TY - CHAP
T1 - Expressed Sequence Tags
T2 - Normalization and subtraction of cDNA libraries
AU - Soares, Marcelo Bento
AU - De Fatima Bonaldo, Maria
AU - Hackett, Jeremiah D.
AU - Bhattacharya, Debashish
PY - 2009
Y1 - 2009
N2 - Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.
AB - Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.
KW - cDNA library
KW - dinoflagellate
KW - expressed sequence tag (EST)
KW - normalization
KW - reassociation kinetics
KW - redundancy
KW - subtracted cDNA library
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U2 - 10.1007/978-1-60327-136-3_6
DO - 10.1007/978-1-60327-136-3_6
M3 - Chapter
C2 - 19277560
AN - SCOPUS:67650641732
SN - 9781588297594
T3 - Methods in Molecular Biology
SP - 109
EP - 123
BT - Expressed Sequence Tags (ESTs)
PB - Humana Press Inc.
ER -