Structured Review

GE Healthcare amplicons
Ablation of ADAR2 autoediting and alternative splicing in ΔECS mice. (A) Extent of editing at the −1 site in ADAR2 pre-mRNA isolated from different tissues of wild-type (open columns) and ΔECS (filled columns) mice was determined by primer extension analysis. Values were obtained from tissue isolated from three animals per genotype (mean ± SEM). (B) Schematic diagram of four possible ADAR2 mRNA isoforms resulting from alternative splicing of exon 4 and inclusion or exclusion of 47-nt cassette (+47 and −47, respectively) at the 5′ boundary of exon 5. (C) Quantitative analysis of ADAR2 alternative splicing. The positions of RT-PCR <t>amplicons</t> corresponding to the four alternatively spliced ADAR2 transcripts described for panel B are indicated. The percentages of total ADAR2 transcripts containing the 47-nt cassette but not exon 4 (3/5 +47) are shown (mean ± SEM; n = 3).
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1) Product Images from "Altered RNA Editing in Mice Lacking ADAR2 Autoregulation §"

Article Title: Altered RNA Editing in Mice Lacking ADAR2 Autoregulation §

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.2.480-488.2006

Ablation of ADAR2 autoediting and alternative splicing in ΔECS mice. (A) Extent of editing at the −1 site in ADAR2 pre-mRNA isolated from different tissues of wild-type (open columns) and ΔECS (filled columns) mice was determined by primer extension analysis. Values were obtained from tissue isolated from three animals per genotype (mean ± SEM). (B) Schematic diagram of four possible ADAR2 mRNA isoforms resulting from alternative splicing of exon 4 and inclusion or exclusion of 47-nt cassette (+47 and −47, respectively) at the 5′ boundary of exon 5. (C) Quantitative analysis of ADAR2 alternative splicing. The positions of RT-PCR amplicons corresponding to the four alternatively spliced ADAR2 transcripts described for panel B are indicated. The percentages of total ADAR2 transcripts containing the 47-nt cassette but not exon 4 (3/5 +47) are shown (mean ± SEM; n = 3).
Figure Legend Snippet: Ablation of ADAR2 autoediting and alternative splicing in ΔECS mice. (A) Extent of editing at the −1 site in ADAR2 pre-mRNA isolated from different tissues of wild-type (open columns) and ΔECS (filled columns) mice was determined by primer extension analysis. Values were obtained from tissue isolated from three animals per genotype (mean ± SEM). (B) Schematic diagram of four possible ADAR2 mRNA isoforms resulting from alternative splicing of exon 4 and inclusion or exclusion of 47-nt cassette (+47 and −47, respectively) at the 5′ boundary of exon 5. (C) Quantitative analysis of ADAR2 alternative splicing. The positions of RT-PCR amplicons corresponding to the four alternatively spliced ADAR2 transcripts described for panel B are indicated. The percentages of total ADAR2 transcripts containing the 47-nt cassette but not exon 4 (3/5 +47) are shown (mean ± SEM; n = 3).

Techniques Used: Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

Targeting strategy for selective ablation of ADAR2 autoediting. (A) Schematic diagram of a portion of the mouse ADAR2 gene, extending from exon 3 through exon 6, before and after targeted gene modification. The positions of the positive selectable marker (PGK-neo), the ECS, and the alternative 3′ splice site generated by ADAR2 autoediting (*) are indicated. The positions of primer pairs used for genotype analysis (→), introduced loxP recombination sites for selective deletion of the PGK-neo cassette and the ECS region (▹), and DNA sequences outside the region of targeting vector homology (---) are shown. (B) Analysis of mouse genotype by PCR amplification of mouse tail genomic DNA. The migration positions for PCR amplicons corresponding to the wild-type (+/+; 343 bp) and ΔECS (−/−; 172 bp) alleles are indicated.
Figure Legend Snippet: Targeting strategy for selective ablation of ADAR2 autoediting. (A) Schematic diagram of a portion of the mouse ADAR2 gene, extending from exon 3 through exon 6, before and after targeted gene modification. The positions of the positive selectable marker (PGK-neo), the ECS, and the alternative 3′ splice site generated by ADAR2 autoediting (*) are indicated. The positions of primer pairs used for genotype analysis (→), introduced loxP recombination sites for selective deletion of the PGK-neo cassette and the ECS region (▹), and DNA sequences outside the region of targeting vector homology (---) are shown. (B) Analysis of mouse genotype by PCR amplification of mouse tail genomic DNA. The migration positions for PCR amplicons corresponding to the wild-type (+/+; 343 bp) and ΔECS (−/−; 172 bp) alleles are indicated.

Techniques Used: Modification, Marker, Generated, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Migration

2) Product Images from "Large-scale mitogenomics enables insights into Schizophora (Diptera) radiation and population diversity"

Article Title: Large-scale mitogenomics enables insights into Schizophora (Diptera) radiation and population diversity

Journal: Scientific Reports

doi: 10.1038/srep21762

Coverage plots. ( A ) Coverage of mitogenomes from 16 species of Schizophora generated by long-range PCR and shotgun sequencing. Highlighted regions indicate an overlap (green) and the 16S gap (orange) region between the two amplicons. Gaps in 16S sequence were further closed through standard PCR and Sanger sequencing. ( B ) 16 mitogenomes assembled from short reads generated by whole genome sequencing. Both strategies used short reads from MiSeq or HiSeq Illumina platforms to generate high-quality assemblies. ( C ) Coverage plot of the complete mtDNA assembled with long reads generated with SMRT sequencing technology. The scheme shows the complete mtDNA of C. megacephala (sample F03) assembled with 15,835 bp. First track shows the low GC content (23.5%). Orange bars on the second track refer to the coverage. The innermost track shows gene order in each mtDNA strand. Yellow arrows denote PCGs, green arrows show rRNA subunits and orange arrows refer to tRNAs.
Figure Legend Snippet: Coverage plots. ( A ) Coverage of mitogenomes from 16 species of Schizophora generated by long-range PCR and shotgun sequencing. Highlighted regions indicate an overlap (green) and the 16S gap (orange) region between the two amplicons. Gaps in 16S sequence were further closed through standard PCR and Sanger sequencing. ( B ) 16 mitogenomes assembled from short reads generated by whole genome sequencing. Both strategies used short reads from MiSeq or HiSeq Illumina platforms to generate high-quality assemblies. ( C ) Coverage plot of the complete mtDNA assembled with long reads generated with SMRT sequencing technology. The scheme shows the complete mtDNA of C. megacephala (sample F03) assembled with 15,835 bp. First track shows the low GC content (23.5%). Orange bars on the second track refer to the coverage. The innermost track shows gene order in each mtDNA strand. Yellow arrows denote PCGs, green arrows show rRNA subunits and orange arrows refer to tRNAs.

Techniques Used: Generated, Polymerase Chain Reaction, Shotgun Sequencing, Sequencing

3) Product Images from "Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR"

Article Title: Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR

Journal: Journal of Biomedicine and Biotechnology

doi: 10.1155/2010/854737

ANKIB1 gene breakpoint determination by RT-QPCR (± standard error mean). Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed square (■), and a closed circle (●), respectively. The ANKIB1 exons on the left of the vertical dotted lines (exon1–10) show the copy number loss of the associated amplicons.
Figure Legend Snippet: ANKIB1 gene breakpoint determination by RT-QPCR (± standard error mean). Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed square (■), and a closed circle (●), respectively. The ANKIB1 exons on the left of the vertical dotted lines (exon1–10) show the copy number loss of the associated amplicons.

