long range polymerase chain reaction  (TaKaRa)


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    Name:
    LA PCR Kit
    Description:
    The LA PCR Kit Version 2 1 contains all the reagents needed for amplification of long DNA templates enabling routine amplification of products up to 20 kb in length including GC rich amplicons For some DNA templates amplification of up to 48 kb is possible This long range PCR kit contains TaKaRa LA Taq DNA Polymerase buffers MgCl2 dNTPs molecular weight markers and control templates plus corresponding primers to ensure optimal PCR performance during long PCR
    Catalog Number:
    rr013b
    Price:
    None
    Category:
    LA Taq PCR kit LA Taq products Long range PCR PCR
    Size:
    100 Rxns
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    Structured Review

    TaKaRa long range polymerase chain reaction
    The LA PCR Kit Version 2 1 contains all the reagents needed for amplification of long DNA templates enabling routine amplification of products up to 20 kb in length including GC rich amplicons For some DNA templates amplification of up to 48 kb is possible This long range PCR kit contains TaKaRa LA Taq DNA Polymerase buffers MgCl2 dNTPs molecular weight markers and control templates plus corresponding primers to ensure optimal PCR performance during long PCR
    https://www.bioz.com/result/long range polymerase chain reaction/product/TaKaRa
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    long range polymerase chain reaction - by Bioz Stars, 2021-03
    86/100 stars

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    Related Articles

    Synthesized:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: Total cellular RNA was isolated from stromal cells using RNAzol-B (Cinna/Biotex Laboratories, Houston, TX) according to the manufacturer's instructions. .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Random Hexamer Labeling:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: Total cellular RNA was isolated from stromal cells using RNAzol-B (Cinna/Biotex Laboratories, Houston, TX) according to the manufacturer's instructions. .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Amplification:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: Total cellular RNA was isolated from stromal cells using RNAzol-B (Cinna/Biotex Laboratories, Houston, TX) according to the manufacturer's instructions. .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: A Further Analysis of the Relationship between Yellow Ripe-Fruit Color and the Capsanthin-Capsorubin Synthase Gene in Pepper (Capsicum sp.) Indicated a New Mutant Variant in C. annuum and a Tandem Repeat Structure in Promoter Region
    Article Snippet: Cloning of Ccs gene and promoter sequences Sixteen genetically distinct pepper cultivars were selected based on the color types in fully ripe fruit (eight red and eight yellow), including CK6 (C. annuum , red), CK8 (C. annuum , red), R29 (C. frutescens , red), R37 (C. annuum , red), R28 (C. chinense , red), R30 (C. chinense , red), R26 (C. baccatum , red), R38 (C. baccatum , red), P123-1-1 (C. annuum , yellow), CK4 (C. annuum , yellow), CK4-1 (C. annuum , yellow), CK18 (C. annuum , yellow), CK7 (C. annuum , yellow), CK26-1 (C. annuum , yellow), R12 (C. annuum , yellow), and R19 (C. annuum , yellow). .. The CDS (coding sequence) and promoter fragments of the Ccs gene from all sixteen cultivars were amplified using a Long-Distance PCR Kit (Takara, China) with gene-specific primers ( ). ..

    Article Title: AID assists DNMT1 to attenuate BCL6 expression through DNA methylation in diffuse large B-cell lymphoma cell lines
    Article Snippet: .. The fragments containing the first intron of BCL6 gene were amplified from genomic DNA by long-range PCR according to the manufacturer’s protocol of TaKaRa LA PCR™ Kit Ver.2.1 (TaKaRa, #RR013A). ..

    Article Title: Extraocular Muscle Reveals Selective Vulnerability of Type IIB Fibers to Respiratory Chain Defects Induced by Mitochondrial DNA Alterations
    Article Snippet: Long range PCR was used to screen for the presence of mtDNA deletions accumulated in muscles of mutant mice. .. The deleted mtDNA molecules were then amplified using TAKARA LA PCR Kit (TAKARA Bio), as described previously. .. The PCR products were then resolved on a 0.8% agarose gel in TAE buffer, stained with ethidium bromide (0.8 µg/mL), and visualized under ultraviolet (UV)-light (EL Logic 200 Imaging system).

