Analytical Biochemistry 351 (2006) 311–313 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.01.049 ANALYTICAL BIOCHEMISTRY Notes & Tips Assembling and cloning genes for fusion proteins using reverse transcription one-step overlap extension PCR method Zsolt B. Nagy a , Zoltán Varga-Orvos a , Barnabás Szakál b , László Tamás c,¤ , László G. Puskás a a Laboratory of Functional Genomics, Biological Research Center, Hungarian Academy of Sciences, Szeged H-6701, Hungary b Institute of Genetics, Biological Research Center, Szeged H-6701, Hungary c Department of Plant Physiology and Molecular Plant Biology, Eötvös Loránd University, Budapest H-1117, Hungary Received 11 November 2005 Available online 20 February 2006 The need to construct fusion genes for diVerent purposes arises in various Welds of molecular biology [1,2]. PCR is one of the most popular methods to generate fusions of diVerent DNA sequences. Examples include constructs where (i) diVerent lengths of a particular promoter sequence are fused to the coding region of another gene to determine which sequences are involved in the regulation of gene expression or which promoter expresses a particular gene more eYciently, (ii) fusion genes are created to code for hybrid proteins, and (iii) DNA sequences representing precise deletion mutants are constructed. The construction of such fused or deleted genes usually involves ligation of diVerent restriction fragments, a tedious and time-consum- ing process that frequently is restricted by the availability of proper cleavage sites [3,4]. The overlap extension (OE) 1 technique is widely used in genetic engineering to overcome such limitations. Specially designed oligonucleotides and PCR methods are applied to generate two DNA fragments with overlapping ends. These fragments are combined in a fusion reaction to anneal the overlapping ends of the appropriate strands. Both 3' ends serve as primers extending the complementary strands. The resulting fusion product is ampliWed further by subsequent PCR. SpeciWc alterations can be introduced into the PCR product through incorporation of changes into the overlap- ping primers [5]. A variation of the OE techniques, the one- step overlap extension PCR (OOE–PCR), was described by Urban and coworkers [6]. The OOE–PCR method runs in only one reaction by choosing a high dilution of mutagenic primers to generate desired mutations of diVerent genes. So far, only one reverse transcription (RT)–PCR-based mutagenesis method, using total RNA as a template, has been described by Nagy and coworkers [7]. The starting point of this method is RNA, but it is not based on an OE strategy. Here we describe an OE strategy that is reduced to only one RT–PCR reaction and therefore is named reverse tran- scription one-step overlap extension PCR (RT–OOE– PCR). Our method works with extracted RNA, so there is no need for cDNA cloning and/or puriWcation of interme- diate PCR products. This method uses universal primers and only one gene-speciWc overlapping primer. The fused sequence can be easily detected and distinguished from the nonfused sequences by a simple digestion reaction. The method is described by reference to the fusion of the human -synuclein gene to the red Xuorescent protein 1 reporter gene (DsRed1) from Discosoma sp. Total RNA was isolated with a GenElute Mammalian Total RNA Miniprep Kit (Sigma–Aldrich, St. Louis, MO, USA). The RNA concentration was assessed spectrophoto- metrically by a NanoDrop (Rockland, DE, USA), and the quality was monitored by an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Hep2 cells (HeLa derivative, human negroid cervical car- cinoma cells) were maintained in Dulbecco’s modiWed Eagle’s media (Invitrogen, Carlsbad, CA, USA) supple- mented with 10% fetal bovine serum (Gibco, Germany), 2 mM glutamine (Gibco), and 1% nonessential amino acids (Gibco) in a 5% CO 2 environment at 37 °C. Sterile 24 £ 32- mm cover slips (Menzel, Hamburg, Germany) were placed in 60-mm plastic Petri dishes (Corning, USA), and 2.5£ 10 5 cells were plated. After a 24-h culture period, transfections were made with 4 g of circular plasmid DNA using Lipo- fectamine 2000 transfection reagent (Invitrogen) according * Corresponding author. Fax: +36 1 381 2164. E-mail address: tamasl@ludens.elte.hu (L. Tamás). 1 Abbreviations used: OE, overlap extension; OOE–PCR, one-step over- lap extension PCR; RT, reverse transcription; RT–OOE–PCR, reverse transcription one-step overlap extension PCR; PBS, phosphate-buVered saline.