Techniques Used: Quantitative RT-PCR

(a) Haplotype analysis of microsatellites markers from chromosome 7q21.1. The haplotype shared by the affected individuals (black-filled symbol) is “boxed”. In the affected patients, the symbol (∗) indicates the indefinite alleles, located in the deletion. Since the hemizygosity for the non shared alleles in the affected patients, the analysis, shows “homozygosity” for three markers (D7S2409, D7S1813, D7S1789), thus, the deleted alleles are indicated with the same symbol (∗). (b) Measurements of copy number status (± standard error mean) of genes mapping in the 7q21 chromosomal region determined by RT-QPCR. On the Top: the genomic organization of genes. Genes are shown above the horizontal axis, which also indicates the extension and orientation of each gene. Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed quare (■), and a closed circle (●), respectively. The genes between the two vertical gray dotted lines show a hemizigous deletion detectable by the copy number loss of the associated amplicons.
Figure Legend Snippet: (a) Haplotype analysis of microsatellites markers from chromosome 7q21.1. The haplotype shared by the affected individuals (black-filled symbol) is “boxed”. In the affected patients, the symbol (∗) indicates the indefinite alleles, located in the deletion. Since the hemizygosity for the non shared alleles in the affected patients, the analysis, shows “homozygosity” for three markers (D7S2409, D7S1813, D7S1789), thus, the deleted alleles are indicated with the same symbol (∗). (b) Measurements of copy number status (± standard error mean) of genes mapping in the 7q21 chromosomal region determined by RT-QPCR. On the Top: the genomic organization of genes. Genes are shown above the horizontal axis, which also indicates the extension and orientation of each gene. Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed quare (■), and a closed circle (●), respectively. The genes between the two vertical gray dotted lines show a hemizigous deletion detectable by the copy number loss of the associated amplicons.

Techniques Used: Quantitative RT-PCR

4) Product Images from "Diagnosis of Contagious Bovine Pleuropneumonia by PCR-Laser- Induced Fluorescence and PCR-Restriction Endonuclease Analysis Based on the 16S rRNA Genes of Mycoplasma mycoides subsp. mycoides SC"

Article Title: Diagnosis of Contagious Bovine Pleuropneumonia by PCR-Laser- Induced Fluorescence and PCR-Restriction Endonuclease Analysis Based on the 16S rRNA Genes of Mycoplasma mycoides subsp. mycoides SC

Journal: Journal of Clinical Microbiology

doi:

Electropherogram of samples analyzed by PCR-LIF. The amplicon from M. capricolum subsp. capripneumoniae appeared as a single peak in the electropherogram, and the size was calculated in relation to internal size standards (not shown) as 128.0 bp. Amplicons from M. mycoides SC were clearly detected as two peaks, and the sizes were calculated as 127.0 and 128.9 bp.
Figure Legend Snippet: Electropherogram of samples analyzed by PCR-LIF. The amplicon from M. capricolum subsp. capripneumoniae appeared as a single peak in the electropherogram, and the size was calculated in relation to internal size standards (not shown) as 128.0 bp. Amplicons from M. mycoides SC were clearly detected as two peaks, and the sizes were calculated as 127.0 and 128.9 bp.

Techniques Used: Polymerase Chain Reaction, Amplification

5) Product Images from "Transcription Analysis of Genes Encoding Homologues of Reductive Dehalogenases in "Dehalococcoides" sp. Strain CBDB1 by Using Terminal Restriction Fragment Length Polymorphism and Quantitative PCR ▿" sp. Strain CBDB1 by Using Terminal Restriction Fragment Length Polymorphism and Quantitative PCR ▿ †"

Article Title: Transcription Analysis of Genes Encoding Homologues of Reductive Dehalogenases in "Dehalococcoides" sp. Strain CBDB1 by Using Terminal Restriction Fragment Length Polymorphism and Quantitative PCR ▿" sp. Strain CBDB1 by Using Terminal Restriction Fragment Length Polymorphism and Quantitative PCR ▿ †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01042-08

Composite figure showing sections of gels with RT-PCR products obtained with 13 different degenerate primer pairs from cultures supplemented at time point 0 with 1,2,3-TCB (A) or 1,2,4-TCB (B). The approximate sizes of the respective amplicons according
Figure Legend Snippet: Composite figure showing sections of gels with RT-PCR products obtained with 13 different degenerate primer pairs from cultures supplemented at time point 0 with 1,2,3-TCB (A) or 1,2,4-TCB (B). The approximate sizes of the respective amplicons according

Techniques Used: Reverse Transcription Polymerase Chain Reaction

6) Product Images from "Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection"

Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection

Journal: Viruses

doi: 10.3390/v10080422

A spectrum of ZIKV genetic diversity is represented by three historically important and spatiotemporally distinct strains: MR-766, P6-740, and PRVABC-59. The consensus nucleotide sequence for each of their full-length genomic RNAs was determined by sequencing three overlapping uncloned cDNA amplicons collectively representing the entire genomic RNA, except for the 5′ and 3′ termini, which were subsequently defined by performing both 5′- and 3′-rapid amplification of cDNA ends (RACE); each of these RACEs was followed by cDNA cloning and sequencing of ~20 randomly picked clones. ( A ) Genomic organization of the three ZIKV strains; ( B ) Pairwise comparison of the complete nucleotide (nt) and deduced amino acid (aa) sequences of the three ZIKV genomes; ( C ) Phylogenetic tree based on the nucleotide sequence of 29 ZIKV genomes, including the 15 complete (MR-766, green; P6-740, orange; PRVABC-59, red; and 12 others, black) and 14 near-complete (gray) genomes, with Japanese encephalitis virus (JEV) K87P39 included as an outgroup. Bootstrap values from 1000 replicates are shown at each node of the tree. The scale bar represents the number of nucleotide substitutions per site. The strain name is followed by a description in parenthesis of the country, year, and host of isolation and the GenBank accession numbers. Note that MR-766 has been fully sequenced in this study and by three other groups (designated MR-766/CDC, MR-766/NIID, and MR-766/USAMRIID).
Figure Legend Snippet: A spectrum of ZIKV genetic diversity is represented by three historically important and spatiotemporally distinct strains: MR-766, P6-740, and PRVABC-59. The consensus nucleotide sequence for each of their full-length genomic RNAs was determined by sequencing three overlapping uncloned cDNA amplicons collectively representing the entire genomic RNA, except for the 5′ and 3′ termini, which were subsequently defined by performing both 5′- and 3′-rapid amplification of cDNA ends (RACE); each of these RACEs was followed by cDNA cloning and sequencing of ~20 randomly picked clones. ( A ) Genomic organization of the three ZIKV strains; ( B ) Pairwise comparison of the complete nucleotide (nt) and deduced amino acid (aa) sequences of the three ZIKV genomes; ( C ) Phylogenetic tree based on the nucleotide sequence of 29 ZIKV genomes, including the 15 complete (MR-766, green; P6-740, orange; PRVABC-59, red; and 12 others, black) and 14 near-complete (gray) genomes, with Japanese encephalitis virus (JEV) K87P39 included as an outgroup. Bootstrap values from 1000 replicates are shown at each node of the tree. The scale bar represents the number of nucleotide substitutions per site. The strain name is followed by a description in parenthesis of the country, year, and host of isolation and the GenBank accession numbers. Note that MR-766 has been fully sequenced in this study and by three other groups (designated MR-766/CDC, MR-766/NIID, and MR-766/USAMRIID).

