polymerase chain reaction restriction fragment length polymorphism pcr rflp assay  (Thermo Fisher)


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    Thermo Fisher polymerase chain reaction restriction fragment length polymorphism pcr rflp assay
    Polymerase Chain Reaction Restriction Fragment Length Polymorphism Pcr Rflp Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polymerase Chain Reaction:

    Article Title: Investigation on the role of VEGF gene polymorphisms in the risk of osteosarcoma
    Article Snippet: .. The VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C gene polymorphisms were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay according to manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). .. The PCR primers of VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C were designed by Sequenom Assay Design 3.1 software (Sequenom Inc., San Diego, CA).

    Article Title: Strong association between the interleukin-8-251A/T polymorphism and coronary artery disease risk
    Article Snippet: .. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay was used in 6 studies, 2 studies used TaqMan (Applied Biosystems, Foster City, CA) method, and 1 study used Matrix Adsorbed Laser Desorption-Ionisation-Time of Flight (MALDI-TOF) to detect the genotypes. ..

    Article Title: Association of GSTP1 and XRCC1 gene polymorphisms with clinical outcome of advanced non-small cell lung cancer patients with cisplatin-based chemotherapy
    Article Snippet: .. Genotypes of GSTP1 A313G, XRCC1 Arg194Trp, Arg280His and Arg399Gln were conducted using Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) assay (Applied Biosystems, Foster City, CA, USA). ..

    Article Title: Polymorphism of the dopamine transporter type 1 gene modifies the treatment response in Parkinson’s disease
    Article Snippet: .. The catechol-O-methyltransferase ( COMT ) VAL158MET variant (rs4608) was analysed using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, as described previously ( ) The MAOB rs1799836 was analysed using a PCR-RFLP assay in a total volume of 25 µl of AmpliTaq PCR buffer containing 50 ng genomic DNA, one unit of AmpliTaq polymerase (Applied Biosystems), 1.50 mM MgCl2 , 250 µM dNTPs, and 15 pmol of forward primer (5′-TTCTGGCCTTTACCTTGGTG-3′) and reverse primer (5′-GCCAGATTTCATCCTCTGGA-3′). .. Amplification products were resolved in 5% acrylamide gels.

    Variant Assay:

    Article Title: Polymorphism of the dopamine transporter type 1 gene modifies the treatment response in Parkinson’s disease
    Article Snippet: .. The catechol-O-methyltransferase ( COMT ) VAL158MET variant (rs4608) was analysed using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, as described previously ( ) The MAOB rs1799836 was analysed using a PCR-RFLP assay in a total volume of 25 µl of AmpliTaq PCR buffer containing 50 ng genomic DNA, one unit of AmpliTaq polymerase (Applied Biosystems), 1.50 mM MgCl2 , 250 µM dNTPs, and 15 pmol of forward primer (5′-TTCTGGCCTTTACCTTGGTG-3′) and reverse primer (5′-GCCAGATTTCATCCTCTGGA-3′). .. Amplification products were resolved in 5% acrylamide gels.

