long-range pcr Search Results


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  • 88
    TaKaRa long range polymerase chain reaction pcr
    An SVA retrotransposal insertion induces abnormal splicing in <t>FCMD</t> a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of <t>RT-PCR</t> product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).
    Long Range Polymerase Chain Reaction Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Illumina Inc long range polymerase chain reaction pcr
    Laboratory procedure used to obtain MHC II <t>DRB</t> short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: <t>PCR</t> primers
    Long Range Polymerase Chain Reaction Pcr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Eurofins special long range polymerase chain reaction
    Laboratory procedure used to obtain MHC II <t>DRB</t> short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: <t>PCR</t> primers
    Special Long Range Polymerase Chain Reaction, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Illumina Inc sequencing long range polymerase chain reaction pcr
    Laboratory procedure used to obtain MHC II <t>DRB</t> short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: <t>PCR</t> primers
    Sequencing Long Range Polymerase Chain Reaction Pcr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche long range pcr
    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective <t>HLA</t> gene haplotype phasing. The size selection also determines an actual molar concentration for bridge <t>PCR</t> to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
    Long Range Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 695 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen long range pcr
    <t>RT-PCR</t> analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the <t>MHO_710–770</t> gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).
    Long Range Pcr, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche long range pcr kit
    IL-1β induction and chromatin structure of cPLA 2 <t>α</t> in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time <t>RT-PCR.</t> Relative fold induction was calculated
    Long Range Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher long range pcr kit
    IL-1β induction and chromatin structure of cPLA 2 <t>α</t> in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time <t>RT-PCR.</t> Relative fold induction was calculated
    Long Range Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche long range pcr kits
    IL-1β induction and chromatin structure of cPLA 2 <t>α</t> in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time <t>RT-PCR.</t> Relative fold induction was calculated
    Long Range Pcr Kits, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher long range pcr system
    Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern. The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the <t>CNVs</t> were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified <t>PCR</t> product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.
    Long Range Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher long range phusion pcr
    Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern. The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the <t>CNVs</t> were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified <t>PCR</t> product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.
    Long Range Phusion Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa long range pcr enzyme
    Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern. The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the <t>CNVs</t> were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified <t>PCR</t> product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.
    Long Range Pcr Enzyme, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche long expand range pcr
    Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern. The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the <t>CNVs</t> were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified <t>PCR</t> product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.
    Long Expand Range Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    An SVA retrotransposal insertion induces abnormal splicing in FCMD a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of RT-PCR product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).

    Journal: Nature

    Article Title: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

    doi: 10.1038/nature10456

    Figure Lengend Snippet: An SVA retrotransposal insertion induces abnormal splicing in FCMD a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of RT-PCR product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).

    Article Snippet: To detect abnormally-spliced RT-PCR products from FCMD, ARH, and NLSDM patients, and from human brain AB627340 cDNA, long range PCR was performed using LA Taq with LA Taq Buffer II (Takara), adding dimethyl sulfoxide and 7-deaza-dGTP (Roche).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Northern Blot, Mouse Assay

    AON cocktail rescues normal fukutin mRNA a, RT-PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black closed arrow, a common forward primer located on fukutin coding region; black open arrow, a reverse primer to detect the abnormal RT-PCR product (161 bp); gray closed arrow, the other reverse primer to detect the restored normal RT-PCR product (129 bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated in Hp/Hp mice and Hp/− mice. Local injection of AED cocktail into TA (n=3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n=2) and myotubes (right, n=2). The Y axis shows the percent recovery of normal mRNA (* p

    Journal: Nature

    Article Title: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

    doi: 10.1038/nature10456

    Figure Lengend Snippet: AON cocktail rescues normal fukutin mRNA a, RT-PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black closed arrow, a common forward primer located on fukutin coding region; black open arrow, a reverse primer to detect the abnormal RT-PCR product (161 bp); gray closed arrow, the other reverse primer to detect the restored normal RT-PCR product (129 bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated in Hp/Hp mice and Hp/− mice. Local injection of AED cocktail into TA (n=3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n=2) and myotubes (right, n=2). The Y axis shows the percent recovery of normal mRNA (* p

    Article Snippet: To detect abnormally-spliced RT-PCR products from FCMD, ARH, and NLSDM patients, and from human brain AB627340 cDNA, long range PCR was performed using LA Taq with LA Taq Buffer II (Takara), adding dimethyl sulfoxide and 7-deaza-dGTP (Roche).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Injection, Negative Control

