long range polymerase chain reaction Search Results


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  • 99
    New England Biolabs long range pcr
    Long Range Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins special long range polymerase chain reaction
    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).
    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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    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 990 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc long range 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 Pcr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    TaKaRa la polymerase chain reaction pcr kit
    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
    La Polymerase Chain Reaction Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc long range polymerase chain reaction trusight hla v2 sequencing panel
    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 Trusight Hla V2 Sequencing Panel, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche long expand range 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 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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    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 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kapa Biosystems kapa long range pcr
    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
    Kapa Long Range Pcr, supplied by Kapa Biosystems, 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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    Thermo Fisher long range phusion pcr
    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 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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    TaKaRa long range pcr enzyme
    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 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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    Thermo Fisher long range pcr system
    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 System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen long range pcr
    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, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 89/100, based on 20 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

    Assessment of duplications of mitochondrial genome. Duplication PCR assay was carried out using 1056F forward (‘F’) and 1144R reverse (‘R’) primers (schematic of mtDNA molecule) on muscle homogenate DNA samples available from seven sIBM patients (‘P5’, ‘P6’, ‘P7’, ‘P8’, ‘P10’, ‘P11’ and ‘P13’). Two control DNA samples known to contain duplications (‘c1’ and ‘c2’) and two samples from patients with single mtDNA deletion (‘s1’ and ‘s2’) were also tested. Agarose gels from several independent experiments are presented. Large products indicate presence of duplicated or partially-duplicated mitochondrial genomes.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Assessment of duplications of mitochondrial genome. Duplication PCR assay was carried out using 1056F forward (‘F’) and 1144R reverse (‘R’) primers (schematic of mtDNA molecule) on muscle homogenate DNA samples available from seven sIBM patients (‘P5’, ‘P6’, ‘P7’, ‘P8’, ‘P10’, ‘P11’ and ‘P13’). Two control DNA samples known to contain duplications (‘c1’ and ‘c2’) and two samples from patients with single mtDNA deletion (‘s1’ and ‘s2’) were also tested. Agarose gels from several independent experiments are presented. Large products indicate presence of duplicated or partially-duplicated mitochondrial genomes.

    Article Snippet: Long range PCR Long range PCR was carried out using LA Taq DNA polymerase or PrimeSTAR GXL DNA polymerase (TaKaRa, Clontech) on DNA samples extracted from individual myofibres and homogenate muscle samples.

    Techniques: Polymerase Chain Reaction

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw382

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

    Article Snippet: Long range PCR Long range PCR was carried out using LA Taq DNA polymerase or PrimeSTAR GXL DNA polymerase (TaKaRa, Clontech) on DNA samples extracted from individual myofibres and homogenate muscle samples.

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

    Long range PCR analysis of single myofibre mtDNA. Agarose gel images of 16kb long range PCRs carried out with single cell DNA extracts. The cells were selected based on triplex real-time PCR results; mostly MT-ND1 and MT-ND1 MT-ND4 deletions were targeted. Each lane is labelled with patient number/cell number (e.g. ‘P9/1’) and each amplified PCR product has a unique identifier: ‘A, B, C’ etc. which is maintained throughout the manuscript. In order to ensure amplification of full-length products with this PCR, whole blood DNA from a healthy individual was used (‘wt’).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw382

    Figure Lengend Snippet: Long range PCR analysis of single myofibre mtDNA. Agarose gel images of 16kb long range PCRs carried out with single cell DNA extracts. The cells were selected based on triplex real-time PCR results; mostly MT-ND1 and MT-ND1 MT-ND4 deletions were targeted. Each lane is labelled with patient number/cell number (e.g. ‘P9/1’) and each amplified PCR product has a unique identifier: ‘A, B, C’ etc. which is maintained throughout the manuscript. In order to ensure amplification of full-length products with this PCR, whole blood DNA from a healthy individual was used (‘wt’).

    Article Snippet: Long range PCR Long range PCR was carried out using LA Taq DNA polymerase or PrimeSTAR GXL DNA polymerase (TaKaRa, Clontech) on DNA samples extracted from individual myofibres and homogenate muscle samples.

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw382

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

    Article Snippet: Long range PCR Long range PCR was carried out using LA Taq DNA polymerase or PrimeSTAR GXL DNA polymerase (TaKaRa, Clontech) on DNA samples extracted from individual myofibres and homogenate muscle samples.

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw382

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

    Article Snippet: Long range PCR Long range PCR was carried out using LA Taq DNA polymerase or PrimeSTAR GXL DNA polymerase (TaKaRa, Clontech) on DNA samples extracted from individual myofibres and homogenate muscle samples.

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw382

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

    Article Snippet: Long range PCR Long range PCR was carried out using LA Taq DNA polymerase or PrimeSTAR GXL DNA polymerase (TaKaRa, Clontech) on DNA samples extracted from individual myofibres and homogenate muscle samples.

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

    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

    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

    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