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    Thermo Fisher rflp analysis
    Detection of SNP profile and haplotype distribution within the ANXA5 promoter region in Estonia and Denmark. (A) Genomic context of the ANXA5 gene. Chromosomal positions are based on hg19. (B) Location of identified single nucleotide polymorphisms (SNPs) within the resequenced region of ANXA5 core promoter. SNP positions are given according to the initially reported [ 10 ] first transcription start site (arrow) of the non-conserved untranslated first exon (blue box). Common SNPs are denoted with black and rare singleton SNPs with white triangles. SNP nomenclature according to dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ) is the following: -19G/A, rs112782763; 1A/C, rs28717001; 27T/C, rs28651243; 76G/A, rs113588187. In the current human genome assembly ( http://www.ensembl.org/ ), the determined transcription start site is shifted for all annotated ANXA5 transcripts and only rs113588187 is located within the transcribed region. (C) Linkage disequilibrium (r 2 ) between pairs of SNPs within the resequenced region of the ANXA5 promoter. The order of SNPs is given according to the direction of transcription. Black box indicates complete LD between a pair of SNPs. (D) Distribution of haplotypes identified within the core promoter of Estonian subjects based on haplotype reconstruction analysis with all common SNPs (n = 5) or four common SNPs within the LD block. Haplotype phasing of either five or four SNPs yielded identical results. (E) Detection and observed prevalence of M2 haplotype by Restriction Fragment Length Polymorphism <t>(RFLP)</t> analysis targeting the M2 tag-SNP 76G/A (rs113588187). In case of a GG homozygote at position 76, RFLP analysis results in two fragments (188 bp and 106 bp), three fragments are detected in subjects heterozygous for the M2 haplotype (294 bp, 188 bp and 106 bp), whereas one uncut fragment is observed in homozygous carriers of M2 haplotype (294 bp). Minor allele of the 76G/A tag-SNP defining the M2 haplotype is denoted in red. M, molecular weight marker, 100 bp DNA Ladder (Solid Biodyne). <t>PCR,</t> uncut PCR product not subjected to RFLP analysis. N, number of subjects. *Fisher’s exact P -value.
    Rflp Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher lbcpf1 grna
    DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated exon skipping. ( A ) Two gRNAs [either <t>gRNA</t> (g2 or g3), which target intron 50, and the other (g1), which targets exon 51] were used to direct Cpf1-mediated removal of the exon 51 splice acceptor site. ( B ) T7E1 assay using 293T cells transfected with <t>LbCpf1</t> and gRNA2 (g2) or gRNA3 (g3) shows cleavage of the DMD locus at intron 50. Red arrowheads denote cleavage products. M, marker; Ctrl, control. ( C ) PCR products of genomic DNA isolated from DMD-iPSCs transfected with a plasmid expressing LbCpf1, g1 + g2, and GFP. The lower band (red arrowhead) indicates removal of the exon 51 splice acceptor site. ( D ) Sequence of the lower PCR band from (C) shows a 200-bp deletion, spanning from the 3′ end of intron 50 to the 5′ end of exon 51. This confirms removal of the “ag” splice acceptor of exon 51. The sequence of the uncorrected allele is shown above that of the LbCpf1-edited allele. ( E ) RT-PCR of iPSC-derived cardiomyocytes using primer sets described in Fig. 2B . The 700-bp band in the WT lane is the dystrophin transcript from exons 47 to 52; the 300-bp band in the uncorrected lane is the dystrophin transcript from exons 47 to 52 with exon 48 to 50 deletion; and the lower band in the g1 + g2 mixture lane (edited by LbCpf1) shows exon 51 skipping. ( F )Sequence of the lower band from (E) (g1 + g2 mixture lane) confirms skipping of exon 51, which reframed the DMD ORF. ( G ) Western blot analysis shows dystrophin protein expression in iPSC-derived cardiomyocyte mixtures after exon 51 skipping by LbCpf1 with g1 + g2. αMHC is loading control. ( H ) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures following Cpf1-mediated exon skipping with g1 + g2 gRNA compared to WT and uncorrected cardiomyocyte mixtures. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm.
    Lbcpf1 Grna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher its1 pcr
    <t>PCR</t> patterns of amplified <t>ITS1</t> ribosomal region. Lane M: 50 bp ladder molecular weight marker; lane 1 (a, b): negative control; lanes 2-8 (a) and 2-7 (b) denoted to different fluke samples amplified as a single band of 680 bp of bovine and buffalo in Nghe An province.
    Its1 Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection of SNP profile and haplotype distribution within the ANXA5 promoter region in Estonia and Denmark. (A) Genomic context of the ANXA5 gene. Chromosomal positions are based on hg19. (B) Location of identified single nucleotide polymorphisms (SNPs) within the resequenced region of ANXA5 core promoter. SNP positions are given according to the initially reported [ 10 ] first transcription start site (arrow) of the non-conserved untranslated first exon (blue box). Common SNPs are denoted with black and rare singleton SNPs with white triangles. SNP nomenclature according to dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ) is the following: -19G/A, rs112782763; 1A/C, rs28717001; 27T/C, rs28651243; 76G/A, rs113588187. In the current human genome assembly ( http://www.ensembl.org/ ), the determined transcription start site is shifted for all annotated ANXA5 transcripts and only rs113588187 is located within the transcribed region. (C) Linkage disequilibrium (r 2 ) between pairs of SNPs within the resequenced region of the ANXA5 promoter. The order of SNPs is given according to the direction of transcription. Black box indicates complete LD between a pair of SNPs. (D) Distribution of haplotypes identified within the core promoter of Estonian subjects based on haplotype reconstruction analysis with all common SNPs (n = 5) or four common SNPs within the LD block. Haplotype phasing of either five or four SNPs yielded identical results. (E) Detection and observed prevalence of M2 haplotype by Restriction Fragment Length Polymorphism (RFLP) analysis targeting the M2 tag-SNP 76G/A (rs113588187). In case of a GG homozygote at position 76, RFLP analysis results in two fragments (188 bp and 106 bp), three fragments are detected in subjects heterozygous for the M2 haplotype (294 bp, 188 bp and 106 bp), whereas one uncut fragment is observed in homozygous carriers of M2 haplotype (294 bp). Minor allele of the 76G/A tag-SNP defining the M2 haplotype is denoted in red. M, molecular weight marker, 100 bp DNA Ladder (Solid Biodyne). PCR, uncut PCR product not subjected to RFLP analysis. N, number of subjects. *Fisher’s exact P -value.

