nspi  (New England Biolabs)


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    New England Biolabs nspi
    Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the <t>PCR</t> product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with <t>NspI.</t> Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.
    Nspi, 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
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    Images

    1) Product Images from "Copy Number Variation at the APOL1 Locus"

    Article Title: Copy Number Variation at the APOL1 Locus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0125410

    Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the PCR product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with NspI. Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.
    Figure Legend Snippet: Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the PCR product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with NspI. Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.

    Techniques Used: Sequencing, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, TaqMan Copy Number Assay, Variant Assay

    2) Product Images from "tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci"

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx853

    Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.
    Figure Legend Snippet: Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

    Techniques Used: Ligation, Sequencing, Amplification, Polymerase Chain Reaction

    3) Product Images from "Tunable Genotyping-By-Sequencing (tGBS®) Enables Reliable Genotyping of Heterozygous Loci"

    Article Title: Tunable Genotyping-By-Sequencing (tGBS®) Enables Reliable Genotyping of Heterozygous Loci

    Journal: bioRxiv

    doi: 10.1101/100461

    Diagram of tGBS. Digestion. Genomic DNA is digested with two restriction enzymes: NspI leaves a 3’overhang and BfuCI leaves a 5’ overhang. Ligation. Two single-strand oligos are ligated to the complementary 3’ and 5’ overhangs. The oligo matching the 3’ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5’ overhang is universal and present in every reaction for later amplification. Selective PCR. Target sites are selected using a selective primer with variable selective bases (“CA”) that match selective sites in the digested genome fragments and a non-selective primer. When properly amplified, the selective site is complementary to the selective bases. Final PCR. Primers matching the amplification primer and the selective primer which contain the full Proton adapter sequence are used for amplification of the final library. Final on-target sequence. The final sequence contains the 5’ Proton adapter sequence, an internal barcode, the NspI restriction enzyme site, the target molecule, selective bases, the BfuCI restriction enzyme site and the 3’ Proton adapter sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.
    Figure Legend Snippet: Diagram of tGBS. Digestion. Genomic DNA is digested with two restriction enzymes: NspI leaves a 3’overhang and BfuCI leaves a 5’ overhang. Ligation. Two single-strand oligos are ligated to the complementary 3’ and 5’ overhangs. The oligo matching the 3’ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5’ overhang is universal and present in every reaction for later amplification. Selective PCR. Target sites are selected using a selective primer with variable selective bases (“CA”) that match selective sites in the digested genome fragments and a non-selective primer. When properly amplified, the selective site is complementary to the selective bases. Final PCR. Primers matching the amplification primer and the selective primer which contain the full Proton adapter sequence are used for amplification of the final library. Final on-target sequence. The final sequence contains the 5’ Proton adapter sequence, an internal barcode, the NspI restriction enzyme site, the target molecule, selective bases, the BfuCI restriction enzyme site and the 3’ Proton adapter sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

    Techniques Used: Ligation, Sequencing, Amplification, Polymerase Chain Reaction

    4) Product Images from "Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements"

