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Isolation of human transcripts expressed in hamster cells from YACs by cDNA representational difference analysis
1Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-4442 USA.
Genome Research
|February 19, 1999
Summary
This study introduces a new method using cDNA representational difference analysis (RDA) to efficiently isolate expressed genes from yeast artificial chromosomes (YACs). This approach aids in gene discovery for genetic disorders like Niemann-Pick type C (NP-C).
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Positional cloning often relies on bacterial or cosmid clones for gene isolation.
- Yeast artificial chromosomes (YACs) are frequently used for initial physical mapping but are less optimal for gene isolation.
Purpose of the Study:
- To develop a strategy for identifying gene sequences within YACs.
- To directly isolate expressed transcripts from YACs in mammalian cells using cDNA representational difference analysis (RDA).
Main Methods:
- Generated tester cDNAs from a hamster cell line with a stable 590-kb YAC (911D5) expressing the NPC1 gene.
- Generated driver cDNAs from a control hamster cell line lacking the YAC.
- Utilized cDNA RDA to isolate expressed gene fragments from the YAC.
Main Results:
- NPC1 was the most abundant product identified by RDA.
- Two non-NPC1 fragments, 7-R and 2-R, were isolated and mapped to the 911D5 YAC.
- Fragment 7-R showed high sequence identity to a known human EST, while 2-R localized to an overlapping YAC region.
Conclusions:
- Stable YAC transfer followed by cDNA RDA is an effective strategy for expediting human gene cloning.
- This method facilitates gene isolation from YAC contigs in candidate genomic regions.
- The approach aids in identifying novel expressed sequence tags and mapping them to specific genomic locations.
