apoi  (New England Biolabs)


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    Name:
    ApoI
    Description:
    ApoI 5 000 units
    Catalog Number:
    R0566L
    Price:
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    Category:
    Restriction Enzymes
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    5 000 units
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    New England Biolabs apoi
    ApoI
    ApoI 5 000 units
    https://www.bioz.com/result/apoi/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    apoi - by Bioz Stars, 2021-06
    96/100 stars

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    1) Product Images from "Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome"

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

    Journal: Nucleic Acids Research

    doi:

    Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.
    Figure Legend Snippet: Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Techniques Used: Flow Cytometry, Mutagenesis, Polymerase Chain Reaction, Isolation

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia
    Article Snippet: A 778 bp fragment was amplified with the 1/13 primer pair (Supplementary Table ). .. Double digestion of the PCR products with Apo I and Hpy 188I (New England Biolabs) identified three groups of sequencing: cid A_IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp); cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 123; 83; 57; 51; and 24 bp); and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). .. For cidB _IV variants, only the second polymorphic region was found to be correlated with crossing types, so the PCR-RFLP test was designed to discriminate between the cidB _IV(1), cidB _IV(2), and cidB _IV(3) sequences (Fig. ).

    Article Title: Chloroquine-Resistant Haplotype Plasmodium falciparum Parasites, Haiti
    Article Snippet: The nested PCR protocol used primers CRTP1 and CRTP2 for the first round of amplification and primers CRTD1 and CRTD2 for the second round ( , ). .. Digestion of Amplicons from pfcrt with Apo I For each sample positive for SSU DNA, an aliquot (10 μL) of the pfcrt gene PCR product was digested with 10 U of Apo I (New England Biolabs, Beverly, MA, USA) according to the manufacturer’s instructions. .. Briefly, 10 U of Apo I in 1× NE buffer 3 (100 mol/L NaCl, 50 mmol/L Tris-HCl, 10 mmol/L MgCl2 , 1 mmol/L dithiothreitol) and bovine serum albumin (100 μg/μL) were incubated overnight with 10 μL of the PCR product at 50°C ( – ).

    Article Title: Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia
    Article Snippet: A 778 bp fragment was amplified with the 1/13 primer pair (Supplementary Table ). .. Double digestion of the PCR products with Apo I and Hpy 188I (New England Biolabs) identified three groups of sequencing: cid A_IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp); cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 123; 83; 57; 51; and 24 bp); and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). .. For cidB _IV variants, only the second polymorphic region was found to be correlated with crossing types, so the PCR-RFLP test was designed to discriminate between the cidB _IV(1), cidB _IV(2), and cidB _IV(3) sequences (Fig. ).

    Sequencing:

    Article Title: Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia
    Article Snippet: A 778 bp fragment was amplified with the 1/13 primer pair (Supplementary Table ). .. Double digestion of the PCR products with Apo I and Hpy 188I (New England Biolabs) identified three groups of sequencing: cid A_IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp); cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 123; 83; 57; 51; and 24 bp); and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). .. For cidB _IV variants, only the second polymorphic region was found to be correlated with crossing types, so the PCR-RFLP test was designed to discriminate between the cidB _IV(1), cidB _IV(2), and cidB _IV(3) sequences (Fig. ).

    Article Title: Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia
    Article Snippet: A 778 bp fragment was amplified with the 1/13 primer pair (Supplementary Table ). .. Double digestion of the PCR products with Apo I and Hpy 188I (New England Biolabs) identified three groups of sequencing: cid A_IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp); cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 123; 83; 57; 51; and 24 bp); and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). .. For cidB _IV variants, only the second polymorphic region was found to be correlated with crossing types, so the PCR-RFLP test was designed to discriminate between the cidB _IV(1), cidB _IV(2), and cidB _IV(3) sequences (Fig. ).

    Produced:

    Article Title: Streptococcal Histone-Like Protein: Primary Structure of hlpA and Protein Binding to Lipoteichoic Acid and Epithelial Cells
    Article Snippet: The regions upstream and downstream of the hlpA gene were cloned by inverse PCR. .. Five micrograms of DNA from D471 was digested to completion with Apo I (New England Biolabs), which produced 0.5- to 3-kb fragments but did not cut within hlpA . .. After the fragments were religated to form circular molecules, they were precipitated with ethanol and used as templates for PCR amplification (30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 2 min, and extension at 72°C for 3 min) with primers generated to internal regions of the hlpA structural gene (Table ).