    DNA methylation profiles of three CpG regions in bovine RTEL . Three regions of CpG island were chosen for DNA methylation analysis, CpG region 1 (R1) and 2 (R2) are located in promoter region and CpG region control (RC) is in exon 2 and intron 2. A. Relative positions of three CpG regions be analyzed. Position of the islands are indicated by thick lines on the top, related genomic region and ATG site are indicated by boxes and arrow (not to scale), exon1 and exon1' mean alternative first exon and the arrows bellow indicate two putative transcription start sites. B. DNA methylation profile of the three selected regions. The cycles in a string mean the average methylation level of specific sites in each CpG region, the numbers above indicate the relative position of each CpG dinucleotides. Analyzed tissues (testis, spleen and heart) are indicated on the left. For the convenience to compare, CpG regions in the promoter regions (R1 and R2) and internal region are show separately. In each string, the open cycles indicate that the CpG sites are hypomethylated in all the clones that analyzed whereas closed cycles mean fully methylated CpG dinucleotides, other partially blacked cycles indicate different methylation level, details are shown in the right of the bottom. The average methylation level was calculated by sequencing ten individual clones of the PCRs. Besides the methylation level of each CpG sites, the percentages of DNA methylation in the indicated region of the CpG regions are also shown on the right (the data of R1 and R2 are incorporated to represent the methylation level of the promoter region). Only differential methylated CpG sites in three detected CpG regions are shown in the figure.

    Journal: BMC Molecular Biology

    Article Title: Identification and characterization of bovine regulator of telomere length elongation helicase gene (RTEL): molecular cloning, expression distribution, splice variants and DNA methylation profile

    doi: 10.1186/1471-2199-8-18

    Figure Lengend Snippet: DNA methylation profiles of three CpG regions in bovine RTEL . Three regions of CpG island were chosen for DNA methylation analysis, CpG region 1 (R1) and 2 (R2) are located in promoter region and CpG region control (RC) is in exon 2 and intron 2. A. Relative positions of three CpG regions be analyzed. Position of the islands are indicated by thick lines on the top, related genomic region and ATG site are indicated by boxes and arrow (not to scale), exon1 and exon1' mean alternative first exon and the arrows bellow indicate two putative transcription start sites. B. DNA methylation profile of the three selected regions. The cycles in a string mean the average methylation level of specific sites in each CpG region, the numbers above indicate the relative position of each CpG dinucleotides. Analyzed tissues (testis, spleen and heart) are indicated on the left. For the convenience to compare, CpG regions in the promoter regions (R1 and R2) and internal region are show separately. In each string, the open cycles indicate that the CpG sites are hypomethylated in all the clones that analyzed whereas closed cycles mean fully methylated CpG dinucleotides, other partially blacked cycles indicate different methylation level, details are shown in the right of the bottom. The average methylation level was calculated by sequencing ten individual clones of the PCRs. Besides the methylation level of each CpG sites, the percentages of DNA methylation in the indicated region of the CpG regions are also shown on the right (the data of R1 and R2 are incorporated to represent the methylation level of the promoter region). Only differential methylated CpG sites in three detected CpG regions are shown in the figure.

    Article Snippet: To amplify RTEL, long-range PCRs (TaKaRa LA Taq™) were performed using five sets of primers complement to the exonic regions.

    Techniques: DNA Methylation Assay, Methylation, Clone Assay, Sequencing

    Laboratory procedure used to obtain MHC II DRB short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: PCR primers

    Journal: Heredity

    Article Title: A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)

    doi: 10.1038/s41437-018-0070-5

    Figure Lengend Snippet: Laboratory procedure used to obtain MHC II DRB short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: PCR primers

    Article Snippet: More in detail, we designed primers on DRB exons 1 and 3 and set-up a long-range PCR coupled with Illumina NGS sequencing to a very high coverage.

    Techniques: Amplification, Sequencing, Gel Extraction, Polymerase Chain Reaction

    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Journal: BMC Genomics

    Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

    doi: 10.1186/1471-2164-14-355

    Figure Lengend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Article Snippet: The six highly polymorphic HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1 ) were amplified by long-range PCR and the PCR amplicons covering full sequences of the genes were subjected to the MiSeq sequencer via the transposase-based library preparation.

