Construction and characterization of a normalized cDNA library
- New York State Psychiatric Institute, New York, NY (United States)
The authors have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, they used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C{sub 0}t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG02-91ER61233
- OSTI ID:
- 86531
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, Issue 20; Other Information: PBD: 27 Sep 1994
- Country of Publication:
- United States
- Language:
- English
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