Journal: Blood Cancer Journal

Article Title: Identification of cell-type-specific mutations in nodal T-cell lymphomas

doi: 10.1038/bcj.2016.122

Figure Lengend Snippet: Distribution of newly identified gene mutations in nodal T-cell lymphomas. The results of Sanger sequencing and/or amplicon-based deep sequencing for some newly identified gene mutations in whole tumor, PD1+ cells and CD20+ cells are shown. The numeric values indicate allele frequencies of mutations defined by deep sequencing. The AITL samples are indicated in black letters. The PTCL-NOS/nodal PTCL with TFH phenotype sample is indicated in blue letters. NA, not analyzed by deep sequencing. The filled and dashed red arrows indicate mutations and no mutations, respectively.

Article Snippet: Amplicon-based sequencing The libraries were prepared using the Ion Plus Fragment Library Kit according to the protocol for preparing short amplicon libraries (Life Technologies).

Techniques: Sequencing, Amplification

Journal: Blood Cancer Journal

Article Title: Identification of cell-type-specific mutations in nodal T-cell lymphomas

doi: 10.1038/bcj.2016.122

Figure Lengend Snippet: B-cell-specific mutations in nodal T-cell lymphomas. The results of Sanger sequencing and/or amplicon-based deep sequencing for some newly identified gene mutations in whole tumor, PD1+ cells and CD20+ cells are shown. The numeric values indicate allele frequencies of mutations defined by deep sequencing. The AITL samples are indicated in black letters. The PTCL-NOS/nodal PTCL with TFH phenotype sample is indicated in blue letters. NA, not analyzed by deep sequencing. The filled and dashed red arrows indicate mutations and no mutations, respectively. NOTCH1 is marked by red letters because this is repetitive.

Article Snippet: Amplicon-based sequencing The libraries were prepared using the Ion Plus Fragment Library Kit according to the protocol for preparing short amplicon libraries (Life Technologies).

Techniques: Sequencing, Amplification

Journal: Blood Cancer Journal

Article Title: Identification of cell-type-specific mutations in nodal T-cell lymphomas

doi: 10.1038/bcj.2016.122

Figure Lengend Snippet: RHOA mutations are specific to PD1+ cells. ( a ) An example of the immunostaining pattern for PD1 and CD20 in AITL. Left, PD1+ cells; right, CD20+ cells. ( b ) Sequences of G17V RHOA mutations in whole tumor, PD1+ cells and CD20+ cells. The numeric values indicate allele frequencies of mutations defined by amplicon-based deep sequencing. The AITL samples are indicated in black letters. The nodal PTCL with TFH phenotype sample is indicated in red letters *: RHOA c.A51T:p.G17V, silent mutation. The filled and dashed red arrows indicate mutations and no mutations, respectively.

Article Snippet: Amplicon-based sequencing The libraries were prepared using the Ion Plus Fragment Library Kit according to the protocol for preparing short amplicon libraries (Life Technologies).

Techniques: Immunostaining, Amplification, Sequencing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

doi: 10.1093/nar/gkw382

Figure Lengend Snippet: Long range PCR analysis of mtDNA from individual myofibres from a sIBM patient. A representative agarose gel image of 10-kb long range PCR analysis of DNA extracted from four individual COX-normal (‘cell 1–4’) and COX-deficient myofibres (‘cell 5–8’) from a sIBM patient (‘P8’) is presented. A positive control of DNA extracted from whole-blood of a healthy individual (‘c’) was used to ensure detection of full-length amplicons whereas a no-template (‘nt’) control sample served as a control for contamination. A 10-kb product was amplified with wild-type mtDNA (‘c’) whereas shorter products were formed with mtDNA molecules harbouring deletions (‘cell1-4’). A single deletion species was found in ‘cell 1’ and ‘cell 2’ (blue arrows) whilst ‘cell 3’ and ‘cell 4’ contained two or three deletions of different sizes respectively (red arrows). The location of the forward (‘F’) and reverse (‘R’) primers used in the assay (F6358 and R001) is shown in a schematic representation of an mtDNA molecule on the right.

Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Amplification

Journal: Nucleic Acids Research

Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

doi: 10.1093/nar/gkw382

Figure Lengend Snippet: Map of mtDNA deletions. Size and position within the mitochondrial genome of 20 types of mtDNA deletions identified by 16-kb long range PCR are presented as coloured curved lines. Line endings on the left and right mark the 5′ and 3′ breakpoints, respectively. Identifiers ‘A-P3’ correspond to the respective PCR amplicons in Figure 6 .

Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

Techniques: Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

doi: 10.1093/nar/gkw382

Figure Lengend Snippet: Map of mtDNA deletions. Size and position within the mitochondrial genome of eight types of mtDNA deletions identified by smPCR are presented as coloured curved lines. Line endings on the left and right mark the 5′ and 3′ breakpoints, respectively. Numbers ‘1–8’ correspond to the respective PCR amplicons in Figure 2 .

Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

Techniques: Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

doi: 10.1093/nar/gkw382

Figure Lengend Snippet: Analysis of individual molecules of mitochondrial genome using single molecule PCR amplification. Representative smPCR gel images from single cell lysates obtained from three sIBM patients (‘P13’, ‘P9’ and ‘P7’), where each amplified product came from a single molecule of mtDNA. In order to ensure that each amplicon was indeed created from one template, the fraction of positives had to be equal or lower than 0.25 (no more than 1 out of 4 PCR reactions contained a product). Primers hybridizing with the D-Loop region were designed to amplify ∼16 kb of mtDNA and are marked as forward (‘F’) and reverse (‘R’) in a schematic of mtDNA molecule on right. One fragment was generated from samples: ‘P13/6’, ‘P13/7’, ‘P9/9’ and ‘P7/1’ signifying one type of mtDNA deletion (additional smaller amplicons, equidistant from the main amplicon in each lane, are a consequence of mispriming). Samples ‘P9/10’ and ‘P9/11’ both gave rise to three amplicons each indicating three different deletion species per cell (blue horizontal lines indicate different sizes of individual fragments). All 20 amplicons marked with numbers (‘1–8’) were sequenced using Sanger sequencing, whereas two products ‘7’ and two products ‘8’ were also verified using next generation deep sequencing. Amplicons ‘*’ and ‘#’ were detected by agarose gel electrophoresis but not sequenced.

Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

Techniques: Polymerase Chain Reaction, Amplification, Generated, Sequencing, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

doi: 10.1093/nar/gkw382

Figure Lengend Snippet: Validation of P8/1 single myofibre long-range PCR result. A total of 16-kb long range PCR produced two products from ‘P8/1’ DNA extract indicating two types of deletion. This was shown by sequencing of the PCR amplicon. In order to validate it further stepping-in PCR method was applied. ( A ) ‘PCR 3’ used primers flanking the shorter deletion (primers indicated by red arrows). Three outcomes were considered: (i) product of 651 bp as a consequence of amplification of molecule ‘M1’; (ii) absence of product should molecule ‘M2’ harbour the only genuine deletion in the sample; (iii) a wild-type product of 7512 bp. ‘PCR 4’ contained primers flanking the larger deletion (green arrows). The possible outcomes were: (i) a single product of 1645 bp if the only deletion molecule present in the cell was ‘M1’; (ii) two products measuring 1645 and 736 bp if both ‘M1’ and ‘M2’ were present; (iii) wild-type band of 8506 bp. ( B ) Agarose gel showing PCR products from ‘reaction 3’ and ‘reaction 4’ carried out using whole-cell lysates from ‘P8/1’ and healthy control homogenate DNA ‘c5’. The amplicons are labelled with appropriate molecular sizes.

Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

Techniques: Polymerase Chain Reaction, Produced, Sequencing, Amplification, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

doi: 10.1093/nar/gkw382

Figure Lengend Snippet: Detection of mtDNA deletions in homogenate DNA samples by next generation sequencing. ( A ) DNA extracted from ten 20-μm muscle cryosections from two sIBM patients (P1 and P5) and two controls (c6 and c8) was subjected to long range PCR. For the patients, four serial dilutions were used in four reactions to find the concentration that would result in the highest number of amplicons. Selected samples are depicted by red arrows. The schematic on the right shows position of PCR primers used (261F and 16291R). ( B ) Graphical representation of read depth for all sequenced samples. Both controls show almost identical peaks and troughs, whereas the patients differ from controls and one another. Low read depth, clearly visible in certain areas of the mitochondrial genome, indicates mtDNA deletions (depicted by red arrows). ( C ) Graphical representation of all deletions detected in the patients’ mtDNA by analysis of chimeric sequencing fragments. Each horizontal bar shows the deleted portion of the mtDNA genome as represented by the chimeric reads that align to two distinct parts of the mtDNA reference sequence. The x axis shows the nucleotide position on the mitochondrial genome (0–16 569 bp). The y axis depicts cumulative read count, with individual reads ordered from top to bottom by 5' and then 3' breakpoints. All chimeric reads are depicted; those in grey have read counts below 5 and are unlikely to represent deletion species. All other multiple read deletions are colour coded for clarity, with breakpoints annotated on both the main figure and in the legend and the height of each bar representing the read count.

Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

Techniques: Next-Generation Sequencing, Polymerase Chain Reaction, Concentration Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

doi: 10.1093/nar/gkw382

Figure Lengend Snippet: PCR-based validation of deletion breakpoints. Sequencing of 16kb PCR products ‘E’ and ‘F’ derived from two separate cells from the same patient (‘P13/1’ and ‘P13/2’) revealed an identical double-deletion in their mitochondrial genome. In order to confirm this phenomenon, stepping-in PCR was designed. ( A ) ‘PCR 1’ used primers flanking the outer breakpoints of the double-deletion, as indicated by red arrows. Three possible scenarios were considered: (i) the double-deletion was real, in which case the expected product would measure 1246 bp; (ii) there was only a single deletion contained between the outer breakpoints, in which case the product would be 617-bp long; (iii) the PCR would only generate a wild-type amplicon of 10 449 bp. ‘PCR 2’ used a forward primer binding to the short fragment of DNA in between the inner breakpoints and the same reverse primer as used in ‘PCR 1’ (primers indicated by green arrows). Again, three scenarios were investigated: (i) the double-deletion was genuine, in which case the product would measure 850 bp; (ii) a single deletion was real, in which case there would be no amplicon as the binding site for the forward primer would have been removed; (iii) only wild-type would amplify, producing a fragment of 6239 bp. ( B ) Agarose gel showing amplified products from ‘PCR 1’ and ‘PCR 2’ performed on whole-cell lysate from ‘P13/1’ and ‘P13/2’. The amplicons are labelled with the molecular sizes expected given identical double-deletion in both molecules. ( C ) Cells ‘P13/1’ and ‘P13/2’ visualized using COX/SDH histochemistry prior to laser-microdissection for downstream mtDNA analyses. ( D ) Five healthy control muscle homogenate DNA samples (‘c3-7’) and homogenate DNA from the same sIBM patient (‘P13’) were subjected to ‘PCRs 1 and 2’. Control samples ‘c3-6’ produced only wild-type amplicons, whereas control ‘c7’ and ‘P13’ produced additional smaller products indicating presence of deleted species of mtDNA.