    Journal: PLoS ONE

    Article Title: Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe

    doi: 10.1371/journal.pone.0131606

    Figure Lengend Snippet: Detection of SNP profile and haplotype distribution within the ANXA5 promoter region in Estonia and Denmark. (A) Genomic context of the ANXA5 gene. Chromosomal positions are based on hg19. (B) Location of identified single nucleotide polymorphisms (SNPs) within the resequenced region of ANXA5 core promoter. SNP positions are given according to the initially reported [ 10 ] first transcription start site (arrow) of the non-conserved untranslated first exon (blue box). Common SNPs are denoted with black and rare singleton SNPs with white triangles. SNP nomenclature according to dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ) is the following: -19G/A, rs112782763; 1A/C, rs28717001; 27T/C, rs28651243; 76G/A, rs113588187. In the current human genome assembly ( http://www.ensembl.org/ ), the determined transcription start site is shifted for all annotated ANXA5 transcripts and only rs113588187 is located within the transcribed region. (C) Linkage disequilibrium (r 2 ) between pairs of SNPs within the resequenced region of the ANXA5 promoter. The order of SNPs is given according to the direction of transcription. Black box indicates complete LD between a pair of SNPs. (D) Distribution of haplotypes identified within the core promoter of Estonian subjects based on haplotype reconstruction analysis with all common SNPs (n = 5) or four common SNPs within the LD block. Haplotype phasing of either five or four SNPs yielded identical results. (E) Detection and observed prevalence of M2 haplotype by Restriction Fragment Length Polymorphism (RFLP) analysis targeting the M2 tag-SNP 76G/A (rs113588187). In case of a GG homozygote at position 76, RFLP analysis results in two fragments (188 bp and 106 bp), three fragments are detected in subjects heterozygous for the M2 haplotype (294 bp, 188 bp and 106 bp), whereas one uncut fragment is observed in homozygous carriers of M2 haplotype (294 bp). Minor allele of the 76G/A tag-SNP defining the M2 haplotype is denoted in red. M, molecular weight marker, 100 bp DNA Ladder (Solid Biodyne). PCR, uncut PCR product not subjected to RFLP analysis. N, number of subjects. *Fisher’s exact P -value.

    Article Snippet: The PCR product (294 bp) was subjected to RFLP analysis using BamHI restriction enzyme (Thermo Scientific, USA) that exhibits no sensitivity to DNA methylation and specifically cuts in case of the major allele G at position 76 ( ; ).

    Techniques: Blocking Assay, Molecular Weight, Marker, Polymerase Chain Reaction

    PCR–RFLP restriction products of the GSTP1 gene. Lanes 1 and 2 represent products of wild-type (Ile/Ile) genotype, lanes 3, 4, and 5 represent heterozygous (Ile/Val) while lane 6 indicates homozygous (Val/Val) genotype; M, DNA Q2 marker; N, negative control.

    Journal: Frontiers in Psychiatry

    Article Title: Autism Spectrum Disorders and Perinatal Complications—Is Oxidative Stress the Connection?

    doi: 10.3389/fpsyt.2019.00675

    Figure Lengend Snippet: PCR–RFLP restriction products of the GSTP1 gene. Lanes 1 and 2 represent products of wild-type (Ile/Ile) genotype, lanes 3, 4, and 5 represent heterozygous (Ile/Val) while lane 6 indicates homozygous (Val/Val) genotype; M, DNA Q2 marker; N, negative control.

