Isolation of genes from complex sources of mammalian genomic DNA using exon amplification

Abstract

Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.

A 1.1-Mb Transcript Map of the Hereditary Hemochromatosis Locus

“…Exon-trapping experiments were carried out as described previously (Buckler et al 1991;Church et al 1994) with minor modifications on the largeinsert bacterial clone contig. Preliminary experiments suggested that to detect rare splice events, 96 trapped products per large-insert bacterial clone needed to be sequenced (data not shown).…”

“…CsCl-purified genomic P1, BAC, and PAC DNAs were digested with BamHI, BglII, PstI, SacI, and XhoI, and 125 ng of each digest ligated into 500 ng of pSPL3 (Church et al 1994) (Life Technologies, Gaithersburg, MD) digested with the appropriate restriction enzyme and phosphatased with CIAP (U.S. Biochemical, Cleveland, OH). One-tenth of the ligation was used to transform XL1-Blue MRFЈ cells (Stratagene, La Jolla, CA) by electroporation.…”

“…Cytoplasmic RNA was isolated 48 hr after transfection. RT-PCR was carried out as described (Church et al 1994) using commercially available reagents (Life Technologies, Gaithersburg, MD). The resulting CUA-tailed PCR fragments for each restriction-digested bacterial clone were pooled and UDG cloned into pSP72-U.…”

“…It is extremely sensitive and capable of isolating portions of rare transcripts. Exon-trapping (Buckler et al 1991;Church et al 1994) recovers spliced exons from in vivo-expressed genomic DNA clones and produces candidate exons without any prior knowledge of the target gene's expression. Two additional approaches, sample sequencing and complete genomic sequencing, use the highthroughput capabilities of genomic DNA sequencing and subsequent comparisons of these sequence data to public databases of expressed sequences.…”

A new candidate region for the positional cloning of the XLP gene

“…After Cos cells transfection, a mini library of the RT-PCR products was constructed for each cosmid as described by Church et al 11 The cosmids covering the XLP-D deletion were subcloned on the basis of a detailed restriction map 8 (and unpublished data, Yin L, 1998) and sequenced using Dye Primer cycle sequencing chemistry with an Applied Biosystems 377A automated DNA sequencer. Plasmid and cosmid end sequencing was performed using Dye Terminator sequencing chemistry with Taq polymerase together with T3/T7/Sp6 primers.…”