Sanger method

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The Sanger method is a DNA sequencing technique used to determine the sequence of nucleotides in a DNA molecule. It is a widely used method for DNA sequencing and is a core function of the lab equipment.

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15 protocols using sanger method

Bat-Borne Flavivirus Detection in Colombia

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Tissues of brain, heart, lung, spleen, liver, and kidney of 286 captured bats from Cordoba and Sucre departments were analyzed. The RNA was extracted with Trizol and cDNA was synthesized using a reverse a reverse transcriptase enzyme M-MLV (Invitrogen), a cDNA was obtained. Nested RT-PCR was performed with a first-round to get an amplicon product of 1360 bp using the primers: Flavi 1+(5′-GAYYTIGGITGYGGIGIGGIRGITGG-3′) and Flavi 1-(5′-TCCCAICCIGCIRTRTCRTCIGC-3′), and Flavi 2+(5′-GYRTIYAYAWCAYSAT GGG-3′) and Flavi 2-(5′-CCARTGITCYKYRTTIAIRAA ICC-3′) for a second-round to obtain an amplicon product of 143 bp. Degenerated primers were designed based on conserved from a region of gene NS5, which encodes for the polymerase, to align with known flaviviruses sequences [20 (link)]. As a control for each species, complementary primers were used to sequence a mitochondrial gene mt DNA from bats [21 ]. The Yellow Fever Virus (YFV) vaccine prepared with an attenuated live virus strain 17D-204 (Sanofi-Pasteur, Lyon, France) was used as a positive control. As a negative control, molecular water grade was used. The obtained amplicons (Figure 1) were sequenced in both forward and reverse directions by Sanger method at Macrogen (Korea)
The obtained amplicons (Figure 1) were sequenced by the Sanger method at Macrogen (Korea).

Calderón A., Guzmán C., Oviedo-Socarras T., Mattar S., Rodríguez V., Castañeda V, & Moraes Figueiredo L.T. (2021). Two Cases of Natural Infection of Dengue-2 Virus in Bats in the Colombian Caribbean. Tropical Medicine and Infectious Disease, 6(1), 35.

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Microbial Diversity Analysis of C. viridis and H. columella

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Samples of C. viridis and H. columella were submersed in absolute ethanol immediately after collection and taken to the laboratory in a cooled container. H. columella was extracted using Qiamp DNA stool kit (Qiagen) and C. viridis with DNeasy blood and tissue kit (Qiagen). One 16 S rRNA gene fragment, ca. 1,450 nt. in size, was amplified using universal primers 26 F and 1492R58 . PCR conditions were as described previously59 . PCR products were purified using QIAquick PCR Purification kit (Qiagen) and cloned using TOPO^® TA Cloning^® Kit for sequencing using One Shot^® TOP10 Chemically Competent E. coli (Invitrogen), according to the manufacturer instructions. Following colony growth, correct-size inserts were identified using PCR with T3–T7 primers, and purified and sequenced using the Sanger method (Macrogen Europe). Sequences containing the 273 nt. calcibacteria fragment15 (link) were selected and aligned with the SILVA database using SINA web aligner. The alignment was merged into ARB software and improved using the Fast Aligner tool according to the secondary structure.

Garate L., Sureda J., Agell G, & Uriz M.J. (2017). Endosymbiotic calcifying bacteria across sponge species and oceans. Scientific Reports, 7, 43674.

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Genotyping CYP2D6*10 Polymorphism

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The PCR products of exon1 were digested by HphI (Fermantas, Germany) at 37˚C for at least 6 hours to detect CYP2D6*10 (P34S) (C/T, rs1065852). RFLP patterns showed 363, 71, and 69 bp fragments for C allele as well as 263, 100, 71, and 69 bp fragments for T allele. The size of the digestion products was evaluated by 1.52% agarose gel electrophoresis that was followed by ethidium bromide staining.
Some samples of exon 1 were selected randomly to be sequenced in order to confirm the genotypes obtained by PCRRFLP analysis.
Purified PCR products of exon 4, including part of intron 3 and intron 4, were sequenced by an applied biosystem automated DNA sequencing Sanger method (Macrogen Inc., Korea). Also some samples of exon 1 were randomly sequenced. Finch TV software version 1.4.0 was used to analyze the sequencing diagram results (http://www.geospiza.com/Products/finchtv.shtml). In addition, results of each exon or intron were blasted against the ancestral sequence in NCBI (http://blast.ncbi.nlm.nih.gov.com).

Modaresi-nejad M., Shiva M, & Afsharian P. (2015). Novel +90G>A Intronic Polymorphism of CYP2D6. Cell Journal (Yakhteh), 17(1), 83-88.