    Ligation:

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome
    Article Snippet: Subsequent desalting was accomplished by drop dialysis ( ) to prevent high salt concentration from interfering with loading of DNA into capillaries, and ∼107 copies [(1.5 × 107 ) × 0.8 (efficiency of digestion) × 0.8 (yield of dialysis)] were estimated for the restriction fragment of AhdI and HinfI. .. To generate the HPRT target with the ligated clamp suitable for CDCE analysis (Fig. ), restriction digestion by ApoI (New England Biolabs) was followed by clamp ligation. ..

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    New England Biolabs apo i
    c idA _IV gene polymorphic regions and <t>PCR-RFLP</t> tests for specific variants. a Schematic representation of the architecture of cidA _IV polymorphism. The black line represents the cidA_IV sequence with a focus on the two polymorphic regions. Non-polymorphic regions were shortened and represented as a dashed line. Numbers in red under the line represent nucleotide positions, and the numbers in blue indicate amino-acid positions. The overlapping oligonucleotides used for PCR amplification are represented by arrows numbered as in Supplementary Table 2 . Both polymorphic regions were studied, but only the upstream matched the compatibility profile; its four different sequences (α, β γ, and δ) were followed by one of the two possible sequences (1 or 2) in the downstream polymorphic region. A different color code was used for α (purple), β (light blue), γ (light green), and δ (yellow) sequences in the upstream part of the cidA gene. b The repertoire of cidA _IV variants is different in “compatible” and “incompatible” lines. cidA _IV(δ) is present only in lines with “incompatible” crossing type. The names of the C . pipiens lines used to set-up the PCR-RFLP ( c ) test are highlighted in red. c PCR-RFLP tests for distinguishing between cidA _IV variants on the basis of the upstream polymorphic region. A 778 bp fragment was amplified with primers 1/13. Double digestion with <t>Apo</t> I and Hpy 188I distinguished between cidA _IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp), cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 122; 83; 57; 51; and 24 bp), and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). Panel to the left of the electrophoresis gel: PCR-RFLP on DNA from clones; right panel: PCR-RFLP on DNA from the Istanbul, Harash, Ichkeul 13, and Ichkeul 09 lines. On the right of the gel, a schematic representation of the PCR-RFLP profiles of these lines. Digestion bands that are specific of the variants are represented with the color code established in a . Bands that are not used to discriminate between the variants are represented in black on the bottom of the schematic gel
    Apo I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    c idA _IV gene polymorphic regions and PCR-RFLP tests for specific variants. a Schematic representation of the architecture of cidA _IV polymorphism. The black line represents the cidA_IV sequence with a focus on the two polymorphic regions. Non-polymorphic regions were shortened and represented as a dashed line. Numbers in red under the line represent nucleotide positions, and the numbers in blue indicate amino-acid positions. The overlapping oligonucleotides used for PCR amplification are represented by arrows numbered as in Supplementary Table 2 . Both polymorphic regions were studied, but only the upstream matched the compatibility profile; its four different sequences (α, β γ, and δ) were followed by one of the two possible sequences (1 or 2) in the downstream polymorphic region. A different color code was used for α (purple), β (light blue), γ (light green), and δ (yellow) sequences in the upstream part of the cidA gene. b The repertoire of cidA _IV variants is different in “compatible” and “incompatible” lines. cidA _IV(δ) is present only in lines with “incompatible” crossing type. The names of the C . pipiens lines used to set-up the PCR-RFLP ( c ) test are highlighted in red. c PCR-RFLP tests for distinguishing between cidA _IV variants on the basis of the upstream polymorphic region. A 778 bp fragment was amplified with primers 1/13. Double digestion with Apo I and Hpy 188I distinguished between cidA _IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp), cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 122; 83; 57; 51; and 24 bp), and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). Panel to the left of the electrophoresis gel: PCR-RFLP on DNA from clones; right panel: PCR-RFLP on DNA from the Istanbul, Harash, Ichkeul 13, and Ichkeul 09 lines. On the right of the gel, a schematic representation of the PCR-RFLP profiles of these lines. Digestion bands that are specific of the variants are represented with the color code established in a . Bands that are not used to discriminate between the variants are represented in black on the bottom of the schematic gel

