long range pcr  (TaKaRa)

 
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    Name:
    TaKaRa LA Taq DNA Polymerase
    Description:
    TaKaRa LA Taq DNA Polymerase combines Taq DNA polymerase and a DNA proofreading polymerase with 3 →5 exonuclease activity to enable PCR amplification of very long DNA templates long range PCR This mixture of enzymes allows for long and accurate LA PCR of targets from a variety of templates including genomic DNA LA Taq DNA polymerase is supplied with optimized LA PCR Buffer II with or without Mg2 and dNTPs
    Catalog Number:
    rr002a
    Price:
    None
    Size:
    125 Units
    Category:
    LA Taq DNA polymerase LA Taq products Long range PCR PCR
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    Structured Review

    TaKaRa long range 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).
    TaKaRa LA Taq DNA Polymerase combines Taq DNA polymerase and a DNA proofreading polymerase with 3 →5 exonuclease activity to enable PCR amplification of very long DNA templates long range PCR This mixture of enzymes allows for long and accurate LA PCR of targets from a variety of templates including genomic DNA LA Taq DNA polymerase is supplied with optimized LA PCR Buffer II with or without Mg2 and dNTPs
    https://www.bioz.com/result/long range pcr/product/TaKaRa
    Average 99 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    long range pcr - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy"

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

    Journal: Nature

    doi: 10.1038/nature10456

    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).
    Figure Legend 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).

    Techniques Used: 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
    Figure Legend 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

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

    2) Product Images from "Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells"

    Article Title: Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.022590

    TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P
    Figure Legend Snippet: TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P

    Techniques Used: Expressing, Polymerase Chain Reaction, Amplification

    3) Product Images from "Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease"

    Article Title: Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2018020180

    MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.
    Figure Legend Snippet: MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.

    Techniques Used: Sequencing, Generated, Polymerase Chain Reaction, Amplification, Mutagenesis, Genotyping Assay, Mass Spectrometry, Primer Extension Assay

    4) Product Images from "Novel Endothelial Cell-Specific AQP1 Knockout Mice Confirm the Crucial Role of Endothelial AQP1 in Ultrafiltration during Peritoneal Dialysis"

    Article Title: Novel Endothelial Cell-Specific AQP1 Knockout Mice Confirm the Crucial Role of Endothelial AQP1 in Ultrafiltration during Peritoneal Dialysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145513

