RGD Reference Report - Normalization and subtraction: two approaches to facilitate gene discovery. - Rat Genome Database
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Normalization and subtraction: two approaches to facilitate gene discovery.

Authors: Bonaldo, MF  Lennon, G  Soares, MB 
Citation: Bonaldo MF, etal., Genome Res 1996 Sep;6(9):791-806.
RGD ID: 69939
Pubmed: (View Article at PubMed) PMID:8889548

Large-scale sequencing of cDNAs randomly picked from libraries has proven to be a very powerful approach to discover (putatively) expressed sequences that, in turn, once mapped, may greatly expedite the process involved in the identification and cloning of human disease genes. However, the integrity of the data and the pace at which novel sequences can be identified depends to a great extent on the cDNA libraries that are used. Because altogether, in a typical cell, the mRNAs of the prevalent and intermediate frequency classes comprise as much as 50-65% of the total mRNA mass, but represent no more than 1000-2000 different mRNAs, redundant identification of mRNAs of these two frequency classes is destined to become overwhelming relatively early in any such random gene discovery programs, thus seriously compromising their cost-effectiveness. With the goal of facilitating such efforts, previously we developed a method to construct directionally cloned normalized cDNA libraries and applied it to generate infant brain (INIB) and fetal liver/spleen (INFLS) libraries, from which a total of 45,192 and 86,088 expressed sequence tags, respectively, have been derived. While improving the representation of the longest cDNAs in our libraries, we developed three additional methods to normalize cDNA libraries and generated over 35 libraries, most of which have been contributed to our integrated Molecular Analysis of Genomes and Their Expression (IMAGE) Consortium and thus distributed widely and used for sequencing and mapping. In an attempt to facilitate the process of gene discovery further, we have also developed a subtractive hybridization approach designed specifically to eliminate (or reduce significantly the representation of) large pools of arrayed and (mostly) sequenced clones from normalized libraries yet to be (or just partly) surveyed. Here we present a detailed description and a comparative analysis of four methods that we developed and used to generate normalize cDNA libraries from human (15), mouse (3), rat (2), as well as the parasite Schistosoma mansoni (1). In addition, we describe the construction and preliminary characterization of a subtracted liver/spleen library (INFLS-SI) that resulted from the elimination (or reduction of representation) of -5000 INFLS-IMAGE clones from the INFLS library.

Annotation

Objects referenced in this article
0 Adam19 ADAM metallopeptidase domain 19 All species
0 Adam22 ADAM metallopeptidase domain 22 All species
0 Adam23 ADAM metallopeptidase domain 23 All species
0 Adam9 ADAM metallopeptidase domain 9 All species
0 Amacr alpha-methylacyl-CoA racemase All species
0 Aplp2 amyloid beta precursor like protein 2 All species
0 ARL8A ADP ribosylation factor like GTPase 8A All species
0 Cct4 chaperonin containing TCP1 subunit 4 All species
0 CLEC4F C-type lectin domain family 4 member F All species
0 EXOC6 exocyst complex component 6 All species
0 Fen1 flap structure-specific endonuclease 1 All species
0 Folr1 folate receptor alpha All species
0 Fos Fos proto-oncogene, AP-1 transcription factor subunit All species
0 Fxyd3 FXYD domain-containing ion transport regulator 3 All species
0 Fxyd7 FXYD domain-containing ion transport regulator 7 All species
0 Gimap1 GTPase, IMAP family member 1 All species
0 Gimap7 GTPase, IMAP family member 7 All species
0 Gimap8 GTPase, IMAP family member 8 All species
0 Gpx2 glutathione peroxidase 2 All species
0 Hck HCK proto-oncogene, Src family tyrosine kinase All species
0 Hmox1 heme oxygenase 1 All species
0 Klk1c8 kallikrein 1-related peptidase C8 All species
0 Kyat1 kynurenine aminotransferase 1 All species
0 Llgl1 LLGL scribble cell polarity complex component 1 All species
0 Mrpl19 mitochondrial ribosomal protein L19 All species
0 Prodh1 proline dehydrogenase 1 All species
0 Ptpmt1 protein tyrosine phosphatase, mitochondrial 1 All species
0 Rpl39 ribosomal protein L39 All species
0 Rps15 ribosomal protein S15 All species
0 Rps5 ribosomal protein S5 All species
0 Slc25a1 solute carrier family 25 member 1 All species
0 Sp3 Sp3 transcription factor All species
0 Sprn shadow of prion protein All species
0 Vkorc1 vitamin K epoxide reductase complex, subunit 1 All species

Additional Information