    Article Snippet: For RFLP analysis, 5 μl of PCR product was digested overnight at 37°C with 2 U of restriction enzyme EarI and 1xTango Buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in total volume of 15 μl.

    Techniques: Polymerase Chain Reaction, Marker, Negative Control

    PCR-RFLP restriction products of the GSTA1 gene. Lanes 1 and 2 represent PCR products of GSTA1 *CC genotype (400 bp bands); lanes 3, 5, 6, and 7 represent PCR-RFLP restriction products of GSTA1 *CT genotype (400 bp, 308 bp, 92 bp bands); Lane 4 comprises RFLP-PCR restriction products of GSTA1 *TT genotype (308 bp, 92 bp bands); M, DNA marker; N, negative control without a DNA content.

    Journal: Frontiers in Psychiatry

    Article Title: Autism Spectrum Disorders and Perinatal Complications—Is Oxidative Stress the Connection?

    doi: 10.3389/fpsyt.2019.00675

    Figure Lengend Snippet: PCR-RFLP restriction products of the GSTA1 gene. Lanes 1 and 2 represent PCR products of GSTA1 *CC genotype (400 bp bands); lanes 3, 5, 6, and 7 represent PCR-RFLP restriction products of GSTA1 *CT genotype (400 bp, 308 bp, 92 bp bands); Lane 4 comprises RFLP-PCR restriction products of GSTA1 *TT genotype (308 bp, 92 bp bands); M, DNA marker; N, negative control without a DNA content.

    Article Snippet: For RFLP analysis, 5 μl of PCR product was digested overnight at 37°C with 2 U of restriction enzyme EarI and 1xTango Buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) in total volume of 15 μl.

    Techniques: Polymerase Chain Reaction, Marker, Negative Control

    DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated exon skipping. ( A ) Two gRNAs [either gRNA (g2 or g3), which target intron 50, and the other (g1), which targets exon 51] were used to direct Cpf1-mediated removal of the exon 51 splice acceptor site. ( B ) T7E1 assay using 293T cells transfected with LbCpf1 and gRNA2 (g2) or gRNA3 (g3) shows cleavage of the DMD locus at intron 50. Red arrowheads denote cleavage products. M, marker; Ctrl, control. ( C ) PCR products of genomic DNA isolated from DMD-iPSCs transfected with a plasmid expressing LbCpf1, g1 + g2, and GFP. The lower band (red arrowhead) indicates removal of the exon 51 splice acceptor site. ( D ) Sequence of the lower PCR band from (C) shows a 200-bp deletion, spanning from the 3′ end of intron 50 to the 5′ end of exon 51. This confirms removal of the “ag” splice acceptor of exon 51. The sequence of the uncorrected allele is shown above that of the LbCpf1-edited allele. ( E ) RT-PCR of iPSC-derived cardiomyocytes using primer sets described in Fig. 2B . The 700-bp band in the WT lane is the dystrophin transcript from exons 47 to 52; the 300-bp band in the uncorrected lane is the dystrophin transcript from exons 47 to 52 with exon 48 to 50 deletion; and the lower band in the g1 + g2 mixture lane (edited by LbCpf1) shows exon 51 skipping. ( F )Sequence of the lower band from (E) (g1 + g2 mixture lane) confirms skipping of exon 51, which reframed the DMD ORF. ( G ) Western blot analysis shows dystrophin protein expression in iPSC-derived cardiomyocyte mixtures after exon 51 skipping by LbCpf1 with g1 + g2. αMHC is loading control. ( H ) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures following Cpf1-mediated exon skipping with g1 + g2 gRNA compared to WT and uncorrected cardiomyocyte mixtures. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm.