    Generation and validation of the floxed AQP1 allele. (A) Both 5' and 3' homologous recombinants were screened by long-range PCR with neo-specific primers P1F/1R and P2F/2R. PCR of representative embryonic stem (ES) clones showed the targeted 5'-(9,1kb) and the 3'- (5.3kb) homologous recombination events, respectively. (B) Targeted ES clones in (A) were confirmed by Southern blot analysis. Using the 5' probe (upper panel), the 3' probe (middle panel), and the neo probe (lower panel), the representative resulting Southern hybridisation signals appeared upon digestion of genomic DNA from ES clones with the Xho I (3' probe and neo probe) or Nsi I (5' probe). The genotypes of WT (+) and targeted ES clones with neo cassette (fl-neo) and the size of the detected fragments are indicated. Detection of a single 7.3 kb fragment with the neo probe indicates a singular integration event, whereas one clone (#) showed an additional integration of the neo cassette. Germline transmission was obtained from the clone indicated with an asterisk following blastocyst injection. (C) Genotyping of AQP1 +/+ (+), AQP1 fl-neo/+ (fl-neo) and AQP1 flox/+ (fl) mice by PCR using primers 3F and 3R. (D) WT (+/+), heterozygous (fl/+), homozygous (fl/fl) floxed alleles were distinguished by PCR with primers 4F and 4R. (E) Immunoblot of total protein fractions from visceral peritoneal (VP) homogenate probed with AQP1 antibody. Equal loading (40 μg of protein from each sample was verified using an anti-β-actin antibody. (F) AQP1 immunostaining showed normal localisation in the microvascular endothelium (arrows, stained in red) in VP and no apparent difference was observed between the AQP1 +/+ and AQP1 fl/fl mice. Calibration bar: 50μM.
    Figure Legend Snippet: Generation and validation of the floxed AQP1 allele. (A) Both 5' and 3' homologous recombinants were screened by long-range PCR with neo-specific primers P1F/1R and P2F/2R. PCR of representative embryonic stem (ES) clones showed the targeted 5'-(9,1kb) and the 3'- (5.3kb) homologous recombination events, respectively. (B) Targeted ES clones in (A) were confirmed by Southern blot analysis. Using the 5' probe (upper panel), the 3' probe (middle panel), and the neo probe (lower panel), the representative resulting Southern hybridisation signals appeared upon digestion of genomic DNA from ES clones with the Xho I (3' probe and neo probe) or Nsi I (5' probe). The genotypes of WT (+) and targeted ES clones with neo cassette (fl-neo) and the size of the detected fragments are indicated. Detection of a single 7.3 kb fragment with the neo probe indicates a singular integration event, whereas one clone (#) showed an additional integration of the neo cassette. Germline transmission was obtained from the clone indicated with an asterisk following blastocyst injection. (C) Genotyping of AQP1 +/+ (+), AQP1 fl-neo/+ (fl-neo) and AQP1 flox/+ (fl) mice by PCR using primers 3F and 3R. (D) WT (+/+), heterozygous (fl/+), homozygous (fl/fl) floxed alleles were distinguished by PCR with primers 4F and 4R. (E) Immunoblot of total protein fractions from visceral peritoneal (VP) homogenate probed with AQP1 antibody. Equal loading (40 μg of protein from each sample was verified using an anti-β-actin antibody. (F) AQP1 immunostaining showed normal localisation in the microvascular endothelium (arrows, stained in red) in VP and no apparent difference was observed between the AQP1 +/+ and AQP1 fl/fl mice. Calibration bar: 50μM.

    Techniques Used: Polymerase Chain Reaction, Homologous Recombination, Clone Assay, Southern Blot, Hybridization, Transmission Assay, Injection, Mouse Assay, Immunostaining, Staining

    Targeting construct and screening strategies. A part of the wild type (+) allele of mouse AQP1 is shown with indicated exons (black boxes) and restriction enzyme sites. The targeting allele (fl-neo) is indicated with 3’ and 5’ targeting arms (thick lines), loxP / FRT sites and pro- and eukaryotic neomycin selection cassette (neo-gb2-PGK). The 5’ probe and 3’ probe for Southern blot located outside the targeting vector detect 11.1-kb (+) and or 7.1-kb (fl-neo) fragments from Nsi I-digested genomic DNA and 22.6-kb (+) and or 7.3-kb (fl-neo) fragments following Xho I-digestion genomic DNA, respectively. Mice carrying the floxed allele (AQP fl-neo ) were crossed to FLPeR mice for excision of the FRT-flanked neo cassette. The resulting floxed mice (AQP fl ) were crossed to Cdh5 (PAC)-CreERT2 (Cdh5-Cre) transgenic mice to excise exons 2 and 3 following tamoxifen induction, and then generate the AQP1 null allele (AQP1 del ) in endothelial cells. The P1F/1R, P2F/2R, P3F/3R and P4F/4R primers for PCR-based genotype analyses and the lengths of their responding PCR products are indicated.
    Figure Legend Snippet: Targeting construct and screening strategies. A part of the wild type (+) allele of mouse AQP1 is shown with indicated exons (black boxes) and restriction enzyme sites. The targeting allele (fl-neo) is indicated with 3’ and 5’ targeting arms (thick lines), loxP / FRT sites and pro- and eukaryotic neomycin selection cassette (neo-gb2-PGK). The 5’ probe and 3’ probe for Southern blot located outside the targeting vector detect 11.1-kb (+) and or 7.1-kb (fl-neo) fragments from Nsi I-digested genomic DNA and 22.6-kb (+) and or 7.3-kb (fl-neo) fragments following Xho I-digestion genomic DNA, respectively. Mice carrying the floxed allele (AQP fl-neo ) were crossed to FLPeR mice for excision of the FRT-flanked neo cassette. The resulting floxed mice (AQP fl ) were crossed to Cdh5 (PAC)-CreERT2 (Cdh5-Cre) transgenic mice to excise exons 2 and 3 following tamoxifen induction, and then generate the AQP1 null allele (AQP1 del ) in endothelial cells. The P1F/1R, P2F/2R, P3F/3R and P4F/4R primers for PCR-based genotype analyses and the lengths of their responding PCR products are indicated.

