cheek/anatomy & histology , DNA/blood , DNA/chemistry , Deoxyribonuclease HpaII , Deoxyribonucleases, Type II Site-Specific , Female , Gene Amplification , Genotype , Humans , Male , Molecular Sequence Data , Mutation , Pedigree , Polymorphism, Genetic , Polymorphism, Restriction Fragment LengthABSTRACT
The gene responsible for cystic fibrosis (CF) has recently been identified. Coding sequence for the cystic fibrosis transmembrane conductance regulator (CFTR) spans at least 230 kb of the human genome. Although all 27 exons of the gene are represented in cosmid or bacteriophage clones, there are still several gaps in the physical map of this region. It should be possible to complete the map and to clone the entire CFTR gene in a single fragment of DNA using a yeast artificial chromosome (YAC) vector. Herein we describe the construction and physical mapping of a 1.5-Mb YAC contig which encompasses D7S8 (J3.11) and D7S23 (KM19), two genetic loci flanking the CF locus. One of the clones in the contig, 37AB12, contains a 310-kb YAC which includes the entire CFTR gene and flanking sequence in both the 5' and 3' directions.