    Article Title: Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements

    Journal: Nature genetics

    doi: 10.1038/ng.613

    Androgen stimulation induces AR/TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human PCa. a–b , Sites closest to TMPRSS2-ERG breakpoints from 8 PCa cases determined from various studies (arrows) were significantly enriched for high KSDS enrichment of TOP2 catalytic cleavage (p = 0.010 and 0.013 respectively) in LAPC4 cells. Labeled sites (e.g., T8, T23, E5, E13, etc.) are analyzed in subsequent experiments. c , DHT and TOP2B dependent TOP2 catalytic cleavage in LAPC4 cells around the case 24 breakpoint aligning with region T8 (upper panel). The lack of KSDS enrichment at region E47 at ERG was re-confirmed in an independent KSDS experiment (lower panel). d , SLOT assay showed that DHT-induced TOP2 catalytic activity in LAPC4 cells was significantly higher at an NspI fragment most proximal to the TMPRSS2 breakpoint observed in case 24 than to the adjacent, more distal NspI fragment (see Supplementary Fig. 8, 9 for overview of SLOT). e , ChIP enrichment of AR and TOP2B at representative TOP2 catalytic cleavage sites in DHT-stimulated LAPC4 cells relative to untreated controls. f–g , 3C analysis reveals DHT-dependent spatial chromatin interaction of TMPRSS2 enhancer and promoter with region T8 (see first lane vs. fourth lane). Omission of NspI restriction enzyme and/or DNA ligase served as assay negative controls. Inhibition with Mer prevented these DHT-induced interactions. Error bars indicate ± SE of two to three experiments.
    Figure Legend Snippet: Androgen stimulation induces AR/TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human PCa. a–b , Sites closest to TMPRSS2-ERG breakpoints from 8 PCa cases determined from various studies (arrows) were significantly enriched for high KSDS enrichment of TOP2 catalytic cleavage (p = 0.010 and 0.013 respectively) in LAPC4 cells. Labeled sites (e.g., T8, T23, E5, E13, etc.) are analyzed in subsequent experiments. c , DHT and TOP2B dependent TOP2 catalytic cleavage in LAPC4 cells around the case 24 breakpoint aligning with region T8 (upper panel). The lack of KSDS enrichment at region E47 at ERG was re-confirmed in an independent KSDS experiment (lower panel). d , SLOT assay showed that DHT-induced TOP2 catalytic activity in LAPC4 cells was significantly higher at an NspI fragment most proximal to the TMPRSS2 breakpoint observed in case 24 than to the adjacent, more distal NspI fragment (see Supplementary Fig. 8, 9 for overview of SLOT). e , ChIP enrichment of AR and TOP2B at representative TOP2 catalytic cleavage sites in DHT-stimulated LAPC4 cells relative to untreated controls. f–g , 3C analysis reveals DHT-dependent spatial chromatin interaction of TMPRSS2 enhancer and promoter with region T8 (see first lane vs. fourth lane). Omission of NspI restriction enzyme and/or DNA ligase served as assay negative controls. Inhibition with Mer prevented these DHT-induced interactions. Error bars indicate ± SE of two to three experiments.

    Techniques Used: Labeling, Activity Assay, Chromatin Immunoprecipitation, Inhibition

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    New England Biolabs nspi
    Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the <t>PCR</t> product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with <t>NspI.</t> Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.
    Nspi, 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/nspi/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nspi - by Bioz Stars, 2022-05
    93/100 stars
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    Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the PCR product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with NspI. Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.

    Journal: PLoS ONE

    Article Title: Copy Number Variation at the APOL1 Locus

    doi: 10.1371/journal.pone.0125410

    Figure Lengend Snippet: Copy number variation at APOL1 locus. a. Sequence coverage analysis of samples from 1000 Genomes Project. Grey lines are used to depict coverage of each of 2,527 samples at genomic loci to which sequence reads map. Red lines indicate coverage for 8 samples with apparent duplication. The black line depicts median coverage for all samples showing elevated coverage over a region of this locus, including APOL1, suggesting duplication. Coordinates of the genome are plotted on the X-axis against normalized read depth on the Y-axis. Coordinates of APOL1 gene are chr22: 36,649,117–36,663,571 (hg19). b. Pictorial representation of the duplication event. An approximately 100kb region of chromosome 22, beginning from a region adjacent to APOL2, and involving all of APOL1 and part of MYH9 is duplicated (chr22:36,612,938–36,714,262, hg19). c. Gel electrophoresis (3% agarose) of the PCR product of the qualitative PCR assay used for detection of duplication. The PCR assay amplifies sequence across the junction between the duplicated segments, and results in a 291 bp size product, if present. Lane 1 corresponds to 100 bp ladder with size of each fragment indicated. Lanes 2 and 3 represent samples from individuals of European ancestry, who are negative for duplication. Lanes 4, 5 and 6 show a band of the expected size and indicate the presence of a duplication in these three unrelated African American individuals. Lanes 7, 8 and 9 show the absence of a PCR product in three other African American individuals. One of these three (lane 7) showed 3 copies using the Taqman copy number assay, indicating an insertional variant of different structure. Lane 10 is a non-template control reaction. d. Allelic discrimination digest assay: Gel electrophoresis of the PCR products obtained during allelic discrimination assay. Lane 1 shows a 100 bp ladder. Lane 2 (a, b and c) corresponds to sample NA 19701, Lane 3 (a, b and c) corresponds to sample NA 19372, Lane 4 (a, b and c) corresponds to sample NA 19700 and lane 5 corresponds to sample NA 19702. The band in the lanes 2a, 3a, 4a and 5a represent PCR products not subjected to any digestion (314 bp in size). Lanes 2b, 3b, 4b and 5b represent PCR products after digestion with HindIII. Lanes 2c, 3c, 4c and 5c represent PCR products after digestion with NspI. Since sample NA 19700 is homozygous for G0, there are only 2 bands in lane 4b and 1 band in lane 4c. Of note, with HindIII digestion, which cleaves the PCR product only if G0 allele is present, bands are expected to be 100 bp and 214 bp in size. With NspI, which cleaves the PCR product only in the presence of I384M G1 allele, the bands are expected to be 228 and 86 bp in size.