    Techniques Used: Construct, Selection, Southern Blot, Plasmid Preparation, Mouse Assay, Transgenic Assay, Polymerase Chain Reaction

    5) Product Images from "Molecular Heterogeneity in Acute Promyelocytic Leukemia - a Single Center Experience from India"

    Article Title: Molecular Heterogeneity in Acute Promyelocytic Leukemia - a Single Center Experience from India

    Journal: Mediterranean Journal of Hematology and Infectious Diseases

    doi: 10.4084/MJHID.2018.002

    Cellular Morphology and PML-RARA fusion transcript detection. Abnormal promyelocytes with characteristic bilobed nuclei (B) and presence of abundant cytoplasmic granules which showed strong cytochemical myeloperoxidase positivity (D) is seen in the uppermost panel. Some unusual myeloid differentiation is also seen (A C). PCR amplicon size (480 bp) of novel Bcr3 PML-RARA transcript on agarose gel (middle panel) and capillary (lower panel) electrophoresis.
    Figure Legend Snippet: Cellular Morphology and PML-RARA fusion transcript detection. Abnormal promyelocytes with characteristic bilobed nuclei (B) and presence of abundant cytoplasmic granules which showed strong cytochemical myeloperoxidase positivity (D) is seen in the uppermost panel. Some unusual myeloid differentiation is also seen (A C). PCR amplicon size (480 bp) of novel Bcr3 PML-RARA transcript on agarose gel (middle panel) and capillary (lower panel) electrophoresis.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis

    6) Product Images from "The transcriptome of the novel dinoflagellate Oxyrrhis marina (Alveolata: Dinophyceae): response to salinity examined by 454 sequencing"

    Article Title: The transcriptome of the novel dinoflagellate Oxyrrhis marina (Alveolata: Dinophyceae): response to salinity examined by 454 sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-12-519

    Sequence alignment of 3' and 5' ends of the HSP90 gene and contiguous intergenic regions . Coding regions have been translated to amino acid sequences to highlight the position of synonymous (open circles) and non-synonymous (filled circles) substitutions. Note a single non synonymous substitution occurred at amino acid position -28. The lengths of 3'/5' UTRs (blue) and intergenic regions (red) were inferred from comparison of genomic and cDNA sequences. IG1-6 refer to genomic sequence classes derived from PCR and cloning of the intergenic region.
    Figure Legend Snippet: Sequence alignment of 3' and 5' ends of the HSP90 gene and contiguous intergenic regions . Coding regions have been translated to amino acid sequences to highlight the position of synonymous (open circles) and non-synonymous (filled circles) substitutions. Note a single non synonymous substitution occurred at amino acid position -28. The lengths of 3'/5' UTRs (blue) and intergenic regions (red) were inferred from comparison of genomic and cDNA sequences. IG1-6 refer to genomic sequence classes derived from PCR and cloning of the intergenic region.

    Techniques Used: Sequencing, Derivative Assay, Polymerase Chain Reaction, Clone Assay

    7) Product Images from "Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination"