Techniques Used: Sequencing, Rapid Amplification of cDNA Ends, Clone Assay, Isolation

7) Product Images from "Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency"

Article Title: Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency

Journal: Journal of Virology

doi: 10.1128/JVI.75.15.6977-6988.2001

Proviral loads in sheep infected with BLV mutant proviruses. The wild-type (pBLV-WT) provirus and each recombinant (pBLV-Δ21-bp, pBLV-NF2, pBLV-GRE, pBLV-CRE3x, pBLV-Ebox, and pBLV-IRF) provirus was injected into two different sheep as indicated. Three and six months after seroconversion, blood was isolated by jugular venipuncture and the corresponding lysates were amplified by 25 cycles of PCR using two primers flanking the tax gene. Under these conditions, PCR amplifications yielded semiquantitative estimation of the proviral loads as shown by 10-fold dilutions (10× and 100×) of a lysate from an infected sheep (sheep 2664). As a negative control, a lysate from an uninfected sheep was tested in parallel (sheep 115). After PCR, the resulting amplicons were analyzed by Southern blotting using a tax probe.
Figure Legend Snippet: Proviral loads in sheep infected with BLV mutant proviruses. The wild-type (pBLV-WT) provirus and each recombinant (pBLV-Δ21-bp, pBLV-NF2, pBLV-GRE, pBLV-CRE3x, pBLV-Ebox, and pBLV-IRF) provirus was injected into two different sheep as indicated. Three and six months after seroconversion, blood was isolated by jugular venipuncture and the corresponding lysates were amplified by 25 cycles of PCR using two primers flanking the tax gene. Under these conditions, PCR amplifications yielded semiquantitative estimation of the proviral loads as shown by 10-fold dilutions (10× and 100×) of a lysate from an infected sheep (sheep 2664). As a negative control, a lysate from an uninfected sheep was tested in parallel (sheep 115). After PCR, the resulting amplicons were analyzed by Southern blotting using a tax probe.

Techniques Used: Infection, Mutagenesis, Recombinant, Injection, Isolation, Amplification, Polymerase Chain Reaction, Negative Control, Southern Blot

8) Product Images from "HiDRA-seq: High-Throughput SARS-CoV-2 Detection by RNA Barcoding and Amplicon Sequencing"

Article Title: HiDRA-seq: High-Throughput SARS-CoV-2 Detection by RNA Barcoding and Amplicon Sequencing

Journal: bioRxiv

doi: 10.1101/2020.06.02.130484

(A) Matched pairs of normalized Ct values and alignment rate. Ct values (y-axis-left) for sets of RT-qPCR technical replicates were normalized and correlated to the alignment rate (y-axis-right) for the different amplicons using the Wilcoxon-Signed-Rank test for matched pairs (p
Figure Legend Snippet: (A) Matched pairs of normalized Ct values and alignment rate. Ct values (y-axis-left) for sets of RT-qPCR technical replicates were normalized and correlated to the alignment rate (y-axis-right) for the different amplicons using the Wilcoxon-Signed-Rank test for matched pairs (p

Techniques Used: Quantitative RT-PCR

(A) Schematic representation of the protocol. Using a low volume liquid handling robot, the patients’ RNA is distributed in 384-well plates containing indexed oligo-dT primers. Barcoded cDNA is generated by reverse transcription and pooled into a single tube. Libraries are produced from SARS-CoV-2-specific amplicons and sequenced. Reads are de-multiplexed based on both a plate and a sample barcode and used for downstream diagnostic analysis. (B) Amplicon primer design. The table contains the sequences of the forward primers used to generate the different amplicons. The reverse primer is common to all amplicons and it anneals to the 3’-end of the barcoded oligo-dT primers used to generate cDNA in the reverse transcription. The primer labels contain the position of the first sequenced base. Insert length was calculated using the start of the poly-A sequence. SARS-CoV-2 NC_045512_2 sequence and GAPDH NM_002046.4 sequence were used as reference. For the theoretical size of the amplicon after PCR 1 the Nextera Tag (34bp), the length of the barcoded Oligo-dT-primer (80bp) and the primer sequence was added.
Figure Legend Snippet: (A) Schematic representation of the protocol. Using a low volume liquid handling robot, the patients’ RNA is distributed in 384-well plates containing indexed oligo-dT primers. Barcoded cDNA is generated by reverse transcription and pooled into a single tube. Libraries are produced from SARS-CoV-2-specific amplicons and sequenced. Reads are de-multiplexed based on both a plate and a sample barcode and used for downstream diagnostic analysis. (B) Amplicon primer design. The table contains the sequences of the forward primers used to generate the different amplicons. The reverse primer is common to all amplicons and it anneals to the 3’-end of the barcoded oligo-dT primers used to generate cDNA in the reverse transcription. The primer labels contain the position of the first sequenced base. Insert length was calculated using the start of the poly-A sequence. SARS-CoV-2 NC_045512_2 sequence and GAPDH NM_002046.4 sequence were used as reference. For the theoretical size of the amplicon after PCR 1 the Nextera Tag (34bp), the length of the barcoded Oligo-dT-primer (80bp) and the primer sequence was added.

Techniques Used: Generated, Produced, Diagnostic Assay, Amplification, Sequencing, Polymerase Chain Reaction

(A) Read distribution in the different wells of the plate. The histogram shows the read numbers obtained in the different wells of the plate for the different amplicons. As the input for the protocols was not normalized, a wide distribution in the number of reads is observed. Wells with
Figure Legend Snippet: (A) Read distribution in the different wells of the plate. The histogram shows the read numbers obtained in the different wells of the plate for the different amplicons. As the input for the protocols was not normalized, a wide distribution in the number of reads is observed. Wells with

Techniques Used:

9) Product Images from "Size Polymorphism in Alleles of the Myoglobin Gene from Biomphalaria Mollusks"

Article Title: Size Polymorphism in Alleles of the Myoglobin Gene from Biomphalaria Mollusks

Journal: Genes

doi: 10.3390/genes1030357

Schematic representation of the supposed alleles from (A) B. glabrata, (B) B. straminea,, (C) B. tenagophila according to amplicons obtained. Exons (white) were represented only to delimit introns (black).
Figure Legend Snippet: Schematic representation of the supposed alleles from (A) B. glabrata, (B) B. straminea,, (C) B. tenagophila according to amplicons obtained. Exons (white) were represented only to delimit introns (black).

Techniques Used:

Annealing position of the primers related to the myoglobin gene from B. glabrata (U89283.1). Solid line: Primers BIO 1F/R. Dashed line: BIO 4F/R. Grey box: BIO2/3. Tick line: Initiation, stop codon. I, II, III: introns 1, 2, 3 respectively. Below the figure, the sequence of the primers, expected sizes of amplicons according to the gene U89283.1 are shown.
Figure Legend Snippet: Annealing position of the primers related to the myoglobin gene from B. glabrata (U89283.1). Solid line: Primers BIO 1F/R. Dashed line: BIO 4F/R. Grey box: BIO2/3. Tick line: Initiation, stop codon. I, II, III: introns 1, 2, 3 respectively. Below the figure, the sequence of the primers, expected sizes of amplicons according to the gene U89283.1 are shown.

Techniques Used: Sequencing

Polyacrylamide gel electrophoresis with silver stain. PCR-RFLP of the amplicons of the introns from Biomphalaria myoglobin. 1. B. glabrata; 2. B. straminea; 3. B. tenagophila. MW = molecular weight (base pairs) of φX174/Hae III (Amersham Biosciences). Above the lanes, restriction endonucleases are indicated.
Figure Legend Snippet: Polyacrylamide gel electrophoresis with silver stain. PCR-RFLP of the amplicons of the introns from Biomphalaria myoglobin. 1. B. glabrata; 2. B. straminea; 3. B. tenagophila. MW = molecular weight (base pairs) of φX174/Hae III (Amersham Biosciences). Above the lanes, restriction endonucleases are indicated.