Article Snippet: Briefly, selected amplicons, measuring not more than 9 kb, were purified using Agencourt AMPure XP reagent and assessed for concentration and quality using Agilent 12 000 DNA kit on a Bioanalyzer (Agilent 2100, Agilent Technologies).

Techniques: Polymerase Chain Reaction, Sequencing, Derivative Assay, Amplification, Binding Assay, Agarose Gel Electrophoresis, Laser Capture Microdissection, Produced

Journal: PLoS ONE

Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

doi: 10.1371/journal.pone.0040438

Figure Lengend Snippet: DPMT validation for MOB P3 and MOB P4 relaxases. A) Phylogenetic tree of MOB P3 and MOB P4 relaxase families. B) Alignment of the relaxase motifs used to design the MOB P3 and MOB P4 degenerate primers (P3-f+P3-r, continuous black; and P4-f+P4-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB P31 (P3-f and P3-r). D) Amplicons obtained with primers for subfamily MOB P42 (P4-f and P4-r). Symbols, colour codes and lanes as in Figure 1 .

Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

Techniques:

Journal: PLoS ONE

Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

doi: 10.1371/journal.pone.0040438

Figure Lengend Snippet: DPMT validation for MOB P1 relaxases. A) Phylogenetic tree of MOB P1 relaxases. B) Alignment of the relaxase motifs used to design the MOB P1 degenerate primers (P11-f, continuous black; P12-f, continuous dark grey; P131-f, continuous grey; P14-f, continuous light grey; and P1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB P11 (P11-f and P1-r). D) Amplicons obtained with primers for subfamily MOB P12 (P12-f and P1-r). E) Amplicons obtained with primers for subfamily MOB P13 (P131-f and P1-r). F) Amplicons obtained with primers for subfamily MOB P14 (P14-f and P1-r). Symbols, colour codes and lanes as in Figure 1 .

Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

Techniques:

Journal: PLoS ONE

Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

doi: 10.1371/journal.pone.0040438

Figure Lengend Snippet: DPMT validation for MOB C relaxases. A) Phylogenetic tree of MOB C relaxase family. B) Alignment of the relaxase motifs used to design the MOB C degenerate primers (C11-f+C11-r, continuous black; C12-f+C12-r, continuous dark grey). C) Amplicons obtained with primers for subfamily MOB C11 (C11-f and C11-r). D) Amplicons obtained with primers for subfamily MOB C12 (C12-f and C12-r). Symbols, colour codes and lanes as in Figure 1 .

Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

Techniques:

Journal: PLoS ONE

Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

doi: 10.1371/journal.pone.0040438

Figure Lengend Snippet: DPMT validation for MOB P5 relaxases. A) Phylogenetic tree of MOB P5 relaxase family. B) Alignment of the relaxase motifs used to design the MOB P5 degenerate primers (P51-f, continuous black; P52-f, continuous dark grey; P5-r, dashed black; and P53-f+P53-r, continuous grey) C) Amplicons obtained with primers for subfamily MOB P51 (P51-f and P5-r). D) Amplicons obtained with primers for subfamily MOB P52 (P52-f and P5-r). E) Amplicons obtained with primers for subfamily MOB P53 (P53-f and P53-r). Symbols, colour codes and lanes as in Figure 1 .

Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

Techniques:

Journal: PLoS ONE

Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

doi: 10.1371/journal.pone.0040438

Figure Lengend Snippet: DPMT validation for MOB F relaxases. A) Phylogenetic tree of MOB F relaxases. Triangles at the end of the branches represent a compressed group of very similar relaxases ( > 95%). A solid black arrow points to the prototype plasmid for each subfamily. Arrows point to plasmids that experimentally amplified, in spite of containing at least one mismatch in the 12 nucleotides of the CORE sequence. Relaxases contained in our reference collection ( Table 1 ) are denoted by an asterisk. Plasmids detectable by PBRT amplification [19] , [20] , [21] , [22] , [24] , [25] , [27] , [28] , [29] are underlined. New relaxase sequences uncovered by DPMT are shown in red. B) Alignment of the relaxase motifs used to design the MOB F degenerate primers. Colour code: red on yellow = invariant amino acids; blue on blue = strongly conserved; black on green = similar; green on white = weakly similar; black on white = not conserved. Black arrowheads point to the key residues that define the relaxase motifs. Different rectangles embrace the conserved amino acids used to infer the 3′ degenerate core of each oligonucleotide (F11-f, continuous black; F12-f, continuous dark grey; and F1-r, dashed black). C) Amplicons obtained with primers for subfamily MOB F11 (F11-f and F1-r). Lane 1, pSU1588; 2, pSU4280; 3, pSU10013; 4, pSU10014; 5, pSU10017; 6, pSU10018; 7, pSU10021; 8, pSU316; 9, pSU10022; 10, pSU10010; 11, R751; 12, pSU10028; 13, pSU10029; 14, pSU10056; 15, pSU10055; 16, pSU10001; 17, pSU10012; 18, pSU10011; 19, pSU10009; 20, pSU4601; 21, pSU10006; 22, pSU10007; 23, pSU10064; 24, pSU10059; 25, pSU10008; 26, pSU10039; 27, pSU10040; 28, pSU10041; 29, pSU10004; 30, pSU10003; 31, pSU10043; 32, pSU4830; 33, pSU10002; 34, negative control. Lane M, molecular mass marker, HyperLadder IV (Bioline). D) Amplicons obtained with primers for subfamily MOB F12 (F12-f and F1-r). Lanes as in (C).

Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

Techniques: Plasmid Preparation, Amplification, Sequencing, Negative Control, Marker

Journal: PLoS ONE

Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

doi: 10.1371/journal.pone.0040438

Figure Lengend Snippet: DPMT validation for MOB Q relaxases. A) Phylogenetic tree of MOB Q relaxase family. B) Alignment of the relaxase motifs used to design the MOB Q degenerate primers (Q11-f+Q11-r, continuous black; Q12-f+Q12-r, continuous dark grey; and Qu-f+Qu-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB Q11 (Q11-f and Q11-r). D) Amplicons obtained with primers for subfamily MOB Q12 (Q12-f and Q12-r). E) Amplicons obtained with primers for subfamily MOB Qu (Qu-f and Qu-r). Symbols, colour codes and lanes as in Figure 1 .

Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

Techniques:

Journal: PLoS ONE

Article Title: A Degenerate Primer MOB Typing (DPMT) Method to Classify Gamma-Proteobacterial Plasmids in Clinical and Environmental Settings

doi: 10.1371/journal.pone.0040438

Figure Lengend Snippet: DPMT validation for MOB H relaxases. A) Phylogenetic tree of MOB H relaxase family. B) Alignment of the relaxase motifs used to design the MOB H degenerate primers (H11-f+H11-r, continuous black; H121-f+H121-r, continuous dark grey; and H2-f+H2-r, continuous grey). C) Amplicons obtained with primers for subfamily MOB H11 (H11-f and H11-r). D) Amplicons obtained with primers for subfamily MOB H121 (H121-f and H121-r). E) Amplicons obtained with primers for subfamily MOB H2 (H2-f and H2-r). Symbols, colour codes and lanes as in Figure 1 .

Article Snippet: Amplicons were visualized after 2% agarose gel electrophoresis, using a GelDoc (BioRad Laboratories) and, when appropriate, sequenced by Macrogen Laboratories (Seoul, South Korea).

Techniques:

Journal: PLoS ONE

Article Title: An efficient and reliable DNA-based sex identification method for archaeological Pacific salmonid (Oncorhynchus spp.) remains

doi: 10.1371/journal.pone.0193212

Figure Lengend Snippet: Negative images of electrophoresis gels showing the (A) Clock1a / sdY and (B) D-loop/ sdY PCR assay results for modern male and female samples from five Pacific salmonid species. The approximate location of the IPC and sdY amplicons are indicated by the labelled arrows. The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA).

Article Snippet: The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA). (PDF) Click here for additional data file.

Techniques: Electrophoresis, Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: An efficient and reliable DNA-based sex identification method for archaeological Pacific salmonid (Oncorhynchus spp.) remains

doi: 10.1371/journal.pone.0193212

Figure Lengend Snippet: Negative images of electrophoresis gels showing the (A) Clock1a / sdY (B) D-loop/ sdY assay results for nine of the analyzed archaeological salmonid samples. The approximate location of the IPC and sdY amplicons are indicated by the labelled arrows. The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA). Note: For SB11, the D-loop/ sdY assay (B) produced two weak nonspecific bands only slightly smaller than the predicted size of the sdY amplicon, suggesting they might represent sdY . However, the Clock1a / sdY assay (A) only yielded a fragment of Clock1a , confirming the nonspecific bands likely do not represent sdY , verifying SB11’s female identity.

Article Snippet: The 100 bp ladder used to estimate the size of the amplicons is from Invitrogen (Waltham, MA, USA). (PDF) Click here for additional data file.

Techniques: Electrophoresis, Produced, Amplification

Journal: BMC Genomics

Article Title: Deep sequencing of HPV E6/E7 genes reveals loss of genotypic diversity and gain of clonal dominance in high-grade intraepithelial lesions of the cervix

doi: 10.1186/s12864-017-3612-y

Figure Lengend Snippet: HPV DNA detection by PCR amplification, capillary electrophoresis and dideoxy (Sanger) sequencing. a Gel image and electropherogram of amplicon detection by high-resolution capillary electrophoresis. Representative samples #311, 312, 319, and 330 (HSIL) reveal 1 or 2 amplicons after using consensus primers (GP-E6/E7 F/B) to amplify an E6/E7 segment with an expected fragment size of ~660 bp (range, 619-819 bp). Amplicon size variability reflects sequence differences between HPV genotypes. In general, deep sequencing resolved a greater number of HPV genotypes than capillary electrophoresis per sample. Sample #330 illustrates this with detection of 2 amplicons on electrophoresis, but 8 genotypes by deep sequencing. b Representative sample (#311) with a single HPV infection revealing 1 amplicon (699 bp peak on electropherogram) and clean sequencing chromatogram. Representative sample (#319) with multiple HPV infections revealing 2 amplicons (619 and 656 bp peaks on electropherogram) and “noisy” overlapping peaks on the chromatogram. AM, alignment marker; B, buffer; bp, base pair; M, molecular-weight marker

Article Snippet: Dideoxy sequencing of the amplicons (~200 ng DNA/sample) was performed using primer GP-E6-3 F at Eurofins Operon (USA).

