Construction of a genomic DNA 'feature map' by sequencing from nested deletions: application to the HLA class I region.
We are applying a transposon-based approach for detecting and mapping features of special interest to construct 'feature maps' of currently uncharacterized portions of the human leukocyte antigen (HLA) complex on chromosome 6. Such feature maps should facilitate identifying regions for high resolution analysis. Here we describe the feature mapping of a 35 kb DNA fragment located between the HLA-C and HLA-E loci. This fragment was cloned into a transposon gamma delta-based cosmid vector designed for generating nested deletions in vivo. Seventy informative nested deletions extending into the cloned fragment were isolated, and DNA adjacent to the deletion endpoints was sequenced by fluorescent automated technology. These islands of DNA sequences constituted the foundation of the feature map, and (i) identified putative exons, (ii) determined the positions of Alu elements, (iii) determined the span of the keratinocyte-specific S gene, and (iv) localized evolutionarily conserved sequences. The construction of feature maps using this in vivo nested deletion-sequencing approach provides a rapid and efficient means to identify DNA regions that merit more detailed analysis.
Krishnan
B R
BR
Department of Internal Medicine, Howard Hughes Medical Institute and Department of Molecular Microbiology, Washington University School of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110.
Jamry
I
I
Berg
D E
DE
Berg
C M
CM
Chaplin
D D
DD
D001483:Base Sequence; D005075:Biological Evolution; D002874:Chromosome Mapping; D002896:Chromosomes, Human, Pair 6; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003360:Cosmids; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements; D005091:Exons; D005822:Genetic Vectors; D015894:Genome, Human; D006680:HLA Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion
D017931:DNA Primers; D004251:DNA Transposable Elements; D006680:HLA Antigens; D004247:DNA