Techniques Used: Polyacrylamide Gel Electrophoresis, Silver Staining, Polymerase Chain Reaction, Molecular Weight

10) Product Images from "A Novel Missense Variant Associated with A Splicing Defect in A Myopathic Form of PGK1 Deficiency in The Spanish Population"

Article Title: A Novel Missense Variant Associated with A Splicing Defect in A Myopathic Form of PGK1 Deficiency in The Spanish Population

Journal: Genes

doi: 10.3390/genes10100785

Analysis of the PGK1 patient’s blood cDNA. ( A ). Electrophoresis on 1% agarose gel. Lane 1: Index case showing a transcript of normal size (b band), one higher molecular weight cDNA species (a band), and an apparently more abundant transcript of lower molecular weight (c band); Lane 2: Healthy control showing a transcript of normal size (b band). ( B ). Sanger Sequencing of the gel purified amplicons. ( 1a ): Sequence of patient’s a band showing the retention of intron 9; ( 1b ): Sequence of the patient’s b band displaying the junction of exons 9 and 10, and the variant c.1114G > A (indicated by an arrow). Healthy control revealed the same pattern for b band (not shown). The hemizygous c.1114G > A variant was detected in the patient’s sequence of a transcript of normal size (1b); ( 1c ): Sequence of patient’s c band showing the skipping of exon 9 in the cDNA.
Figure Legend Snippet: Analysis of the PGK1 patient’s blood cDNA. ( A ). Electrophoresis on 1% agarose gel. Lane 1: Index case showing a transcript of normal size (b band), one higher molecular weight cDNA species (a band), and an apparently more abundant transcript of lower molecular weight (c band); Lane 2: Healthy control showing a transcript of normal size (b band). ( B ). Sanger Sequencing of the gel purified amplicons. ( 1a ): Sequence of patient’s a band showing the retention of intron 9; ( 1b ): Sequence of the patient’s b band displaying the junction of exons 9 and 10, and the variant c.1114G > A (indicated by an arrow). Healthy control revealed the same pattern for b band (not shown). The hemizygous c.1114G > A variant was detected in the patient’s sequence of a transcript of normal size (1b); ( 1c ): Sequence of patient’s c band showing the skipping of exon 9 in the cDNA.

Techniques Used: Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Sequencing, Purification, Variant Assay

11) Product Images from "The Schistosoma mansoni Tegumental-Allergen-Like (TAL) Protein Family: Influence of Developmental Expression on Human IgE Responses"

Article Title: The Schistosoma mansoni Tegumental-Allergen-Like (TAL) Protein Family: Influence of Developmental Expression on Human IgE Responses

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001593

Expression of SmTAL4 in cercarial tail only. For PCR analysis ( A ), total RNA from isolated from heads (H) and tails (T) after mechanical separation and used to prepare cDNA for use with specific primers to generate amplicons for Sm ß-actin (203 bp), SmTAL4 (152 bp), SmTAL5(228 bp) and SmTAL13(206 bp). Products were separated on a 2% agarose gel and detected with ethidium bromide. For immunostaining ( B ) 8 µ sections of frozen sections of whole cercariae were fixed and stained with rat anti-SmTAL4 antiserum and TRITC -anti-rat antibody (red). Nuclei were counterstained with DAPI (blue). In the negative control (insert) anti- SmTAL4 antiserum was pre-absorbed with recombinant SmTAL4.
Figure Legend Snippet: Expression of SmTAL4 in cercarial tail only. For PCR analysis ( A ), total RNA from isolated from heads (H) and tails (T) after mechanical separation and used to prepare cDNA for use with specific primers to generate amplicons for Sm ß-actin (203 bp), SmTAL4 (152 bp), SmTAL5(228 bp) and SmTAL13(206 bp). Products were separated on a 2% agarose gel and detected with ethidium bromide. For immunostaining ( B ) 8 µ sections of frozen sections of whole cercariae were fixed and stained with rat anti-SmTAL4 antiserum and TRITC -anti-rat antibody (red). Nuclei were counterstained with DAPI (blue). In the negative control (insert) anti- SmTAL4 antiserum was pre-absorbed with recombinant SmTAL4.

Techniques Used: Expressing, Polymerase Chain Reaction, Isolation, Agarose Gel Electrophoresis, Immunostaining, Staining, Negative Control, Recombinant

12) Product Images from "Characterisation of parapoxviruses isolated from Norwegian semi-domesticated reindeer (Rangifer tarandus tarandus)"

Article Title: Characterisation of parapoxviruses isolated from Norwegian semi-domesticated reindeer (Rangifer tarandus tarandus)

Journal: Virology Journal

doi: 10.1186/1743-422X-2-79

Maximum Parsimony tree based on the translated nucleotide sequences of the Norwegian and Finnish reindeer vIL-10 gene amplicons obtained in this study, compared with corresponding amino acid sequences from mammals published in Genebank. Black numbers (branch length) describe the genetic distance/number of changes along the branch. Blue numbers (bootstrap values) describe the reliability for each clade in percent.
Figure Legend Snippet: Maximum Parsimony tree based on the translated nucleotide sequences of the Norwegian and Finnish reindeer vIL-10 gene amplicons obtained in this study, compared with corresponding amino acid sequences from mammals published in Genebank. Black numbers (branch length) describe the genetic distance/number of changes along the branch. Blue numbers (bootstrap values) describe the reliability for each clade in percent.

Techniques Used:

13) Product Images from "A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA"

Article Title: A Novel Approach for Determining Cancer Genomic Breakpoints in the Presence of Normal DNA

Journal: PLoS ONE

doi: 10.1371/journal.pone.0000380

Breakpoint identification by PAMP with an INK4A minigenomic tiling array. (A) Five groups of primers (F A , F B , R x , R Y and R Z , the small arrows and arrow heads) near the potential breakpoints were generated for PAMP based on our previous mapping [3] . The mapped CDKN2A breakpoints of the Detroit 562 cell line ( Figure 5 ) are indicated for clarification. The “E1”, “E2” and “E3” designations (blue fonts) are the relative positions of INK4A exons. The first exon of ARF is further to the right of this diagram and is not covered by this array. The tiling probes for the array are indicated with two alternating colors (short black and orange lines) for ease of identification. (B) The first row of the INK4A minigenomic array was spotted with the tiling probes shown in panel A. Cot-1 DNA (repetitive sequence of genomic DNA) spots are indicated on this array. The rest of the spots are herring sperm DNA. Both Cot-1 and herring sperm DNA are used as nonspecific controls. This array was hybridized with labeled samples derived from two cell lines. The same sets of primers (F A , F B , R Y and R Z ) were used for PAMP reactions on Detroit 562 (mutant) and HEK293 (wild type) genomic DNA to map the potential CDKN2A breakpoints. The amplicons were labeled with different dyes, yielding a green signal (Cy-3) for the mutant sample and a red signal (Cy-5) for the wild type sample, to be simultaneously hybridized on the array (two-color array). The two green spots on the first row revealed the breakpoint location as been discussed in Figure 2 .
Figure Legend Snippet: Breakpoint identification by PAMP with an INK4A minigenomic tiling array. (A) Five groups of primers (F A , F B , R x , R Y and R Z , the small arrows and arrow heads) near the potential breakpoints were generated for PAMP based on our previous mapping [3] . The mapped CDKN2A breakpoints of the Detroit 562 cell line ( Figure 5 ) are indicated for clarification. The “E1”, “E2” and “E3” designations (blue fonts) are the relative positions of INK4A exons. The first exon of ARF is further to the right of this diagram and is not covered by this array. The tiling probes for the array are indicated with two alternating colors (short black and orange lines) for ease of identification. (B) The first row of the INK4A minigenomic array was spotted with the tiling probes shown in panel A. Cot-1 DNA (repetitive sequence of genomic DNA) spots are indicated on this array. The rest of the spots are herring sperm DNA. Both Cot-1 and herring sperm DNA are used as nonspecific controls. This array was hybridized with labeled samples derived from two cell lines. The same sets of primers (F A , F B , R Y and R Z ) were used for PAMP reactions on Detroit 562 (mutant) and HEK293 (wild type) genomic DNA to map the potential CDKN2A breakpoints. The amplicons were labeled with different dyes, yielding a green signal (Cy-3) for the mutant sample and a red signal (Cy-5) for the wild type sample, to be simultaneously hybridized on the array (two-color array). The two green spots on the first row revealed the breakpoint location as been discussed in Figure 2 .