Techniques: Polymerase Chain Reaction, Amplification, Electrophoresis, Sequencing, Infection, Marker, Molecular Weight

Journal: BMC Genomics

Article Title: Deep sequencing of HPV E6/E7 genes reveals loss of genotypic diversity and gain of clonal dominance in high-grade intraepithelial lesions of the cervix

doi: 10.1186/s12864-017-3612-y

Figure Lengend Snippet: HPV genotype composition found in LSIL and HSIL samples. Deep sequencing of HPV E6/E7 amplicons derived from each LSIL or HSIL sample identified 1 to 8 HPV genotypes and quantitated their composition (%) based on number of mapped reads to total mapped reads. The top three dominant (highest proportion) genotypes found in LSIL were HPV-39, -16, and -35 [ red , solid/hashed]. The carcinogenicity of LSIL dominant genotypes were: carcinogenic 29/43 (67%, red ); possibly carcinogenic 6/43 (14%, blue ); and not classifiable/probably not carcinogenic 8/43 (19%, green ). For HSIL, the dominant genotype was primarily HPV-16 (21/29, 72%); and the dominant genotypes were all carcinogenic 29/29 (100%, red ). The HPV carcinogenicity is based on IARC’s classification of human carcinogens [ 8 ]. HSIL, high-grade squamous intraepithelial lesion; IARC, International Agency for Research on Cancer; LSIL, low-grade squamous intraepithelial lesion; ID, identification

Article Snippet: Dideoxy sequencing of the amplicons (~200 ng DNA/sample) was performed using primer GP-E6-3 F at Eurofins Operon (USA).

Techniques: Sequencing, Derivative Assay

Journal: Nature biotechnology

Article Title: Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

doi: 10.1038/nbt.3290

Figure Lengend Snippet: Chemically modified sgRNAs facilitate high frequencies of gene disruption in stimulated primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). ( a ) 1 million primary human T cells were nucleofected with 10 μg of the indicated synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μg Cas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies for three different donors + s.e.m., n = 6, as measured by TIDE analysis of PCR amplicons spanning the sgRNA target site, and using a mock-treated sample as control reference. ( b ) Stimulated T cells were nucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencies were measured by TIDE analysis as above. Bars represent average indel frequencies for three different donors + s.e.m., n = 6. ( c ) 500,000 mobilized human peripheral blood CD34 + HSPCs were nucleofected with 10 μg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1 μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9 protein was included for comparison. Bars represent average indel frequencies + s.e.m., n = 3, as measured by T7 endonuclease cleavage assay. ( d ) 1 million stimulated T cells or mobilized human peripheral blood CD34 + HSPCs were nucleofected with 15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 ). Bars represent average indel frequencies + s.e.m., n = 3.

Article Snippet: PCR amplicons spanning the sgRNA genomic target sites were generated using the iProof High-Fidelity Master Mix (Bio-Rad, Hercules, CA, USA) with the following primer pairs: IL2RG _fw: 5′-TCACACAGCACATATTTGCCACACCCTCTG-3′; IL2RG _RV: 5′-TGCCCACATGATTGTAATGGCCAGTGG-3′; HBB _fw: 5′-CCAACTCCT AAGCCAGTGCCAGAAGAG-3′; HBB _rv: 5′-AGTCAGTGCCTATCAGAAAC CCAAGAG-3′; CCR5 _fw: 5′-GCACAGGGTGGAACAAGATGG-3′; CCR5_rv: 5′-CACCACCCCAAAGGTGACCGT-3′.

Techniques: Modification, Plasmid Preparation, Polymerase Chain Reaction, Cleavage Assay

Journal: Nature biotechnology

Article Title: Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

doi: 10.1038/nbt.3290

Figure Lengend Snippet: Synthesized and chemically modified sgRNAs facilitate high frequencies of indels and HR in the human cell line K562. ( a ) Sequence and schematic secondary structure of the IL2RG sgRNA loaded into Cas9 and bound to its genomic target site. Nucleotides with chemical modifications are marked with red flags. ( b ). ( c , d amplicons ( c ) or gene addition by HR at the three loci IL2RG , HBB and CCR5 with synthetic sgRNAs ( d ). The synthetic sgRNAs were delivered at 1 μg (light shade) or 20 μg (dark shade) per 1 million cells. Cas9 was expressed from a plasmid (2 μg) and for HR experiments 5 μg of GFP-encoding donor plasmid was included. As a positive control, 2 μg of sgRNA plasmid encoding both the sgRNA and the Cas9 protein was used (gray bars). Bars represent average values + s.e.m., n = 3. ( e ) Specificity of targeted cleavage mediated by synthetic sgRNAs as performed in c for 20 μg of sgRNA. Indel frequencies were measured by deep sequencing of PCR amplicons of the targeted genomic loci and three bioinformatically predicted off-target loci for each gene. Bars represent average values + s.e.m., n = 3. ( f ) Staggered delivery of 15 μg Cas9 mRNA and 10 μg IL2RG synthetic sgRNAs into 1 million K562 cells using electroporation. Bars represent average indel frequencies + s.e.m., n = 3, as measured by tracking of indels by decomposition (TIDE) analysis of PCR amplicons spanning the sgRNA target sites, using a mock-treated sample as reference control. ( g ) Cas9 protein was complexed with a 2.5 molar excess of the indicated synthetic IL2RG sgRNAs and nucleofected into 1 million K562 cells at the indicated amounts. Indel frequencies were measured by TIDE analysis as above and bars represent average indel frequencies + s.e.m., n = 3.

Article Snippet: PCR amplicons spanning the sgRNA genomic target sites were generated using the iProof High-Fidelity Master Mix (Bio-Rad, Hercules, CA, USA) with the following primer pairs: IL2RG _fw: 5′-TCACACAGCACATATTTGCCACACCCTCTG-3′; IL2RG _RV: 5′-TGCCCACATGATTGTAATGGCCAGTGG-3′; HBB _fw: 5′-CCAACTCCT AAGCCAGTGCCAGAAGAG-3′; HBB _rv: 5′-AGTCAGTGCCTATCAGAAAC CCAAGAG-3′; CCR5 _fw: 5′-GCACAGGGTGGAACAAGATGG-3′; CCR5_rv: 5′-CACCACCCCAAAGGTGACCGT-3′.

