Abi prism 3130xl genetic analyser

Amplification of faecal 16S rDNA, digestion using restriction enzymes, size fractionation of terminal restriction fragments, and analyses of T-RFLP data were done according to the method used by Nagashima et al., with modifications [23 (link),24 (link)]. Briefly, PCR was carried out using total faecal DNA with primers for 5′-carboxy-fluorescein-labelled 516f and 1510r. The resulting 16S rDNA amplicons were treated for 3 h at 55 °C with 10 U of Bs/I (5′-CCNNNNN|NNGG-3′) (New England Biolabs, MA, USA). The fluorescently labelled terminal restriction fragments produced by digestion with Bs/I were analysed by electrophoresis on an ABI PRISM 3130xl Genetic Analyser (Applied Biosystems, CA, USA) in GeneScan mode (injection time was 30 s, and run time was 40 min).

For CmMAX1 degenerate primers were developed based on MAX1 amino acid sequences from Arabidopsis, tomato, poplar, soy, Medicago, vine and RicinusS1 Table. Furthermore, a BLAST search was done to the chrysanthemum transcriptome database of Xu et al. [62 (link)] using Arabidopsis genes known to be involved in branching (corresponding accession numbers are indicated in Table S.2). This resulted in orthologous sequences for CmMAX3, CmDRM1, CmSTM, CmRR1, CmHK3a, CmHK3b, CmAXR1, CmAXR2, CmIAA16, CmAXR6, CmIAA12, CmPIN1, CmTIR1 and CmTIR3. Reference genes were identified by BLAST against the NCBI database. These included CmACT2, CmATUB, CmUBQ10, CmUBC, CmEF1α, CmCACS, CmEXP5, CmEXP6, CmPGK, CmPSAA and CmHH3. Sequences of CmBTUB_AB608732 and CmMTP_AB542716.1. were available for Chrysanthemum. Primers S2 Table. were developed using Primer3 PLUS. Genes were cloned using a pGEM-T kit (Promega) and sequenced according to the protocol of the Big Dye Terminator Cycle Sequencing kit version 1.1 on an ABI Prism 3130 xl Genetic analyser (Applied Biosystems). BlastX [63 (link)] was used to validate isolated fragment identity.

Genomic DNA from control samples was extracted from peripheral leukocytes using the Puregene Kit according to the manufacturer’s instructions (Gentra Systems, Inc., Minneapolis, MN, USA). Mutation screening was achieved through Sanger sequencing. Reference sequence NM_020193.3 was obtained from the USCS genome browser [32 , 33 (link)]. The genome build GRCh37 (hg19) was used. Whole coding regions and exon-intron boundaries were analysed. EMSY primer sequences and PCR conditions are available upon request. Sequencing was performed using the Big Dye Terminator v.3.1 Cycle Sequencing Kit and the ABIPRISM 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The sequences were analysed using Sequencher v.5.1 software (Gene Codes Corporation, Ann Arbor, MI, USA). The RefSNP number for the identified variants was obtained from the NCBI Single Nucleotide Polymorphism database (dbSNP) [34 ]. Two variants, rs1044265 and rs2513513, were analysed from controls using TaqMan SNP Genotyping Assays according to the manufacturer’s instructions with the ABI Prism® 7900HT instrument and SDS2.2.2 software (Applied Biosystems). The genotyping call rates of the SNPs were ≥0.98.

The BigDye Terminator Cycle Sequencing kit was used according to the manufacturer’s instructions. The sequencing run was carried out using the 16-capillary ABI PRISM 3130 xl Genetic Analyser, (Applied Biosystems).

Purified PCR products (10–20 ng per community sample, 1 ng per sample for clone PCR products) were digested over night at 37°C in a 10 μl reaction volume with 5 U of either the restriction endonuclease HaeIII or MspI (New England Biolabs, Germany). Samples were ethanol precipitated, and dried DNA pellets were re-suspended in 10 μl HiDi formamide containing 1.5% (v/v) MapMarker 1000 (Bioventures, USA) or GeneScan-500 ROX standard (Applied Biosystems, Germany) in case of mcrA/mrtA products. Fluorescently labeled terminal restriction fragments (T-RFs) were separated on an ABI PRISM 3130xl Genetic Analyser (Applied Biosystems, Germany) with POP-7 polymer. The lengths of the terminal fragments were determined using GeneMapper V3.7 software (Applied Biosystems, Germany) and fluorescent data for the range of 50–1000 bp was exported (50–500 in case of mcrA/mrtA) to R script, and peak areas were normalized and noise filtering was applied (σ = 5) according to Abdo and colleagues (2006 (link)). A multivariate cluster analysis with the Bray–Curtis dissimilarity index was performed in program past (Hammer et al., 2001 ). In addition to the presence and absence of certain T-RFLP peaks, the calculations take the relative abundance ratios into consideration.

DNA was isolated from culture isolates with Genolyse buffer (Hain life sciences). Amplification of 305bp band of rpoB gene was done using primer sequence described previously [10 (link)]. Sequencing of 81-bp rpoB gene was carried out with ABI prism 3130xl genetic analyser (Applied Biosystems) and BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). The sequencing results were analyzed by using BioEdit software and alignment was done by using clustalW. Data obtained were compared with standard H37Rv sequence.