Techniques Used: Generated, Clarification Assay, Sequencing, Labeling, Derivative Assay, Mutagenesis

14) Product Images from "Environmental Occurrence of Madurella mycetomatis, the Major Agent of Human Eumycetoma in Sudan"

Article Title: Environmental Occurrence of Madurella mycetomatis, the Major Agent of Human Eumycetoma in Sudan

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.40.3.1031-1036.2002

Molecular detection of M. mycetomatis in soils and thorn samples. (A) Results of the broad-spectrum ITS PCR employing primers ITS4 and ITS5. Note that amplicons can be observed in all lanes. (B) Result of the nested PCR using M. mycetomatis -specific primers. Lanes 1 to 6, amplicons derived from Dutch control samples: lanes 7 to 14, amplified material from Sudanese samples; lane 15, results obtained with purified M. mycetomatis DNA; lanes 16 and 17, negative controls; lanes M, 100-bp molecule sizing ladder, and the 600-bp fragment is indicated (arrow).
Figure Legend Snippet: Molecular detection of M. mycetomatis in soils and thorn samples. (A) Results of the broad-spectrum ITS PCR employing primers ITS4 and ITS5. Note that amplicons can be observed in all lanes. (B) Result of the nested PCR using M. mycetomatis -specific primers. Lanes 1 to 6, amplicons derived from Dutch control samples: lanes 7 to 14, amplified material from Sudanese samples; lane 15, results obtained with purified M. mycetomatis DNA; lanes 16 and 17, negative controls; lanes M, 100-bp molecule sizing ladder, and the 600-bp fragment is indicated (arrow).

Techniques Used: Polymerase Chain Reaction, Nested PCR, Derivative Assay, Amplification, Purification

15) Product Images from "Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinomas"

Article Title: Expression of multiple forms of 3'-end variant CCK2 receptor mRNAs in human pancreatic adenocarcinomas

Journal: BMC Research Notes

doi: 10.1186/1756-0500-4-131

Representative Ethidium-bromide stained agarose gel visualising PCR amplicons derived from 17 archival biopsy specimens (lane 1 to 17) . A) Quality control of total RNA (free of contaminating genomic DNA), and B) single-stranded cDNA by means of β-actin PCR amplification after DNase treatment. C) Visualised amplicons from nested CCK2R, D) gastrin, and E) CCK2i4svR specific PCR assays as described in methods. The position of the 500 bp size marker in each 100 bp ladder (lane M) is indicated. Black arrows in the left margin indicate the position of a) amplicons derived from genomic DNA or cDNA retaining introns, b) wild-type CCK2 receptor and gastrin mRNA amplicons, and c) CCK2i4svR splice variant amplicons derived from ss-cDNA. White arrows (lane 6, 10, and 17) indicate confirmed CCK2 receptor splice variants by DNA sequencing. Stripped arrows indicate the position of the CCK2i4svR amplicon in sample No. 15. In all amplification assays human genomic DNA (+) was included as control. (-) NTC (no DNA template added to the master mix).
Figure Legend Snippet: Representative Ethidium-bromide stained agarose gel visualising PCR amplicons derived from 17 archival biopsy specimens (lane 1 to 17) . A) Quality control of total RNA (free of contaminating genomic DNA), and B) single-stranded cDNA by means of β-actin PCR amplification after DNase treatment. C) Visualised amplicons from nested CCK2R, D) gastrin, and E) CCK2i4svR specific PCR assays as described in methods. The position of the 500 bp size marker in each 100 bp ladder (lane M) is indicated. Black arrows in the left margin indicate the position of a) amplicons derived from genomic DNA or cDNA retaining introns, b) wild-type CCK2 receptor and gastrin mRNA amplicons, and c) CCK2i4svR splice variant amplicons derived from ss-cDNA. White arrows (lane 6, 10, and 17) indicate confirmed CCK2 receptor splice variants by DNA sequencing. Stripped arrows indicate the position of the CCK2i4svR amplicon in sample No. 15. In all amplification assays human genomic DNA (+) was included as control. (-) NTC (no DNA template added to the master mix).

Techniques Used: Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Derivative Assay, Amplification, Marker, Variant Assay, DNA Sequencing

16) Product Images from "A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing"

Article Title: A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing

Journal: BMC Cancer

doi: 10.1186/s12885-016-2589-2

Sensitivity of HRM. Dilutions of genomic DNA from the KRAS mutant cell line PL45 (codon 12 GGT → GAT, heterozygous mutation) in WT genomic DNA was analysed by HRM. Normalised fluorescence and difference graphs are indicated. Clear discrimination of WT and mutant amplicons is possible using either graph if the fraction of PL45-DNA in the sample exceeds 25 %, corresponding to a mutant allele frequency of 12.5 %
Figure Legend Snippet: Sensitivity of HRM. Dilutions of genomic DNA from the KRAS mutant cell line PL45 (codon 12 GGT → GAT, heterozygous mutation) in WT genomic DNA was analysed by HRM. Normalised fluorescence and difference graphs are indicated. Clear discrimination of WT and mutant amplicons is possible using either graph if the fraction of PL45-DNA in the sample exceeds 25 %, corresponding to a mutant allele frequency of 12.5 %

Techniques Used: Mutagenesis, Fluorescence

17) Product Images from "Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol"

Article Title: Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol

Journal: PLoS ONE

doi: 10.1371/journal.pone.0181491

Electropherogram from Sanger sequencing of rRNA amplicons for a clonal isolate of Haematococcus pluvialis . Arrows indicate sites of putative intragenomic variation in a relatively short stretch of primary sequence.
Figure Legend Snippet: Electropherogram from Sanger sequencing of rRNA amplicons for a clonal isolate of Haematococcus pluvialis . Arrows indicate sites of putative intragenomic variation in a relatively short stretch of primary sequence.

Techniques Used: Sequencing

Related Articles

Clone Assay:

Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
Article Snippet: .. In all cases, a defined region of the ZIKV ORF was amplified by PCR using pBac/PRVABC-59 as a template and the appropriate pair of primers listed in : (i) Frag-zC (147 bp), ZikaC-F + ZikaC-R; (ii) Frag-zM (120 bp), ZikaM-F + ZikaM-R; (iii) Frag-zE (147 bp), ZikaE-F + ZikaE-R; (iv) Frag-zNS4A (177 bp), ZikaNS4A-F + ZikaNS4A-R; and (v) Frag-zNS4B (177 bp), ZikaNS4B-F + ZikaNS4B-R. Each of the resulting amplicons was cloned into pGex-4T-1 (GE Healthcare, Piscataway, NJ, USA) by ligating the 4954 bp Eco RI-Xho I fragment of the pGex-4T-1 vector with 135, 108, 135, 165, and 165 bp Eco RI-Xho I fragments of the Frag-zC, -zM, -zE, -zNS4A, and -zNS4B amplicons, respectively. ..

