acu i  (New England Biolabs)


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    AcuI
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    AcuI 1 500 units
    Catalog Number:
    r0641l
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    1 500 units
    Category:
    Restriction Enzymes
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    New England Biolabs acu i
    AcuI
    AcuI 1 500 units
    https://www.bioz.com/result/acu i/product/New England Biolabs
    Average 96 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    acu i - by Bioz Stars, 2020-07
    96/100 stars

    Images

    1) Product Images from "A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism"

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

    Journal: Science Advances

    doi: 10.1126/sciadv.1600699

    gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.
    Figure Legend Snippet: gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Fractionation, Marker

    2) Product Images from "Recursive Directional Ligation by Plasmid Reconstruction allows Rapid and Seamless Cloning of Oligomeric Genes"

    Article Title: Recursive Directional Ligation by Plasmid Reconstruction allows Rapid and Seamless Cloning of Oligomeric Genes

    Journal: Biomacromolecules

    doi: 10.1021/bm901387t

    (A) Recursive directional ligation by plasmid reconstruction (PRe-RDL). One round in PRe-RDL involves: (1) purifying the ELP-containing DNA fragment from the parent vector that is digested with AcuI and BglI; and (2) purifying the ELP-containing fragment
    Figure Legend Snippet: (A) Recursive directional ligation by plasmid reconstruction (PRe-RDL). One round in PRe-RDL involves: (1) purifying the ELP-containing DNA fragment from the parent vector that is digested with AcuI and BglI; and (2) purifying the ELP-containing fragment

    Techniques Used: Ligation, Plasmid Preparation

    3) Product Images from "Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures"

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.02.068

    Identification of Targeted Dinucleotide Signatures Using DTECT (A) Schematic representation of DTECT. The targeted genomic locus containing a hypothetical targeted dinucleotide (N = A, C, G, or T; green) is PCR amplified using a forward AcuI-tagging primer juxtaposed to the targeted dinucleotide and a locus-specific DNA primer (AcuI-tagging primer design and PCR, steps I and II). The AcuI-tagging primer (60 nt) consists of DNA sequences complementary to the genomic locus (purple) interrupted by a hairpin containing an AcuI recognition site (green), and a non-complementary DNA sequence (blue). The locus-specific reverse primer (red) is located > 100 bp from the targeted dinucleotide. The obtained PCR product is subsequently cleaved by the AcuI restriction enzyme in a position adjacent to the targeted dinucleotide, resulting in the generation of two DNA fragments of 60 and > 100 bp (AcuI digestion, step III). The 60 bp fragment containing the exposed signature of the targeted dinucleotide is then isolated using SPRI beads, with higher affinity toward > 100 bp DNA products (small fragment isolation, step IV). The 60 bp fragment is then ligated to DNA adaptors containing 3′ overhangs of two bases complementary (specific) or not (non-specific) to the dinucleotide signature (adaptor ligation, step V). The ligated product is then subjected to PCR amplification for analytical or quantitative detection (detection PCR, step VI). The approximate time required for each step is indicated. (B) Schematics of the DTECT adaptor library. Control (green) and mutant (purple) dinucleotide signatures (left panel) are detected using a library of 16 unique adaptors (middle panel). The library contains adaptors with dinucleotides complementary to the control (green) or mutant (purple) signature, as well as non-specific adaptors (blue) (right panel). (C) Schematics of the positive and negative controls used in DTECT experiments to identify signatures of interest (e.g., mutant allele) in allele populations. In gDNA samples containing only the WT dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) serves as a positive control, while the adaptor complementary to the mutant signature of interest (purple) and a non-specific adaptor (blue) are used as negative controls. In gDNA samples containing a mixture of the WT and the mutant dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) is used as a positive control and a non-specific adaptor (blue) serves as a negative control. The adaptor complementary to the mutant dinucleotide signature (purple) is used to detect the presence of the variant of interest and quantify its frequency. See also Figure S1 .
    Figure Legend Snippet: Identification of Targeted Dinucleotide Signatures Using DTECT (A) Schematic representation of DTECT. The targeted genomic locus containing a hypothetical targeted dinucleotide (N = A, C, G, or T; green) is PCR amplified using a forward AcuI-tagging primer juxtaposed to the targeted dinucleotide and a locus-specific DNA primer (AcuI-tagging primer design and PCR, steps I and II). The AcuI-tagging primer (60 nt) consists of DNA sequences complementary to the genomic locus (purple) interrupted by a hairpin containing an AcuI recognition site (green), and a non-complementary DNA sequence (blue). The locus-specific reverse primer (red) is located > 100 bp from the targeted dinucleotide. The obtained PCR product is subsequently cleaved by the AcuI restriction enzyme in a position adjacent to the targeted dinucleotide, resulting in the generation of two DNA fragments of 60 and > 100 bp (AcuI digestion, step III). The 60 bp fragment containing the exposed signature of the targeted dinucleotide is then isolated using SPRI beads, with higher affinity toward > 100 bp DNA products (small fragment isolation, step IV). The 60 bp fragment is then ligated to DNA adaptors containing 3′ overhangs of two bases complementary (specific) or not (non-specific) to the dinucleotide signature (adaptor ligation, step V). The ligated product is then subjected to PCR amplification for analytical or quantitative detection (detection PCR, step VI). The approximate time required for each step is indicated. (B) Schematics of the DTECT adaptor library. Control (green) and mutant (purple) dinucleotide signatures (left panel) are detected using a library of 16 unique adaptors (middle panel). The library contains adaptors with dinucleotides complementary to the control (green) or mutant (purple) signature, as well as non-specific adaptors (blue) (right panel). (C) Schematics of the positive and negative controls used in DTECT experiments to identify signatures of interest (e.g., mutant allele) in allele populations. In gDNA samples containing only the WT dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) serves as a positive control, while the adaptor complementary to the mutant signature of interest (purple) and a non-specific adaptor (blue) are used as negative controls. In gDNA samples containing a mixture of the WT and the mutant dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) is used as a positive control and a non-specific adaptor (blue) serves as a negative control. The adaptor complementary to the mutant dinucleotide signature (purple) is used to detect the presence of the variant of interest and quantify its frequency. See also Figure S1 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Isolation, Ligation, Mutagenesis, Positive Control, Negative Control, Variant Assay

    DTECT-Mediated Identification of Clinically Relevant BRCA1/2 Mutations Generated by Precision Genome Editing and Genotyping of Cell Lines and Animal Models Carrying BRCA1 or BARD1 Mutations (A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios ( Billing et al., 2018 ). (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also Figures S6 , S7 , and S9 .
    Figure Legend Snippet: DTECT-Mediated Identification of Clinically Relevant BRCA1/2 Mutations Generated by Precision Genome Editing and Genotyping of Cell Lines and Animal Models Carrying BRCA1 or BARD1 Mutations (A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios ( Billing et al., 2018 ). (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also Figures S6 , S7 , and S9 .