Techniques: Synthesized, Modification, Sequencing, Plasmid Preparation, Positive Control, Polymerase Chain Reaction, Electroporation

Journal: JHEP Reports

Article Title: Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

doi: 10.1016/j.jhepr.2019.100065

Figure Lengend Snippet: Efficient correction of OTC-deficient patient-derived primary human hepatocytes in vivo . (A) Experimental overview. Cells were engrafted into FRG mice and repopulation established by cycling on and off the drug 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione. 20 Eleven weeks later, when the mice were on day 11 of a 21-day water cycle, animals received 5×10 10 vg/mouse SaCas9-sgRNA4 and 2×10 11 vg/mouse donor vectors, 2.5×10 10 vg/mouse SaCas9-sgRNA4 and 1×10 11 vg/mouse donor vectors or 1.25×10 10 vg/mouse SaCas9-sgRNA4 and 5×10 10 vg/mouse donor vectors packaged in the NP59 capsid via intraperitoneal delivery (n = 3 per treatment group for controls and highest vector dose, and n = 4 for 2 lower vector doses). Livers were analyzed 5 weeks following vector delivery. Next-generation Illumina® sequencing was performed across the OTC locus from (B, C) genomic DNA and (D, E) cDNA isolated from FRG mice transplanted with patient-derived human hepatocytes to quantitate the HDR rates and characterise the unintended modifications found at the SaCas9 cleavage site. Data are plotted as mean ± SEM. Control samples represent PCR amplicons from engrafted FRG mice that did not receive AAV vector treatment. AAV, adeno-associated virus; FRG, Fah -/- Rag2 -/- Il2rg -/ ; HDR, homology-directed repair; InDels, insertions and deletions; ITR, inverted terminal repeat; SaCas9, Staphylococcus aureus Cas9 nuclease; sgRNA, single guide RNA; rAAV, recombinant AAV.

Article Snippet: Pooled amplicons were sent to Genewiz for library preparation and MiSeq paired-read sequencing (Illumina).

Techniques: Derivative Assay, In Vivo, Mouse Assay, Plasmid Preparation, Sequencing, Isolation, Polymerase Chain Reaction, Recombinant

Journal: JHEP Reports

Article Title: Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

doi: 10.1016/j.jhepr.2019.100065

Figure Lengend Snippet: Homology-directed repair of the transposed human OTC locus in Spf ash mice . (A) Experimental overview and timing of intraperitoneal rAAV delivery for CRISPR-SaCas9-mediated HDR in Spf ash mice. Newborn animals received 5×10 10 vg transposase and 1×10 11 vg mutant minigene vectors (packaged in rAAV2/8 capsid) and 3 weeks later, 2×10 11 SaCas9/sgRNA and/or 5×10 11 donor vectors (packaged in rAAV2/rh10 capsid). Mutant minigenes were delivered (intraperitoneal) to newborn spf ash mice using the hybrid AAV/ piggyBac transposase vector system and 3 weeks later animals received genome editing vectors; n = 5 per treatment group. HDR was confirmed 2 weeks later by (B) the presence of functional OTC activity in liver sections (scale bar = 50 μm) and (C) by Sanger sequencing analysis of cloned OTC amplicons amplified using primers with binding sites outside the region of homology with the donor vector. Next-generation Illumina® sequencing was performed across the transposed OTC human minigene locus to more accurately (D) quantitate the HDR rates and (E) characterise the unintended modifications found at the SaCas9 cleavage site. Data are plotted as mean ± SEM. and significance evaluated using the Mann-Whitney non-parametric test (** p

Article Snippet: Pooled amplicons were sent to Genewiz for library preparation and MiSeq paired-read sequencing (Illumina).

Techniques: Mouse Assay, CRISPR, Mutagenesis, Plasmid Preparation, Functional Assay, Activity Assay, Sequencing, Clone Assay, Amplification, Binding Assay, MANN-WHITNEY

Journal: BMC Bioinformatics

Article Title: Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries

doi: 10.1186/s12859-018-2293-0

Figure Lengend Snippet: Sequence accuracy as a function of subread depth. a Accuracy of consensus and b assembly sequences. Data from all the amplicons were pooled together to evaluate the consensus calling accuracy as a function of depth of coverage of SMRT raw reads. The vertical line shows the minimum read depth of the consensus sequences used for assemblies

Article Snippet: Since the amplicons were barcoded using PacBio’s standard barcodes, the default pre-set in SMRT Portal pointing to PacBio barcodes with padding in the reference directory was used.

Techniques: Sequencing

Journal: BMC Bioinformatics

Article Title: Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries

doi: 10.1186/s12859-018-2293-0

Figure Lengend Snippet: Total number of accurate bootstrap assemblies per CCS sample size. At each level of the CCS read depth sample (1-40), the figure shows the total number of bootstrapped assemblies that were 100% identical to the reference sequence. This was determined for the four target regions (25 bootstrap assemblies at each of 4 loci, giving rise to a maximum of 100 on the x-axis) formed from the consensus sequences among the eight overlapping amplicons

Article Snippet: Since the amplicons were barcoded using PacBio’s standard barcodes, the default pre-set in SMRT Portal pointing to PacBio barcodes with padding in the reference directory was used.