    Journal: Science Advances

    Article Title: CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

    doi: 10.1126/sciadv.1602814

    Figure Lengend Snippet: DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated exon skipping. ( A ) Two gRNAs [either gRNA (g2 or g3), which target intron 50, and the other (g1), which targets exon 51] were used to direct Cpf1-mediated removal of the exon 51 splice acceptor site. ( B ) T7E1 assay using 293T cells transfected with LbCpf1 and gRNA2 (g2) or gRNA3 (g3) shows cleavage of the DMD locus at intron 50. Red arrowheads denote cleavage products. M, marker; Ctrl, control. ( C ) PCR products of genomic DNA isolated from DMD-iPSCs transfected with a plasmid expressing LbCpf1, g1 + g2, and GFP. The lower band (red arrowhead) indicates removal of the exon 51 splice acceptor site. ( D ) Sequence of the lower PCR band from (C) shows a 200-bp deletion, spanning from the 3′ end of intron 50 to the 5′ end of exon 51. This confirms removal of the “ag” splice acceptor of exon 51. The sequence of the uncorrected allele is shown above that of the LbCpf1-edited allele. ( E ) RT-PCR of iPSC-derived cardiomyocytes using primer sets described in Fig. 2B . The 700-bp band in the WT lane is the dystrophin transcript from exons 47 to 52; the 300-bp band in the uncorrected lane is the dystrophin transcript from exons 47 to 52 with exon 48 to 50 deletion; and the lower band in the g1 + g2 mixture lane (edited by LbCpf1) shows exon 51 skipping. ( F )Sequence of the lower band from (E) (g1 + g2 mixture lane) confirms skipping of exon 51, which reframed the DMD ORF. ( G ) Western blot analysis shows dystrophin protein expression in iPSC-derived cardiomyocyte mixtures after exon 51 skipping by LbCpf1 with g1 + g2. αMHC is loading control. ( H ) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures following Cpf1-mediated exon skipping with g1 + g2 gRNA compared to WT and uncorrected cardiomyocyte mixtures. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm.

    Article Snippet: The LbCpf1 gRNA was synthesized using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Derivative Assay, Transfection, Marker, Polymerase Chain Reaction, Isolation, Plasmid Preparation, Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunocytochemistry, Staining

    DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated genome editing by reframing. ( A ) DMD skin fibroblast-derived iPSCs were edited by Cpf1 using gRNA (corrected DMD iPSCs) and then differentiated into cardiomyocytes (corrected cardiomyocytes) for analysis of genetic correction of the DMD mutation. ( B ) A DMD deletion of exons 48 to 50 results in splicing of exon 47 to 51, generating an out-of-frame mutation of dystrophin. Forward primer (F) targeting exon 47 and reverse primer (R) targeting exon 52 were used in RT-PCR to confirm the reframing strategy by Cpf1-meditated genome editing in cardiomyocytes. Uncorrected cardiomyocytes lack exons 48 to 50. In contrast, after reframing, exon 51 is placed back in frame with exon 47. ( C ) Sequencing of representative RT-PCR products shows that uncorrected DMD iPSC-derived cardiomyocytes have a premature stop codon in exon 51, which creates a nonsense mutation. After Cpf1-mediated reframing, the ORF of dystrophin is restored. Dashed red line denotes exon boundary. ( D ) Western blot analysis shows dystrophin expression in a mixture of DMD iPSC-derived cardiomyocytes edited by reframing with LbCpf1 or AsCpf1 and g1 gRNA. Even without clonal selection, Cpf1-mediated reframing is efficient and sufficient to restore dystrophin expression in the cardiomyocyte mixture. α-Myosin heavy chain (αMHC) is loading control. ( E ) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte (CM) mixtures following LbCpf1- or AsCpf1-mediated reframing. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm. ( F ) Western blot analysis shows dystrophin expression in single clones (#2 and #5) of iPSC-derived cardiomyocytes following clonal selection after LbCpf1-mediated reframing. αMHC is loading control. ( G ) Immunocytochemistry shows dystrophin expression in clone #2 LbCpf1-edited iPSC-derived cardiomyocytes. Scale bar, 100 μm. ( H ) Quantification of mtDNA copy number in single clones (#2 and #5) of LbCpf1-edited iPSC-derived cardiomyocytes. Data are means ± SEM ( n = 3). P