    Polymerase Chain Reaction:

    Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro
    Article Snippet: Total cellular RNA was isolated from stromal cells using RNAzol-B (Cinna/Biotex Laboratories, Houston, TX) according to the manufacturer's instructions. .. Single-stranded cDNA was synthesized by reverse transcriptase (RT) with random hexamer oligonucleotides as primers, and the desired cDNA was amplified by polymerase chain reaction (PCR) using a TaKaRa RNA LA PCR kit (TaKaRa Biomedicals, Kyoto, Japan). .. Primers (sense and antisense) for the amplification of each cytokine cDNA were as follows: β-actin sense, TGG AAT CCT GTG GCA TCC ATG AAA C; β-actin antisense, TAA AAC GCA GCT CAG TAA CAG TCC G; IL-1α sense, CTC TAG AGC ACC ATG CTA CAG AC; IL-1α antisense, TGG AAT CCA GGG GAA ACA CTG; IL-2 sense, TGA TGG ACC TAC AGG AGC TCC TGA G; IL-2 antisense, GAG TCA AAT CCA GAA CAT GCC GCA G; IL-3 sense, GAA GTG GAT CCT GAG GAC AGA TAC G; IL-3 antisense, GAC CAT GGG CCA TGA GGA ACA TTC; IL-4 sense, CGA AGA ACA CCA CAG AGA GTG AGC T; IL-4 antisense, GAC TCA TTC ATG GTG CAG CTT ATC G; IL-5 sense, CTC TAG TAA GCC CAC TTC TA; IL-5 antisense, TGA TAC ATG AAT AAC ATC CC; IL-6 sense, TGG AGT CAC AGA AGG AGT GGC TAA G; IL-6 antisense, TCT GAC CAC AGA AGG AGT GGC TAA G; IL-7 sense, AAA TGC AGC TGA CTG CTG GC; IL-7 antisense, TCT CCA GTC TAA AAC AGG AC; IL-10 sense, TAC CTG GTA GAA GTG ATG CC; IL-10 antisense, CAT CAT GTA TGC TTC TAT GC; tumour necrosis factor-α (TNF-α) sense, GGC AGG TCT ACT TTG GAG TCA TTG C; TNF-α antisense, ACA TTC GAG GCT CCA GTG AAT TCG G; lymphotoxin (LT)-α sense, TGG CTG GGA ACA GGG GAA GGT TGA C; LT-α antisense, CGT GCT TTC TTC TAG AAC CCC TTG G. The conditions for PCR were 1 min at 94°, 2 min at 55° and 3 min at 72° for 30 cycles.

    Article Title: A Further Analysis of the Relationship between Yellow Ripe-Fruit Color and the Capsanthin-Capsorubin Synthase Gene in Pepper (Capsicum sp.) Indicated a New Mutant Variant in C. annuum and a Tandem Repeat Structure in Promoter Region
    Article Snippet: Cloning of Ccs gene and promoter sequences Sixteen genetically distinct pepper cultivars were selected based on the color types in fully ripe fruit (eight red and eight yellow), including CK6 (C. annuum , red), CK8 (C. annuum , red), R29 (C. frutescens , red), R37 (C. annuum , red), R28 (C. chinense , red), R30 (C. chinense , red), R26 (C. baccatum , red), R38 (C. baccatum , red), P123-1-1 (C. annuum , yellow), CK4 (C. annuum , yellow), CK4-1 (C. annuum , yellow), CK18 (C. annuum , yellow), CK7 (C. annuum , yellow), CK26-1 (C. annuum , yellow), R12 (C. annuum , yellow), and R19 (C. annuum , yellow). .. The CDS (coding sequence) and promoter fragments of the Ccs gene from all sixteen cultivars were amplified using a Long-Distance PCR Kit (Takara, China) with gene-specific primers ( ). ..

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis
    Article Snippet: .. Primer pairs specific for Tn4351 (primers TN-1 and IS4351-F) were used to amplify the sequences adjacent to the insertion site using the LA PCR kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). .. The nucleotide sequence was compared to sequence in the National Center for Biotechnology Information database using the BLASTX program [ ].