Techniques: Sequencing

Journal: BMC Bioinformatics

Article Title: Clustering of circular consensus sequences: accurate error correction and assembly of single molecule real-time reads from multiplexed amplicon libraries

doi: 10.1186/s12859-018-2293-0

Figure Lengend Snippet: Graphical representation of the C3S-LAA process and pipeline. a Raw reads comprised of multiple subreads are depicted for three different amplicons [green, fuchsia and blue boxes; different shades of color are used to portray variable subread sequence qualities (darker shading portrays higher quality)]. Subreads are separated by a shared adapter sequence (grey boxes). The higher quality CCS read for each raw read is used to cluster the corresponding raw reads into CCS-based cluster groups. Error correction is performed per CCS-based cluster, producing top quality consequences sequences, followed by assembly of any overlapping consensus sequences. b A single run parameters file is used by all components of the pipeline. The grey highlighted rectangles represent two main steps of C3S-LAA. (i) Using the CCS reads generated by the SMRT analysis reads of insert protocol, C3S clusters the raw reads according to each barcode-primer pair combination, producing files of read identifiers to whitelist the corresponding raw reads. (ii) Raw read clusters are passed to Quiver to generate amplicon-specific consensus sequences, which are then passed to Minimus for sequence assembly. Rectangles with folded corners represent single files or multiple files (depicted as stacks of files) and those with rounded edges represent scripts and tools. Arrows indicates output files that are generated. Connecting lines with dots at one end depict input files, with the dot corresponding to the source data for the connected script or tool

Article Snippet: Since the amplicons were barcoded using PacBio’s standard barcodes, the default pre-set in SMRT Portal pointing to PacBio barcodes with padding in the reference directory was used.

Techniques: Sequencing, Generated, Amplification

Journal: Journal of Clinical Microbiology

Article Title: Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

doi: 10.1128/JCM.01764-16

Figure Lengend Snippet: (A) SYBR green dissociation curve analyses of LSG-qPCR amplicons from the Stratagene Mx3000P platform. G1, L . ( V .) braziliensis , L . ( V .) guyanensis , and L . ( V .) panamensis ; G2A/B, L . ( L .) infantum/L . ( L .) chagasi and L . ( L .) tropica ; G3, L . ( L .) amazonensis

Article Snippet: On the basis of the amplicons' Tm values observed with the Stratagene Mx3000P and ABI 7500 platforms, Leishmania spp. (as identified by conventional ITS2-PCR followed by DNA sequencing analysis) were classified into four groups, referred to here as G1, G2A, G2B, and G3 ( and ).

Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Clinical Microbiology

Article Title: Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

doi: 10.1128/JCM.01764-16

Figure Lengend Snippet: For reference sizes, amplicons from the LSG-qPCR and QC-qPCR were resolved on a 1.5% agarose gel. Lanes 1 and 5, 100-bp ladder size standards; lane 2, L . ( V .) panamensis ; lane 3, L . ( L .) tropica (G2B); lane 4, L . ( L .) aethiopica ; lane 6, human beta-actin

Article Snippet: On the basis of the amplicons' Tm values observed with the Stratagene Mx3000P and ABI 7500 platforms, Leishmania spp. (as identified by conventional ITS2-PCR followed by DNA sequencing analysis) were classified into four groups, referred to here as G1, G2A, G2B, and G3 ( and ).

Techniques: Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Molecular Cancer

Article Title: Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients

doi: 10.1186/s12943-015-0381-6

Figure Lengend Snippet: Silencing of KLK6 expression in FaDu cells promotes tumor cell proliferation. ( A ) KLK6 expression in human HNSCC (FaDu, Cal27, SCC25) and HeLa cervix carcinoma cells was monitored on protein level by Western blot analysis with cell culture supernatants (upper panel), and on transcript level by semi-quantitative (middle panels) as well as quantitative RT-PCR (lower graph). Detection of LMNB1 amplicons served as control for cDNA quality and quantity for semi-quantitative RT-PCR (lower panel), while transcript levels of three independent reference genes ( ACTB, LMNB1, TBP ) were used for quantitative RT-PCR data. ( B ) KLK6 expression in stable FaDu-Mock and FaDu-shKLK6 clones is given by semi-quantitative (upper panel) and quantitative RT-PCR (lower graph) and was determined as described in ( A ). Differences in tumor cell proliferation between stable FaDu-Mock and FaDu-shKLK6 clones was monitored by quantification of cell counts over a time period of six days ( C ) and a BrdU incorporation assay ( D ). The graph represents mean values and standard deviations (SD) of the percentage total of BrdU-positive cells from three independent FaDu-shKLK6 clones and mock controls, respectively

Article Snippet: Amplicons were separated by 1 % agarose gel electrophoresis and visualized with GelRed (Biotium, USA) using the UV documentation system for agarose gel (peqlab, Germany).

