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A modified GLGI method for identification of novel porcine genes from long serial analysis of gene expression tags

Published online by Cambridge University Press:  02 August 2007

Tang Zhong-Lin
Affiliation:
Key Laboratory of Animal Heredity and Breeding, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100094, China
Li Yong
Affiliation:
Key Laboratory of Animal Heredity and Breeding, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
Zhao Shu-Hong
Affiliation:
Key Laboratory of Animal Heredity and Breeding, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
Liu Bang
Affiliation:
Key Laboratory of Animal Heredity and Breeding, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
Fan Bin
Affiliation:
Key Laboratory of Animal Heredity and Breeding, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China
Li Kui*
Affiliation:
Key Laboratory of Animal Heredity and Breeding, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100094, China
*
*Corresponding author. E-mail: likuihau@yahoo.com

Abstract

Combining the long serial analysis of gene expression (LongSAGE) and the generation of longer cDNA fragments from serial analysis of gene expression tags for gene identification (GLGI) technique, a new strategy called modified GLGI (M-GLGI) was developed to isolate unknown 3′ expressed sequence tags (ESTs) and discover novel genes. A 17 bp LongSAGE tag was used as sense primer instead of a 10-base SAGE tag; PCR reaction was performed under an appropriate annealing temperature for each tag; universal DNA polymerase was used in PCR amplification instead of Pfu enzyme; a common cloning strategy using pMD-18T vector and Escherichia coli DH5α cells were used instead of a special vector and competent cells. Moreover, ESTs isolated by M-GLGI had 3′ ends with the polyadenylation signals and poly(dA) tails. This method is more sensitive for identifying genes expressed in low abundance than conventional EST sequencing.

Type
Research Article
Copyright
Copyright © China Agricultural University and Cambridge University Press 2007

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Footnotes

First published in Journal of Agricultural Biotechnology 2007, 15(1): 15–19

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