    Journal: Nature Communications

    Article Title: Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia

    doi: 10.1038/s41467-017-02749-w

    Figure Lengend Snippet: c idA _IV gene polymorphic regions and PCR-RFLP tests for specific variants. a Schematic representation of the architecture of cidA _IV polymorphism. The black line represents the cidA_IV sequence with a focus on the two polymorphic regions. Non-polymorphic regions were shortened and represented as a dashed line. Numbers in red under the line represent nucleotide positions, and the numbers in blue indicate amino-acid positions. The overlapping oligonucleotides used for PCR amplification are represented by arrows numbered as in Supplementary Table 2 . Both polymorphic regions were studied, but only the upstream matched the compatibility profile; its four different sequences (α, β γ, and δ) were followed by one of the two possible sequences (1 or 2) in the downstream polymorphic region. A different color code was used for α (purple), β (light blue), γ (light green), and δ (yellow) sequences in the upstream part of the cidA gene. b The repertoire of cidA _IV variants is different in “compatible” and “incompatible” lines. cidA _IV(δ) is present only in lines with “incompatible” crossing type. The names of the C . pipiens lines used to set-up the PCR-RFLP ( c ) test are highlighted in red. c PCR-RFLP tests for distinguishing between cidA _IV variants on the basis of the upstream polymorphic region. A 778 bp fragment was amplified with primers 1/13. Double digestion with Apo I and Hpy 188I distinguished between cidA _IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp), cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 122; 83; 57; 51; and 24 bp), and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). Panel to the left of the electrophoresis gel: PCR-RFLP on DNA from clones; right panel: PCR-RFLP on DNA from the Istanbul, Harash, Ichkeul 13, and Ichkeul 09 lines. On the right of the gel, a schematic representation of the PCR-RFLP profiles of these lines. Digestion bands that are specific of the variants are represented with the color code established in a . Bands that are not used to discriminate between the variants are represented in black on the bottom of the schematic gel

    Article Snippet: Double digestion of the PCR products with Apo I and Hpy 188I (New England Biolabs) identified three groups of sequencing: cid A_IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp); cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 123; 83; 57; 51; and 24 bp); and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp).

    Techniques: Polymerase Chain Reaction, Sequencing, Amplification, Electrophoresis, Clone Assay

    Features of i3C performed in HUVEC s Overview of the i3C protocol. Living cells are harvested in a close‐to‐physiological buffer (PB; step 1); intact nuclei isolated by mild NP‐40 treatment (step 2); chromatin digested using Apo I or Nla III, nuclei spun to release unattached chromatin (step 3); and leave cut chromatin bound to the nuclear substructure (step 4). Then, ligation takes places in situ , and DNA is isolated (step 5). Percentage of total cell chromatin present at the different steps of the procedure (± SD; n = 2). Relative contribution of the different HUVEC ChromHMM features in each i3C fraction. i4C‐seq (blue shades) and conventional 4C (gray shades) were performed side by side in HUVECs, using Apo I and the SAMD4A TSS as a viewpoint (triangle); profiles from two replicates are overlaid. The browser view shows interactions in the ˜1 Mbp around SAMD4A . The zoom‐in shows interactions in the SAMD4A TAD (gray rectangle). Strong (red) and intermediate (brown) interactions called by fourSig , RefSeq gene models, and ENCODE ChIP‐seq data are shown below.