    Article Title: Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156636

    Targeting BLG in goat fibroblasts. (A) Schematic overview depicting the targeting strategy for the BLG locus. Blue boxes, exons of BLG; Diagonal lines above, TALEN1/2 pair binding sites; Level arrow lines, primers used for PCR; The predicted size of southern hybridization bands with BamHI digestion, for both the endogenous BLG locus and the BLG targeted locus, is indicated. (B) PCR-based measurements of TALEN-driven exogenous gene integration into the BLG locus in goat fibroblasts. Cells were right untransfected (lane 4, for negative control) or were transfected with an expression cassette for TALENs that induce a DSB at exon1 of BLG locus (lane 3), and donor plasmids carrying a foreign gene flanked by two homology arms, in the absence (lane 2) and presence (lane 1) of the TALEN. (C) 3’ Junction PCR results from cell lysis templates. TALEN-mediated transgene insertion yielded 2,300-bp PCR products using primers B31 and B32, which were specific to the neo gene and the BLG locus, respectively. (D) 5’ Junction PCR analysis performed on genomic DNA of 3’ junction PCR-positive colonies using primers B51 and B52 to amplify the 1,800-bp left-hand junction between the endogenous BLG locus and the exogenous hLA.
    Figure Legend Snippet: Targeting BLG in goat fibroblasts. (A) Schematic overview depicting the targeting strategy for the BLG locus. Blue boxes, exons of BLG; Diagonal lines above, TALEN1/2 pair binding sites; Level arrow lines, primers used for PCR; The predicted size of southern hybridization bands with BamHI digestion, for both the endogenous BLG locus and the BLG targeted locus, is indicated. (B) PCR-based measurements of TALEN-driven exogenous gene integration into the BLG locus in goat fibroblasts. Cells were right untransfected (lane 4, for negative control) or were transfected with an expression cassette for TALENs that induce a DSB at exon1 of BLG locus (lane 3), and donor plasmids carrying a foreign gene flanked by two homology arms, in the absence (lane 2) and presence (lane 1) of the TALEN. (C) 3’ Junction PCR results from cell lysis templates. TALEN-mediated transgene insertion yielded 2,300-bp PCR products using primers B31 and B32, which were specific to the neo gene and the BLG locus, respectively. (D) 5’ Junction PCR analysis performed on genomic DNA of 3’ junction PCR-positive colonies using primers B51 and B52 to amplify the 1,800-bp left-hand junction between the endogenous BLG locus and the exogenous hLA.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Hybridization, Negative Control, Transfection, Expressing, TALENs, Lysis

    Identification for BLG bi-allelic modification clones. (A) Long-range PCR analysis performed on genomic DNA by primers LRF and LRR. Lane WT, non-transgenic cells; Lanes 1–22, gene-targeted clones. The wild-type allele PCR product was 0.5 kb and the targeted allele PCR product was 4.7 kb. (B) Southern blot analysis for gene targeted clones. Lane WT, non-transgenic cells; Lanes 1–8, BLG bi-allelic modification clones identified by LR-PCR; Lanes 9–14, BLG mono-allelic modification clones identified by LR-PCR.
    Figure Legend Snippet: Identification for BLG bi-allelic modification clones. (A) Long-range PCR analysis performed on genomic DNA by primers LRF and LRR. Lane WT, non-transgenic cells; Lanes 1–22, gene-targeted clones. The wild-type allele PCR product was 0.5 kb and the targeted allele PCR product was 4.7 kb. (B) Southern blot analysis for gene targeted clones. Lane WT, non-transgenic cells; Lanes 1–8, BLG bi-allelic modification clones identified by LR-PCR; Lanes 9–14, BLG mono-allelic modification clones identified by LR-PCR.

    Techniques Used: Modification, Polymerase Chain Reaction, Transgenic Assay, Clone Assay, Southern Blot

    8) Product Images from "ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy"