    Techniques Used: Generated, Next-Generation Sequencing, CRISPR, Expressing, Transfection, Introduce, Amplification, Sequencing, Polymerase Chain Reaction, Mutagenesis, Clone Assay, Derivative Assay, Knock-In, Mouse Assay

    Detection and Quantification of Dinucleotide Signatures Using DTECT (A) Design of AcuI-tagging primers that allow the capture of two dinucleotide signatures (CC and TT, blue) on opposite DNA strands. (B) PCR amplification (22 cycles) of the AcuI-digested DNA products containing the CC and TT signatures shown in (A), which have been captured using GG or AA adaptors. (C) PCR amplification (22 cycles) of DNA fragments captured as in (B) with or without dephosphorylation of the AcuI-digested products by the shrimp alkaline phosphatase (rSAP). (D) PCR amplification (22 cycles) of DNA fragments captured as in (B) in the absence or presence of AcuI, DNA adaptors (GG adaptor for signature CC and AA adaptor for signature TT), or T4 DNA ligase. (E) Schematic representation of the AcuI-tagging primer design for detecting four possible dinucleotide signatures (1–4) containing the same targeted base (C:G, red) in the PIK3R1 gene. (F) Detection of the four dinucleotide signatures shown in (E) by DTECT (18 PCR cycles) using specific (green) and non-specific (blue) adaptors. (G) Quantification by DTECT of the relative abundance of SMARCAL1 , SPRTN , and PIK3R1 WT (green) and STOP (purple) dinucleotide signatures in mixtures of WT and STOP alleles at predefined ratios. Graphs (left) represent the correlation between the frequency of WT and STOP variants determined by DTECT and the expected frequency of the same variants in the mixed populations for each of the preceding 3 genes. Error bars represent the SD of independent experiments (n = 2). Pearson correlation (r) was determined by comparing expected and DTECT-based frequency. Comparison of the mean frequency of STOP and WT signatures determined by DTECT and their expected frequency is shown in the right panel (n = 3 independent genes, SMARCAL1 , SPRTN , and PIK3R1 ). (H) Representation of the AcuI-tagging primers used to detect the WT and STOP alleles of the PIK3R1 gene. The targeted dinucleotides are shown in blue, the edited base is indicated with an asterisk, and part of the AcuI-tagging primer sequence is shown in purple. (I) PCR amplification (25 cycles) of WT and STOP PIK3R1 alleles (arrow) captured using DTECT from WT:STOP allele mixtures (i.e., 100:0 and 99:1). An adaptor (CG) specific for the WT allele is used as a positive control, and a non-specific adaptor (TT) is used as a negative control. An adaptor that captures the STOP PIK3R1 allele (CA) serves as an additional negative control in the reaction containing only the WT allele. Background non-specific PCR products are indicated with an asterisk. (J) Fold change variation in the frequency of capture of each of the 16 dinucleotide signatures relative to the mean dinucleotide capture frequency. Oligonucleotides containing distinct dinucleotide signatures are captured using specific adaptors. The fraction of captured material is then quantified by qPCR and normalized to the mean value obtained from the capture of all 16 dinucleotide signatures. Error bars indicate the SD of 4 independent experiments. Dots represent individual data points. (K) Fold change variation in the frequency of capture of dinucleotide signatures with 1 A/T + 1 C/G, 2 A/T, or 2 C/G bases relative to the mean dinucleotide capture frequency, determined as described in (J). Error bars represent the SD of 8 mean values for dinucleotides with 1 A/T + 1 C/G and 4 mean values for dinucleotides with 2 A/T and 2 C/G, as determined in (J). See also Figures S2 , S3 , and S10 .
    Figure Legend Snippet: Detection and Quantification of Dinucleotide Signatures Using DTECT (A) Design of AcuI-tagging primers that allow the capture of two dinucleotide signatures (CC and TT, blue) on opposite DNA strands. (B) PCR amplification (22 cycles) of the AcuI-digested DNA products containing the CC and TT signatures shown in (A), which have been captured using GG or AA adaptors. (C) PCR amplification (22 cycles) of DNA fragments captured as in (B) with or without dephosphorylation of the AcuI-digested products by the shrimp alkaline phosphatase (rSAP). (D) PCR amplification (22 cycles) of DNA fragments captured as in (B) in the absence or presence of AcuI, DNA adaptors (GG adaptor for signature CC and AA adaptor for signature TT), or T4 DNA ligase. (E) Schematic representation of the AcuI-tagging primer design for detecting four possible dinucleotide signatures (1–4) containing the same targeted base (C:G, red) in the PIK3R1 gene. (F) Detection of the four dinucleotide signatures shown in (E) by DTECT (18 PCR cycles) using specific (green) and non-specific (blue) adaptors. (G) Quantification by DTECT of the relative abundance of SMARCAL1 , SPRTN , and PIK3R1 WT (green) and STOP (purple) dinucleotide signatures in mixtures of WT and STOP alleles at predefined ratios. Graphs (left) represent the correlation between the frequency of WT and STOP variants determined by DTECT and the expected frequency of the same variants in the mixed populations for each of the preceding 3 genes. Error bars represent the SD of independent experiments (n = 2). Pearson correlation (r) was determined by comparing expected and DTECT-based frequency. Comparison of the mean frequency of STOP and WT signatures determined by DTECT and their expected frequency is shown in the right panel (n = 3 independent genes, SMARCAL1 , SPRTN , and PIK3R1 ). (H) Representation of the AcuI-tagging primers used to detect the WT and STOP alleles of the PIK3R1 gene. The targeted dinucleotides are shown in blue, the edited base is indicated with an asterisk, and part of the AcuI-tagging primer sequence is shown in purple. (I) PCR amplification (25 cycles) of WT and STOP PIK3R1 alleles (arrow) captured using DTECT from WT:STOP allele mixtures (i.e., 100:0 and 99:1). An adaptor (CG) specific for the WT allele is used as a positive control, and a non-specific adaptor (TT) is used as a negative control. An adaptor that captures the STOP PIK3R1 allele (CA) serves as an additional negative control in the reaction containing only the WT allele. Background non-specific PCR products are indicated with an asterisk. (J) Fold change variation in the frequency of capture of each of the 16 dinucleotide signatures relative to the mean dinucleotide capture frequency. Oligonucleotides containing distinct dinucleotide signatures are captured using specific adaptors. The fraction of captured material is then quantified by qPCR and normalized to the mean value obtained from the capture of all 16 dinucleotide signatures. Error bars indicate the SD of 4 independent experiments. Dots represent individual data points. (K) Fold change variation in the frequency of capture of dinucleotide signatures with 1 A/T + 1 C/G, 2 A/T, or 2 C/G bases relative to the mean dinucleotide capture frequency, determined as described in (J). Error bars represent the SD of 8 mean values for dinucleotides with 1 A/T + 1 C/G and 4 mean values for dinucleotides with 2 A/T and 2 C/G, as determined in (J). See also Figures S2 , S3 , and S10 .

