Full Length Research Paper

Random amplification of genomic ends (RAGE) as an efficient method for isolation and cloning of promoters and uncloned genomic regions

Boney Kuriakose^1,3*, V. Ganesan^1,4, George Thomas², Arun Viswanathan^1,5 and Narayanaswamy Anand¹

¹Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai -600 025, India.

²Interfield Laboratories, Karuvelipady, Cochin - 680 005, India.

³Floriculture lab, Phytotron D, University of Pretoria Experimental farms, South Street, Hatfield, Pretoria, 0002, South Africa.

⁴Acme ProGen Biotech (India) Private Limited, 260G, Ram Square, Advaitha Ashram Road, Salem -636 004, Tamil Nadu, India.

⁵Department of Biotechnology, Sri Ramachandra University, No.1, Ramachandra Nagar, Porur, Chennai - 600 116, India.

*Corresponding author. Email: boneykuriakose@gmail.com. Tel: +27 792709391. Fax: +27 12 844 0276.

Accepted 23 June, 2009

   Abstract

Isolation of complete coding sequences and regulatory regions is critical for the complete characterization of a gene. Efficient methods to obtain complete genomic or regulatory is important in the process of isolation. The utility of the available genome walking methods are influenced by factors like the size of the genome and the length of the desired sequence. This study utilizes a genome walking method - random amplification of genomic ends (RAGE) efficiently to obtain the 5' – regulatory sequence of a rice stress inducible gene OsAsr1 and to obtain the full length sequence and promoter of the HetR gene of Cylindrospermum stagnale (Cylindrospermum sp. A1345). We demonstrate that this technique can be used for cloning of full length gene and promoters in organisms where whole genome data is unavailable utilising very little sequence information. Our studies show that RAGE can be a strong tool in functional genomics especially in the study of promoters

Key words: RAGE, genome walking, promoter, Oryza rufipogon, Oryza malampuzhaensis, Anabaena, Cylindrospermum, Asr1, HetR.