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    Structured Review

    New England Biolabs bsrbi
    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by <t>AciI</t> or <t>BsrBI</t> restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.
    Bsrbi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsrbi/product/New England Biolabs
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    Images

    1) Product Images from "High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies"

    Article Title: High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

    Journal: Experimental Biology and Medicine

    doi: 10.1177/1535370220968545

    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.
    Figure Legend Snippet: GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.

    Techniques Used: Polymerase Chain Reaction

    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP states other than GYPB DEL1 and DEL2. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. Lanes 22–24 are negative control wells. (a) DEL1 assay; 1.9 kb (V) and 0.8 kb (W) identify the bands expected for a non-DEL1 sample, and 2.2 kb (U) identifies the presence of GYPB DEL1. (b) GYPB DEL2 assay; 2.1 kb (X) band identifies a non-DEL2 sample, and the 1.3 kb (Y) plus 0.8 kb (Z) bands identify the presence of GYPB DEL2. See also Figure 4 . Sample designations identified from Leffler et al. 9
    Figure Legend Snippet: GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP states other than GYPB DEL1 and DEL2. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. Lanes 22–24 are negative control wells. (a) DEL1 assay; 1.9 kb (V) and 0.8 kb (W) identify the bands expected for a non-DEL1 sample, and 2.2 kb (U) identifies the presence of GYPB DEL1. (b) GYPB DEL2 assay; 2.1 kb (X) band identifies a non-DEL2 sample, and the 1.3 kb (Y) plus 0.8 kb (Z) bands identify the presence of GYPB DEL2. See also Figure 4 . Sample designations identified from Leffler et al. 9

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Schematic representation of strategies for amplifying and testing for the GYPB DEL1 and DEL2 structural variants. (a) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers (blue rectangles), putative breakpoint (gold rectangle), and AciI restriction site (yellow rectangle). The forward primer GYP_DEL1_F10 is specific to upstream of GYPE in the GYPE-GYPB region. The reverse primer GYPB_DEL1_R2B5 binds to the upstream of the GYPB gene in the GYB-GYPA region. In a normal or wild type individual, the GYPB_DEL1_R2B5 in the GYPE-GYPB region and the GYP_DEL1_F10 forward primer forms a PCR product made of sequences in the GYPE_GYPB region. In the GYPB DEL1 state, the PCR product formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (b) Alternate schematic representation of the GYPB DEL1 RFLP assay showing a normal chromosome and the GYPB DEL1 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the AciI restriction site and PCR-digestion fragment lengths. (c) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers, putative breakpoint (gold rectangle), and BsrBI restriction site (yellow rectangle). The forward primer GYP_DEL2_F3 is common to the GYPA downstream of the GYPB-GYPA region. The reverse primer GYPB_DEL2_R3 specifically binds to the upstream of the GYPE gene in the GYPE-GYPB region. In a normal or wild type individual, the GYPB_DEL2_F3 in the GYPB-GYPA region and the GYP_DEL2_R3 primer forms a PCR product made of sequences in the GYPB_GYPA region. In the GYPB DEL2 state, the PCR amplicon formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (d) Alternate schematic of the GYPB DEL2 RFLP assay showing a normal chromosome and the GYPB DEL2 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the BsrBI restriction site (red dotted rectangular area) and PCR-digestion fragment lengths. Coordinates of sequences are given with respect to GRCh38.
    Figure Legend Snippet: Schematic representation of strategies for amplifying and testing for the GYPB DEL1 and DEL2 structural variants. (a) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers (blue rectangles), putative breakpoint (gold rectangle), and AciI restriction site (yellow rectangle). The forward primer GYP_DEL1_F10 is specific to upstream of GYPE in the GYPE-GYPB region. The reverse primer GYPB_DEL1_R2B5 binds to the upstream of the GYPB gene in the GYB-GYPA region. In a normal or wild type individual, the GYPB_DEL1_R2B5 in the GYPE-GYPB region and the GYP_DEL1_F10 forward primer forms a PCR product made of sequences in the GYPE_GYPB region. In the GYPB DEL1 state, the PCR product formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (b) Alternate schematic representation of the GYPB DEL1 RFLP assay showing a normal chromosome and the GYPB DEL1 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the AciI restriction site and PCR-digestion fragment lengths. (c) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers, putative breakpoint (gold rectangle), and BsrBI restriction site (yellow rectangle). The forward primer GYP_DEL2_F3 is common to the GYPA downstream of the GYPB-GYPA region. The reverse primer GYPB_DEL2_R3 specifically binds to the upstream of the GYPE gene in the GYPE-GYPB region. In a normal or wild type individual, the GYPB_DEL2_F3 in the GYPB-GYPA region and the GYP_DEL2_R3 primer forms a PCR product made of sequences in the GYPB_GYPA region. In the GYPB DEL2 state, the PCR amplicon formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (d) Alternate schematic of the GYPB DEL2 RFLP assay showing a normal chromosome and the GYPB DEL2 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the BsrBI restriction site (red dotted rectangular area) and PCR-digestion fragment lengths. Coordinates of sequences are given with respect to GRCh38.