    Techniques Used: Polymerase Chain Reaction, Amplification, De-Phosphorylation Assay, Sequencing, Positive Control, Negative Control, Real-time Polymerase Chain Reaction

    4) Product Images from "Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures"

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.02.068

    Identification of Targeted Dinucleotide Signatures Using DTECT (A) Schematic representation of DTECT. The targeted genomic locus containing a hypothetical targeted dinucleotide (N = A, C, G, or T; green) is PCR amplified using a forward AcuI-tagging primer juxtaposed to the targeted dinucleotide and a locus-specific DNA primer (AcuI-tagging primer design and PCR, steps I and II). The AcuI-tagging primer (60 nt) consists of DNA sequences complementary to the genomic locus (purple) interrupted by a hairpin containing an AcuI recognition site (green), and a non-complementary DNA sequence (blue). The locus-specific reverse primer (red) is located > 100 bp from the targeted dinucleotide. The obtained PCR product is subsequently cleaved by the AcuI restriction enzyme in a position adjacent to the targeted dinucleotide, resulting in the generation of two DNA fragments of 60 and > 100 bp (AcuI digestion, step III). The 60 bp fragment containing the exposed signature of the targeted dinucleotide is then isolated using SPRI beads, with higher affinity toward > 100 bp DNA products (small fragment isolation, step IV). The 60 bp fragment is then ligated to DNA adaptors containing 3′ overhangs of two bases complementary (specific) or not (non-specific) to the dinucleotide signature (adaptor ligation, step V). The ligated product is then subjected to PCR amplification for analytical or quantitative detection (detection PCR, step VI). The approximate time required for each step is indicated. (B) Schematics of the DTECT adaptor library. Control (green) and mutant (purple) dinucleotide signatures (left panel) are detected using a library of 16 unique adaptors (middle panel). The library contains adaptors with dinucleotides complementary to the control (green) or mutant (purple) signature, as well as non-specific adaptors (blue) (right panel). (C) Schematics of the positive and negative controls used in DTECT experiments to identify signatures of interest (e.g., mutant allele) in allele populations. In gDNA samples containing only the WT dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) serves as a positive control, while the adaptor complementary to the mutant signature of interest (purple) and a non-specific adaptor (blue) are used as negative controls. In gDNA samples containing a mixture of the WT and the mutant dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) is used as a positive control and a non-specific adaptor (blue) serves as a negative control. The adaptor complementary to the mutant dinucleotide signature (purple) is used to detect the presence of the variant of interest and quantify its frequency. See also Figure S1 .
    Figure Legend Snippet: Identification of Targeted Dinucleotide Signatures Using DTECT (A) Schematic representation of DTECT. The targeted genomic locus containing a hypothetical targeted dinucleotide (N = A, C, G, or T; green) is PCR amplified using a forward AcuI-tagging primer juxtaposed to the targeted dinucleotide and a locus-specific DNA primer (AcuI-tagging primer design and PCR, steps I and II). The AcuI-tagging primer (60 nt) consists of DNA sequences complementary to the genomic locus (purple) interrupted by a hairpin containing an AcuI recognition site (green), and a non-complementary DNA sequence (blue). The locus-specific reverse primer (red) is located > 100 bp from the targeted dinucleotide. The obtained PCR product is subsequently cleaved by the AcuI restriction enzyme in a position adjacent to the targeted dinucleotide, resulting in the generation of two DNA fragments of 60 and > 100 bp (AcuI digestion, step III). The 60 bp fragment containing the exposed signature of the targeted dinucleotide is then isolated using SPRI beads, with higher affinity toward > 100 bp DNA products (small fragment isolation, step IV). The 60 bp fragment is then ligated to DNA adaptors containing 3′ overhangs of two bases complementary (specific) or not (non-specific) to the dinucleotide signature (adaptor ligation, step V). The ligated product is then subjected to PCR amplification for analytical or quantitative detection (detection PCR, step VI). The approximate time required for each step is indicated. (B) Schematics of the DTECT adaptor library. Control (green) and mutant (purple) dinucleotide signatures (left panel) are detected using a library of 16 unique adaptors (middle panel). The library contains adaptors with dinucleotides complementary to the control (green) or mutant (purple) signature, as well as non-specific adaptors (blue) (right panel). (C) Schematics of the positive and negative controls used in DTECT experiments to identify signatures of interest (e.g., mutant allele) in allele populations. In gDNA samples containing only the WT dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) serves as a positive control, while the adaptor complementary to the mutant signature of interest (purple) and a non-specific adaptor (blue) are used as negative controls. In gDNA samples containing a mixture of the WT and the mutant dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) is used as a positive control and a non-specific adaptor (blue) serves as a negative control. The adaptor complementary to the mutant dinucleotide signature (purple) is used to detect the presence of the variant of interest and quantify its frequency. See also Figure S1 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Isolation, Ligation, Mutagenesis, Positive Control, Negative Control, Variant Assay