    Journal: Science Advances

    Article Title: CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

    doi: 10.1126/sciadv.1602814

    Figure Lengend Snippet: DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated genome editing by reframing. ( A ) DMD skin fibroblast-derived iPSCs were edited by Cpf1 using gRNA (corrected DMD iPSCs) and then differentiated into cardiomyocytes (corrected cardiomyocytes) for analysis of genetic correction of the DMD mutation. ( B ) A DMD deletion of exons 48 to 50 results in splicing of exon 47 to 51, generating an out-of-frame mutation of dystrophin. Forward primer (F) targeting exon 47 and reverse primer (R) targeting exon 52 were used in RT-PCR to confirm the reframing strategy by Cpf1-meditated genome editing in cardiomyocytes. Uncorrected cardiomyocytes lack exons 48 to 50. In contrast, after reframing, exon 51 is placed back in frame with exon 47. ( C ) Sequencing of representative RT-PCR products shows that uncorrected DMD iPSC-derived cardiomyocytes have a premature stop codon in exon 51, which creates a nonsense mutation. After Cpf1-mediated reframing, the ORF of dystrophin is restored. Dashed red line denotes exon boundary. ( D ) Western blot analysis shows dystrophin expression in a mixture of DMD iPSC-derived cardiomyocytes edited by reframing with LbCpf1 or AsCpf1 and g1 gRNA. Even without clonal selection, Cpf1-mediated reframing is efficient and sufficient to restore dystrophin expression in the cardiomyocyte mixture. α-Myosin heavy chain (αMHC) is loading control. ( E ) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte (CM) mixtures following LbCpf1- or AsCpf1-mediated reframing. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm. ( F ) Western blot analysis shows dystrophin expression in single clones (#2 and #5) of iPSC-derived cardiomyocytes following clonal selection after LbCpf1-mediated reframing. αMHC is loading control. ( G ) Immunocytochemistry shows dystrophin expression in clone #2 LbCpf1-edited iPSC-derived cardiomyocytes. Scale bar, 100 μm. ( H ) Quantification of mtDNA copy number in single clones (#2 and #5) of LbCpf1-edited iPSC-derived cardiomyocytes. Data are means ± SEM ( n = 3). P

    Article Snippet: The LbCpf1 gRNA was synthesized using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Derivative Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Expressing, Selection, Immunocytochemistry, Staining, Clone Assay

    Correction of DMD mutations by Cpf1-mediated genome editing. ( A ) A DMD deletion of exons 48 to 50 results in splicing of exons 47 to 51, generating an out-of-frame mutation of dystrophin. Two strategies were used for the restoration of dystrophin expression by Cpf1. In the “reframing” strategy, small INDELs in exon 51 restore the protein reading frame of dystrophin. The “exon skipping” strategy is achieved by disruption of the splice acceptor of exon 51, which results in splicing of exons 47 to 52 and restoration of the protein reading frame. ( B ) The 3′ end of an intron is T-rich, which generates Cpf1 PAM sequences, enabling genome cleavage by Cpf1. ( C ) Illustration of Cpf1 gRNA targeting DMD exon 51. The T-rich PAM (red line) is located upstream of exon 51 near the splice acceptor site. The sequence of the Cpf1 g1 gRNA targeting exon 51 is shown, highlighting the complementary nucleotides in blue. Cpf1 cleavage produces a staggered end distal to the PAM site (demarcated by red arrowheads). The 5′ region of exon 51 is shaded in light blue. Exon sequence is in uppercase letters. Intron sequence is in lowercase letters. ( D ) Illustration of a plasmid encoding human codon-optimized Cpf1 (hCpf1) with a nuclear localization signal (NLS) and 2A-GFP, driven by a hybrid form of cytomegalovirus and chicken β-actin promoters (CBh). The plasmid also encodes a Cpf1 gRNA driven by the U6 promoter. Cells transfected with this plasmid express GFP, allowing for selection of Cpf1-expressing cells by FACS. ( E ) T7E1 assays using human 293T cells or DMD iPSCs (Riken51) transfected with plasmid expressing LbCpf1 or AsCpf1, gRNA, and GFP show genome cleavage at DMD exon 51. Red arrowheads point to cleavage products. M, marker; bp, base pair.

