F‐108 polymer and capillary electrophoresis easily resolves complex environmental DNA mixtures and SNPs

Abstract

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 organisms 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.

Resolving human DNA mixtures with F‐108 polymer and capillary electrophoresis single‐strand conformational polymorphism analysis

“…The F-108 polymer has proven to be a suitable sieving matrix for CE-SSCP analysis with varying sample types. It can be comparable, but more rapid, than SNP identification produced from SNaPshot™ multiplex systems [16,17,19,20]. An advantage that the F-108 CE-SSCP method has over SNaPshot™ is that a purification step is not necessary before electrophoresis, making the method less costly per sample, and also less time consuming [16].…”

“…DNA separation with the F-108 pluronic polymer (Millipore Sigma, St. Louis, MO, USA) was performed on an ABI 310 Genetic Analyzer (Applied Biosystems). Polymer and PCR products were prepared for analysis according to previously described parameters [4,17] with modifications including an injection time of 10 s and temperature held at 30 • C. Results were collected on the Data Collection Software v3.1.0 and analyzed with GeneMapper v.4.0 with a minimum threshold of 50 RFU.…”

Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F‐108 tri‐block copolymer and capillary electrophoresis

“…Capillary electrophoresis‐single‐strand conformation polymorphism (CE‐SSCP) allows for the separation of DNA fragments of the same fragment length based on their intrinsic DNA sequence differences as they migrate through a native, non‐denaturing polymer . Recently, F‐108 triblock copolymer, poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide), has been used as a sieving matrix for CE‐SSCP . The increase in resolution is due to the PPO and PEO interactions that result in a high‐density sieving matrix .…”

“…Recently, F‐108 triblock copolymer, poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide), has been used as a sieving matrix for CE‐SSCP . The increase in resolution is due to the PPO and PEO interactions that result in a high‐density sieving matrix . The advantage of the F‐108 polymer when coupled with CE‐SSCP analysis is that it allows for the separation of longer fragments (200–450 bp) and can distinguish the intrinsic sequence polymorphisms in most cases .…”

“…PCR reactions were performed in 15 μL volume as described previously with modifications incorporating 0.1% BSA and increasing the final concentration of each primer to 0.66 μM. PCR products and Pluronic F‐108 (Sigma‐Aldrich, St. Louis, MO) preparation and separation parameter conditions were prepared as previously described . The separation of each marker was performed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) and analyzed with GeneMapper® 4.0 software (Applied Biosystems, Foster City, CA).…”