Chromosomes have gene locations {DNA mapping}.
chromosome separation
Cell-sorting machines can sort chromosomes. Alternatively, people can separate chromosomes using microscopes and fine instruments. Human cells treated with x-rays can fuse with mouse cells to isolate chromosome pieces, to map with markers {radiation hybrid mapping}. Two-dimensional electrophoresis using variable fields can separate DNA fragments up to three million bases {pulsed field gel electrophoresis} (PFGE), over several days.
protein removal
Proteolytic enzymes remove protein from chromosomes held in gels, leaving DNA.
DNA fragments
NotI, MluI, NruI, and SfiI restriction enzymes have eight-base sites and cut DNA in few places, to make million-base DNA fragments.
methylation
Tissues can have methylated sites, allowing fewer cuts and larger fragments, so researchers must compare different tissues. Large fragments have further processing.
cloning
YACs allow cloning hundred-kilobase DNA fragments. Phages and cosmids allow cloning 40000-base DNA fragments, for genomic libraries.
separation
From clones, electrophoresis separates restriction fragments by size.
markers
Genetic markers can find loci.
hybridization
20-base oligonucleotide probes can hybridize with DNA. Probes can have minor-groove binder to enhance exact hybridization, allowing shorter probes. Probes hybridize with clone DNA fragments. Overlapping DNA fragments hybridize to same probe. Clone-fragment sequence-tagged connector ends hybridize to probes.
sites
Using unique primers, processing identifies unique 200-base to 500-base sequence-tagged sites, which have known locations.
overlapping
DNA fragments overlap to build longer sequences {contig, DNA}, to sequence chromosome DNA.
Biological Sciences>Genetics>Linkage
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Date Modified: 2022.0224