    DTECT-Mediated Identification of Clinically Relevant BRCA1/2 Mutations Generated by Precision Genome Editing and Genotyping of Cell Lines and Animal Models Carrying BRCA1 or BARD1 Mutations (A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios ( Billing et al., 2018 ). (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also Figures S6 , S7 , and S9 .
    Figure Legend Snippet: DTECT-Mediated Identification of Clinically Relevant BRCA1/2 Mutations Generated by Precision Genome Editing and Genotyping of Cell Lines and Animal Models Carrying BRCA1 or BARD1 Mutations (A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios ( Billing et al., 2018 ). (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also Figures S6 , S7 , and S9 .

    Techniques Used: Generated, Next-Generation Sequencing, CRISPR, Expressing, Transfection, Introduce, Amplification, Sequencing, Polymerase Chain Reaction, Mutagenesis, Clone Assay, Derivative Assay, Knock-In, Mouse Assay

    Detection and Quantification of Dinucleotide Signatures Using DTECT (A) Design of AcuI-tagging primers that allow the capture of two dinucleotide signatures (CC and TT, blue) on opposite DNA strands. (B) PCR amplification (22 cycles) of the AcuI-digested DNA products containing the CC and TT signatures shown in (A), which have been captured using GG or AA adaptors. (C) PCR amplification (22 cycles) of DNA fragments captured as in (B) with or without dephosphorylation of the AcuI-digested products by the shrimp alkaline phosphatase (rSAP). (D) PCR amplification (22 cycles) of DNA fragments captured as in (B) in the absence or presence of AcuI, DNA adaptors (GG adaptor for signature CC and AA adaptor for signature TT), or T4 DNA ligase. (E) Schematic representation of the AcuI-tagging primer design for detecting four possible dinucleotide signatures (1–4) containing the same targeted base (C:G, red) in the PIK3R1 gene. (F) Detection of the four dinucleotide signatures shown in (E) by DTECT (18 PCR cycles) using specific (green) and non-specific (blue) adaptors. (G) Quantification by DTECT of the relative abundance of SMARCAL1 , SPRTN , and PIK3R1 WT (green) and STOP (purple) dinucleotide signatures in mixtures of WT and STOP alleles at predefined ratios. Graphs (left) represent the correlation between the frequency of WT and STOP variants determined by DTECT and the expected frequency of the same variants in the mixed populations for each of the preceding 3 genes. Error bars represent the SD of independent experiments (n = 2). Pearson correlation (r) was determined by comparing expected and DTECT-based frequency. Comparison of the mean frequency of STOP and WT signatures determined by DTECT and their expected frequency is shown in the right panel (n = 3 independent genes, SMARCAL1 , SPRTN , and PIK3R1 ). (H) Representation of the AcuI-tagging primers used to detect the WT and STOP alleles of the PIK3R1 gene. The targeted dinucleotides are shown in blue, the edited base is indicated with an asterisk, and part of the AcuI-tagging primer sequence is shown in purple. (I) PCR amplification (25 cycles) of WT and STOP PIK3R1 alleles (arrow) captured using DTECT from WT:STOP allele mixtures (i.e., 100:0 and 99:1). An adaptor (CG) specific for the WT allele is used as a positive control, and a non-specific adaptor (TT) is used as a negative control. An adaptor that captures the STOP PIK3R1 allele (CA) serves as an additional negative control in the reaction containing only the WT allele. Background non-specific PCR products are indicated with an asterisk. (J) Fold change variation in the frequency of capture of each of the 16 dinucleotide signatures relative to the mean dinucleotide capture frequency. Oligonucleotides containing distinct dinucleotide signatures are captured using specific adaptors. The fraction of captured material is then quantified by qPCR and normalized to the mean value obtained from the capture of all 16 dinucleotide signatures. Error bars indicate the SD of 4 independent experiments. Dots represent individual data points. (K) Fold change variation in the frequency of capture of dinucleotide signatures with 1 A/T + 1 C/G, 2 A/T, or 2 C/G bases relative to the mean dinucleotide capture frequency, determined as described in (J). Error bars represent the SD of 8 mean values for dinucleotides with 1 A/T + 1 C/G and 4 mean values for dinucleotides with 2 A/T and 2 C/G, as determined in (J). See also Figures S2 , S3 , and S10 .
    Figure Legend Snippet: Detection and Quantification of Dinucleotide Signatures Using DTECT (A) Design of AcuI-tagging primers that allow the capture of two dinucleotide signatures (CC and TT, blue) on opposite DNA strands. (B) PCR amplification (22 cycles) of the AcuI-digested DNA products containing the CC and TT signatures shown in (A), which have been captured using GG or AA adaptors. (C) PCR amplification (22 cycles) of DNA fragments captured as in (B) with or without dephosphorylation of the AcuI-digested products by the shrimp alkaline phosphatase (rSAP). (D) PCR amplification (22 cycles) of DNA fragments captured as in (B) in the absence or presence of AcuI, DNA adaptors (GG adaptor for signature CC and AA adaptor for signature TT), or T4 DNA ligase. (E) Schematic representation of the AcuI-tagging primer design for detecting four possible dinucleotide signatures (1–4) containing the same targeted base (C:G, red) in the PIK3R1 gene. (F) Detection of the four dinucleotide signatures shown in (E) by DTECT (18 PCR cycles) using specific (green) and non-specific (blue) adaptors. (G) Quantification by DTECT of the relative abundance of SMARCAL1 , SPRTN , and PIK3R1 WT (green) and STOP (purple) dinucleotide signatures in mixtures of WT and STOP alleles at predefined ratios. Graphs (left) represent the correlation between the frequency of WT and STOP variants determined by DTECT and the expected frequency of the same variants in the mixed populations for each of the preceding 3 genes. Error bars represent the SD of independent experiments (n = 2). Pearson correlation (r) was determined by comparing expected and DTECT-based frequency. Comparison of the mean frequency of STOP and WT signatures determined by DTECT and their expected frequency is shown in the right panel (n = 3 independent genes, SMARCAL1 , SPRTN , and PIK3R1 ). (H) Representation of the AcuI-tagging primers used to detect the WT and STOP alleles of the PIK3R1 gene. The targeted dinucleotides are shown in blue, the edited base is indicated with an asterisk, and part of the AcuI-tagging primer sequence is shown in purple. (I) PCR amplification (25 cycles) of WT and STOP PIK3R1 alleles (arrow) captured using DTECT from WT:STOP allele mixtures (i.e., 100:0 and 99:1). An adaptor (CG) specific for the WT allele is used as a positive control, and a non-specific adaptor (TT) is used as a negative control. An adaptor that captures the STOP PIK3R1 allele (CA) serves as an additional negative control in the reaction containing only the WT allele. Background non-specific PCR products are indicated with an asterisk. (J) Fold change variation in the frequency of capture of each of the 16 dinucleotide signatures relative to the mean dinucleotide capture frequency. Oligonucleotides containing distinct dinucleotide signatures are captured using specific adaptors. The fraction of captured material is then quantified by qPCR and normalized to the mean value obtained from the capture of all 16 dinucleotide signatures. Error bars indicate the SD of 4 independent experiments. Dots represent individual data points. (K) Fold change variation in the frequency of capture of dinucleotide signatures with 1 A/T + 1 C/G, 2 A/T, or 2 C/G bases relative to the mean dinucleotide capture frequency, determined as described in (J). Error bars represent the SD of 8 mean values for dinucleotides with 1 A/T + 1 C/G and 4 mean values for dinucleotides with 2 A/T and 2 C/G, as determined in (J). See also Figures S2 , S3 , and S10 .

