Mapping genomic library clones using oligonucleotide arrays.
We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adapters. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density oligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection.
Sapolsky
R J
RJ
Affymetrix, 3380 Central Expressway, Santa Clara, California, 95051, USA.
Lipshutz
R J
RJ
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D015342:DNA Probes; D005819:Genetic Markers; D015698:Genomic Library; D015233:Models, Statistical; D008969:Molecular Sequence Data; D016012:Poisson Distribution; D016133:Polymerase Chain Reaction; D011336:Probability; D015183:Restriction Mapping
D017931:DNA Primers; D015342:DNA Probes; D005819:Genetic Markers; D004247:DNA