Chromosome-specific cDNAs/STSs. Progress report, March 1, 1992--February 28, 1993
This project seeks to construct high-quality normalized cDNA libraries from human tissues, to develop methods for isolation of chromosome-specific cDNAs, and to contribute sequence information to the expanding cDNA/EST database. A human infant brain cDNA library having a very high complexity, short poly (A) tails; long size inserts; undetectable co-cloning events; and low background of non-recombinants was previously described. Over 2,000 ESTs have already been successfully derived from this library, and so become one of the best so far characaterized. Having established the protocol to construct high quality cDNA libraries, a major effort was mounted to develope a method to normalize cDNA libraries constructed in phagemid vectors. Briefly, this method involves priming of the library in the form of single-stranded circles with a Not I-oligo (dT) primer and controlled extensions with Klenow in the presence of dNTPs and ddNTPS. After purification of the partial duplexes over HAP, melting and reannealing to a moderate Cot, unhybridized (normalized) single-stranded circles are purified by HAP and electroporated into bacteria, generating a normalized library. The extent of normalization of the infant brain cDNA library has been determined by a series of screenings with probes that represent mRNAs from the 3 frequency classes.
- Research Organization:
- Columbia Univ., New York, NY (United States)
- Sponsoring Organization:
- USDOE, Washington, DC (United States)
- DOE Contract Number:
- FG02-91ER61233
- OSTI ID:
- 10139502
- Report Number(s):
- DOE/ER/61233-2; ON: DE93011534
- Resource Relation:
- Other Information: PBD: Oct 1992
- Country of Publication:
- United States
- Language:
- English
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