    Article Title: ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007605

    Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase. (A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa -null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa -null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa -null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa -null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total DNA and mtDNA extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa -null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa -null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa -null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table . (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa -null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table . (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.
    Figure Legend Snippet: Inosine incorporation into nucleic acids in human and mouse cells lacking functional ITPase. (A) Bar chart showing a significantly increased inosine base content of RNA in lymphoblastoid cell lines (LCLs) derived from an affected individual (5196 III:3) as compared to that derived from her mother (5196 II:2) (B) Bar chart showing significantly increased inosine base content of RNA in Itpa -null mouse embryonic stem (ES) cells as compared to control ES cells. (C) Bar chart showing increased inosine base content of RNA derived from Itpa -null tissue as compared to controls. Inosine content is significantly higher in RNA derived from Itpa -null hearts than that stage-matched control hearts. There was no significant (ns) difference in IMP content in RNA derived from Itpa -null compared to control kidneys. Error bars ±SEM. (D) Alkaline-gel electrophoresis of total DNA and mtDNA extracted from mouse ES cells untreated or treated with bacterial endonuclease V (EndoV). All lanes shown are on the same gel, and these data are representative of three independent experiments. (E) Densitometry of gels shown in D does not identify any difference between control (green lines) and Itpa -null (red lines) cells for genomic DNA (top panel) but for mtDNA (bottom panel) there is a shift in the migration pattern in the Itpa -null cells suggestive of an increase EndoV digestion compared to the controls. (F) Long-range PCR (LR-PCR) of the mitochondrial genome shows no evidence for increased deletions in Itpa -null ES cells as compared to controls. The data shown are representative of three independent experiments. The primers used are listed in S2 Table . (G) Quantitative RT-PCR (qPCR) on total DNA shows that ratios of mtDNA to genomic DNA are comparable between control and Itpa -null cells. The data shown are derived from analysis of six individual DNA preparations per genotype, each analysed in triplicate. All the primers used are listed in S2 Table . (H,I) Alkaline comet assays on LCLs derived from an affected individual (5196 III:3) and her mother (5196 II:2) and null and parental mouse ESC respectively with cells exposed to hydrogen peroxide as a positive control. Neither cell type shows evidence for increase single or double strand breaks in genomic DNA. Quantitation of DNA damage is by Olive tail moment (the product of the tail length and the fraction of total DNA in the tail) and is a measure of both the extent of DNA fragmentation and size of fragmented DNA.

    Techniques Used: Functional Assay, Derivative Assay, Nucleic Acid Electrophoresis, Migration, Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Positive Control, Quantitation Assay

    9) Product Images from "Complete Unique Genome Sequence, Expression Profile, and Salivary Gland Tissue Tropism of the Herpesvirus 7 Homolog in Pigtailed Macaques"

    Article Title: Complete Unique Genome Sequence, Expression Profile, and Salivary Gland Tissue Tropism of the Herpesvirus 7 Homolog in Pigtailed Macaques

    Journal: Journal of Virology

    doi: 10.1128/JVI.00651-16

    Schematic overview of MneHV7 genomic sequences obtained by de novo assembly. (A) A large contiguous sequence (contig) of 122 kb covering most of the unique MneHV7 genome segment and a smaller contig of 6.8 kb with similarity to the HHV-7 U95 and U100 genes were aligned to the HHV-7 (strain RK) reference genome. The gap between the two contigs consisted mostly of the R2 repeat region and was resolved by long-range PCR and Sanger sequencing. (B) An additional contig of 2.4 kb with similarity to HHV-7 end-terminal repeat elements (DR-L/DR-R) was also identified and contained the sequence for the DR1 gene and the first exon as well as most of the intron of the DR6 gene. (C) Sequences at both ends of the 122-kb contig extended into the end-terminal sequences and exhibited strong similarity with the conserved cleavage-packaging motifs, Pac-1 and Pac-2, of human roseoloviruses flanked by the telomeric repeat regions, T1 and T2. The segments containing conserved nucleotide reiterations (highlighted or shaded) for Pac-1 and Pac-2 and flanking telomeric repeat regions (T1, T2) are indicated. All sequences identified to be part of an end-terminal repeat element were duplicated at both genomic ends, on the basis of homology with HHV-7. The MneHV7 unique genome sequence and a segment of the DRs at the genomic termini, including the DR1 gene and the DR6 first exon, are available in GenBank under accession number KU351741 .
    Figure Legend Snippet: Schematic overview of MneHV7 genomic sequences obtained by de novo assembly. (A) A large contiguous sequence (contig) of 122 kb covering most of the unique MneHV7 genome segment and a smaller contig of 6.8 kb with similarity to the HHV-7 U95 and U100 genes were aligned to the HHV-7 (strain RK) reference genome. The gap between the two contigs consisted mostly of the R2 repeat region and was resolved by long-range PCR and Sanger sequencing. (B) An additional contig of 2.4 kb with similarity to HHV-7 end-terminal repeat elements (DR-L/DR-R) was also identified and contained the sequence for the DR1 gene and the first exon as well as most of the intron of the DR6 gene. (C) Sequences at both ends of the 122-kb contig extended into the end-terminal sequences and exhibited strong similarity with the conserved cleavage-packaging motifs, Pac-1 and Pac-2, of human roseoloviruses flanked by the telomeric repeat regions, T1 and T2. The segments containing conserved nucleotide reiterations (highlighted or shaded) for Pac-1 and Pac-2 and flanking telomeric repeat regions (T1, T2) are indicated. All sequences identified to be part of an end-terminal repeat element were duplicated at both genomic ends, on the basis of homology with HHV-7. The MneHV7 unique genome sequence and a segment of the DRs at the genomic termini, including the DR1 gene and the DR6 first exon, are available in GenBank under accession number KU351741 .