    Article Snippet: 10 μL of each PCR product was digested with HindIII or NspI (cleaving G0 at S342G and G1 at I384M, respectively) according to manufacturer’s directions (New England Biolabs) for 3–4 hrs at 37C.

    Techniques: Sequencing, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, TaqMan Copy Number Assay, Variant Assay

    Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

    Journal: Nucleic Acids Research

    Article Title: tGBS® genotyping-by-sequencing enables reliable genotyping of heterozygous loci

    doi: 10.1093/nar/gkx853

    Figure Lengend Snippet: Diagram of tGBS. Digestion . Genomic DNA is digested with two REs: NspI leaves a 3′overhang and BfuCI leaves a 5′ overhang. Ligation . Two distinct oligos are ligated to the complementary 3′ and 5′ overhangs. The oligo matching the 3′ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5′ overhang is universal and present in every reaction for later amplification. Selective PCR . Target sites are selected using a selective primer with variable selective bases (‘CA’) that match selected sequences in the digested genome fragments and a non-selective primer. When properly amplified, the selected sequence is complementary to the selective bases. Final PCR . Primers matching the amplification primer and the selective primer which contain the full Proton adaptor sequence are used for amplification of the final library. Final on-target sequence . The final sequence contains the 5′ Proton adaptor sequence, an internal barcode, the NspI RE site, the target molecule, selective bases, the BfuCI RE site and the 3′ Proton adaptor sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

    Article Snippet: tGBS procedure Approximately 120 ng of genomic DNA from each sample was digested with 100 units of NspI [New England Biolabs (Beverly, MA, USA), No. R0602L] and 400 units of BfuCI [New England Biolabs (Beverly, MA, USA), No. R0636L] in NEB CutSmart Buffer 4 in a 30-μl volume at 37°C for 1.5 h following the manufacturer’s protocol.

    Techniques: Ligation, Sequencing, Amplification, Polymerase Chain Reaction

    Diagram of tGBS. Digestion. Genomic DNA is digested with two restriction enzymes: NspI leaves a 3’overhang and BfuCI leaves a 5’ overhang. Ligation. Two single-strand oligos are ligated to the complementary 3’ and 5’ overhangs. The oligo matching the 3’ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5’ overhang is universal and present in every reaction for later amplification. Selective PCR. Target sites are selected using a selective primer with variable selective bases (“CA”) that match selective sites in the digested genome fragments and a non-selective primer. When properly amplified, the selective site is complementary to the selective bases. Final PCR. Primers matching the amplification primer and the selective primer which contain the full Proton adapter sequence are used for amplification of the final library. Final on-target sequence. The final sequence contains the 5’ Proton adapter sequence, an internal barcode, the NspI restriction enzyme site, the target molecule, selective bases, the BfuCI restriction enzyme site and the 3’ Proton adapter sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

    Journal: bioRxiv

    Article Title: Tunable Genotyping-By-Sequencing (tGBS®) Enables Reliable Genotyping of Heterozygous Loci

    doi: 10.1101/100461

    Figure Lengend Snippet: Diagram of tGBS. Digestion. Genomic DNA is digested with two restriction enzymes: NspI leaves a 3’overhang and BfuCI leaves a 5’ overhang. Ligation. Two single-strand oligos are ligated to the complementary 3’ and 5’ overhangs. The oligo matching the 3’ overhang contains a sample-specific internal barcode sequence for sample identification. The oligo matching the 5’ overhang is universal and present in every reaction for later amplification. Selective PCR. Target sites are selected using a selective primer with variable selective bases (“CA”) that match selective sites in the digested genome fragments and a non-selective primer. When properly amplified, the selective site is complementary to the selective bases. Final PCR. Primers matching the amplification primer and the selective primer which contain the full Proton adapter sequence are used for amplification of the final library. Final on-target sequence. The final sequence contains the 5’ Proton adapter sequence, an internal barcode, the NspI restriction enzyme site, the target molecule, selective bases, the BfuCI restriction enzyme site and the 3’ Proton adapter sequence. It is possible to adapt the tGBS protocol for sequencing on an Illumina instrument by redesigning the ligation oligos and PCR primers.