    Techniques Used: Polymerase Chain Reaction, RFLP Assay, Amplification

    2) Product Images from "Somatic Mosaicism for a Lethal TRPV4 Mutation Results in Non-Lethal Metatropic Dysplasia"

    Article Title: Somatic Mosaicism for a Lethal TRPV4 Mutation Results in Non-Lethal Metatropic Dysplasia

    Journal: American journal of medical genetics. Part A

    doi: 10.1002/ajmg.a.37942

    Somatic mosaicism for the TRPV4 mutation. (A) Ethidium bromide-stained 2% agarose gel showing BsrBI-cleaved PCR products containing TRPV4 exon 12. The p.L618P mutation created a BsrBI site which was detected in both the heterozygous lethal MD control,
    Figure Legend Snippet: Somatic mosaicism for the TRPV4 mutation. (A) Ethidium bromide-stained 2% agarose gel showing BsrBI-cleaved PCR products containing TRPV4 exon 12. The p.L618P mutation created a BsrBI site which was detected in both the heterozygous lethal MD control,

    Techniques Used: Mutagenesis, Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction

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    New England Biolabs bsrbi
    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by <t>AciI</t> or <t>BsrBI</t> restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.
    Bsrbi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsrbi/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsrbi - by Bioz Stars, 2022-05
    93/100 stars
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    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.

    Journal: Experimental Biology and Medicine

    Article Title: High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

    doi: 10.1177/1535370220968545

    Figure Lengend Snippet: GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP States. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. (a) DEL1 assay; The first seven wells (1–7) contain wild-type cell lines giving bands at 1.9 kb and 0.3 kb (V and W, respectively), five wells (11–15) contain homozygous cell-lines which give a single uncut band at 2.2 kb (U), while three cell-lines heterozygous for DEL1 (lanes 8–10) show two upper bands and one small band (2.3 kb [U], 1.9 kb [V], and 0.3 kb [W]). Lanes 16–19 are GYPB DEL2 positive cell lines that are all cut by the AciI enzyme (1.9 kb [V] and 0.3 kb [W]) indicating “normal” or non-DEL1. (b) GYPB DEL2 assay; The first seven wells (1–7) contain wild-type cell lines giving a single uncut band at 2.1 kb (X); lane (19) contains a GYPB DEL2 homozygous cell line giving two bands (1.3 kb [Y] and 0.8 kb [Z]); three wells (16–18) contain heterozygous cell-lines which give three bands (2.1 kb [X], 1.3 kb [Y], and 0.8 kb [Z]). Lanes 8–15 are DEL1 positive cell lines that are not cut by BsrBI indicating “normal” or non-DEL2.

    Article Snippet: Restriction enzymes AciI (Catalog number: R0551L, NEB, UK) and BsrBI (Catalog number: R0102L, NEB, UK) were purchased ( ).