Amplification:

Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
Article Snippet: .. In all cases, a defined region of the ZIKV ORF was amplified by PCR using pBac/PRVABC-59 as a template and the appropriate pair of primers listed in : (i) Frag-zC (147 bp), ZikaC-F + ZikaC-R; (ii) Frag-zM (120 bp), ZikaM-F + ZikaM-R; (iii) Frag-zE (147 bp), ZikaE-F + ZikaE-R; (iv) Frag-zNS4A (177 bp), ZikaNS4A-F + ZikaNS4A-R; and (v) Frag-zNS4B (177 bp), ZikaNS4B-F + ZikaNS4B-R. Each of the resulting amplicons was cloned into pGex-4T-1 (GE Healthcare, Piscataway, NJ, USA) by ligating the 4954 bp Eco RI-Xho I fragment of the pGex-4T-1 vector with 135, 108, 135, 165, and 165 bp Eco RI-Xho I fragments of the Frag-zC, -zM, -zE, -zNS4A, and -zNS4B amplicons, respectively. ..

Agarose Gel Electrophoresis:

Article Title: Altered RNA Editing in Mice Lacking ADAR2 Autoregulation §
Article Snippet: .. Amplicons corresponding to alternatively spliced ADAR2 variants were resolved by 2.5% agarose gel electrophoresis and quantified by phosphorimager analysis (Amersham Biosciences). .. To determine the extents of site-selective editing for different ADAR substrates, first-strand cDNA was synthesized and amplified using gene-specific primers and assessed using a modified primer extension analysis ( ); the primers and mixtures of deoxy- and dideoxynucleotides used in each primer extension assay are listed in Table S1 in the supplemental material.

Purification:

Article Title: Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR
Article Snippet: .. Amplicons with an abnormal elution profile were purified using the GFX PCR and Band Purification Kit (Ge HealthCare, Buckinghamshire, UK), sequenced with the BigDye Terminator Cycle Sequencing Kit v. 1.1 (Applied Biosystems), loaded on ABI 3100 capillaries (Applied Biosystems) and analysed using the Sequencing Analysis software v2.0. .. RT-QPCR Gene Copy Number Analysis A new protocol of RT-QPCR was developed to provide a sensitive method for detecting large deletions encompassing the KRIT1 gene and the 7q21 locus.

Article Title: Large-scale mitogenomics enables insights into Schizophora (Diptera) radiation and population diversity
Article Snippet: .. Amplicons were purified with illustra GFX™ Purification Kit (GE Healthcare) and quantified by Qubit® (Life Technologies). ..

Article Title: Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency
Article Snippet: .. The amplicons were purified with a Sephaglass bandprep kit (Amersham Pharmacia Biotech) and sequenced by PCR with primers regularly distributed along the LTR using the double-stranded DNA cycle sequencing system (Life Technologies). .. Comparing the different BLV LTR sequences reported in the data banks as well as our unpublished results, we were intrigued by the very high conservation of the transcription factor binding sites among the various isolates.

Electrophoresis:

Article Title: Diagnosis of Contagious Bovine Pleuropneumonia by PCR-Laser- Induced Fluorescence and PCR-Restriction Endonuclease Analysis Based on the 16S rRNA Genes of Mycoplasma mycoides subsp. mycoides SC
Article Snippet: .. Electrophoresis and analysis of the amplicons were carried out with the ALFexpress DNA Sequencer and Fragment Manager software (FM v. 1.2; Amersham Pharmacia Biotech). .. A denaturing acrylamide gel (8% acrylamide and 7 M urea) was prepared by adding 1.6 ml of a stock solution of 40% acrylamide to 25 ml of ALF-grade Ready Mix Gel (Amersham Pharmacia Biotech), and the gel was cast in a short gel cassette with a vertical length of 15 cm and a thickness of 0.3 mm.

Polymerase Chain Reaction:

Article Title: Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR
Article Snippet: .. Amplicons with an abnormal elution profile were purified using the GFX PCR and Band Purification Kit (Ge HealthCare, Buckinghamshire, UK), sequenced with the BigDye Terminator Cycle Sequencing Kit v. 1.1 (Applied Biosystems), loaded on ABI 3100 capillaries (Applied Biosystems) and analysed using the Sequencing Analysis software v2.0. .. RT-QPCR Gene Copy Number Analysis A new protocol of RT-QPCR was developed to provide a sensitive method for detecting large deletions encompassing the KRIT1 gene and the 7q21 locus.

Article Title: Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency
Article Snippet: .. The amplicons were purified with a Sephaglass bandprep kit (Amersham Pharmacia Biotech) and sequenced by PCR with primers regularly distributed along the LTR using the double-stranded DNA cycle sequencing system (Life Technologies). .. Comparing the different BLV LTR sequences reported in the data banks as well as our unpublished results, we were intrigued by the very high conservation of the transcription factor binding sites among the various isolates.

Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
Article Snippet: .. In all cases, a defined region of the ZIKV ORF was amplified by PCR using pBac/PRVABC-59 as a template and the appropriate pair of primers listed in : (i) Frag-zC (147 bp), ZikaC-F + ZikaC-R; (ii) Frag-zM (120 bp), ZikaM-F + ZikaM-R; (iii) Frag-zE (147 bp), ZikaE-F + ZikaE-R; (iv) Frag-zNS4A (177 bp), ZikaNS4A-F + ZikaNS4A-R; and (v) Frag-zNS4B (177 bp), ZikaNS4B-F + ZikaNS4B-R. Each of the resulting amplicons was cloned into pGex-4T-1 (GE Healthcare, Piscataway, NJ, USA) by ligating the 4954 bp Eco RI-Xho I fragment of the pGex-4T-1 vector with 135, 108, 135, 165, and 165 bp Eco RI-Xho I fragments of the Frag-zC, -zM, -zE, -zNS4A, and -zNS4B amplicons, respectively. ..

Article Title: HiDRA-seq: High-Throughput SARS-CoV-2 Detection by RNA Barcoding and Amplicon Sequencing
Article Snippet: .. Once the PCR was concluded, the amplicons were cleaned up using Sera-Mag Select beads (GE Healthcare) with a ratio 1:0.8 (DNA:beads). .. The size and concentration of the amplicons was analyzed in a 4200 TapeStation System (Agilent) using a D1000 ScreenTape.

Sequencing:

Article Title: Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR
Article Snippet: .. Amplicons with an abnormal elution profile were purified using the GFX PCR and Band Purification Kit (Ge HealthCare, Buckinghamshire, UK), sequenced with the BigDye Terminator Cycle Sequencing Kit v. 1.1 (Applied Biosystems), loaded on ABI 3100 capillaries (Applied Biosystems) and analysed using the Sequencing Analysis software v2.0. .. RT-QPCR Gene Copy Number Analysis A new protocol of RT-QPCR was developed to provide a sensitive method for detecting large deletions encompassing the KRIT1 gene and the 7q21 locus.

Article Title: Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency
Article Snippet: .. The amplicons were purified with a Sephaglass bandprep kit (Amersham Pharmacia Biotech) and sequenced by PCR with primers regularly distributed along the LTR using the double-stranded DNA cycle sequencing system (Life Technologies). .. Comparing the different BLV LTR sequences reported in the data banks as well as our unpublished results, we were intrigued by the very high conservation of the transcription factor binding sites among the various isolates.

