Optimisation of the degenerate oligonucleotide primed PCR (DOP- PCR) for capillary thermocycler Csaba Kiss *, Maria Kost-Alimova, George Klein, Laszlo Szekely Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Nobels vag 16, S-171 77 Stockholm, Sweden Received 10 August 2001; received in revised form 11 March 2002; accepted 18 March 2002 Abstract DOP-PCR was optimised for capillary thermocycler. Five primers with five or six nucleotide anchor sequences and different restriction enzyme recognition sites were tested. The efficiency of the amplification was confirmed by gel electrophoresis and fluorescence in situ hybridisation on metaphase chromosomes. # 2002 Elsevier Science B.V. All rights reserved. Keywords: DOP-PCR; Capillary PCR; PCR optimisation The degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) was developed in order to representatively amplify DNA for cytogenetic studies [1]. DOP-PCR is a reliable tool to amplify DNA accurately and uniformly. It has been widely used to amplify DNA from FACS sorted chromosomes [2] or from microdissected chromosome bands [3]. The proto- col has also been optimised for comparative genomic hybridisation (CGH) experiments [4]. The DOP-PCR protocol is a combination of two different cycling programs. During the first five cycles, the sample is amplified under low stringency conditions, then 30 /40 cycles of high stringency PCR is performed. DOP-PCR primers are usually 22 bp long and contain a 6 bp well- defined 3? end, which is followed by 6 bp of degenerate nucleotides. The 5? end contains a rare restriction- endonuclease recognition site. During the low stringency cycles, only the 3? end of the primer initiates elongation from the sites that contain the 6 bp well-defined sequence. The degenerate part of the primer ensures that all possible combination of the 12 bp will be recognised and extended. To our knowledge, all pub- lished protocols use the same primer (DOP-ORI-Xho). We tested this primer and four new primers that differ both in their 5? end restriction sites and a 3? well-defined sequence. The capillary PCR was designed to provide the best thermal conductivity parameters that match the kinetics of DNA denaturation and annealing [5]. It is also the fastest way of performing DNA amplification. The PCR is performed in small volumes in glass capillaries. The heating and cooling are conducted by high-speed turbulent air. The combination of the small heat capacity of air and the high surface/volume ratio of the sample in the capillary allows the temperature to change rapidly. As a result, the required incubation times for denaturation and annealing during the PCR reaction are very short [6]. Capillary cycling requires specific reaction conditions that are generally different from PCR performed in plastic tubes. The PCR buffer contains a relatively high concentration (50 mg/ml) of bovine serum albumin (BSA) to prevent the denatura- tion of the Taq polymerase on the large glass surface of the capillary. The optimal Mg concentration tends to be higher (3 mM). The DOP-PCR is performed with a highly degenerate primer that contains a mixture of 4096 different variant. Such a complex amplification requires very specific reaction conditions to avoid primer dimers and other PCR artefacts. The PCR was performed in a 10-ml volume (50 mM Tris /HCl pH 8.6, 50 mg/ml BSA (Sigma), 3 mM MgCl 2 , 200 mM of each dNTP, 0.4 U Platinum Taq polymerase (GIBCO Lifetechnology)) using 10 mM of the appropriate DOP-PCR primer, if otherwise mentioned. The cycling was performed in a Rapidcycler (Idaho Tech.): 15 s at 94 8C, followed by * Corresponding author. Tel.: /46-87-286-259; fax: /46-833-0498 E-mail address: csaba.kiss@mtc.ki.se (C. Kiss). Biomolecular Engineering 19 (2002) 31 /34 www.elsevier.com/locate/geneanbioeng 1389-0344/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S1389-0344(02)00008-4