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Identification of Non-Helicobacter Bacterial Isolates

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Single colonies of non-Helicobacter pylori appearing on the same blood-agar plates with H. pylori were selected from the agar surface and purified by sub-culturing several times using TSA agar plates with 5% sheep blood, incubated under anaerobic and aerobic conditions, and subsequently incubated at 37°C for 48 hours prior to DNA extraction. We next performed PCR of the 16s rRNA gene using bacteria universal primers previously described [27 (link)]. Next we performed purification of the PCR product, and 16S rRNA sequencing using Sanger method (Macrogen, NYC, US) [22 (link), 26 (link)]. DNA was extracted using genomic DNA purification kit (Fermentas K0722).

Guillermo Ignacio P.P., Van T.N., Thu H.D., Zhan G., Do Nguyet A., Nguyet N.T., Thi L.T., Vinh N.T, & Nguyen Thi H.H. (2016). Isolation and characterization of Helicobacter pylori recovered from gastric biopsies under anaerobic conditions. Diagnostic microbiology and infectious disease, 86(2), 136-140.

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Sequencing and Alignment of DENV-2 E Protein

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For the DENV-2 strains used in this study, E protein was amplified using SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions and previously reported primer sequences and protocols [54 (link)]. Amplicons were sequenced using the Sanger method (Macrogen Inc., Seoul, Korea). Sequences were assembled and edited with the Lasergene software, and a BLAST search was performed using the online platform Dengue Virus Typing Tool v.3.82. Subsequently, reference sequences retrieved from NCBI and the sequences under study were aligned with CLUSTAL W using MEGA v7. To determine nucleotide differences between strains, 1308 nucleotides corresponding to 435 amino acids of the E protein of the DENV-2 New Guinea C (AB609589) and DENV-2 16,681 (NC001474) strains were compared with those of the strains assessed here (DENV-2/NG and DENV-2/16681, respectively).

Monsalve-Escudero L.M., Loaiza-Cano V., Zapata-Cardona M.I., Quintero-Gil D.C., Hernández-Mira E., Pájaro-González Y., Oliveros-Díaz A.F., Diaz-Castillo F., Quiñones W., Robledo S, & Martinez-Gutierrez M. (2021). The Antiviral and Virucidal Activities of Voacangine and Structural Analogs Extracted from Tabernaemontana cymosa Depend on the Dengue Virus Strain. Plants, 10(7), 1280.

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Bacterial and Archaeal 16S rRNA Gene Amplification

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Pure metagenoms were PCR amplified for bacterial and archaeal 16S rRNA genes using the universal primers (Table 1, Supplementary data). One hundred Nano gram of DNA was used in a PCR reaction mixture (final volume of 50 µL)
containing 1.5 mM MgCl₂, 1X Reaction buffer, 0.2 mM dNTP, 5 pmoL of each primer and 2.5 U Taq DNA polymerase. PCR was performed with an initial denaturation at 95 °C for 5 min, followed by 30 cycles of 95 °C for 60 s (denaturation), 50-54 °C for 60 s (annealing), 72 °C for 1.5 min (extension) and 72 °C for 10 min (final extension). The PCR products were visualized on 1% agarose gel in TAE buffer and then purified using the Roche High pure PCR purification kit.
The amplicons were ligated into pTz57 R/T vector, according to the Fermentas’s protocol. Ligation products were transformed into E. coli DH5α cells by heat shock transformation method ( 16
) and screened on LB/Ampicillin/IPTG/X-Gal plates in 37 °C for 16 h. The positive clones were selected based on the blue-white screening method; accordingly, white colonies were considered as recombinant clones and confirmed by PCR using vector specific primers M13F (5ˊ-GTAAAACGACGGCCAG-3ˊ) and M13R (5ˊ- CAGGAAACAGCTATGAC-3ˊ) ( 17 (link)
). Then, plasmids were extracted from positive clones by plasmid extraction Kit (Roche, Germany) for sequencing (Sanger method, Macrogen, South Korea).

Mousavi Maleki M.S., Yakhchali B., Karkhaneh A.A., Rezvani M, & Ahmadpour F. (2022). A Cross-section Metagenomics and 16S Ribosomal DNA Based Evaluation of the Bacterial and Archaeal Communities Resident in the Forumad Chromite Mine, Northeastern of Iran. Iranian Journal of Biotechnology, 20(4), e2818.