    Journal: Molecular Systems Biology

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

    doi: 10.15252/msb.20167311

    Figure Lengend Snippet: Features of i3C performed in HUVEC s Overview of the i3C protocol. Living cells are harvested in a close‐to‐physiological buffer (PB; step 1); intact nuclei isolated by mild NP‐40 treatment (step 2); chromatin digested using Apo I or Nla III, nuclei spun to release unattached chromatin (step 3); and leave cut chromatin bound to the nuclear substructure (step 4). Then, ligation takes places in situ , and DNA is isolated (step 5). Percentage of total cell chromatin present at the different steps of the procedure (± SD; n = 2). Relative contribution of the different HUVEC ChromHMM features in each i3C fraction. i4C‐seq (blue shades) and conventional 4C (gray shades) were performed side by side in HUVECs, using Apo I and the SAMD4A TSS as a viewpoint (triangle); profiles from two replicates are overlaid. The browser view shows interactions in the ˜1 Mbp around SAMD4A . The zoom‐in shows interactions in the SAMD4A TAD (gray rectangle). Strong (red) and intermediate (brown) interactions called by fourSig , RefSeq gene models, and ENCODE ChIP‐seq data are shown below.

    Article Snippet: Next, chromatin is digested with 500 units of Apo I or Nla III (New England Biolabs; 33°C, 30–45 min) without shaking.

    Techniques: Isolation, Ligation, In Situ, Chromatin Immunoprecipitation

    TALE ‐ iD verifies native looping at the human ZFPM 2 locus An overview of TALE‐iD. A construct encoding a TALE DNA‐binding domain that targets an active enhancer in the ZFPM2 first intron is fused to a bacterial Dam methylase and introduced into K562 cells. Cells are harvested 48 h after transfection; genomic DNA is isolated and digested with Dpn I to reveal sites specifically methylated by the Dam activity. Finally, qPCR using primers flanking different Dpn I sites is used as readout. i4C performed in K562 cells using Apo I and the ZFPM2 TSS as a viewpoint (triangle). i4C interaction in the 458‐kbp ZFPM2 locus is shown, and the enhancer targeted by the TALE‐iD construct is indicated (red triangle). K562 ENCODE ChIP‐seq data are also shown below. qPCR readout at different Dpn I sites. Dpn I sites at the ZFPM2 promoter (p1–p4) and enhancer (e1–e3; positions in panel B) were targeted in qPCRs after restriction digest. Bar plots show log 2 ‐fold enrichment of cut sites (1/ΔΔ C t ) over background Dpn I cutting levels in untransfected K562 cells. Regions c1–c4 serve as controls; region m1 (an enhancer shown to interact with the TSS by i4C) is also methylated as part of a multi‐loop structure. The same Dpn I sites were also tested in transfections involving a construct encoding either a non‐targeting (“scrambled”) TALE domain or the targeting domain fused to an inactive Dam protein (“ΔDam”). * P

    Journal: Molecular Systems Biology

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

    doi: 10.15252/msb.20167311

    Figure Lengend Snippet: TALE ‐ iD verifies native looping at the human ZFPM 2 locus An overview of TALE‐iD. A construct encoding a TALE DNA‐binding domain that targets an active enhancer in the ZFPM2 first intron is fused to a bacterial Dam methylase and introduced into K562 cells. Cells are harvested 48 h after transfection; genomic DNA is isolated and digested with Dpn I to reveal sites specifically methylated by the Dam activity. Finally, qPCR using primers flanking different Dpn I sites is used as readout. i4C performed in K562 cells using Apo I and the ZFPM2 TSS as a viewpoint (triangle). i4C interaction in the 458‐kbp ZFPM2 locus is shown, and the enhancer targeted by the TALE‐iD construct is indicated (red triangle). K562 ENCODE ChIP‐seq data are also shown below. qPCR readout at different Dpn I sites. Dpn I sites at the ZFPM2 promoter (p1–p4) and enhancer (e1–e3; positions in panel B) were targeted in qPCRs after restriction digest. Bar plots show log 2 ‐fold enrichment of cut sites (1/ΔΔ C t ) over background Dpn I cutting levels in untransfected K562 cells. Regions c1–c4 serve as controls; region m1 (an enhancer shown to interact with the TSS by i4C) is also methylated as part of a multi‐loop structure. The same Dpn I sites were also tested in transfections involving a construct encoding either a non‐targeting (“scrambled”) TALE domain or the targeting domain fused to an inactive Dam protein (“ΔDam”). * P

    Article Snippet: Next, chromatin is digested with 500 units of Apo I or Nla III (New England Biolabs; 33°C, 30–45 min) without shaking.