    Techniques Used: Polymerase Chain Reaction, Amplification, De-Phosphorylation Assay, Sequencing, Positive Control, Negative Control, Real-time Polymerase Chain Reaction

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Single nucleotide polymorphisms and haplotypes of the genes encoding the CYP1B1 in Korean women: No association with advanced endometriosis
    Article Snippet: .. In order, each PCR products were digested with 10 U/μl of Ngo MIV, 5 U/μl of Fok I, 10 U/μl of Acu I and 10 U/μl of Mwo I restriction enzymes (New England Biolabs Inc.). .. The DNA fragments were then seperated and visualized by electrophoresis on 2% agarose gel containing ethidium bromide.

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
    Article Snippet: .. Acu I/Xba I digestion The PCR product was digested with 2 μl of Acu I (5 U/μl; NEB) and 2 μl of Xba I (20 U/μl; NEB) in 1× CutSmart Buffer containing 40 μM S -adenosylmethionine in a 60-μl volume at 37°C overnight. ..

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures
    Article Snippet: .. The purified PCR products were digested by 0.25 μl AcuI (NEB #0641L) in a 20 μl reaction containing 1X CutSmart Buffer (NEB) supplemented with 40 μM S-adenosylmethionine (SAM) and 100 ng of purified PCR product. .. The reaction was incubated for 1 hour at 37°C with heat inactivation at 65°C for 20 min. IV) Isolation of the AcuI-digested genomic amplicon by solid phase reversible immobilization (SPRI) .

    Clone Assay:

    Article Title: Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen
    Article Snippet: .. Restriction enzymes — BamHI, XbaI, AcuI, BseRI and Bg1I; alkaline phosphatase; T4 polynucleotide kinase (3′ phosphatase minus) and T4 DNA ligase were obtained from New England Biolabs (MA, USA). pET-24 a (+) cloning vector was purchased from Novagen Inc. (WI, USA). .. NEB 10-beta competent Escherichia coli (high effciency) cells, and BL21 (DE3) competent E. coli cells were purchased from New England Biolabs (MA, USA).

    Positron Emission Tomography:

    Article Title: Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen
    Article Snippet: .. Restriction enzymes — BamHI, XbaI, AcuI, BseRI and Bg1I; alkaline phosphatase; T4 polynucleotide kinase (3′ phosphatase minus) and T4 DNA ligase were obtained from New England Biolabs (MA, USA). pET-24 a (+) cloning vector was purchased from Novagen Inc. (WI, USA). .. NEB 10-beta competent Escherichia coli (high effciency) cells, and BL21 (DE3) competent E. coli cells were purchased from New England Biolabs (MA, USA).

    Article Title: Bio-Inspired Synthesis of Hybrid Silica Nanoparticles Templated from Elastin-like Polypeptide Micelles
    Article Snippet: .. Restriction enzymes BglI, NdeI, BseRI, AcuI, BamHI and XbaI, T4 DNA Ligase and calf intestinal phosphatase (CIP) were purchased from New England Biolabs (Ipswich, MA). pET-24a(+) vector was purchased from Novagen Inc. (Madison, WI). .. BL21 E. coli competent cells were purchased from EdgeBio (Gaithersburg, MD).

    Plasmid Preparation:

    Article Title: Synthesis and Immunogenicity Assessment of Elastin-Like Polypeptide-M2e Construct as an Influenza Antigen
    Article Snippet: .. Restriction enzymes — BamHI, XbaI, AcuI, BseRI and Bg1I; alkaline phosphatase; T4 polynucleotide kinase (3′ phosphatase minus) and T4 DNA ligase were obtained from New England Biolabs (MA, USA). pET-24 a (+) cloning vector was purchased from Novagen Inc. (WI, USA). .. NEB 10-beta competent Escherichia coli (high effciency) cells, and BL21 (DE3) competent E. coli cells were purchased from New England Biolabs (MA, USA).

    Article Title: Bio-Inspired Synthesis of Hybrid Silica Nanoparticles Templated from Elastin-like Polypeptide Micelles
    Article Snippet: .. Restriction enzymes BglI, NdeI, BseRI, AcuI, BamHI and XbaI, T4 DNA Ligase and calf intestinal phosphatase (CIP) were purchased from New England Biolabs (Ipswich, MA). pET-24a(+) vector was purchased from Novagen Inc. (Madison, WI). .. BL21 E. coli competent cells were purchased from EdgeBio (Gaithersburg, MD).