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Molecular Characterization of Key Genes

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A set of four genes were selected, including succinyl CoA ligase (SCL), pyruvate decarboxylase-1 (PDC-1), acetyl-CoA synthetase (ACS), and histone deacetylase-6 (HDA-6).
The mRNA sequence of SCL, ACS, PCD-1, and HDA-6 genes was not available for these two species; therefore, the conserved domain of known sequences of the Lamiids clade was used for designing of degenerate primers (Table 1).
PCR was used for amplification of the selected segment of the mentioned genes. PCR amplification reactions (25 μL) contained 2.5 μL of 10x enzyme buffer, 1 mM MgCl₂, 200 μM each of dATP, dCTP, dGTP, and dTTP (Sinaclon, Tehran, Iran), 0.4 μM each primer (Macrogen, Seoul, South Korea) (Table 1), 1 unit of Taq polymerase (Sinaclon, Tehran, Iran), and 100 ng of cDNA. The PCR cycles consisted of initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 46–56 °C for 45 s (Table 1), and extension at 72 °C for 1 min with a final extension at 72 °C for 5 min. The amplicons were later resolved on 1.2% agarose gel and then reverse primers were used for direct sequencing using the Sanger method (Macrogen, Seoul, South Korea). Next, these sequences were deposited on the NCBI database (SCL: MH444601, MH444602; ACS: MH444599, MH444600; PDC-1: MH602964, MH602965; and HDA-6: MH444603, MH570448).

Ashrafi M., Azimi-Moqadam M.R., MohseniFard E., Shekari F., Jafary H., Moradi P., Pucci M., Abate G, & Mastinu A. (2022). Physiological and Molecular Aspects of Two Thymus Species Differently Sensitive to Drought Stress. BioTech, 11(2), 8.

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Genomic DNA Extraction and Sequencing

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The genomic DNA was extracted from the cells harvested after transformation, or the individual colonized mutants selected from TAP agar plate, for targeted deep sequencing and Sanger sequencing, respectively. To isolate genomic DNA, cells were harvested, resuspended in a microprep buffer containing 2.5× extract buffer (0.35 M Sorbitol, 0.1 M Tris/HCl pH 7.5, and 5 mM EDTA), 2.5× nuclei lysis buffer (0.2 M Tris/HCl pH 7.5, 0.05 M EDTA, 2 M NaCl, and 2% (w/v) CTAB), and 1 × 5% N-Lauroylsarcosine, and incubated for 2 hr at 65 °C. The genomic DNA was extracted with Chloroform:Isoamylalcohol 24:1 and precipitated with Isopropanol. For Sanger sequencing, the target regions were PCR amplified with specific primers (Supplementary Table 2). The PCR products were verified by agarose gel electrophoresis, eluted from the gel and sequenced using the Sanger method (Macrogen, South Korea).

Baek K., Kim D.H., Jeong J., Sim S.J., Melis A., Kim J.S., Jin E, & Bae S. (2016). DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Scientific Reports, 6, 30620.

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Phylogenetic Analysis of Microbial Isolates

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PCR products of the five isolates were purified using the DNA Clean & Concentrator Kit (ZymoResearch, US), and the clean products were sequenced using the Sanger method (Macrogen, Korea). Chromatogram files of the three successfully sequenced isolates were imported into Chromas v2.6, and low-quality regions for each of the forward and reverse reads were trimmed independently and a consensus sequence generated based on the forward and reverse sequences for each of the samples. Sequences generated were compared to those available in Genbank using Blast and phylogenetic analysis was performed using a set of reference sequences obtained from Genbank. Sequences generated were submitted to Genbank under the following ccession numbers: MT992066, MT992067 and MT992068.

Lutomiah J., Mulwa F., Mutisya J., Koskei E., Langat S., Nyunja A., Koka H., Konongoi S., Chepkorir E., Ofula V., Owaka S., Eyase F, & Sang R. (2021). Probable contribution of Culex quinquefasciatus mosquitoes to the circulation of chikungunya virus during an outbreak in Mombasa County, Kenya, 2017–2018. Parasites & Vectors, 14, 138.

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Screening for LCYE Knockout Mutants

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Colonies from TAP agar plates were transferred to 96-well plates, and the individual cells were incubated in TAP media with hygromycin-B for 3 days. Transformed cells were subjected to colony PCR with specific primers adjacent to sgRNA target sites in LCYE (F: 5′-TGG TGA AAT CTA GCG TCG GCT-3′, R: 5′-GAC GCA ATT GCC GCT TGA GA-3′) for mutant screening. Knockout mutants with the aph7 DNA inserted were selected, their genomic DNA was isolated, and the target region was PCR-amplified for sequence confirmation. The PCR products were separated on an agarose gel and sequenced using the Sanger method (Macrogen, Seoul, South Korea).

Song I., Kim J., Baek K., Choi Y., Shin B, & Jin E. (2020). The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii. Microbial Cell Factories, 19, 220.

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