A comparison of direct selection and exon amplification techniques in the derivation of a transcription map of chromosome 5q35

Journal Article · Thu Sep 01 04:00:00 EDT 1994 · American Journal of Human Genetics

OSTI ID:133464

Del Mastro, R; Lovett, M ^[1]; Stotler, C ^[2]

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1. Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)
2. Massachusetts General Hospital, Boston, MA (United States); and others

Direct cDNA selection and exon amplification are two widely used gene identification techniques. We have embarked on a comparison of these using a collection of 410 cosmids from human chromosome 5q35. Direct selection was performed using three cDNA pools, from placenta, Hela cells and activated T-cells. Six hundred cloned cDNAs were picked from the selected material. For exon amplification, the 410 cosmids were divided into forty-one pools of ten. From these 41 pools, two thousand exons were cloned and arrayed. The first validation step consisted of hybridizing 285 cDNAs and 102 exons to the 410 cosmids. A total of 62% of the cDNAs and 70% of the exons identified 244 and 99 cosmids respectively. A total of 264 cosmids were detected by the combination of exon and cDNA probes. Further comparisons included hybridizing cDNAs to exons and exons to cDNAs. Here 8% of the cDNAs identified exons and 30% of exons detected cDNAs. The low number of exons detecting cDNAs is possibly due to the limited spectrum of cDNA pools used in this direct selection. However, the high number of cDNAs that fail to detect any trapped exons is surprising and unexpected. Trivial explanations, such as a high frequency of repeat-containing cDNAs or a bias towards 5{prime} or 3{prime} ends in the cDNAs, were discounted by testing all of the cDNAs on whole genome Southern blots and by deriving full length clones for some of the selected cDNAs. Sequence comparisons of these cDNAs and over 100 selected cDNAs and exons has further confirmed the observed overlaps, low level of redundancy in the selected cDNAs, and has also revealed several interesting homologies with previously described genes. This comparative study suggests that the construction of detailed long range transcription maps will require a mixture of expression and structure-based approaches.

• OSTI ID: 133464
• Report Number(s): CONF-941009--
• Journal Information: American Journal of Human Genetics, Journal Name: American Journal of Human Genetics Journal Issue: Suppl.3 Vol. 55; ISSN AJHGAG; ISSN 0002-9297
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
COMPARATIVE EVALUATIONS
DETECTION
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
GENE AMPLIFICATION
GENES
GENETIC MAPPING
HUMAN CHROMOSOME 5
TRANSCRIPTION