Efficient selection of 3'-terminal exons from vertebrate DNA.

Identification of expressed sequences within genomic DNA is a hurdle in the characterization of complex genomes. We developed an exon trapping scheme that provides a positive selection for vertebrate 3'-terminal exons. A copy of the trapped exon sequence is obtained by RT/PCR amplification. The technique detects valid terminal exons without interference from partial exons or non-specific sequences, including simple human repeated sequences. Application to random human cosmids yielded one unique trapped terminal exon per cosmid on average. Because vertebrate terminal exons average 600-700 nucleotides in length, the technique provides transcribed sequences of sufficient length to assist further mapping efforts.     

Identification of an amplified gene cluster in glioma including two novel amplified genes isolated by exon trapping

Gene amplification, which occurs in more than 50% of malignant gliomas, is considered to play a pivotal role in tumorigenesis. There are, however, few studies aimed toward the isolation of novel genes from amplified sequences. Previously, we reported amplification of the protooncogene MET (hepatocyte growth factor receptor; 7q31) in more than 20% of glioblastomas. For an approximate size estimation of the amplification unit we analyzed three glioblastomas all of which carried an amplified MET gene, by Southern blot analysis and/or competitive polymerase chain reaction using eight DNA markers. Although the extent of the amplified domain varied, the close vicinity of the MET gene was the only region consistently amplified in these glioblastomas. A yeast artificial chromosome (YAC) contig of 900 kb was refined spanning the amplified region flanking the MET gene. The YAC inserts were subcloned into 59 cosmids, which were used for exon trapping. Eight sequences were identical to parts of the genes MET and CAPZA2 (human actin capping protein α-subunit). Two newly identified exons and the CAPZA2 exons were amplified in tumor TX3095, which retains an amplified MET gene. The new exons were localized close to MET and CAPZA2. Characterization of the clones, which were termed glioma-amplified sequence (GAS)7-1 and GAS7-2, showed an open reading frame and a different expression pattern in multiple human tissues. This study reports the identification of a cluster of amplified genes including two novel genes in a region amplified in more than 20% of glioblastomas. Received: 27 June 1997 / Accepted: 18 July 1997     

Direct selection of DNA sequences conserved between species.

An essential requirement in the analysis of genomes is the identification of functionally important sequence elements, which are often evolutionarily conserved. We describe here the development of a novel procedure for the selective isolation of conserved sequences which is based on hybridization of PCR-amplifiable DNA fragments from the whole genome of one species to biotinylated DNA from a genomic region of another species. The interspecies DNA hybrids are immobilized and the PCR-amplifiable DNA fragments are eluted, amplified and after further hybridization-amplification rounds cloned. This method was used for the generation of sublibraries of conserved sequences from mouse and pig DNA from regions corresponding to cosmids from the human Xq28 region. Mouse and pig homologs of sequences containing exons of known human genes, as well as exons from novel genes have been identified.     

High resolution deletion breakpoint mapping in the DMD gene by whole cosmid hybridization.

The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.     

Specific isolation of 3'-terminal exons of human genes by exon trapping.

Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing trapping of internal exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient trapping of 3'-terminal exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' exons in addition to a great reduction of non-specific background. As a test for the system, 3' exon trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' exon trapping strategy should facilitate the cloning of genes from large genomic regions.     

Exon concatenation to increase the efficiency of mutation screening by DGGE

For genes that have a substantial number of exons and long intronic sequences, mutation screening by denaturing gradient gel electrophoresis (DGGE) requires the amplification of each exon from genomic DNA by PCR. This results in a high number of fragments to be analyzed by DGGE so that the analysis of large sample sets becomes labor intensive and time consuming. To address this problem, we have developed a new strategy for mutation analysis, lexon-DGGE, which combines the joining of different exons by PCR (also known as lexons) with a highly sensitive technique such as DGGE to screen for mutations. The lexon technique is based on the concatenation of several exons, adjacent or not, from genomic DNA into a single DNA fragment so that this approach could simultaneously be used to check the mutational status of several small genes. To show the feasibility of the approach, we have used the lexon-DGGE technique to analyze all coding exons, intron-exon junctions, noncoding exon 1, and part of the noncoding region of exon 11 of the TP53 gene. The validity and performance of the technique were confirmed by using negative and positive controls for each of the DNAfragments analyzed.     

Sequence analysis of a total of three megabases of DNA in two regions of chromosome 8p.

Large-scale sequencing of genomic regions and in silico gene trapping together represent a highly efficient and powerful approach for identifying novel genes. We performed megabase-level sequence analyses of two genomic regions on human chromosome 8p (8p11.2 and 8p22-->p21.3), after covering those segments with sequence-ready contigs composed of 74 cosmids, 14 BACs, and three PAC clones. We determined continuous nucleotide sequences of 1,856,753 bases on 8p11.2 and 1,210,381 bases on 8p22-->p21.3 by combining the shotgun and primer-walking methods. In silico gene trapping identified four novel genes in the 8p11.2 region and, in the 8p22-->p21.3 region, six known genes (PRLTS, PCM1, MTAMR7, HCAT2, HFREP-1 and PHP) and three novel genes. The distribution of Alu and LINE1 repetitive elements and the densities of predicted exons were different in each region, and Alu-rich portions contained more exonic sequences than LINE1-rich areas.     

A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1

The exon-amplification method was used to identify putative transcribed sequences from an 800-kb region that includes the genes for phospholipase Cβ3 and PYGM on human chromosome 11q13. The clone contig consisted of ten cosmids, three bacterial artificial chromosomes, and one P1 artificial chromosome. A total of 83 exons were generated of which 23 were derived from known genes and expressed sequence tags (ESTs). Five different EST cDNA clones were identified and mapped on the contig. One is a homolog of the human p70S6 kinase (p70s6 k) gene whose function involves the translational regulation of ribosomal protein synthesis and thereby impacts on ribosomal biogenesis. The gene for p70s6 k is expressed universally, including within adipose cells and retina, and it could play a role in Bardet-Biedl syndrome type 1, which has been mapped to 11q13. Received: 22 July 1998 / Accepted: 24 August 1998     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

Isolation of coding sequences from bovine cosmids by means of exon trapping

Exon trapping was employed to identify coding sequences from a collection of 46 bovine cosmids, previously characterized for the presence of microsatellite markers and physically mapped to chromosomes by FISH. The sequence analysis of 104 clones revealed 18 putative exons, 10 of which showed near identity to known sequences. Among these were the human (cytosine-5)-methyltransferase (DNMT), ATP-citrate lyase (ACLY), the mouse Lbcl1 oncogene, the bovine mitochondrial aconitase (ACO2) and β-arrestin 1 (ARR1). The chromosomal localization of the cloned exons was inferred from the localization of the parent cosmids. DNMT and ACLY were not previously known in cattle, but the physical localization of the cloned bovine exons is in agreement with the published comparative human and bovine maps. The trapping of exons for bovine ACO2 and ARR1 confirms the available mapping information based on synteny and provides a physical assignment for the genes. Received: 23 November 1996 / Accepted: 3 March 1997