    Journal: Science Advances

    Article Title: CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

    doi: 10.1126/sciadv.1602814

    Figure Lengend Snippet: Correction of DMD mutations by Cpf1-mediated genome editing. ( A ) A DMD deletion of exons 48 to 50 results in splicing of exons 47 to 51, generating an out-of-frame mutation of dystrophin. Two strategies were used for the restoration of dystrophin expression by Cpf1. In the “reframing” strategy, small INDELs in exon 51 restore the protein reading frame of dystrophin. The “exon skipping” strategy is achieved by disruption of the splice acceptor of exon 51, which results in splicing of exons 47 to 52 and restoration of the protein reading frame. ( B ) The 3′ end of an intron is T-rich, which generates Cpf1 PAM sequences, enabling genome cleavage by Cpf1. ( C ) Illustration of Cpf1 gRNA targeting DMD exon 51. The T-rich PAM (red line) is located upstream of exon 51 near the splice acceptor site. The sequence of the Cpf1 g1 gRNA targeting exon 51 is shown, highlighting the complementary nucleotides in blue. Cpf1 cleavage produces a staggered end distal to the PAM site (demarcated by red arrowheads). The 5′ region of exon 51 is shaded in light blue. Exon sequence is in uppercase letters. Intron sequence is in lowercase letters. ( D ) Illustration of a plasmid encoding human codon-optimized Cpf1 (hCpf1) with a nuclear localization signal (NLS) and 2A-GFP, driven by a hybrid form of cytomegalovirus and chicken β-actin promoters (CBh). The plasmid also encodes a Cpf1 gRNA driven by the U6 promoter. Cells transfected with this plasmid express GFP, allowing for selection of Cpf1-expressing cells by FACS. ( E ) T7E1 assays using human 293T cells or DMD iPSCs (Riken51) transfected with plasmid expressing LbCpf1 or AsCpf1, gRNA, and GFP show genome cleavage at DMD exon 51. Red arrowheads point to cleavage products. M, marker; bp, base pair.

    Article Snippet: The LbCpf1 gRNA was synthesized using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Mutagenesis, Expressing, Sequencing, Plasmid Preparation, Transfection, Selection, FACS, Marker

    CRISPR-Cpf1–mediated editing of exon 23 of the mouse Dmd gene. ( A ) Illustration of mouse Dmd locus highlighting the mutation at exon 23. Sequence shows the nonsense mutation caused by C-to-T transition, which creates a premature stop codon. ( B ) Illustration showing the targeting location of gRNAs (g1, g2, and g3) (in light blue) in exon 23 of the Dmd gene. Red line represents LbCpf1 PAM. ( C ) T7E1 assay using mouse 10T1/2 cells transfected with LbCpf1 or AsCpf1 with different gRNAs (g1, g2, or g3) targeting exon 23 shows that LbCpf1 and AsCpf1 have different cleavage efficiency at the Dmd exon 23 locus. Red arrowheads show cleavage products of genome editing. M, marker. ( D ) Illustration of LbCpf1-mediated gRNA (g2) targeting of Dmd exon 23. Red arrowheads indicate the cleavage site. The ssODN HDR template contains the mdx correction, four silent mutations (green), and a Tse I restriction site (underlined).

    Journal: Science Advances

    Article Title: CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

    doi: 10.1126/sciadv.1602814

    Figure Lengend Snippet: CRISPR-Cpf1–mediated editing of exon 23 of the mouse Dmd gene. ( A ) Illustration of mouse Dmd locus highlighting the mutation at exon 23. Sequence shows the nonsense mutation caused by C-to-T transition, which creates a premature stop codon. ( B ) Illustration showing the targeting location of gRNAs (g1, g2, and g3) (in light blue) in exon 23 of the Dmd gene. Red line represents LbCpf1 PAM. ( C ) T7E1 assay using mouse 10T1/2 cells transfected with LbCpf1 or AsCpf1 with different gRNAs (g1, g2, or g3) targeting exon 23 shows that LbCpf1 and AsCpf1 have different cleavage efficiency at the Dmd exon 23 locus. Red arrowheads show cleavage products of genome editing. M, marker. ( D ) Illustration of LbCpf1-mediated gRNA (g2) targeting of Dmd exon 23. Red arrowheads indicate the cleavage site. The ssODN HDR template contains the mdx correction, four silent mutations (green), and a Tse I restriction site (underlined).

    Article Snippet: The LbCpf1 gRNA was synthesized using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: CRISPR, Mutagenesis, Sequencing, Transfection, Marker

