3208 Electrophoresis 2014, 35, 3208–3211 Natalie Damaso 1,2,3 Lauren Martin 1,2,3 Priyanka Kushwaha 3 DeEtta Mills 1,2 1 Department of Biological Sciences, Florida International University, Miami, FL, USA 2 International Forensic Research Institute, Florida International University, Miami, FL, USA 3 Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA Received February 10, 2014 Revised August 21, 2014 Accepted August 22, 2014 Short Communication F-108 polymer and capillary electrophoresis easily resolves complex environmental DNA mixtures and SNPs Ecological studies of microbial communities often use profiling methods but the true community diversity can be underestimated in methods that separate amplicons based on sequence length using performance optimized polymer 4. Taxonomically, unrelated or- ganisms can produce the same length amplicon even though the amplicons have different sequences. F-108 polymer has previously been shown to resolve same length amplicons by sequence polymorphisms. In this study, we showed F-108 polymer, using the ABI Prism 310 Genetic Analyzer and CE, resolved four bacteria that produced the same length amplicon for the 16S rRNA domain V3 but have variable nucleotide content. Second, a microbial mat community profile was resolved and supported by NextGen sequencing where the number of peaks in the F-108 profile was in concordance with the confirmed species numbers in the mat. Third, equine DNA was analyzed for SNPs. The F-108 polymer was able to distinguish heterozygous and homozygous individuals for the melanocortin 1 receptor coat color gene. The method proved to be rapid, inexpensive, reproducible, and uses common CE instruments. The potential for F-108 to resolve DNA mixtures or SNPs can be applied to various sample types—from SNPs to forensic mixtures to ecological communities. Keywords: Capillary electrophoresis / F-108 polymer / Microbial community / SNP / SSCP DOI 10.1002/elps.201400069 Environmental microbial community mixtures are difficult to analyze and current molecular profiling methods do not always support sequence queries of differences within or between samples. For community DNA analyses, the cur- rent practices for differentiating mixtures require either tra- ditional cloning and sequencing, or NextGen sequencing, both of which can be expensive and time consuming [1, 2]. Therefore, rapid DNA profiling techniques such as termi- nal RFLP [3], denaturing gradient gel electrophoresis [4], and amplicon length heterogeneity PCR methods [5] using dena- turing polymers or conditions are often used to query DNA community mixtures. Amplicon separation using denatur- ing conditions and polymers such as performance optimized polymer 4 (POP-4), can underestimate the true sequence di- versity hidden within each amplicon. This is because taxo- nomically unrelated organisms can produce a length ampli- Correspondence: Dr. DeEtta Mills, Department of Biological Sci- ences, Florida International University, 11200 SW 8th Street, OE 167, Miami, FL 33199, USA E-mail: millsd@fiu.edu Fax: +1-305-348-1986 Abbreviations: F-108, Pluronic F-108 (tri-block copolymer); mc1r, melanocortin 1 receptor; POP-4, performance opti- mized polymer 4; POP-CAP, performance optimized polymer conformation analysis polymer con that is not representative of the varied base sequence contained within the amplicon [6, 7]. SSCP is a technique that has been highly applicable in community profiling [8], but can often be difficult to optimize for CE. This technique has commonly been used to detect un- known mutations or SNPs in short amplicons. During sep- aration ssDNA fragments partially renature and form folded conformations due to intramolecular interactions between the bases under nondenaturing gel electrophoresis. Different electrophoretic mobility based on the secondary DNA struc- tures can result in separation of DNA strands with a resolu- tion of one base [9]. A polymer that has recently been used as a CE-SSCP sieving matrix to enhance resolution is the F-108 tri- block copolymer, poly(ethyleneoxide)-poly(propyleneoxide)- poly(ethyleneoxide). The PPO forms hydrophobic micelles that do not directly interact with DNA, but induce micelle formation resulting in a high-density sieving matrix that will separate DNA amplicons with different base content. Previ- ous study by Shin et al. 2010 discovered that the PEO chain length increased the resolution due to the hydrophilic mesh that interacts with DNA and, together, it results in a high- density sieving matrix [10]. The objectives of this research were as follows: First, to validate the F-108 polymer for the ABI CE using four model organisms that display the same length amplicon for 16S rRNA hypervariable domains under denaturing conditions but have varying base composition; second, was to assess the C  2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com