    Purification:

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures
    Article Snippet: .. The purified PCR products were digested by 0.25 μl AcuI (NEB #0641L) in a 20 μl reaction containing 1X CutSmart Buffer (NEB) supplemented with 40 μM S-adenosylmethionine (SAM) and 100 ng of purified PCR product. .. The reaction was incubated for 1 hour at 37°C with heat inactivation at 65°C for 20 min. IV) Isolation of the AcuI-digested genomic amplicon by solid phase reversible immobilization (SPRI) .

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    New England Biolabs acu i
    gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, <t>Xba</t> I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.
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    gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

    Journal: Science Advances

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

    doi: 10.1126/sciadv.1600699

    Figure Lengend Snippet: gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

    Article Snippet: Acu I/Xba I digestion The PCR product was digested with 2 μl of Acu I (5 U/μl; NEB) and 2 μl of Xba I (20 U/μl; NEB) in 1× CutSmart Buffer containing 40 μM S -adenosylmethionine in a 60-μl volume at 37°C overnight.

    Techniques: Sequencing, Polymerase Chain Reaction, Fractionation, Marker

    (A) Recursive directional ligation by plasmid reconstruction (PRe-RDL). One round in PRe-RDL involves: (1) purifying the ELP-containing DNA fragment from the parent vector that is digested with AcuI and BglI; and (2) purifying the ELP-containing fragment

    Journal: Biomacromolecules

    Article Title: Recursive Directional Ligation by Plasmid Reconstruction allows Rapid and Seamless Cloning of Oligomeric Genes

    doi: 10.1021/bm901387t

    Figure Lengend Snippet: (A) Recursive directional ligation by plasmid reconstruction (PRe-RDL). One round in PRe-RDL involves: (1) purifying the ELP-containing DNA fragment from the parent vector that is digested with AcuI and BglI; and (2) purifying the ELP-containing fragment

    Article Snippet: Using the dimerization of the 30 repeat fragment of ELP1 in the second round of PRe-RDL as an example, the designated ‘A’ fragment was obtained by digestion of 4 μg of ELP1 -30 with 10 U AcuI and 40 U BglI for 3 hours at 37°C (see ) in NEB Buffer 2 (New England Biolabs; Ipswich, MA).

    Techniques: Ligation, Plasmid Preparation

    Identification of Targeted Dinucleotide Signatures Using DTECT (A) Schematic representation of DTECT. The targeted genomic locus containing a hypothetical targeted dinucleotide (N = A, C, G, or T; green) is PCR amplified using a forward AcuI-tagging primer juxtaposed to the targeted dinucleotide and a locus-specific DNA primer (AcuI-tagging primer design and PCR, steps I and II). The AcuI-tagging primer (60 nt) consists of DNA sequences complementary to the genomic locus (purple) interrupted by a hairpin containing an AcuI recognition site (green), and a non-complementary DNA sequence (blue). The locus-specific reverse primer (red) is located > 100 bp from the targeted dinucleotide. The obtained PCR product is subsequently cleaved by the AcuI restriction enzyme in a position adjacent to the targeted dinucleotide, resulting in the generation of two DNA fragments of 60 and > 100 bp (AcuI digestion, step III). The 60 bp fragment containing the exposed signature of the targeted dinucleotide is then isolated using SPRI beads, with higher affinity toward > 100 bp DNA products (small fragment isolation, step IV). The 60 bp fragment is then ligated to DNA adaptors containing 3′ overhangs of two bases complementary (specific) or not (non-specific) to the dinucleotide signature (adaptor ligation, step V). The ligated product is then subjected to PCR amplification for analytical or quantitative detection (detection PCR, step VI). The approximate time required for each step is indicated. (B) Schematics of the DTECT adaptor library. Control (green) and mutant (purple) dinucleotide signatures (left panel) are detected using a library of 16 unique adaptors (middle panel). The library contains adaptors with dinucleotides complementary to the control (green) or mutant (purple) signature, as well as non-specific adaptors (blue) (right panel). (C) Schematics of the positive and negative controls used in DTECT experiments to identify signatures of interest (e.g., mutant allele) in allele populations. In gDNA samples containing only the WT dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) serves as a positive control, while the adaptor complementary to the mutant signature of interest (purple) and a non-specific adaptor (blue) are used as negative controls. In gDNA samples containing a mixture of the WT and the mutant dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) is used as a positive control and a non-specific adaptor (blue) serves as a negative control. The adaptor complementary to the mutant dinucleotide signature (purple) is used to detect the presence of the variant of interest and quantify its frequency. See also Figure S1 .

    Journal: Cell reports

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

    doi: 10.1016/j.celrep.2020.02.068

    Figure Lengend Snippet: Identification of Targeted Dinucleotide Signatures Using DTECT (A) Schematic representation of DTECT. The targeted genomic locus containing a hypothetical targeted dinucleotide (N = A, C, G, or T; green) is PCR amplified using a forward AcuI-tagging primer juxtaposed to the targeted dinucleotide and a locus-specific DNA primer (AcuI-tagging primer design and PCR, steps I and II). The AcuI-tagging primer (60 nt) consists of DNA sequences complementary to the genomic locus (purple) interrupted by a hairpin containing an AcuI recognition site (green), and a non-complementary DNA sequence (blue). The locus-specific reverse primer (red) is located > 100 bp from the targeted dinucleotide. The obtained PCR product is subsequently cleaved by the AcuI restriction enzyme in a position adjacent to the targeted dinucleotide, resulting in the generation of two DNA fragments of 60 and > 100 bp (AcuI digestion, step III). The 60 bp fragment containing the exposed signature of the targeted dinucleotide is then isolated using SPRI beads, with higher affinity toward > 100 bp DNA products (small fragment isolation, step IV). The 60 bp fragment is then ligated to DNA adaptors containing 3′ overhangs of two bases complementary (specific) or not (non-specific) to the dinucleotide signature (adaptor ligation, step V). The ligated product is then subjected to PCR amplification for analytical or quantitative detection (detection PCR, step VI). The approximate time required for each step is indicated. (B) Schematics of the DTECT adaptor library. Control (green) and mutant (purple) dinucleotide signatures (left panel) are detected using a library of 16 unique adaptors (middle panel). The library contains adaptors with dinucleotides complementary to the control (green) or mutant (purple) signature, as well as non-specific adaptors (blue) (right panel). (C) Schematics of the positive and negative controls used in DTECT experiments to identify signatures of interest (e.g., mutant allele) in allele populations. In gDNA samples containing only the WT dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) serves as a positive control, while the adaptor complementary to the mutant signature of interest (purple) and a non-specific adaptor (blue) are used as negative controls. In gDNA samples containing a mixture of the WT and the mutant dinucleotide signature, the adaptor complementary to the WT dinucleotide signature (green) is used as a positive control and a non-specific adaptor (blue) serves as a negative control. The adaptor complementary to the mutant dinucleotide signature (purple) is used to detect the presence of the variant of interest and quantify its frequency. See also Figure S1 .