    CRISPR-LbCpf1–mediated Dmd correction in mdx mice. ( A ) Strategy of gene correction in mdx mice by LbCpf1-mediated germline editing. Zygotes from intercrosses of mdx parents were injected with gene editing components (LbCpf1 mRNA, g2 gRNA, and ssODN) and reimplanted into pseudopregnant mothers, which gave rise to pups with gene correction ( mdx -C). ( B ) Illustration showing LbCpf1 correction of mdx allele by HDR or NHEJ. ( C ) Genotyping results of LbCpf1-edited mdx mice. Top: T7E1 assay. Blue arrowhead denotes uncleaved DNA, and red arrowhead shows T7E1-cleaved DNA. Bottom: Tse I RFLP assay. Blue arrowhead denotes uncorrected DNA, and red arrowhead points to Tse I cleavage, indicating HDR correction. mdx -C1 to mdx -C5 denote LbCpf1-edited mdx mice. ( D ) Top: Sequence of WT Dmd exon 23. Middle: Sequence of mdx Dmd exon 23 with C-to-T mutation, which generates a stop codon. Bottom: Sequence of Dmd exon 23 with HDR correction by LbCpf1-mediated editing. Black arrows point to silent mutations introduced by the ssODN HDR template. ( E ) H E staining of tibialis anterior (TA) and gastrocnemius/plantaris (G/P) muscles from WT, mdx , and LbCpf1-edited mice ( mdx -C). Scale bar, 100 μm. ( F ) Immunohistochemistry of tibialis anterior and gastrocnemius/plantaris muscles from WT, mdx , and LbCpf1-edited mice ( mdx -C) using an antibody to dystrophin (red). mdx muscle showed fibrosis and inflammatory infiltration, whereas mdx -C muscle showed normal muscle structure.

    Journal: Science Advances

    Article Title: CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

    doi: 10.1126/sciadv.1602814

    Figure Lengend Snippet: CRISPR-LbCpf1–mediated Dmd correction in mdx mice. ( A ) Strategy of gene correction in mdx mice by LbCpf1-mediated germline editing. Zygotes from intercrosses of mdx parents were injected with gene editing components (LbCpf1 mRNA, g2 gRNA, and ssODN) and reimplanted into pseudopregnant mothers, which gave rise to pups with gene correction ( mdx -C). ( B ) Illustration showing LbCpf1 correction of mdx allele by HDR or NHEJ. ( C ) Genotyping results of LbCpf1-edited mdx mice. Top: T7E1 assay. Blue arrowhead denotes uncleaved DNA, and red arrowhead shows T7E1-cleaved DNA. Bottom: Tse I RFLP assay. Blue arrowhead denotes uncorrected DNA, and red arrowhead points to Tse I cleavage, indicating HDR correction. mdx -C1 to mdx -C5 denote LbCpf1-edited mdx mice. ( D ) Top: Sequence of WT Dmd exon 23. Middle: Sequence of mdx Dmd exon 23 with C-to-T mutation, which generates a stop codon. Bottom: Sequence of Dmd exon 23 with HDR correction by LbCpf1-mediated editing. Black arrows point to silent mutations introduced by the ssODN HDR template. ( E ) H E staining of tibialis anterior (TA) and gastrocnemius/plantaris (G/P) muscles from WT, mdx , and LbCpf1-edited mice ( mdx -C). Scale bar, 100 μm. ( F ) Immunohistochemistry of tibialis anterior and gastrocnemius/plantaris muscles from WT, mdx , and LbCpf1-edited mice ( mdx -C) using an antibody to dystrophin (red). mdx muscle showed fibrosis and inflammatory infiltration, whereas mdx -C muscle showed normal muscle structure.

    Article Snippet: The LbCpf1 gRNA was synthesized using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: CRISPR, Mouse Assay, Injection, Non-Homologous End Joining, RFLP Assay, Sequencing, Mutagenesis, Staining, Immunohistochemistry

    PCR patterns of amplified ITS1 ribosomal region. Lane M: 50 bp ladder molecular weight marker; lane 1 (a, b): negative control; lanes 2-8 (a) and 2-7 (b) denoted to different fluke samples amplified as a single band of 680 bp of bovine and buffalo in Nghe An province.

    Journal: Journal of Parasitology Research

    Article Title: Identification of Fasciola Species Isolates from Nghe An Province, Vietnam, Based on ITS1 Sequence of Ribosomal DNA Using a Simple PCR-RFLP Method

    doi: 10.1155/2018/2958026

    Figure Lengend Snippet: PCR patterns of amplified ITS1 ribosomal region. Lane M: 50 bp ladder molecular weight marker; lane 1 (a, b): negative control; lanes 2-8 (a) and 2-7 (b) denoted to different fluke samples amplified as a single band of 680 bp of bovine and buffalo in Nghe An province.

    Article Snippet: To perform RFLP assay, total volume of 16 μ l, including 5 μ l of ITS1 PCR product, was added with 1 μ l of Rsa I, 1 μ l of 10X Tango buffer (Thermo Fisher Scientific, USA), and 9 μ l of distilled water.

    Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Negative Control