Techniques: Expressing, Western Blot, Cell Culture, Quantitative RT-PCR, Clone Assay, BrdU Incorporation Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: A multistrain approach to studying the mechanisms underlying compatibility in the interaction between Biomphalaria glabrata and Schistosoma mansoni

doi: 10.1371/journal.pntd.0005398

Figure Lengend Snippet: Diverse Sm PoMucs are expressed by the different strains of S . mansoni . (A) Sm PoMuc cDNA structure. The 5’ region is characterized by a variable number of tandem repeats. Two different types of repeats (r1 and r2) were mainly identified in previous studies [ 19 , 20 ]. SmPoMuc cDNA structure is shared by previously published Sm BRE, and Sm GH2 strains [ 19 , 20 ] and the new ones, Sm LE and Sm VEN. The 3’ region (exon 3 to exon 15) harbors sequence differences that enable the proteins to be classified into the only three groups (groups 1, 2, and 3.1) known to be expressed [ 19 , 20 ]. (B) Transcription patterns of Sm PoMucs in 11 individuals of each schistosome strain. Nested RT-PCR amplicons were obtained from individual sporocysts (1 to 11) of Sm LE, Sm VEN, Sm BRE, and Sm GH2, and resolved by agarose gel electrophoresis. PCR was performed using consensus primers that amplified the complete coding sequence of all Sm PoMuc transcripts (located in exon 1 and exon 15; vertical and dashed lines represent the positions of the primers used for PCR and nested PCR, respectively). “C-” denotes the negative control. Sm BRE and Sm GH2 nested PCR results were from [ 21 ] (C) Inter-strain polymorphism at the protein level. Protein extracts (8 μg) were obtained from sporocysts of each S . mansoni strain, and Western blot analysis was performed using anti- Sm PoMuc polyclonal antibodies. Actin was detected as a control.

Article Snippet: Two μl of the RT reaction was then used for PCR (Advantage 2 PCR system, Invitrogen, Carlsbad, CA, USA) with primers that were designed to specially target and amplify novel predicted transcripts, and amplicons were sequenced (GATC Biotech, Konstanz, Germany).

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Nested PCR, Negative Control, Western Blot

Journal: Journal of Neuro-Oncology

Article Title: Aberrant DNA methylation of alternative promoter of DLC1 isoform 1 in meningiomas

doi: 10.1007/s11060-016-2261-3

Figure Lengend Snippet: a Genomic location of 5 transcription variants of DLC1. b, c DNA methylation level of the DLC1 locus at CpGs measured by the HumanMethylation 450K array (Illumina) in meningiomas and normal meninges. Each blue / red dot represents an individual CpG. d DNA methylation levels in meningiomas of different grade at CpGs within differentially methylated regions (DMR) (HumanMethylation 450K microarray data). d, e DNA methylation level of DMR at 5′ region of DLC1 locus determined by targeted bisulfite sequencing of two PCR amplicons. d Comparison of average methylation levels of each CpGs in meningiomas and normal samples. Each dot represents the mean value and error bars represent standard errors of the mean. e Comparison of average DNA methylation levels for two amplicons in normal meninges and different grade meningiomas. Horizontal lines denote the mean value

Article Snippet: Amplicons were pooled, diluted 1:100 and then amplified using barcoded adaptor-linkers from Fluidigm, according to manufacturer’s protocols.

Techniques: DNA Methylation Assay, Methylation, Microarray, Methylation Sequencing, Polymerase Chain Reaction

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

Article Title: Genome-Wide Development and Use of Microsatellite Markers for Large-Scale Genotyping Applications in Foxtail Millet [Setaria italica (L.)]

doi: 10.1093/dnares/dst002

Figure Lengend Snippet: Representative gel showing amplification profiles of one microsatellite marker SiGMS 3261 and its fragment length polymorphism among foxtail millet and related species. The amplicons are resolved in 2% agarose gel along with 100 bp DNA size standard.

Article Snippet: For cloning, the amplicons amplified by microsatellite marker were eluted using AccuPrep DNA Gel Purification Kit (Bioneer, Korea) and cloned into pGEM-T Easy vector (Promega, USA) following the manufacturer's instructions.

Techniques: Amplification, Marker, Agarose Gel Electrophoresis

Journal: BMC Research Notes

Article Title: DNA analysis of molluscs from a museum wet collection: a comparison of different extraction methods

doi: 10.1186/s13104-016-2147-7

Figure Lengend Snippet: Representative image of an agarose gel electrophoresis for the amplicons COI-lf and 16S-sf ( 1 = A. brevispinosa 37,334 A2, 2 = A. brevispinosa 37,334 A1, 3 = A. arbustorum 86,813 A2, 4 = A. arbustorum 100,810 A1, 5 = D. polymorpha 101,850, 6 = D. polymorpha 83,846 B1, 7 = H. pomatia 74,402 B2, 8 = P. planorbis 84,060 A2, C = Negative control)

Article Snippet: To test whether the amplified amplicons are the expected gene sequences, a subset of samples was sequenced (LGC Genomics, Berlin) using the same primers used for producing the amplicons.

Techniques: Agarose Gel Electrophoresis, Negative Control

Journal: Applied and Environmental Microbiology

Article Title: PCR-Restriction Fragment Length Polymorphism Method for Detection of Cyclospora cayetanensis in Environmental Waters without Microscopic Confirmation

doi: 10.1128/AEM.69.8.4662-4669.2003

Figure Lengend Snippet: RFLP analysis with Alu I of PCR (primers CYCAI2 and CYCAR1) amplicons from selected environmental samples. Lanes: M, DNA molecular weight marker VIII; +, C . cayetanensis oocysts with clear bands at 98, 88, and 50 bp and a faint band at 15 bp; 1, sample collected from the SDC on 21 April 1998; 2 to 5, individual water samples collected from the SARVB on 28 August 1998; 6 and 7, individual water samples collected from the SARYL on 28 September 1998. The term “Uncut” identifies amplicons that do not contain Alu I sites.

Article Snippet: Because of the probability of multiple similar-size but dissimilar-sequence products, amplicons from PCR (primers CYCAI2 and CYCAR1) were separated by denaturing high-performance liquid chromatography (Wave System, Transgenomic, San Jose, Calif.).

Techniques: Polymerase Chain Reaction, Environmental Sampling, Molecular Weight, Marker