Rapid screening of microsatellite markers for polymorphisms using SYBR green I and a DNA sequencer.

Vol. 36, No. 3 (2004) BioTechniques 409 The process of microsatellite development and profiling involves three primary steps: (i) isolating regions of genomic DNA that contain microsatellite loci; (ii) developing strategies for screening each locus, which requires designing primers for amplification, optimizing reaction conditions, and screening for variation; and (iii) genotyping sampled individuals. The protocols for cloning genomic DNA and isolating microsatellite loci have previously been based on those of Sambrook et al. (1) and Rassman et al. (2). However, recent techniques have been developed that greatly streamline this process and reduce the time and money involved in cloning and isolating microsatellite loci (3). Significant methodological and technical advances have also streamlined the process of genotyping individuals. This revolution began when fluorescent molecules replaced radioisotopes as the primary method of labeling DNA fragments for visualization in sequencing analyses (4). Moreover, fluorescent techniques are safer because they remove the need for radioisotopes. Although methodological advances have recently occurred in the first and third steps of microsatellite development and profiling, the process of screening markers for polymorphisms has changed little, and this step has become limiting. To reduce the time and cost and increase the safety involved in screening microsatellites for polymorphisms, several nonradioactive methods have been developed (5–8). However, these techniques still represent additional costs because they require the use of equipment that is unnecessary for other steps in fluorescent-based microsatellite development and profiling. Purchasing fluorescently labeled primers for this step is not economical due to the large cost involved in labeling primers with fluorescent molecules and because a portion of the loci will not be polymorphic. Therefore, a method for screening microsatellite markers for polymorphisms using unlabeled primers and fluorescent detection systems is necessary to reduce the cost and further streamline the process of microsatellite analysis. Here we describe a method for screening microsatellite markers for polymorphisms using unlabeled primers, an ABI PTM 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA), and the DNA stain SYBR® Green I (Cambrex, Rockland, ME, USA). Right whale (Eubalaena sp.) samples used to develop this technique were collected as previously described (9), and DNA extraction also followed previously described protocols (10). PCR amplification of the dinucleotide microsatellite loci TextVet19, TextVet20 (11), and RW31 (12) was performed on individuals whose genotypes had previously been determined using the methRapid screening of microsatellite markers for polymorphisms using SYBR® Green I and a DNA sequencer