    Techniques: Polymerase Chain Reaction

    GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP states other than GYPB DEL1 and DEL2. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. Lanes 22–24 are negative control wells. (a) DEL1 assay; 1.9 kb (V) and 0.8 kb (W) identify the bands expected for a non-DEL1 sample, and 2.2 kb (U) identifies the presence of GYPB DEL1. (b) GYPB DEL2 assay; 2.1 kb (X) band identifies a non-DEL2 sample, and the 1.3 kb (Y) plus 0.8 kb (Z) bands identify the presence of GYPB DEL2. See also Figure 4 . Sample designations identified from Leffler et al. 9

    Journal: Experimental Biology and Medicine

    Article Title: High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

    doi: 10.1177/1535370220968545

    Figure Lengend Snippet: GYPB DEL1 (a) and DEL2 (b) assays on cell lines with known GYP states other than GYPB DEL1 and DEL2. PCR using the GYPB DEL1 (a) or DEL2 (b) primers was carried out on the samples followed by AciI or BsrBI restriction enzyme digestion, respectively. Lanes 22–24 are negative control wells. (a) DEL1 assay; 1.9 kb (V) and 0.8 kb (W) identify the bands expected for a non-DEL1 sample, and 2.2 kb (U) identifies the presence of GYPB DEL1. (b) GYPB DEL2 assay; 2.1 kb (X) band identifies a non-DEL2 sample, and the 1.3 kb (Y) plus 0.8 kb (Z) bands identify the presence of GYPB DEL2. See also Figure 4 . Sample designations identified from Leffler et al. 9

    Article Snippet: Restriction enzymes AciI (Catalog number: R0551L, NEB, UK) and BsrBI (Catalog number: R0102L, NEB, UK) were purchased ( ).

    Techniques: Polymerase Chain Reaction, Negative Control

    Schematic representation of strategies for amplifying and testing for the GYPB DEL1 and DEL2 structural variants. (a) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers (blue rectangles), putative breakpoint (gold rectangle), and AciI restriction site (yellow rectangle). The forward primer GYP_DEL1_F10 is specific to upstream of GYPE in the GYPE-GYPB region. The reverse primer GYPB_DEL1_R2B5 binds to the upstream of the GYPB gene in the GYB-GYPA region. In a normal or wild type individual, the GYPB_DEL1_R2B5 in the GYPE-GYPB region and the GYP_DEL1_F10 forward primer forms a PCR product made of sequences in the GYPE_GYPB region. In the GYPB DEL1 state, the PCR product formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (b) Alternate schematic representation of the GYPB DEL1 RFLP assay showing a normal chromosome and the GYPB DEL1 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the AciI restriction site and PCR-digestion fragment lengths. (c) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers, putative breakpoint (gold rectangle), and BsrBI restriction site (yellow rectangle). The forward primer GYP_DEL2_F3 is common to the GYPA downstream of the GYPB-GYPA region. The reverse primer GYPB_DEL2_R3 specifically binds to the upstream of the GYPE gene in the GYPE-GYPB region. In a normal or wild type individual, the GYPB_DEL2_F3 in the GYPB-GYPA region and the GYP_DEL2_R3 primer forms a PCR product made of sequences in the GYPB_GYPA region. In the GYPB DEL2 state, the PCR amplicon formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (d) Alternate schematic of the GYPB DEL2 RFLP assay showing a normal chromosome and the GYPB DEL2 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the BsrBI restriction site (red dotted rectangular area) and PCR-digestion fragment lengths. Coordinates of sequences are given with respect to GRCh38.