    Article Snippet: To mimic the 5′ phosphorylation induced by AcuI in DTECT experiments, the reverse oligonucleotide (PB1449: 5′-gtagttcgccagttCTTCAGaatagtttgcgcaCAGGACGGGTCTCGTGAGGAATTGC-3′) was phosphorylated with PNK (NEB).

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Isolation, Ligation, Mutagenesis, Positive Control, Negative Control, Variant Assay

    DTECT-Mediated Identification of Clinically Relevant BRCA1/2 Mutations Generated by Precision Genome Editing and Genotyping of Cell Lines and Animal Models Carrying BRCA1 or BARD1 Mutations (A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios ( Billing et al., 2018 ). (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also Figures S6 , S7 , and S9 .

    Journal: Cell reports

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

    doi: 10.1016/j.celrep.2020.02.068

    Figure Lengend Snippet: DTECT-Mediated Identification of Clinically Relevant BRCA1/2 Mutations Generated by Precision Genome Editing and Genotyping of Cell Lines and Animal Models Carrying BRCA1 or BARD1 Mutations (A) Schematic representation of the human BRCA1 protein. BRCA1 domains and ClinVar BRCA1 mutations generated in this study are indicated. (B) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 10 BRCA1 mutations are introduced into HEK293T cells by CRISPR-mediated base editing. Experiments were conducted in cells expressing the base editor FNLS-BE3 upon transfection of sgRNAs to introduce the indicated mutations. Histograms show the mean frequency of the indicated variants estimated by DTECT, and error bars represent the SD from 2 independent DTECT assays for the same AcuI-tagged amplicon. ND, not determined due to sequencing failure. (C) Analytical detection of the indicated BRCA1 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. (D) Schematic representation of the human BRCA2 protein. BRCA2 domains and ClinVar BRCA2 mutations generated in this study are indicated. (E) Quantification using DTECT (red) and NGS (green) of the editing efficiency by which 13 BRCA2 mutations are introduced into HEK293T cells by CRISPR-mediated base editing, as described in (B). (F) Analytical detection of the indicated BRCA2 mutations in HEK293T cell populations by DTECT (21 PCR cycles) using adaptors specific for WT (green) or mutant (purple) alleles. Experiments were conducted as in (C). (G) Genotyping by DTECT-based analytical PCR (18 cycles) of single clones carrying WT and/or BRCA1 E638K mutant alleles derived from the BRCA1 E638K mutant cell population shown in (C). WT (4, not edited), heterozygous (1), and homozygous (2) BRCA1 mutant clones identified by DTECT are indicated. (H) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (G). The targeted dinucleotide is indicated in green, and part of the sequence of the AcuI-tagging primer is indicated in purple. (I) Genotyping by DTECT-based analytical PCR of Bard1 S563F (left) and Brca1 S1598F (right) knockin mutant mice (Bard1, 18 PCR cycles; Brca1, 20 PCR cycles). gDNA for DTECT analysis was obtained from mouse tail samples. WT (Bard1 8 and Brca1 5) mice and heterozygous (Bard1 2 and Brca1 2) and homozygous (Bard1 3) mutant mice identified by DTECT are indicated. No homozygous Brca1 S1598F mutant mice were identified in the analyzed mouse litters due to sub-Mendelian birth ratios ( Billing et al., 2018 ). (J) Sanger sequencing of WT and heterozygous and homozygous mutant amplicons shown in (I). See also Figures S6 , S7 , and S9 .

    Article Snippet: To mimic the 5′ phosphorylation induced by AcuI in DTECT experiments, the reverse oligonucleotide (PB1449: 5′-gtagttcgccagttCTTCAGaatagtttgcgcaCAGGACGGGTCTCGTGAGGAATTGC-3′) was phosphorylated with PNK (NEB).

    Techniques: Generated, Next-Generation Sequencing, CRISPR, Expressing, Transfection, Introduce, Amplification, Sequencing, Polymerase Chain Reaction, Mutagenesis, Clone Assay, Derivative Assay, Knock-In, Mouse Assay

