Prism bigdye terminator v3.1 cycle sequencing kit

Based on the probiotic characteristics, 2 yeast isolates OBS1 and OBS2 were selected and characterized by sequencing of 5.8S rRNA and internal transcribed spacer (ITS) 1 and 2 (Fujita et al. 2001 (link)). Genomic DNA was extracted using an Insta Gene Matrix (BIO RAD, California, USA). The universal primers used for amplification of 5.8S rRNA, ITS1and ITS2 were 5′-TCCGTAGGTGAACCTGCGG-3′ and 5′-TCCTCCGCTTATTGATATGC-3′. The PCR conditions were set to 1 min each for denaturation at 95 °C, annealing at 55 °C, 2 min for an extension at 72 °C and finally finishing with a 10 min step at 72 °C. The amplicons were purified with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA) and sequencing was performed using PRISM BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, California, USA). The amplicons were added to Hi-Di formamide (Applied Biosystems, Foster City, CA) and the mixture was incubated at 95 °C for 5 min, followed by incubation on ice for 5 min and then analyzed by ABI Prism 3730XL DNA analyzer (Applied Biosystems). Complete sequence of 5.8S rRNA and partial sequences of internal transcribed spacer (ITS) 1 and 2 were BLAST searched in NCBI database (www.ncbi.nlm.nih.gov) for similarity. Based on maximum similarity of BLAST results, a phylogenetic dendrogram was constructed using Phylip 3.69.

The 16S rRNA gene sequencing was performed on the genomic DNA extracted from the strong lipase-positive bacterial strains using the InstaGene Matrix kit (Bio-Rad, Seoul, Korea) according to the manufacturer’s instructions. As described by Frank et al. [25 (link)], amplification of the 16S rRNA gene was performed using PCR with primers 27F and 1492R. A multiscreen filter plate (Millipore Corp, Bedford, MA, USA) was used to purify the PCR product, which was then sequenced using the primers 518F (5-CCA GCA GCC GCG GTA ATA CG-3) and 800R (5-TAC CAG GGT ATC TAA TCC-3) with a PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). This process was performed at 95 °C for 5 minutes. The product was cooled on ice for 5 min and analyzed using an ABI Prism 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). Finally, the nearly full-length 16S rRNA sequence was assembled using SeqMan software (DNASTAR Inc., Madison, WI, USA) [25 (link)]. The sequence similarity of each lipase-producing bacterial strain was determined by comparison with the available sequences in the GenBank database using the EZBioCloud server (http://ezbiocloud.net/ (accessed on 13 April 2021).) [26 (link)].

For uniplex PCR, 0.5 μL of extracted DNA, 1.5 mM of MgCl₂, 0.08 mM of dNTPs (Promega, Madison, USA), 0.2 pmol/L of primers (Table-1) [32 –34 (link)], and 0.02 U of GoTaq DNA polymerase (Promega) were mixed in a final volume of 25 μL. The ITS region was amplified using PCR under the following conditions: initial denaturation at 95°C for 5 min; 30 cycles of priming denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; final extension at 72°C for 10 min; for β-tubulin: Initial denaturation at 94°C for 5 min, followed by 35 cycles of initial denaturation at 94°C for 1 min, annealing at 68°C for 1 min, and extension at 68°C for 2 min. A 10 L sample of amplified DNA was separated on a 1.5% agarose gel in 1× TBE, stained with ethidium bromide, and analyzed under UV light. The purified PCR products were sequenced on an automated DNA sequencer (Applied Biosystems, Thermo-Fisher-Scientific Co., USA) using PRISM Big Dye Terminator V3.1 Cycle Sequencing Kit (Cat. No. 4336917; Applied Biosystems).