    Journal: Experimental Biology and Medicine

    Article Title: High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

    doi: 10.1177/1535370220968545

    Figure Lengend Snippet: Schematic representation of strategies for amplifying and testing for the GYPB DEL1 and DEL2 structural variants. (a) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers (blue rectangles), putative breakpoint (gold rectangle), and AciI restriction site (yellow rectangle). The forward primer GYP_DEL1_F10 is specific to upstream of GYPE in the GYPE-GYPB region. The reverse primer GYPB_DEL1_R2B5 binds to the upstream of the GYPB gene in the GYB-GYPA region. In a normal or wild type individual, the GYPB_DEL1_R2B5 in the GYPE-GYPB region and the GYP_DEL1_F10 forward primer forms a PCR product made of sequences in the GYPE_GYPB region. In the GYPB DEL1 state, the PCR product formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (b) Alternate schematic representation of the GYPB DEL1 RFLP assay showing a normal chromosome and the GYPB DEL1 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the AciI restriction site and PCR-digestion fragment lengths. (c) Schematic representation of the alignment for the GYP SDUs showing the location of PCR primers, putative breakpoint (gold rectangle), and BsrBI restriction site (yellow rectangle). The forward primer GYP_DEL2_F3 is common to the GYPA downstream of the GYPB-GYPA region. The reverse primer GYPB_DEL2_R3 specifically binds to the upstream of the GYPE gene in the GYPE-GYPB region. In a normal or wild type individual, the GYPB_DEL2_F3 in the GYPB-GYPA region and the GYP_DEL2_R3 primer forms a PCR product made of sequences in the GYPB_GYPA region. In the GYPB DEL2 state, the PCR amplicon formed is made of sequences in the GYPE and GYPA region because the GYPB is deleted. (d) Alternate schematic of the GYPB DEL2 RFLP assay showing a normal chromosome and the GYPB DEL2 chromosomes aligned. Genes (green, orange, and purple rectangles) and primers (blue rectangle) are indicated as well as the BsrBI restriction site (red dotted rectangular area) and PCR-digestion fragment lengths. Coordinates of sequences are given with respect to GRCh38.

    Article Snippet: Restriction enzymes AciI (Catalog number: R0551L, NEB, UK) and BsrBI (Catalog number: R0102L, NEB, UK) were purchased ( ).

    Techniques: Polymerase Chain Reaction, RFLP Assay, Amplification

    Somatic mosaicism for the TRPV4 mutation. (A) Ethidium bromide-stained 2% agarose gel showing BsrBI-cleaved PCR products containing TRPV4 exon 12. The p.L618P mutation created a BsrBI site which was detected in both the heterozygous lethal MD control,

    Journal: American journal of medical genetics. Part A

    Article Title: Somatic Mosaicism for a Lethal TRPV4 Mutation Results in Non-Lethal Metatropic Dysplasia

    doi: 10.1002/ajmg.a.37942

    Figure Lengend Snippet: Somatic mosaicism for the TRPV4 mutation. (A) Ethidium bromide-stained 2% agarose gel showing BsrBI-cleaved PCR products containing TRPV4 exon 12. The p.L618P mutation created a BsrBI site which was detected in both the heterozygous lethal MD control,

    Article Snippet: The p.L618P mutation created a BsrBI restriction endonuclease cleavage site so presence of the mutant allele in DNA from the proband was confirmed by PCR amplification TRPV4 exon 12 followed by cleavage with BsrBI (New England Biolabs).

    Techniques: Mutagenesis, Staining, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Formation of DNA/RNA heteroduplex by interaction of single-stranded antisense vDNA (vcDNA) with viral RNA. a Schematics of EMCV viral genome. The bottom indicates the positions of the PCR primer pairs used for the detection of vDNA. The arrows above the genome show the sites of restriction endonucleases, BsrB I and Mfe I. b The vDNA is resistant to DNA restrictive endonucleases. The vDNA was extracted from E14TG2a cells infected with EMCV and incubated with restrictive endonuclease BsrB I or Mfe I for 1 h. The integrity of DNA was evaluated by PCR with the indicated primers. The plasmid pCMV-rNJ08, which contains full-length EMCV genomic sequence, was used as positive control of dsDNA. c The vDNA is single-stranded. The vDNA and plasmid pCMV-rNJ08 were incubated with nuclease S1 at 1 U for the indicated time periods. The integrity of DNA was evaluated by PCR with the indicated primers. d The vDNA is complementary with the virus genomic sequence (vcDNA). Unidirectional primer extension was performed as described in Materials and Methods with the indicated primers. The products were then digested by the dsDNA restrictive endonucleases and PCR was performed to evaluate the integrity. e The distribution of vcDNA. The vcDNA was enriched with RNA probes targeting the complementary sequence of the virus genome and was sequenced as described in Materials and Methods, n = 2. f DNA/RNA hybrids were accumulated in virus-infected mESCs. E14TG2a cells were infected with EMCV (MOI = 1, 24 hpi). Cells were mock-treated or pre-treated with RNase H for 1 h at room temperature. The DNA/RNA hybrids (S9.6 antibody, red) and VP1 (green) were visualized by immunofluorescent staining. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. g , h The vcDNA forms DNA/RNA hybrids with viral RNA. The cytoplasmic nucleic acids were extracted from E14TG2a cells infected with EMCV (MOI = 1) for 48 h. The DRIP experiments were performed as described in Materials and Methods with S9.6 antibody. Normal mouse IgG was used as negative control. The viral RNA was analyzed by qRT-PCR ( g ) and the vcDNA level in the supernatant or immunoprecipitates was detected by PCR ( h ). Data are representative of three independent experiments ( b – d , f , h ). In g , the graph represents means ± SD from three independent replicates measured in triplicate.