    Detection and Quantification of Dinucleotide Signatures Using DTECT (A) Design of AcuI-tagging primers that allow the capture of two dinucleotide signatures (CC and TT, blue) on opposite DNA strands. (B) PCR amplification (22 cycles) of the AcuI-digested DNA products containing the CC and TT signatures shown in (A), which have been captured using GG or AA adaptors. (C) PCR amplification (22 cycles) of DNA fragments captured as in (B) with or without dephosphorylation of the AcuI-digested products by the shrimp alkaline phosphatase (rSAP). (D) PCR amplification (22 cycles) of DNA fragments captured as in (B) in the absence or presence of AcuI, DNA adaptors (GG adaptor for signature CC and AA adaptor for signature TT), or T4 DNA ligase. (E) Schematic representation of the AcuI-tagging primer design for detecting four possible dinucleotide signatures (1–4) containing the same targeted base (C:G, red) in the PIK3R1 gene. (F) Detection of the four dinucleotide signatures shown in (E) by DTECT (18 PCR cycles) using specific (green) and non-specific (blue) adaptors. (G) Quantification by DTECT of the relative abundance of SMARCAL1 , SPRTN , and PIK3R1 WT (green) and STOP (purple) dinucleotide signatures in mixtures of WT and STOP alleles at predefined ratios. Graphs (left) represent the correlation between the frequency of WT and STOP variants determined by DTECT and the expected frequency of the same variants in the mixed populations for each of the preceding 3 genes. Error bars represent the SD of independent experiments (n = 2). Pearson correlation (r) was determined by comparing expected and DTECT-based frequency. Comparison of the mean frequency of STOP and WT signatures determined by DTECT and their expected frequency is shown in the right panel (n = 3 independent genes, SMARCAL1 , SPRTN , and PIK3R1 ). (H) Representation of the AcuI-tagging primers used to detect the WT and STOP alleles of the PIK3R1 gene. The targeted dinucleotides are shown in blue, the edited base is indicated with an asterisk, and part of the AcuI-tagging primer sequence is shown in purple. (I) PCR amplification (25 cycles) of WT and STOP PIK3R1 alleles (arrow) captured using DTECT from WT:STOP allele mixtures (i.e., 100:0 and 99:1). An adaptor (CG) specific for the WT allele is used as a positive control, and a non-specific adaptor (TT) is used as a negative control. An adaptor that captures the STOP PIK3R1 allele (CA) serves as an additional negative control in the reaction containing only the WT allele. Background non-specific PCR products are indicated with an asterisk. (J) Fold change variation in the frequency of capture of each of the 16 dinucleotide signatures relative to the mean dinucleotide capture frequency. Oligonucleotides containing distinct dinucleotide signatures are captured using specific adaptors. The fraction of captured material is then quantified by qPCR and normalized to the mean value obtained from the capture of all 16 dinucleotide signatures. Error bars indicate the SD of 4 independent experiments. Dots represent individual data points. (K) Fold change variation in the frequency of capture of dinucleotide signatures with 1 A/T + 1 C/G, 2 A/T, or 2 C/G bases relative to the mean dinucleotide capture frequency, determined as described in (J). Error bars represent the SD of 8 mean values for dinucleotides with 1 A/T + 1 C/G and 4 mean values for dinucleotides with 2 A/T and 2 C/G, as determined in (J). See also Figures S2 , S3 , and S10 .

    Journal: Cell reports

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

    doi: 10.1016/j.celrep.2020.02.068

    Figure Lengend Snippet: Detection and Quantification of Dinucleotide Signatures Using DTECT (A) Design of AcuI-tagging primers that allow the capture of two dinucleotide signatures (CC and TT, blue) on opposite DNA strands. (B) PCR amplification (22 cycles) of the AcuI-digested DNA products containing the CC and TT signatures shown in (A), which have been captured using GG or AA adaptors. (C) PCR amplification (22 cycles) of DNA fragments captured as in (B) with or without dephosphorylation of the AcuI-digested products by the shrimp alkaline phosphatase (rSAP). (D) PCR amplification (22 cycles) of DNA fragments captured as in (B) in the absence or presence of AcuI, DNA adaptors (GG adaptor for signature CC and AA adaptor for signature TT), or T4 DNA ligase. (E) Schematic representation of the AcuI-tagging primer design for detecting four possible dinucleotide signatures (1–4) containing the same targeted base (C:G, red) in the PIK3R1 gene. (F) Detection of the four dinucleotide signatures shown in (E) by DTECT (18 PCR cycles) using specific (green) and non-specific (blue) adaptors. (G) Quantification by DTECT of the relative abundance of SMARCAL1 , SPRTN , and PIK3R1 WT (green) and STOP (purple) dinucleotide signatures in mixtures of WT and STOP alleles at predefined ratios. Graphs (left) represent the correlation between the frequency of WT and STOP variants determined by DTECT and the expected frequency of the same variants in the mixed populations for each of the preceding 3 genes. Error bars represent the SD of independent experiments (n = 2). Pearson correlation (r) was determined by comparing expected and DTECT-based frequency. Comparison of the mean frequency of STOP and WT signatures determined by DTECT and their expected frequency is shown in the right panel (n = 3 independent genes, SMARCAL1 , SPRTN , and PIK3R1 ). (H) Representation of the AcuI-tagging primers used to detect the WT and STOP alleles of the PIK3R1 gene. The targeted dinucleotides are shown in blue, the edited base is indicated with an asterisk, and part of the AcuI-tagging primer sequence is shown in purple. (I) PCR amplification (25 cycles) of WT and STOP PIK3R1 alleles (arrow) captured using DTECT from WT:STOP allele mixtures (i.e., 100:0 and 99:1). An adaptor (CG) specific for the WT allele is used as a positive control, and a non-specific adaptor (TT) is used as a negative control. An adaptor that captures the STOP PIK3R1 allele (CA) serves as an additional negative control in the reaction containing only the WT allele. Background non-specific PCR products are indicated with an asterisk. (J) Fold change variation in the frequency of capture of each of the 16 dinucleotide signatures relative to the mean dinucleotide capture frequency. Oligonucleotides containing distinct dinucleotide signatures are captured using specific adaptors. The fraction of captured material is then quantified by qPCR and normalized to the mean value obtained from the capture of all 16 dinucleotide signatures. Error bars indicate the SD of 4 independent experiments. Dots represent individual data points. (K) Fold change variation in the frequency of capture of dinucleotide signatures with 1 A/T + 1 C/G, 2 A/T, or 2 C/G bases relative to the mean dinucleotide capture frequency, determined as described in (J). Error bars represent the SD of 8 mean values for dinucleotides with 1 A/T + 1 C/G and 4 mean values for dinucleotides with 2 A/T and 2 C/G, as determined in (J). See also Figures S2 , S3 , and S10 .

    Article Snippet: To mimic the 5′ phosphorylation induced by AcuI in DTECT experiments, the reverse oligonucleotide (PB1449: 5′-gtagttcgccagttCTTCAGaatagtttgcgcaCAGGACGGGTCTCGTGAGGAATTGC-3′) was phosphorylated with PNK (NEB).

    Techniques: Polymerase Chain Reaction, Amplification, De-Phosphorylation Assay, Sequencing, Positive Control, Negative Control, Real-time Polymerase Chain Reaction