Construction and characterization of a normalized cDNA library
Abstract
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.
- Authors:
-
- New York State Psychiatric Institute, New York, NY (United States)
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 86531
- DOE Contract Number:
- FG02-91ER61233
- Resource Type:
- Journal Article
- Journal Name:
- Proceedings of the National Academy of Sciences of the United States of America
- Additional Journal Information:
- Journal Volume: 91; Journal Issue: 20; Other Information: PBD: 27 Sep 1994
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 55 BIOLOGY AND MEDICINE, BASIC STUDIES; LIBRARIES; CONSTRUCTION; RECOMBINANT DNA
Citation Formats
Soares, M B, De Fatima Bonaldo, M, Jelene, P, Su, L, Lawton, L, and Efstratiadis, A. Construction and characterization of a normalized cDNA library. United States: N. p., 1994.
Web. doi:10.1073/pnas.91.20.9228.
Soares, M B, De Fatima Bonaldo, M, Jelene, P, Su, L, Lawton, L, & Efstratiadis, A. Construction and characterization of a normalized cDNA library. United States. https://doi.org/10.1073/pnas.91.20.9228
Soares, M B, De Fatima Bonaldo, M, Jelene, P, Su, L, Lawton, L, and Efstratiadis, A. 1994.
"Construction and characterization of a normalized cDNA library". United States. https://doi.org/10.1073/pnas.91.20.9228.
@article{osti_86531,
title = {Construction and characterization of a normalized cDNA library},
author = {Soares, M B and De Fatima Bonaldo, M and Jelene, P and Su, L and Lawton, L and Efstratiadis, A},
abstractNote = {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.},
doi = {10.1073/pnas.91.20.9228},
url = {https://www.osti.gov/biblio/86531},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 20,
volume = 91,
place = {United States},
year = {Tue Sep 27 00:00:00 EDT 1994},
month = {Tue Sep 27 00:00:00 EDT 1994}
}