    Techniques: Construct, Binding Assay, Transfection, Isolation, Methylation, Activity Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    c idA _IV gene polymorphic regions and PCR-RFLP tests for specific variants. a Schematic representation of the architecture of cidA _IV polymorphism. The black line represents the cidA_IV . Both polymorphic regions were studied, but only the upstream matched the compatibility profile; its four different sequences (α, β γ, and δ) were followed by one of the two possible sequences (1 or 2) in the downstream polymorphic region. A different color code was used for α (purple), β (light blue), γ (light green), and δ (yellow) sequences in the upstream part of the cidA gene. b The repertoire of cidA _IV variants is different in “compatible” and “incompatible” lines. cidA _IV(δ) is present only in lines with “incompatible” crossing type. The names of the C . pipiens lines used to set-up the PCR-RFLP ( c ) test are highlighted in red. c PCR-RFLP tests for distinguishing between cidA _IV variants on the basis of the upstream polymorphic region. A 778 bp fragment was amplified with primers 1/13. Double digestion with Apo I and Hpy 188I distinguished between cidA _IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp), cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 122; 83; 57; 51; and 24 bp), and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). Panel to the left of the electrophoresis gel: PCR-RFLP on DNA from clones; right panel: PCR-RFLP on DNA from the Istanbul, Harash, Ichkeul 13, and Ichkeul 09 lines. On the right of the gel, a schematic representation of the PCR-RFLP profiles of these lines. Digestion bands that are specific of the variants are represented with the color code established in a . Bands that are not used to discriminate between the variants are represented in black on the bottom of the schematic gel

    Journal: Nature Communications

    Article Title: Culex pipiens crossing type diversity is governed by an amplified and polymorphic operon of Wolbachia

    doi: 10.1038/s41467-017-02749-w

    Figure Lengend Snippet: c idA _IV gene polymorphic regions and PCR-RFLP tests for specific variants. a Schematic representation of the architecture of cidA _IV polymorphism. The black line represents the cidA_IV . Both polymorphic regions were studied, but only the upstream matched the compatibility profile; its four different sequences (α, β γ, and δ) were followed by one of the two possible sequences (1 or 2) in the downstream polymorphic region. A different color code was used for α (purple), β (light blue), γ (light green), and δ (yellow) sequences in the upstream part of the cidA gene. b The repertoire of cidA _IV variants is different in “compatible” and “incompatible” lines. cidA _IV(δ) is present only in lines with “incompatible” crossing type. The names of the C . pipiens lines used to set-up the PCR-RFLP ( c ) test are highlighted in red. c PCR-RFLP tests for distinguishing between cidA _IV variants on the basis of the upstream polymorphic region. A 778 bp fragment was amplified with primers 1/13. Double digestion with Apo I and Hpy 188I distinguished between cidA _IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp), cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 122; 83; 57; 51; and 24 bp), and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp). Panel to the left of the electrophoresis gel: PCR-RFLP on DNA from clones; right panel: PCR-RFLP on DNA from the Istanbul, Harash, Ichkeul 13, and Ichkeul 09 lines. On the right of the gel, a schematic representation of the PCR-RFLP profiles of these lines. Digestion bands that are specific of the variants are represented with the color code established in a . Bands that are not used to discriminate between the variants are represented in black on the bottom of the schematic gel

    Article Snippet: Double digestion of the PCR products with Apo I and Hpy 188I (New England Biolabs) identified three groups of sequencing: cid A_IV(α) (six fragments: 471; 122; 57; 53; 51; and 24 bp); cidA _IV(β) and cidA _IV(γ) (six fragments: 441; 123; 83; 57; 51; and 24 bp); and cidA _IV(δ) (five fragments: 524; 122; 57; 51; and 24 bp).

    Techniques: Polymerase Chain Reaction, Amplification, Electrophoresis, Clone Assay

    Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Journal: Nucleic Acids Research

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

    doi:

    Figure Lengend Snippet: Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Article Snippet: To generate the HPRT target with the ligated clamp suitable for CDCE analysis (Fig. ), restriction digestion by ApoI (New England Biolabs) was followed by clamp ligation.

    Techniques: Flow Cytometry, Mutagenesis, Polymerase Chain Reaction, Isolation