    Techniques: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

    The chicken/human chimeric IgG expression construct. ( A ) Strategy for the knock-in of the human IgHG1 constant region at the chicken IgHM locus. The chicken IgHM region from the exon C H 1 to C H 3 was cloned to make the targeting vector. The intronic region digested by BseRI was replaced by the human IgHG1 region from H to C H 3 and a selectable marker. Bsd = blasticidin S deaminase. p(A) = polyadenylation sites. Genomic size and distances are not to scale (distance of chicken C H 1 to C H 2 = 4.5 kb; human H-C H 2-C H 3 insert = 1 kb). ( B ) Final genomic structure after integration of the knock-in plasmid and expected mRNA variants produced by alternative splicing. Thick horizontal bars indicate the approximate positions of the primers for PCR and RT–PCR used to confirm genomic integration and expression of IgHM and IgHG1 transcripts. ( C ) Expression of IgHM and of chimeric IgHG1 detected by RT–PCR in the wild-type parental strain (WT) and a positive chimeric transformant (CX13). ( D ) Schematic outline of the ADLib system applied to the selection of chimeric human IgG. (From left to right) The enhancement of sequence diversification at the Ig variable locus in TSA-treated DT40 cultures allows the generation of an autonomously diversifying library of cells expressing various surface IgM; the clones specific for the target antigen are isolated using antigen-coated magnetic beads; CX13 cells co-express secreted-form chicken IgM and chimeric IgG with the same antigen-binding domain; antigen-specific chimeric IgG can therefore be isolated directly from the culture supernatant for further use.

    Journal: Nucleic Acids Research

    Article Title: B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies

    doi: 10.1093/nar/gkq1122

    Figure Lengend Snippet: The chicken/human chimeric IgG expression construct. ( A ) Strategy for the knock-in of the human IgHG1 constant region at the chicken IgHM locus. The chicken IgHM region from the exon C H 1 to C H 3 was cloned to make the targeting vector. The intronic region digested by BseRI was replaced by the human IgHG1 region from H to C H 3 and a selectable marker. Bsd = blasticidin S deaminase. p(A) = polyadenylation sites. Genomic size and distances are not to scale (distance of chicken C H 1 to C H 2 = 4.5 kb; human H-C H 2-C H 3 insert = 1 kb). ( B ) Final genomic structure after integration of the knock-in plasmid and expected mRNA variants produced by alternative splicing. Thick horizontal bars indicate the approximate positions of the primers for PCR and RT–PCR used to confirm genomic integration and expression of IgHM and IgHG1 transcripts. ( C ) Expression of IgHM and of chimeric IgHG1 detected by RT–PCR in the wild-type parental strain (WT) and a positive chimeric transformant (CX13). ( D ) Schematic outline of the ADLib system applied to the selection of chimeric human IgG. (From left to right) The enhancement of sequence diversification at the Ig variable locus in TSA-treated DT40 cultures allows the generation of an autonomously diversifying library of cells expressing various surface IgM; the clones specific for the target antigen are isolated using antigen-coated magnetic beads; CX13 cells co-express secreted-form chicken IgM and chimeric IgG with the same antigen-binding domain; antigen-specific chimeric IgG can therefore be isolated directly from the culture supernatant for further use.

    Article Snippet: Plasmid constructs For the chimeric ch/hu-IgHG1 construct, we first amplified the full-length human IgHG1 constant region (about 3 kb comprising the exons CH 1, H (hinge), CH 2, CH 3 and the downstream polyadenylation signal) and parts of the chicken IgHM constant region from human genomic DNA using the expand long range PCR system (Roche).

    Techniques: Expressing, Construct, Knock-In, Clone Assay, Plasmid Preparation, Marker, Produced, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Selection, Sequencing, Isolation, Magnetic Beads, Binding Assay

    RT-PCR analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Journal: PLoS ONE

    Article Title: Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection

    doi: 10.1371/journal.pone.0181383

    Figure Lengend Snippet: RT-PCR analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Article Snippet: Overlapping regions of the MHO genes 720–770 were amplified using the Long Range PCR Kit (Qiagen, Hilden, Germany) by standard PCR conditions (initial cycle of 3 min at 93°C; 35 cycles of 15 s at 93°C, 30 s at 50°C, 10 min at 68°C).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Schematic representation of of CLDN6, CLDN9, OCLN and SCARB1 genes. Exons are represented by numbered gray rectangles and introns or non coding regions by double blue lines. Positions of primers pairs used for direct genomic sequencing are shown. Reference sequences of the transcripts are NM_021195.4 (CLDN6), NM_020982.3 (CLDN9), NM_002538.3 (OCLN) and NM_005505.4 (SCARB1). The numbering starts at the first base of the initiation codon ATG, and stops at the first base of the termination codon of the corresponding transcripts. The sequences of corresponding primers are shown in Table 1 and Table 2 . F: Forward primer, R: Reverse primer, LR: Long Range PCR primer. SNPs identified by direct genomic sequencing are indicated, in red SNPs specific of the case population HIV+ HCV-.

    Journal: PLoS ONE

    Article Title: Identification of Variants of Hepatitis C Virus (HCV) Entry Factors in Patients Highly Exposed to HCV but Remaining Uninfected: An ANRS Case-Control Study

    doi: 10.1371/journal.pone.0142698

    Figure Lengend Snippet: Schematic representation of of CLDN6, CLDN9, OCLN and SCARB1 genes. Exons are represented by numbered gray rectangles and introns or non coding regions by double blue lines. Positions of primers pairs used for direct genomic sequencing are shown. Reference sequences of the transcripts are NM_021195.4 (CLDN6), NM_020982.3 (CLDN9), NM_002538.3 (OCLN) and NM_005505.4 (SCARB1). The numbering starts at the first base of the initiation codon ATG, and stops at the first base of the termination codon of the corresponding transcripts. The sequences of corresponding primers are shown in Table 1 and Table 2 . F: Forward primer, R: Reverse primer, LR: Long Range PCR primer. SNPs identified by direct genomic sequencing are indicated, in red SNPs specific of the case population HIV+ HCV-.

    Article Snippet: To avoid amplification of the pseudogene for exons 6 to 9 of the OCLN gene, long-range (LR) PCR (Qiagen LongRange PCR Kit) was used as an intermediate step before PCR amplification of the exon.

    Techniques: Genomic Sequencing, Polymerase Chain Reaction

    IL-1β induction and chromatin structure of cPLA 2 α in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time RT-PCR. Relative fold induction was calculated

    Journal: Journal of Lipid Research

    Article Title: A distal enhancer controls cytokine-dependent human cPLA2? gene expression [S]

    doi: 10.1194/jlr.M037382

    Figure Lengend Snippet: IL-1β induction and chromatin structure of cPLA 2 α in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time RT-PCR. Relative fold induction was calculated

    Article Snippet: The −14, −5.5, and −4.1 kb cPLA2 α promoters were amplified from using the Long Range PCR kit (Roche), with the forward primers 5′-AGAGTTGGGATGGAGAAGGTTG-3′, 5′-TGCAAAGTGCCTGCCAGTC-3′, or 5′-GATGGAGATGGCAGTGGCAG-3′, respectively, and the reverse primer 5′-GCTTACAGTTCCCAGAGTTACC-3′, and cloned into the TOPO-XL vector.

    Techniques: Quantitative RT-PCR

    Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern. The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the CNVs were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified PCR product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.

    Journal: PLoS Genetics

    Article Title: A Complex Structural Variation on Chromosome 27 Leads to the Ectopic Expression of HOXB8 and the Muffs and Beard Phenotype in Chickens

    doi: 10.1371/journal.pgen.1006071

    Figure Lengend Snippet: Analyzing the breakpoints of the copy number variations on GGA27 to clarify the rearrangement pattern. The duplicated regions identified by array-CGH are illustrated by green (CNV1), blue (CNV2) and pink (CNV3) boxes, respectively. (A) The boundaries of the CNVs were tested using 8 primers indicated by the arrows located in the known duplicated region of CNV1 and CNV3. All possible amplifications were considered and performed in both Mb and wild-type chickens. (B) The breakpoints of both CNV1_3’ (1,721,521 bp) and CNV3_5’ (4,470,331 bp) were identified after sequencing the specifically-amplified PCR product obtained in Mb chickens using primer F1 and R2. An overlap of two nucleotides was detected in the junction region. Outward facing primers (green and pink arrows) were designed to analyze the other boundary of CNV1 and CNV3. (C) CNV2 was found to be located next to CNV3 in chickens with the Mb phenotype using a genome-walking strategy. The breakpoints of both CNV3_3’ (4,503,417 bp) and CNV2_5’ (3,578,409 bp) were verified by sequencing. A two-nucleotide insertion was found in the junction region. (D) The breakpoints of both CNV2_3’ (3,592,890 bp) and CNV1_5’ (1,702,269 bp) were confirmed through unmapped read alignment of whole genome re-sequencing data. An eight-base insertion was detected in the junction.

    Article Snippet: Re-sequencing of targeted genomic regions Re-sequencing of the three identified CNVs sequences was performed using long-range PCR and Ion Torrent Technology.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction