Efficiency and specificity of gene isolation by exon amplification.
Exon amplification is an increasingly popular approach to the identification of transcribed sequences and will complement other strategies to isolate genes. We have used this system to amplify candidate exons from 32 cosmids, including 8 cosmids which span a well characterized 185-kb region of the human major histocompatibility class II region on Chromosome (Chr) 6. We have examined the efficiency, specificity, and reproducibility of the system in isolating exons from genes known to be present on particular cosmids and have determined the nature and frequency of artefact amplifications in routine cosmid screening. We were able to clone at least one exon from 88% (7/8) of all known genes tested (including exons which are differentially spliced) and obtained artefacts from 19% (6/32) of the cosmids tested. Such artefacts generally arise from the amplification of noncoding sequences flanked by regions with high homology to acceptor and donor splice junctions. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids which have different numbers of genes and gene structures and describe several approaches to the characterization of novel exons cloned in this study.
North
M A
MA
Genome Analysis Laboratory, Imperial Cancer Research Fund, London, UK.
Sanseau
P
P
Buckler
A J
AJ
Church
D
D
Jackson
A
A
Patel
K
K
Trowsdale
J
J
Lehrach
H
H
D000595:Amino Acid Sequence; D016477:Artifacts; D001483:Base Sequence; D002896:Chromosomes, Human, Pair 6; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA; D005091:Exons; D005784:Gene Amplification; D005802:Genes, MHC Class II; D006801:Humans; D008969:Molecular Sequence Data; D012326:RNA Splicing; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid
D004247:DNA