    Article Snippet: tGBS procedureApproximately 120 ng of genomic DNA of each sample was digested with 100 units of NspI [New England Biolabs (Beverly, MA), No. R0602L] and 400 units of BfuCI [New England Biolabs (Beverly, MA), No. R0636L] in a 30 μL volume at 37°C for 1.5 hr following the manufacturer’s protocol.

    Techniques: Ligation, Sequencing, Amplification, Polymerase Chain Reaction

    Androgen stimulation induces AR/TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human PCa. a–b , Sites closest to TMPRSS2-ERG breakpoints from 8 PCa cases determined from various studies (arrows) were significantly enriched for high KSDS enrichment of TOP2 catalytic cleavage (p = 0.010 and 0.013 respectively) in LAPC4 cells. Labeled sites (e.g., T8, T23, E5, E13, etc.) are analyzed in subsequent experiments. c , DHT and TOP2B dependent TOP2 catalytic cleavage in LAPC4 cells around the case 24 breakpoint aligning with region T8 (upper panel). The lack of KSDS enrichment at region E47 at ERG was re-confirmed in an independent KSDS experiment (lower panel). d , SLOT assay showed that DHT-induced TOP2 catalytic activity in LAPC4 cells was significantly higher at an NspI fragment most proximal to the TMPRSS2 breakpoint observed in case 24 than to the adjacent, more distal NspI fragment (see Supplementary Fig. 8, 9 for overview of SLOT). e , ChIP enrichment of AR and TOP2B at representative TOP2 catalytic cleavage sites in DHT-stimulated LAPC4 cells relative to untreated controls. f–g , 3C analysis reveals DHT-dependent spatial chromatin interaction of TMPRSS2 enhancer and promoter with region T8 (see first lane vs. fourth lane). Omission of NspI restriction enzyme and/or DNA ligase served as assay negative controls. Inhibition with Mer prevented these DHT-induced interactions. Error bars indicate ± SE of two to three experiments.

    Journal: Nature genetics

    Article Title: Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements

    doi: 10.1038/ng.613

    Figure Lengend Snippet: Androgen stimulation induces AR/TOP2B recruitment and TOP2B catalytic cleavage at genomic breakpoints of TMPRSS2 and ERG observed in human PCa. a–b , Sites closest to TMPRSS2-ERG breakpoints from 8 PCa cases determined from various studies (arrows) were significantly enriched for high KSDS enrichment of TOP2 catalytic cleavage (p = 0.010 and 0.013 respectively) in LAPC4 cells. Labeled sites (e.g., T8, T23, E5, E13, etc.) are analyzed in subsequent experiments. c , DHT and TOP2B dependent TOP2 catalytic cleavage in LAPC4 cells around the case 24 breakpoint aligning with region T8 (upper panel). The lack of KSDS enrichment at region E47 at ERG was re-confirmed in an independent KSDS experiment (lower panel). d , SLOT assay showed that DHT-induced TOP2 catalytic activity in LAPC4 cells was significantly higher at an NspI fragment most proximal to the TMPRSS2 breakpoint observed in case 24 than to the adjacent, more distal NspI fragment (see Supplementary Fig. 8, 9 for overview of SLOT). e , ChIP enrichment of AR and TOP2B at representative TOP2 catalytic cleavage sites in DHT-stimulated LAPC4 cells relative to untreated controls. f–g , 3C analysis reveals DHT-dependent spatial chromatin interaction of TMPRSS2 enhancer and promoter with region T8 (see first lane vs. fourth lane). Omission of NspI restriction enzyme and/or DNA ligase served as assay negative controls. Inhibition with Mer prevented these DHT-induced interactions. Error bars indicate ± SE of two to three experiments.

    Article Snippet: Biotin-labeled DNA was precipitated using Strepavidin coated magnetic beads (NEB), digested with NspI (NEB), and unbound fragments were removed by washing with wash Buffer (10 mM Tris HCl, 1mM EDTA, 2 M NaCl).

    Techniques: Labeling, Activity Assay, Chromatin Immunoprecipitation, Inhibition