    Journal: Cell Research

    Article Title: Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells

    doi: 10.1038/s41422-021-00524-7

    Figure Lengend Snippet: Formation of DNA/RNA heteroduplex by interaction of single-stranded antisense vDNA (vcDNA) with viral RNA. a Schematics of EMCV viral genome. The bottom indicates the positions of the PCR primer pairs used for the detection of vDNA. The arrows above the genome show the sites of restriction endonucleases, BsrB I and Mfe I. b The vDNA is resistant to DNA restrictive endonucleases. The vDNA was extracted from E14TG2a cells infected with EMCV and incubated with restrictive endonuclease BsrB I or Mfe I for 1 h. The integrity of DNA was evaluated by PCR with the indicated primers. The plasmid pCMV-rNJ08, which contains full-length EMCV genomic sequence, was used as positive control of dsDNA. c The vDNA is single-stranded. The vDNA and plasmid pCMV-rNJ08 were incubated with nuclease S1 at 1 U for the indicated time periods. The integrity of DNA was evaluated by PCR with the indicated primers. d The vDNA is complementary with the virus genomic sequence (vcDNA). Unidirectional primer extension was performed as described in Materials and Methods with the indicated primers. The products were then digested by the dsDNA restrictive endonucleases and PCR was performed to evaluate the integrity. e The distribution of vcDNA. The vcDNA was enriched with RNA probes targeting the complementary sequence of the virus genome and was sequenced as described in Materials and Methods, n = 2. f DNA/RNA hybrids were accumulated in virus-infected mESCs. E14TG2a cells were infected with EMCV (MOI = 1, 24 hpi). Cells were mock-treated or pre-treated with RNase H for 1 h at room temperature. The DNA/RNA hybrids (S9.6 antibody, red) and VP1 (green) were visualized by immunofluorescent staining. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. g , h The vcDNA forms DNA/RNA hybrids with viral RNA. The cytoplasmic nucleic acids were extracted from E14TG2a cells infected with EMCV (MOI = 1) for 48 h. The DRIP experiments were performed as described in Materials and Methods with S9.6 antibody. Normal mouse IgG was used as negative control. The viral RNA was analyzed by qRT-PCR ( g ) and the vcDNA level in the supernatant or immunoprecipitates was detected by PCR ( h ). Data are representative of three independent experiments ( b – d , f , h ). In g , the graph represents means ± SD from three independent replicates measured in triplicate.

    Article Snippet: Characterization of vDNA The cytoplasmic DNA was purified from mESCs after being infected with EMCV (MOI = 1) for 48 h. The DNA samples were incubated with dsDNA restrictive endonuclease, BsrB I (New England Biolabs, R0102V) or MfeI (New England Biolabs, R0589V), at 37 °C for 30 min, separately.

    Techniques: Polymerase Chain Reaction, Infection, Incubation, Plasmid Preparation, Sequencing, Positive Control, Staining, Negative Control, Quantitative RT-PCR