Plasmid Preparation:

Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
Article Snippet: .. In all cases, a defined region of the ZIKV ORF was amplified by PCR using pBac/PRVABC-59 as a template and the appropriate pair of primers listed in : (i) Frag-zC (147 bp), ZikaC-F + ZikaC-R; (ii) Frag-zM (120 bp), ZikaM-F + ZikaM-R; (iii) Frag-zE (147 bp), ZikaE-F + ZikaE-R; (iv) Frag-zNS4A (177 bp), ZikaNS4A-F + ZikaNS4A-R; and (v) Frag-zNS4B (177 bp), ZikaNS4B-F + ZikaNS4B-R. Each of the resulting amplicons was cloned into pGex-4T-1 (GE Healthcare, Piscataway, NJ, USA) by ligating the 4954 bp Eco RI-Xho I fragment of the pGex-4T-1 vector with 135, 108, 135, 165, and 165 bp Eco RI-Xho I fragments of the Frag-zC, -zM, -zE, -zNS4A, and -zNS4B amplicons, respectively. ..

Software:

Article Title: Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR
Article Snippet: .. Amplicons with an abnormal elution profile were purified using the GFX PCR and Band Purification Kit (Ge HealthCare, Buckinghamshire, UK), sequenced with the BigDye Terminator Cycle Sequencing Kit v. 1.1 (Applied Biosystems), loaded on ABI 3100 capillaries (Applied Biosystems) and analysed using the Sequencing Analysis software v2.0. .. RT-QPCR Gene Copy Number Analysis A new protocol of RT-QPCR was developed to provide a sensitive method for detecting large deletions encompassing the KRIT1 gene and the 7q21 locus.

Article Title: Diagnosis of Contagious Bovine Pleuropneumonia by PCR-Laser- Induced Fluorescence and PCR-Restriction Endonuclease Analysis Based on the 16S rRNA Genes of Mycoplasma mycoides subsp. mycoides SC
Article Snippet: .. Electrophoresis and analysis of the amplicons were carried out with the ALFexpress DNA Sequencer and Fragment Manager software (FM v. 1.2; Amersham Pharmacia Biotech). .. A denaturing acrylamide gel (8% acrylamide and 7 M urea) was prepared by adding 1.6 ml of a stock solution of 40% acrylamide to 25 ml of ALF-grade Ready Mix Gel (Amersham Pharmacia Biotech), and the gel was cast in a short gel cassette with a vertical length of 15 cm and a thickness of 0.3 mm.

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    GE Healthcare amplicons
    Coverage plots. ( A ) Coverage of mitogenomes from 16 species of Schizophora generated by long-range PCR and shotgun sequencing. Highlighted regions indicate an overlap (green) and the 16S gap (orange) region between the two <t>amplicons.</t> Gaps in 16S sequence were further closed through standard PCR and Sanger sequencing. ( B ) 16 mitogenomes assembled from short reads generated by whole genome sequencing. Both strategies used short reads from MiSeq or HiSeq Illumina platforms to generate high-quality assemblies. ( C ) Coverage plot of the complete mtDNA assembled with long reads generated with SMRT sequencing technology. The scheme shows the complete mtDNA of C. megacephala (sample F03) assembled with 15,835 bp. First track shows the low GC content (23.5%). Orange bars on the second track refer to the coverage. The innermost track shows gene order in each mtDNA strand. Yellow arrows denote PCGs, green arrows show rRNA subunits and orange arrows refer to tRNAs.
    Amplicons, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 296 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare amplicon library preparation
    Taxonomic composition of the kelp biofilm community . Pie charts showing the relative abundances of sequences associated with bacterial taxa within each sequencing dataset down to order level. Visualizations were done using KRONA (Ondov et al., 2011 ). (A) 16 S rRNA <t>amplicon</t> dataset. Relative abundances were determined using the SILVA NGS pipeline (Quast et al., 2013 ; Yilmaz et al., 2014 ). (B,C) Metagenomic shotgun libraries of KelpA KelpB, respectively. KelpA (B) was derived from the same sample as the amplicon library, while KelpB (C) was derived from a separate sample obtained from a different leaf of the same Kelp specimen, using a slightly modified DNA extraction protocol. Relative abundances were determined based on sequencing read classifications obtained by using the Least Common Ancestor (LCA) approach implemented in MEGAN5 (Huson and Weber, 2013 ). In order to maximize coverage for low abundant community members, both datasets were treated as a composite dataset KelpAB (D) for assembly.
    Amplicon Library Preparation, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Coverage plots. ( A ) Coverage of mitogenomes from 16 species of Schizophora generated by long-range PCR and shotgun sequencing. Highlighted regions indicate an overlap (green) and the 16S gap (orange) region between the two amplicons. Gaps in 16S sequence were further closed through standard PCR and Sanger sequencing. ( B ) 16 mitogenomes assembled from short reads generated by whole genome sequencing. Both strategies used short reads from MiSeq or HiSeq Illumina platforms to generate high-quality assemblies. ( C ) Coverage plot of the complete mtDNA assembled with long reads generated with SMRT sequencing technology. The scheme shows the complete mtDNA of C. megacephala (sample F03) assembled with 15,835 bp. First track shows the low GC content (23.5%). Orange bars on the second track refer to the coverage. The innermost track shows gene order in each mtDNA strand. Yellow arrows denote PCGs, green arrows show rRNA subunits and orange arrows refer to tRNAs.

    Journal: Scientific Reports

    Article Title: Large-scale mitogenomics enables insights into Schizophora (Diptera) radiation and population diversity

    doi: 10.1038/srep21762

    Figure Lengend Snippet: Coverage plots. ( A ) Coverage of mitogenomes from 16 species of Schizophora generated by long-range PCR and shotgun sequencing. Highlighted regions indicate an overlap (green) and the 16S gap (orange) region between the two amplicons. Gaps in 16S sequence were further closed through standard PCR and Sanger sequencing. ( B ) 16 mitogenomes assembled from short reads generated by whole genome sequencing. Both strategies used short reads from MiSeq or HiSeq Illumina platforms to generate high-quality assemblies. ( C ) Coverage plot of the complete mtDNA assembled with long reads generated with SMRT sequencing technology. The scheme shows the complete mtDNA of C. megacephala (sample F03) assembled with 15,835 bp. First track shows the low GC content (23.5%). Orange bars on the second track refer to the coverage. The innermost track shows gene order in each mtDNA strand. Yellow arrows denote PCGs, green arrows show rRNA subunits and orange arrows refer to tRNAs.

    Article Snippet: Amplicons were purified with illustra GFX™ Purification Kit (GE Healthcare) and quantified by Qubit® (Life Technologies).

    Techniques: Generated, Polymerase Chain Reaction, Shotgun Sequencing, Sequencing

    ANKIB1 gene breakpoint determination by RT-QPCR (± standard error mean). Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed square (■), and a closed circle (●), respectively. The ANKIB1 exons on the left of the vertical dotted lines (exon1–10) show the copy number loss of the associated amplicons.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR

    doi: 10.1155/2010/854737

    Figure Lengend Snippet: ANKIB1 gene breakpoint determination by RT-QPCR (± standard error mean). Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed square (■), and a closed circle (●), respectively. The ANKIB1 exons on the left of the vertical dotted lines (exon1–10) show the copy number loss of the associated amplicons.

    Article Snippet: Amplicons with an abnormal elution profile were purified using the GFX PCR and Band Purification Kit (Ge HealthCare, Buckinghamshire, UK), sequenced with the BigDye Terminator Cycle Sequencing Kit v. 1.1 (Applied Biosystems), loaded on ABI 3100 capillaries (Applied Biosystems) and analysed using the Sequencing Analysis software v2.0.