    Techniques Used: Genomic Sequencing, Sequencing, Polymerase Chain Reaction

    10) Product Images from "The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity"

    Article Title: The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082149

    Structures of the pre- and post-Cre floxed mouse Zip5 gene and integration and genotyping screen designs. ( A ) The mouse Zip5 gene was captured using gap-repair and manipulated using galK recombineering. Exons ( 1–12 ) and the exon encoding transmembrane domain 1 ( TMD1 ) are indicated, as are the positions of LoxP sites (intron 4 and downstream of PGK Neo ), the PGK-neomycin ( PGK Neo ) cassette and the locations of primers used for genotyping. The LoxP site in intron 4 is flanked by an EcoRV restriction enzyme cleavage site. ( B ) The structure of the Zip5 gene after Cre recombination is shown. Recombination eliminates the transmembrane domain of ZIP5. ( C ) The floxed Zip5 gene was targeted into E14 ES cells and properly targeted ES cells were identified by long range PCR using flanking and internal primers. PCR products from the wild-type ( Wt ) and floxed ( Fx ) alleles are indicated. EcoRV cleavage was used to differentiate between the floxed and wild-type alleles in the 5′ PCR screen whereas the 3′ PCR screen yielded the predicted larger product from the wild-type allele. Targeted ES cells were used to generate mice homozygous for the floxed Zip5 allele. ( D ) Mice were genotyped by PCR amplification of the intron 4 region containing the LoxP site. The PCR product from homozygous Zip5 floxed mice before Cre-induced recombination is shown in the left lane while that from control mice is shown in the center lane and that from Zip5 -knockout mice ( Ko ) is shown in the right lane . For the intestine- and pancreas-specific knockout mice, detection of Zip5 mRNA and/or protein was employed to monitor the efficacy of recombination.
    Figure Legend Snippet: Structures of the pre- and post-Cre floxed mouse Zip5 gene and integration and genotyping screen designs. ( A ) The mouse Zip5 gene was captured using gap-repair and manipulated using galK recombineering. Exons ( 1–12 ) and the exon encoding transmembrane domain 1 ( TMD1 ) are indicated, as are the positions of LoxP sites (intron 4 and downstream of PGK Neo ), the PGK-neomycin ( PGK Neo ) cassette and the locations of primers used for genotyping. The LoxP site in intron 4 is flanked by an EcoRV restriction enzyme cleavage site. ( B ) The structure of the Zip5 gene after Cre recombination is shown. Recombination eliminates the transmembrane domain of ZIP5. ( C ) The floxed Zip5 gene was targeted into E14 ES cells and properly targeted ES cells were identified by long range PCR using flanking and internal primers. PCR products from the wild-type ( Wt ) and floxed ( Fx ) alleles are indicated. EcoRV cleavage was used to differentiate between the floxed and wild-type alleles in the 5′ PCR screen whereas the 3′ PCR screen yielded the predicted larger product from the wild-type allele. Targeted ES cells were used to generate mice homozygous for the floxed Zip5 allele. ( D ) Mice were genotyped by PCR amplification of the intron 4 region containing the LoxP site. The PCR product from homozygous Zip5 floxed mice before Cre-induced recombination is shown in the left lane while that from control mice is shown in the center lane and that from Zip5 -knockout mice ( Ko ) is shown in the right lane . For the intestine- and pancreas-specific knockout mice, detection of Zip5 mRNA and/or protein was employed to monitor the efficacy of recombination.

