A strategy for genome-wide gene analysis: Integrated procedure for gene identification

San Ming Wang, Janet D. Rowley

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We have developed a technique called the Integrated Procedure for Gene Identification that modifies and integrates parts from several existing techniques to increase the efficiency for genome- wide gene identification. The procedure has the following features: (i) Only the 3' portion of the expressed templates is used to ensure a match to 3' expressed sequence tag (EST) sequences; (ii) the 3' portion of the cDNA is poly dA/poly dT minus, which maintains complete representation of the expressed copies, particularly the rare copies, which otherwise would be lost heavily because of random poly dA/poly dT hybridization in the subtraction reaction; (iii) redundancy is decreased substantially by the subtraction reaction to reduce the effort for sequencing analysis; (iv) the nonsubtracted templates that largely contain the rare copies are amplified selectively with suppression PCR and are sequenced directly or through serial analysis of gene expression (SAGE); and (v) the identified sequences are matched to databases to determine whether they are cloned genes, ESTs, or novel sequences. Using this procedure in a model system, we showed that the redundant copies were largely removed, and the rates of EST matches and the novel sequence identification were significantly increased. Most of the plasmids containing the matched EST are readily available from the IMAGE consortium. This technique can be used to index genome-wide expressed genes and to identify differentially expressed genes in different cells. Compared with the existing techniques, this procedure is relatively efficient, simple, less expensive, and labor intensive. It is especially useful for standard molecular laboratories to perform genome-wide studies.

Original languageEnglish (US)
Pages (from-to)11909-11914
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume95
Issue number20
DOIs
StatePublished - Sep 29 1998

Fingerprint

Expressed Sequence Tags
Genome
Genes
Plasmids
Complementary DNA
Databases
Gene Expression
Polymerase Chain Reaction
polydeoxyadenylic acid-polythymidylic acid

ASJC Scopus subject areas

  • General

Cite this

A strategy for genome-wide gene analysis : Integrated procedure for gene identification. / Wang, San Ming; Rowley, Janet D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 95, No. 20, 29.09.1998, p. 11909-11914.

Research output: Contribution to journalArticle

@article{33a1ac6d1ff84e47b43c8ee6e364e1ac,
title = "A strategy for genome-wide gene analysis: Integrated procedure for gene identification",
abstract = "We have developed a technique called the Integrated Procedure for Gene Identification that modifies and integrates parts from several existing techniques to increase the efficiency for genome- wide gene identification. The procedure has the following features: (i) Only the 3' portion of the expressed templates is used to ensure a match to 3' expressed sequence tag (EST) sequences; (ii) the 3' portion of the cDNA is poly dA/poly dT minus, which maintains complete representation of the expressed copies, particularly the rare copies, which otherwise would be lost heavily because of random poly dA/poly dT hybridization in the subtraction reaction; (iii) redundancy is decreased substantially by the subtraction reaction to reduce the effort for sequencing analysis; (iv) the nonsubtracted templates that largely contain the rare copies are amplified selectively with suppression PCR and are sequenced directly or through serial analysis of gene expression (SAGE); and (v) the identified sequences are matched to databases to determine whether they are cloned genes, ESTs, or novel sequences. Using this procedure in a model system, we showed that the redundant copies were largely removed, and the rates of EST matches and the novel sequence identification were significantly increased. Most of the plasmids containing the matched EST are readily available from the IMAGE consortium. This technique can be used to index genome-wide expressed genes and to identify differentially expressed genes in different cells. Compared with the existing techniques, this procedure is relatively efficient, simple, less expensive, and labor intensive. It is especially useful for standard molecular laboratories to perform genome-wide studies.",
author = "Wang, {San Ming} and Rowley, {Janet D.}",
year = "1998",
month = "9",
day = "29",
doi = "10.1073/pnas.95.20.11909",
language = "English (US)",
volume = "95",
pages = "11909--11914",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "20",

}

TY - JOUR

T1 - A strategy for genome-wide gene analysis

T2 - Integrated procedure for gene identification

AU - Wang, San Ming

AU - Rowley, Janet D.

PY - 1998/9/29

Y1 - 1998/9/29

N2 - We have developed a technique called the Integrated Procedure for Gene Identification that modifies and integrates parts from several existing techniques to increase the efficiency for genome- wide gene identification. The procedure has the following features: (i) Only the 3' portion of the expressed templates is used to ensure a match to 3' expressed sequence tag (EST) sequences; (ii) the 3' portion of the cDNA is poly dA/poly dT minus, which maintains complete representation of the expressed copies, particularly the rare copies, which otherwise would be lost heavily because of random poly dA/poly dT hybridization in the subtraction reaction; (iii) redundancy is decreased substantially by the subtraction reaction to reduce the effort for sequencing analysis; (iv) the nonsubtracted templates that largely contain the rare copies are amplified selectively with suppression PCR and are sequenced directly or through serial analysis of gene expression (SAGE); and (v) the identified sequences are matched to databases to determine whether they are cloned genes, ESTs, or novel sequences. Using this procedure in a model system, we showed that the redundant copies were largely removed, and the rates of EST matches and the novel sequence identification were significantly increased. Most of the plasmids containing the matched EST are readily available from the IMAGE consortium. This technique can be used to index genome-wide expressed genes and to identify differentially expressed genes in different cells. Compared with the existing techniques, this procedure is relatively efficient, simple, less expensive, and labor intensive. It is especially useful for standard molecular laboratories to perform genome-wide studies.

AB - We have developed a technique called the Integrated Procedure for Gene Identification that modifies and integrates parts from several existing techniques to increase the efficiency for genome- wide gene identification. The procedure has the following features: (i) Only the 3' portion of the expressed templates is used to ensure a match to 3' expressed sequence tag (EST) sequences; (ii) the 3' portion of the cDNA is poly dA/poly dT minus, which maintains complete representation of the expressed copies, particularly the rare copies, which otherwise would be lost heavily because of random poly dA/poly dT hybridization in the subtraction reaction; (iii) redundancy is decreased substantially by the subtraction reaction to reduce the effort for sequencing analysis; (iv) the nonsubtracted templates that largely contain the rare copies are amplified selectively with suppression PCR and are sequenced directly or through serial analysis of gene expression (SAGE); and (v) the identified sequences are matched to databases to determine whether they are cloned genes, ESTs, or novel sequences. Using this procedure in a model system, we showed that the redundant copies were largely removed, and the rates of EST matches and the novel sequence identification were significantly increased. Most of the plasmids containing the matched EST are readily available from the IMAGE consortium. This technique can be used to index genome-wide expressed genes and to identify differentially expressed genes in different cells. Compared with the existing techniques, this procedure is relatively efficient, simple, less expensive, and labor intensive. It is especially useful for standard molecular laboratories to perform genome-wide studies.

UR - http://www.scopus.com/inward/record.url?scp=0009064319&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0009064319&partnerID=8YFLogxK

U2 - 10.1073/pnas.95.20.11909

DO - 10.1073/pnas.95.20.11909

M3 - Article

C2 - 9751764

AN - SCOPUS:0009064319

VL - 95

SP - 11909

EP - 11914

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 20

ER -