    Techniques: Quantitative RT-PCR

    (a) Haplotype analysis of microsatellites markers from chromosome 7q21.1. The haplotype shared by the affected individuals (black-filled symbol) is “boxed”. In the affected patients, the symbol (∗) indicates the indefinite alleles, located in the deletion. Since the hemizygosity for the non shared alleles in the affected patients, the analysis, shows “homozygosity” for three markers (D7S2409, D7S1813, D7S1789), thus, the deleted alleles are indicated with the same symbol (∗). (b) Measurements of copy number status (± standard error mean) of genes mapping in the 7q21 chromosomal region determined by RT-QPCR. On the Top: the genomic organization of genes. Genes are shown above the horizontal axis, which also indicates the extension and orientation of each gene. Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed quare (■), and a closed circle (●), respectively. The genes between the two vertical gray dotted lines show a hemizigous deletion detectable by the copy number loss of the associated amplicons.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Small Deletion at the 7q21.2 Locus in a CCM Family Detected by Real-Time Quantitative PCR

    doi: 10.1155/2010/854737

    Figure Lengend Snippet: (a) Haplotype analysis of microsatellites markers from chromosome 7q21.1. The haplotype shared by the affected individuals (black-filled symbol) is “boxed”. In the affected patients, the symbol (∗) indicates the indefinite alleles, located in the deletion. Since the hemizygosity for the non shared alleles in the affected patients, the analysis, shows “homozygosity” for three markers (D7S2409, D7S1813, D7S1789), thus, the deleted alleles are indicated with the same symbol (∗). (b) Measurements of copy number status (± standard error mean) of genes mapping in the 7q21 chromosomal region determined by RT-QPCR. On the Top: the genomic organization of genes. Genes are shown above the horizontal axis, which also indicates the extension and orientation of each gene. Results from normal genomic DNA (2 normal controls mean), from the unaffected member of family (I : 1 sample), and in one affected members (II : 1) are visualized by a closed triangle (▲), a closed quare (■), and a closed circle (●), respectively. The genes between the two vertical gray dotted lines show a hemizigous deletion detectable by the copy number loss of the associated amplicons.

    Article Snippet: Amplicons with an abnormal elution profile were purified using the GFX PCR and Band Purification Kit (Ge HealthCare, Buckinghamshire, UK), sequenced with the BigDye Terminator Cycle Sequencing Kit v. 1.1 (Applied Biosystems), loaded on ABI 3100 capillaries (Applied Biosystems) and analysed using the Sequencing Analysis software v2.0.

    Techniques: Quantitative RT-PCR

    Safety evaluation of packaging and vector constructs . A) pΔenv1 transportation from transfected to transduced cells as evaluated by gag p25 PCR using the indicated primers. Lane A: DNA from transfected 293T cells; lane B: no template control; lanes C and D: DNA and RNA from transduced 293T cells; lane E: DNA from mock transfected cells. B) Translocation of the U3 deletion to the 5'LTR, as checked by PCR using primers annealing to the beginning U3 and within the R region of LA34 proviral DNA. Lane A: pΔ00 plasmid (full-length LTR), lane B: no template control; lane C: DNA from transduced 293T cells, lane D: DNA from mock transduced cells. C) Analysis of LTR directed transcription as tested by PCR using primers upstream the GFP promoter. Lane A: DNA of transduced 293T cells; lane B: no template control; lanes C and D: RNA from transduced 293T cells with and without DNase treatment prior to reverse transcription; lanes E and F: RNA from mock transduced cells treated as for C and D. Primer nt position referred to the NC_001482 sequence. M 1 : 100 base-pair ladder, M 2 : Gene ruler 1 Kb DNA ladder (GE Healthcare).

    Journal: Genetic Vaccines and Therapy

    Article Title: Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes

    doi: 10.1186/1479-0556-5-8

    Figure Lengend Snippet: Safety evaluation of packaging and vector constructs . A) pΔenv1 transportation from transfected to transduced cells as evaluated by gag p25 PCR using the indicated primers. Lane A: DNA from transfected 293T cells; lane B: no template control; lanes C and D: DNA and RNA from transduced 293T cells; lane E: DNA from mock transfected cells. B) Translocation of the U3 deletion to the 5'LTR, as checked by PCR using primers annealing to the beginning U3 and within the R region of LA34 proviral DNA. Lane A: pΔ00 plasmid (full-length LTR), lane B: no template control; lane C: DNA from transduced 293T cells, lane D: DNA from mock transduced cells. C) Analysis of LTR directed transcription as tested by PCR using primers upstream the GFP promoter. Lane A: DNA of transduced 293T cells; lane B: no template control; lanes C and D: RNA from transduced 293T cells with and without DNase treatment prior to reverse transcription; lanes E and F: RNA from mock transduced cells treated as for C and D. Primer nt position referred to the NC_001482 sequence. M 1 : 100 base-pair ladder, M 2 : Gene ruler 1 Kb DNA ladder (GE Healthcare).

    Article Snippet: The 5'LTR amplicon was cycle sequenced using the automated ALF ExpressII DNA sequencer (GE Healthcare, Cologno Monzese, Italy).

    Techniques: Plasmid Preparation, Construct, Transfection, Polymerase Chain Reaction, Translocation Assay, Sequencing

    Taxonomic composition of the kelp biofilm community . Pie charts showing the relative abundances of sequences associated with bacterial taxa within each sequencing dataset down to order level. Visualizations were done using KRONA (Ondov et al., 2011 ). (A) 16 S rRNA amplicon dataset. Relative abundances were determined using the SILVA NGS pipeline (Quast et al., 2013 ; Yilmaz et al., 2014 ). (B,C) Metagenomic shotgun libraries of KelpA KelpB, respectively. KelpA (B) was derived from the same sample as the amplicon library, while KelpB (C) was derived from a separate sample obtained from a different leaf of the same Kelp specimen, using a slightly modified DNA extraction protocol. Relative abundances were determined based on sequencing read classifications obtained by using the Least Common Ancestor (LCA) approach implemented in MEGAN5 (Huson and Weber, 2013 ). In order to maximize coverage for low abundant community members, both datasets were treated as a composite dataset KelpAB (D) for assembly.

    Journal: Frontiers in Microbiology

    Article Title: Untangling Genomes of Novel Planctomycetal and Verrucomicrobial Species from Monterey Bay Kelp Forest Metagenomes by Refined Binning

    doi: 10.3389/fmicb.2017.00472

    Figure Lengend Snippet: Taxonomic composition of the kelp biofilm community . Pie charts showing the relative abundances of sequences associated with bacterial taxa within each sequencing dataset down to order level. Visualizations were done using KRONA (Ondov et al., 2011 ). (A) 16 S rRNA amplicon dataset. Relative abundances were determined using the SILVA NGS pipeline (Quast et al., 2013 ; Yilmaz et al., 2014 ). (B,C) Metagenomic shotgun libraries of KelpA KelpB, respectively. KelpA (B) was derived from the same sample as the amplicon library, while KelpB (C) was derived from a separate sample obtained from a different leaf of the same Kelp specimen, using a slightly modified DNA extraction protocol. Relative abundances were determined based on sequencing read classifications obtained by using the Least Common Ancestor (LCA) approach implemented in MEGAN5 (Huson and Weber, 2013 ). In order to maximize coverage for low abundant community members, both datasets were treated as a composite dataset KelpAB (D) for assembly.

    Article Snippet: Amplicon library preparation An aliquot of DNA extract A was diluted to 1 ng/μl with PCR-grade water and subjected to Whole Genome Amplification (WGA) using the illustra™ GenomiPhi™ V2 DNA amplification Kit (GE Healthcare) and a Veriti 96-Well thermal cycler (Applied Biosystems).

    Techniques: Sequencing, Amplification, Next-Generation Sequencing, Derivative Assay, Modification, DNA Extraction