    Techniques Used: Polymerase Chain Reaction, Mouse Assay, Amplification, Knock-Out

    11) Product Images from "Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease"

    Article Title: Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2018020180

    MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.
    Figure Legend Snippet: MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.

    Techniques Used: Sequencing, Generated, Polymerase Chain Reaction, Amplification, Mutagenesis, Genotyping Assay, Mass Spectrometry, Primer Extension Assay

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    TaKaRa long range 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).
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    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

    TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P

    Journal: Disease Models & Mechanisms

    Article Title: Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

    doi: 10.1242/dmm.022590

    Figure Lengend Snippet: TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P

    Article Snippet: To detect the SVA, long-range PCR was performed using previously described primers ( ) and Takara's PrimeSTAR GXL DNA polymerase (Clontech, Mountain View, CA, USA) with modified amplification conditions ( Table S1 ).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification

    Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.

    Journal: Frontiers in Genetics

    Article Title: Segmental Maternal UPD of Chromosome 7q in a Patient With Pendred and Silver Russell Syndromes-Like Features

    doi: 10.3389/fgene.2018.00600

    Figure Lengend Snippet: Clinical and molecular characterization of the case. (A) Frontal and lateral view of the proband at the age of 36 months. Note the mild frontal bossing, low-set posteriorly rotated ears, and thin lips. Earing aids are in place. The patient also displays brachydactyly of both hands and feet and clinodactily of V finger of hands. Other clinical findings are listed in the side table, where 3 out of 6 NH-CSS for SRS are in bold characters. (B) The LR-PCR amplicons of the patient (P) and both her father (F) and mother (M) were resolved on a 0.8% agarose gel. The patient (P) displayed a unique shorter band of about 2.5 kb in size, the father (F) showed a unique band of over 10 kb in size, corresponding to the expected 14.7 kb wild type band, whereas her mother (M) showed two bands corresponding to the wild type and the deleted alleles. MW, molecular weight. (C) The proximal and distal breakpoints of the SLC26A4 intragenic deletion were mapped within SLC26A4 IVS16 and the IVS20, respectively. The deletion is about 13 kb long. Sequence alignments of the junction fragments revealed an insertion of a part of CCDC126 IVS3. The rejoining between SLC26A4 IVS20 and CCDC126 IVS3 distal bkp occurred through a de novo 3 bp GCC insertion. (D) Sequencing of the RT-PCR amplicons, extending from exons 13–14 to 3′UTR of SLC26A4 , confirmed the homozygous deletion of exons 17–20 in the child (P). The father (F) showed only the long transcript corresponding to the wild type pendrin, whereas the mother (M) displayed a short transcript and a long one, consistent with a heterozygous state of the deletion. MW, molecular weight.

    Article Snippet: To confirm the deletion and localize its breakpoints (bkp) at nucleotide level, long-range (LR) PCR spanning the SLC26A4 genomic region from exon 16 to exon 21 was carried out with Takara LA Taq (Diatech, Jesi, Italy), according to cycle conditions suggested by the manufacturer.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Sequencing, Reverse Transcription Polymerase Chain Reaction

    MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease

    doi: 10.1681/ASN.2018020180

    Figure Lengend Snippet: MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.

    Article Snippet: The 26_27insG extension product is observed at 5944.85 D. For the 1_16dup and 23delinsAT mutation, the VNTR of MUC1 was amplified from genomic DNA using long-range PCR as described above.

    Techniques: Sequencing, Generated, Polymerase Chain Reaction, Amplification, Mutagenesis, Genotyping Assay, Mass Spectrometry, Primer Extension Assay