    Article Title: Identification and Characterization of a Novel Type III Secretion System in trh-Positive Vibrio parahaemolyticus Strain TH3996 Reveal Genetic Lineage and Diversity of Pathogenic Machinery beyond the Species Level ▿ Strain TH3996 Reveal Genetic Lineage and Diversity of Pathogenic Machinery beyond the Species Level ▿ †
    Article Snippet: PCR conditions for the construction of mutant strains and probes were as follows: after 2 min of denaturation at 94°C, a cycle of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min was repeated 30 times. .. To detect the presence of the T3SS genes, PCR was performed using the EX-PCR kit (Takara Shuzo, Kyoto, Japan). .. The PCR conditions were as follows: after initial denaturation at 94°C for 3 min, a cycle of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min or 2 min was repeated 30 times.

    Article Title: Spermidine Synthase Genes Are Essential for Survival of Arabidopsis
    Article Snippet: .. RT-PCR was conducted by using the RNA LA PCR Kit (Takara, Kyoto) with 0.5 μ g of total RNA. ..

    Article Title: Characterization of a novel germline PALB2 duplication in a hereditary breast and ovarian cancer family
    Article Snippet: Long-range (LR) PCR was performed on genomic DNA from the patient to confirm the large DNA duplication in PALB2 detected by the commercial laboratory. .. LR-PCR was performed using a forward primer in intron 12 and a reverse primer in 3′-untranslated region (UTR) of PALB2 and the TaKaRa LA PCR kit (TaKaRa, Clontech) following the manufacturer’s instructions. ..

    Article Title: Extraocular Muscle Reveals Selective Vulnerability of Type IIB Fibers to Respiratory Chain Defects Induced by Mitochondrial DNA Alterations
    Article Snippet: Long range PCR was used to screen for the presence of mtDNA deletions accumulated in muscles of mutant mice. .. The deleted mtDNA molecules were then amplified using TAKARA LA PCR Kit (TAKARA Bio), as described previously. .. The PCR products were then resolved on a 0.8% agarose gel in TAE buffer, stained with ethidium bromide (0.8 µg/mL), and visualized under ultraviolet (UV)-light (EL Logic 200 Imaging system).

    Sequencing:

    Article Title: A Further Analysis of the Relationship between Yellow Ripe-Fruit Color and the Capsanthin-Capsorubin Synthase Gene in Pepper (Capsicum sp.) Indicated a New Mutant Variant in C. annuum and a Tandem Repeat Structure in Promoter Region
    Article Snippet: Cloning of Ccs gene and promoter sequences Sixteen genetically distinct pepper cultivars were selected based on the color types in fully ripe fruit (eight red and eight yellow), including CK6 (C. annuum , red), CK8 (C. annuum , red), R29 (C. frutescens , red), R37 (C. annuum , red), R28 (C. chinense , red), R30 (C. chinense , red), R26 (C. baccatum , red), R38 (C. baccatum , red), P123-1-1 (C. annuum , yellow), CK4 (C. annuum , yellow), CK4-1 (C. annuum , yellow), CK18 (C. annuum , yellow), CK7 (C. annuum , yellow), CK26-1 (C. annuum , yellow), R12 (C. annuum , yellow), and R19 (C. annuum , yellow). .. The CDS (coding sequence) and promoter fragments of the Ccs gene from all sixteen cultivars were amplified using a Long-Distance PCR Kit (Takara, China) with gene-specific primers ( ). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Spermidine Synthase Genes Are Essential for Survival of Arabidopsis
    Article Snippet: .. RT-PCR was conducted by using the RNA LA PCR Kit (Takara, Kyoto) with 0.5 μ g of total RNA. ..

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  • 86
    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).
    Long Range Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr/product/TaKaRa
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    long range pcr - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

    86
    TaKaRa range pcr
    Estimated CTX prophage region structure of V. <t>cholerae</t> strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and <t>PCR</t> of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.
    Range Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/range pcr/product/TaKaRa
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    range pcr - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

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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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0145513

    Figure Lengend 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.

    Article Snippet: The surviving clones were screened for homologous recombination by long-range PCR with LATaq DNA polymerase (TaKaRa, Otsu, Japan) and the PCR primers are listed in .

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0145513

    Figure Lengend 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.

    Article Snippet: The surviving clones were screened for homologous recombination by long-range PCR with LATaq DNA polymerase (TaKaRa, Otsu, Japan) and the PCR primers are listed in .

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

    Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction