pspgi  (New England Biolabs)


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    Name:
    PspGI
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    PspGI 1 000 units
    Catalog Number:
    r0611s
    Price:
    66
    Category:
    Restriction Enzymes
    Size:
    1 000 units
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    New England Biolabs pspgi
    PspGI
    PspGI 1 000 units
    https://www.bioz.com/result/pspgi/product/New England Biolabs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pspgi - by Bioz Stars, 2021-03
    91/100 stars

    Images

    1) Product Images from "FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations"

    Article Title: FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm809

    Selective PNA-mediated FLAG on cell lines. The FLAG assay was performed on cell lines K562 (GGT/GGT), SW480 (GTT/GTT), PL45 (GGT/GAT) and CALU1 (GGT/TGT), or in absence of DNA target, B. Panel 1: FLAG assay without PspGI restriction endonuclease or PNA (positive controls). An amplification product is produced from all the reactions performed. Panel 2: FLAG assay in presence of PspGI to detect mutant versus wild-type samples. The wild -type sample K562 was digested by the enzyme, while the cell lines containing a mutation are exponentially amplified. Panels 3–5: FLAG assay with addition of PspGI and PNA specific for GTT, GAT or TGT. No amplification product is obtained from wild-type sample K562 (digested by PspGI) and from the cell lines that are suppressed by the fully complementary PNA present in the reaction.
    Figure Legend Snippet: Selective PNA-mediated FLAG on cell lines. The FLAG assay was performed on cell lines K562 (GGT/GGT), SW480 (GTT/GTT), PL45 (GGT/GAT) and CALU1 (GGT/TGT), or in absence of DNA target, B. Panel 1: FLAG assay without PspGI restriction endonuclease or PNA (positive controls). An amplification product is produced from all the reactions performed. Panel 2: FLAG assay in presence of PspGI to detect mutant versus wild-type samples. The wild -type sample K562 was digested by the enzyme, while the cell lines containing a mutation are exponentially amplified. Panels 3–5: FLAG assay with addition of PspGI and PNA specific for GTT, GAT or TGT. No amplification product is obtained from wild-type sample K562 (digested by PspGI) and from the cell lines that are suppressed by the fully complementary PNA present in the reaction.

    Techniques Used: Amplification, Produced, Mutagenesis

    PNA-mediated FLAG –KRAS assay. FLAG-KRAS assay leads to selective amplification of mutant codon 12 KRAS sequences during the real-time fluorescent reaction through a REMS-PCR approach. ( A ) a mutagenic (underlined base) primer introduces a variation that creates the recognition site for PspGI (circle) in the wild-type codon 12 (X = C, ACC) target sequence, thereby suppressing the exponential amplification of the wild-type allele. The mutant alleles escape digestion and this enables their exponential amplification. ( B ) to assess the exact nucleotide alteration of the target amplified, the FLAG-KRAS assay is repeated in four parallel reactions in the presence of PNA probes (dotted lines) specific for the most common mutations of codon 12. When the reaction is conducted in the presence of the fully complementary PNA ( 4 ), no exponential amplification is allowed. In presence of a mismatched PNA ( 1–3 ) the probe is destabilized and the primer can consequently anneal and extend causing an exponential amplification and generation of FLAG fluorescent signal as explained in Figure 1.
    Figure Legend Snippet: PNA-mediated FLAG –KRAS assay. FLAG-KRAS assay leads to selective amplification of mutant codon 12 KRAS sequences during the real-time fluorescent reaction through a REMS-PCR approach. ( A ) a mutagenic (underlined base) primer introduces a variation that creates the recognition site for PspGI (circle) in the wild-type codon 12 (X = C, ACC) target sequence, thereby suppressing the exponential amplification of the wild-type allele. The mutant alleles escape digestion and this enables their exponential amplification. ( B ) to assess the exact nucleotide alteration of the target amplified, the FLAG-KRAS assay is repeated in four parallel reactions in the presence of PNA probes (dotted lines) specific for the most common mutations of codon 12. When the reaction is conducted in the presence of the fully complementary PNA ( 4 ), no exponential amplification is allowed. In presence of a mismatched PNA ( 1–3 ) the probe is destabilized and the primer can consequently anneal and extend causing an exponential amplification and generation of FLAG fluorescent signal as explained in Figure 1.

    Techniques Used: Kras Assay, Amplification, Mutagenesis, Polymerase Chain Reaction, Sequencing

    2) Product Images from "Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators"

    Article Title: Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

    Journal: Nature

    doi: 10.1038/nature25179

    MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).
    Figure Legend Snippet: MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Western Blot, Knock-Out, Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Quantitation Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Northern Blot, Sequencing, Marker

    3) Product Images from "Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides"

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008430

    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.
    Figure Legend Snippet: PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Techniques Used:

    Double digestion of the PEAR product by PspGI and Hpy99I. The sense and the antisense strand are indicated respectively by (+) and ( − ). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“ ^ ”). The antisense strands ( A ) are black boxed, the sense strands ( B and C ) are boxed, and the by-products ( D and E ) are grey boxed. The expected length for each strand is indicated in parenthesis.
    Figure Legend Snippet: Double digestion of the PEAR product by PspGI and Hpy99I. The sense and the antisense strand are indicated respectively by (+) and ( − ). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“ ^ ”). The antisense strands ( A ) are black boxed, the sense strands ( B and C ) are boxed, and the by-products ( D and E ) are grey boxed. The expected length for each strand is indicated in parenthesis.

    Techniques Used:

    Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.
    Figure Legend Snippet: Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Techniques Used: Produced

    HPLC separation and purification of antisense oligonucleotide. Fractions are indicated by letter A to E as shown in Fig. 4 . Fraction A, which contains the antisense strands, was collected in the indicated interval. Sample preparation: 10 µg PEAR product double digested by PspGI and Hpy99I; Column: SOURCE Q PE 4.6/100; Flow rate: 1 ml/min; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH+2M NaCl, pH 12; Gradient: 20–35% B in 50 column volume.
    Figure Legend Snippet: HPLC separation and purification of antisense oligonucleotide. Fractions are indicated by letter A to E as shown in Fig. 4 . Fraction A, which contains the antisense strands, was collected in the indicated interval. Sample preparation: 10 µg PEAR product double digested by PspGI and Hpy99I; Column: SOURCE Q PE 4.6/100; Flow rate: 1 ml/min; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH+2M NaCl, pH 12; Gradient: 20–35% B in 50 column volume.

    Techniques Used: High Performance Liquid Chromatography, Purification, Sample Prep, Flow Cytometry

    4) Product Images from "Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides"

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008430

    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.
    Figure Legend Snippet: PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Techniques Used:

    Double digestion of the PEAR product by PspGI and Hpy99I. The sense and the antisense strand are indicated respectively by (+) and ( − ). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“ ^ ”). The antisense strands ( A ) are black boxed, the sense strands ( B and C ) are boxed, and the by-products ( D and E ) are grey boxed. The expected length for each strand is indicated in parenthesis.
    Figure Legend Snippet: Double digestion of the PEAR product by PspGI and Hpy99I. The sense and the antisense strand are indicated respectively by (+) and ( − ). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“ ^ ”). The antisense strands ( A ) are black boxed, the sense strands ( B and C ) are boxed, and the by-products ( D and E ) are grey boxed. The expected length for each strand is indicated in parenthesis.

    Techniques Used:

    Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.
    Figure Legend Snippet: Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Techniques Used: Produced

    HPLC separation and purification of antisense oligonucleotide. Fractions are indicated by letter A to E as shown in Fig. 4 . Fraction A, which contains the antisense strands, was collected in the indicated interval. Sample preparation: 10 µg PEAR product double digested by PspGI and Hpy99I; Column: SOURCE Q PE 4.6/100; Flow rate: 1 ml/min; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH+2M NaCl, pH 12; Gradient: 20–35% B in 50 column volume.
    Figure Legend Snippet: HPLC separation and purification of antisense oligonucleotide. Fractions are indicated by letter A to E as shown in Fig. 4 . Fraction A, which contains the antisense strands, was collected in the indicated interval. Sample preparation: 10 µg PEAR product double digested by PspGI and Hpy99I; Column: SOURCE Q PE 4.6/100; Flow rate: 1 ml/min; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH+2M NaCl, pH 12; Gradient: 20–35% B in 50 column volume.

    Techniques Used: High Performance Liquid Chromatography, Purification, Sample Prep, Flow Cytometry

    Related Articles

    Synthesized:

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides
    Article Snippet: We are in the process of evaluating a number of different thermostable DNA polymerases such as DyNAzyme II Hot start DNA polymerase and Phusion DNA polymerase that carry the 3′-5′ proof-reading activity and lack the template-independent terminal transferase activity. .. Enzymes and Oligonucleotides Taq DNA Polymerase, PspGI and Hpy99I, were purchased from New England Biolabs (NEB), Inc. A target oligonucleotide and two antisense probes targeting human microRNA miR-375 were custom synthesized and HPLC-purified by Invitrogen Life technologies. .. The sequence of the target oligonucleotide is 5′-TGT TCG TTC GGC TCG CGT GA-3′ .

    High Performance Liquid Chromatography:

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides
    Article Snippet: We are in the process of evaluating a number of different thermostable DNA polymerases such as DyNAzyme II Hot start DNA polymerase and Phusion DNA polymerase that carry the 3′-5′ proof-reading activity and lack the template-independent terminal transferase activity. .. Enzymes and Oligonucleotides Taq DNA Polymerase, PspGI and Hpy99I, were purchased from New England Biolabs (NEB), Inc. A target oligonucleotide and two antisense probes targeting human microRNA miR-375 were custom synthesized and HPLC-purified by Invitrogen Life technologies. .. The sequence of the target oligonucleotide is 5′-TGT TCG TTC GGC TCG CGT GA-3′ .

    Amplification:

    Article Title: Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli
    Article Snippet: .. In DNA amplification reactions such as PCR, the DNA denaturation step is usually set at 95°C for 30 to 60 s. For a 30-cycle PCR, the enzyme would be heated at 95°C for 15 to 30 min. To test the thermostability of Psp GI in DNA polymerase buffers, the enzyme was incubated in 1× Thermopol buffer (NEB) for 30 cycles of thermocycling. .. Psp GI retained 100% activity after the thermocycling (data not shown).

    Polymerase Chain Reaction:

    Article Title: Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli
    Article Snippet: .. In DNA amplification reactions such as PCR, the DNA denaturation step is usually set at 95°C for 30 to 60 s. For a 30-cycle PCR, the enzyme would be heated at 95°C for 15 to 30 min. To test the thermostability of Psp GI in DNA polymerase buffers, the enzyme was incubated in 1× Thermopol buffer (NEB) for 30 cycles of thermocycling. .. Psp GI retained 100% activity after the thermocycling (data not shown).

    Incubation:

    Article Title: Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli
    Article Snippet: .. In DNA amplification reactions such as PCR, the DNA denaturation step is usually set at 95°C for 30 to 60 s. For a 30-cycle PCR, the enzyme would be heated at 95°C for 15 to 30 min. To test the thermostability of Psp GI in DNA polymerase buffers, the enzyme was incubated in 1× Thermopol buffer (NEB) for 30 cycles of thermocycling. .. Psp GI retained 100% activity after the thermocycling (data not shown).

    DNA Purification:

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
    Article Snippet: In this study, we validated the PEAR technique for the preparation of 2′-F-modified and 2′-F/S double modified oligonucleotides using KOD DNA polymerase and Phusion DNA polymerase. .. Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

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    New England Biolabs pspgi
    Selective <t>PNA-mediated</t> FLAG on cell lines. The FLAG assay was performed on cell lines K562 (GGT/GGT), SW480 (GTT/GTT), PL45 (GGT/GAT) and CALU1 (GGT/TGT), or in absence of DNA target, B. Panel 1: FLAG assay without <t>PspGI</t> restriction endonuclease or PNA (positive controls). An amplification product is produced from all the reactions performed. Panel 2: FLAG assay in presence of PspGI to detect mutant versus wild-type samples. The wild -type sample K562 was digested by the enzyme, while the cell lines containing a mutation are exponentially amplified. Panels 3–5: FLAG assay with addition of PspGI and PNA specific for GTT, GAT or TGT. No amplification product is obtained from wild-type sample K562 (digested by PspGI) and from the cell lines that are suppressed by the fully complementary PNA present in the reaction.
    Pspgi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selective PNA-mediated FLAG on cell lines. The FLAG assay was performed on cell lines K562 (GGT/GGT), SW480 (GTT/GTT), PL45 (GGT/GAT) and CALU1 (GGT/TGT), or in absence of DNA target, B. Panel 1: FLAG assay without PspGI restriction endonuclease or PNA (positive controls). An amplification product is produced from all the reactions performed. Panel 2: FLAG assay in presence of PspGI to detect mutant versus wild-type samples. The wild -type sample K562 was digested by the enzyme, while the cell lines containing a mutation are exponentially amplified. Panels 3–5: FLAG assay with addition of PspGI and PNA specific for GTT, GAT or TGT. No amplification product is obtained from wild-type sample K562 (digested by PspGI) and from the cell lines that are suppressed by the fully complementary PNA present in the reaction.

    Journal: Nucleic Acids Research

    Article Title: FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations

    doi: 10.1093/nar/gkm809

    Figure Lengend Snippet: Selective PNA-mediated FLAG on cell lines. The FLAG assay was performed on cell lines K562 (GGT/GGT), SW480 (GTT/GTT), PL45 (GGT/GAT) and CALU1 (GGT/TGT), or in absence of DNA target, B. Panel 1: FLAG assay without PspGI restriction endonuclease or PNA (positive controls). An amplification product is produced from all the reactions performed. Panel 2: FLAG assay in presence of PspGI to detect mutant versus wild-type samples. The wild -type sample K562 was digested by the enzyme, while the cell lines containing a mutation are exponentially amplified. Panels 3–5: FLAG assay with addition of PspGI and PNA specific for GTT, GAT or TGT. No amplification product is obtained from wild-type sample K562 (digested by PspGI) and from the cell lines that are suppressed by the fully complementary PNA present in the reaction.

    Article Snippet: The reaction mix was split into six reactions, performed in parallel and compared to each other: the first reaction is conducted without restriction endonuclease or PNA, while the others are in presence of PspGI (New England Biolabs, Ipswich, MA, USA), 0.5 or 1 U/μl for analysis of DNA extracted from cell lines and clinical samples, respectively.

    Techniques: Amplification, Produced, Mutagenesis

    PNA-mediated FLAG –KRAS assay. FLAG-KRAS assay leads to selective amplification of mutant codon 12 KRAS sequences during the real-time fluorescent reaction through a REMS-PCR approach. ( A ) a mutagenic (underlined base) primer introduces a variation that creates the recognition site for PspGI (circle) in the wild-type codon 12 (X = C, ACC) target sequence, thereby suppressing the exponential amplification of the wild-type allele. The mutant alleles escape digestion and this enables their exponential amplification. ( B ) to assess the exact nucleotide alteration of the target amplified, the FLAG-KRAS assay is repeated in four parallel reactions in the presence of PNA probes (dotted lines) specific for the most common mutations of codon 12. When the reaction is conducted in the presence of the fully complementary PNA ( 4 ), no exponential amplification is allowed. In presence of a mismatched PNA ( 1–3 ) the probe is destabilized and the primer can consequently anneal and extend causing an exponential amplification and generation of FLAG fluorescent signal as explained in Figure 1.

    Journal: Nucleic Acids Research

    Article Title: FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations

    doi: 10.1093/nar/gkm809

    Figure Lengend Snippet: PNA-mediated FLAG –KRAS assay. FLAG-KRAS assay leads to selective amplification of mutant codon 12 KRAS sequences during the real-time fluorescent reaction through a REMS-PCR approach. ( A ) a mutagenic (underlined base) primer introduces a variation that creates the recognition site for PspGI (circle) in the wild-type codon 12 (X = C, ACC) target sequence, thereby suppressing the exponential amplification of the wild-type allele. The mutant alleles escape digestion and this enables their exponential amplification. ( B ) to assess the exact nucleotide alteration of the target amplified, the FLAG-KRAS assay is repeated in four parallel reactions in the presence of PNA probes (dotted lines) specific for the most common mutations of codon 12. When the reaction is conducted in the presence of the fully complementary PNA ( 4 ), no exponential amplification is allowed. In presence of a mismatched PNA ( 1–3 ) the probe is destabilized and the primer can consequently anneal and extend causing an exponential amplification and generation of FLAG fluorescent signal as explained in Figure 1.

    Article Snippet: The reaction mix was split into six reactions, performed in parallel and compared to each other: the first reaction is conducted without restriction endonuclease or PNA, while the others are in presence of PspGI (New England Biolabs, Ipswich, MA, USA), 0.5 or 1 U/μl for analysis of DNA extracted from cell lines and clinical samples, respectively.

    Techniques: Kras Assay, Amplification, Mutagenesis, Polymerase Chain Reaction, Sequencing

    Half-life of recombinant Psp GI at 95°C. One unit of Psp GI was heated at 95°C for 20 min to 4 h, and then the enzyme was used to cleave 1 μg of T7 phage DNA at 75°C. DNA cleavage products were separated on an agarose gel, transferred to a membrane, and detected with a Phototope-star chemiluminescence detection kit. X-ray films were exposed for 10 s, 30 s, 50 s, 1 min, and 2 min to obtain a linear response range and were scanned.

    Journal: Applied and Environmental Microbiology

    Article Title: Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli

    doi:

    Figure Lengend Snippet: Half-life of recombinant Psp GI at 95°C. One unit of Psp GI was heated at 95°C for 20 min to 4 h, and then the enzyme was used to cleave 1 μg of T7 phage DNA at 75°C. DNA cleavage products were separated on an agarose gel, transferred to a membrane, and detected with a Phototope-star chemiluminescence detection kit. X-ray films were exposed for 10 s, 30 s, 50 s, 1 min, and 2 min to obtain a linear response range and were scanned.

    Article Snippet: In DNA amplification reactions such as PCR, the DNA denaturation step is usually set at 95°C for 30 to 60 s. For a 30-cycle PCR, the enzyme would be heated at 95°C for 15 to 30 min. To test the thermostability of Psp GI in DNA polymerase buffers, the enzyme was incubated in 1× Thermopol buffer (NEB) for 30 cycles of thermocycling.

    Techniques: Recombinant, Agarose Gel Electrophoresis

    DNA cleavage activity at different temperatures. One unit of Psp GI was used to cleave 1 μg of T7 phage DNA for 1 h at the specified temperatures. Lanes 1 through 7, 25, 37, 50, 65, 75, 80, and 85°C, respectively; lane 8, native Psp GI digestion of T7 phage DNA at 85°C.

    Journal: Applied and Environmental Microbiology

    Article Title: Characterization of an Extremely Thermostable Restriction Enzyme, PspGI, from a Pyrococcus Strain and Cloning of the PspGI Restriction-Modification System in Escherichia coli

    doi:

    Figure Lengend Snippet: DNA cleavage activity at different temperatures. One unit of Psp GI was used to cleave 1 μg of T7 phage DNA for 1 h at the specified temperatures. Lanes 1 through 7, 25, 37, 50, 65, 75, 80, and 85°C, respectively; lane 8, native Psp GI digestion of T7 phage DNA at 85°C.

    Article Snippet: In DNA amplification reactions such as PCR, the DNA denaturation step is usually set at 95°C for 30 to 60 s. For a 30-cycle PCR, the enzyme would be heated at 95°C for 15 to 30 min. To test the thermostability of Psp GI in DNA polymerase buffers, the enzyme was incubated in 1× Thermopol buffer (NEB) for 30 cycles of thermocycling.

    Techniques: Activity Assay

    MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).

    Journal: Nature

    Article Title: Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators

    doi: 10.1038/nature25179

    Figure Lengend Snippet: MORC2, MPP8 and TASOR silence L1 transcription. a. Relative genomic copy number of newly integrated L1-GFP reporters in the indicated mutant K562 pools after dox-induction. PspGI-assisted qPCR assay used here was designed to selectively detect spliced GFP rather than the unspliced version (see Methods section). The L1-GFP copies were normalized to beta-actin DNAs; data then normalized to Ctrl. As a putative L1 activator, SLTM shows an opposite effect on the DNA copy number, compared with L1 suppressors. Center value as median. n = 3 technical replicates per gene. b. RNA-seq data in Ctrl K562 cells showing that most heterochromatin regulators in Fig. 2a are expressed, supporting the selective effect of HUSH and MORC2 in L1 regulation. c. Western blots validating the knockout (KO) effects in independent KO K562 cell clones. Ctrl samples were loaded at 4 different amounts (200%, 100%, 50%, 25% of KO clones). Three experiments repeated independently with similar results. To obtain KO clones, we sorted mutant K562 pools (cells used in Fig. 1e,f ) into 96-well plates, expanded cells and screened for KO clones through western blotting. Of note, all K562 KO clones were derived from the same starting L1-GFP reporter line, and thus do not differ in reporter transgene integrations among the clones. d. Representative images of single molecule FISH (smFISH) assays targeting ACTB mRNAs and RNA transcripts from L1-GFP reporters in Ctrl and KO K562 clones after 5 days of dox-induction. No signal was observed from L1-GFP reporters without dox-induction (data not shown). Two experiments repeated independently with similar results. See also panel e and Fig. 2b (showing L1-GFP mRNA only). e. Quantitation of the L1-GFP transcription level from the indicated number of K562 cells, determined by smFISH assays (panel d and Fig. 2b ). The number of L1-GFP mRNA transcripts is normalized to the number of beta-actin mRNAs within each K562 cell. Box plots show median and interquartile range (IQR), whiskers are 1.5× IQR. P-value, two-sided Wilcoxon test. 95% CI for median from 1,000× bootstrap: Control: 0.059-0.082; MORC2: 0.106-0.123; MPP8: 0.264-0.410; TASOR: 0.514-0.671. f. MORC2, MPP8, and TASOR KOs increase the genomic copy number of newly integrated L1-GFP reporters. PspGI-assisted qPCR assays were performed as in panel a), but using clonal KO K562 clones instead of mutant cell pools. Data normalized to Ctrl. n = 3 technical replicates, center value as median. g. MORC2 KO, MPP8 KO, and TASOR KO increase the expression of endogenous L1s. RT-qPCR experiments were performed as in ( Fig. 1f ), but using clonal KO K562 clones instead of mutant cell pools. n = 2 biological replicates × 3 technical replicates (center value as median). The primers do not target the L1-GFP reporter and the cell lines were not dox-induced, so these RT-qPCR assays will not detect L1-GFP transcripts. h. Western blots showing depletion effects of MORC2, MPP8 and TASOR in the mutant pools of K562 cells (left) and in the mutant pools of H9 hESCs without transgenic L1 reporters (right). Two experiments repeated independently with similar results. i. Northern blots showing increased transcription from the L1-GFP reporter in KO K562 clones (same cell lines as in panel c) after 5 days’ dox-induction. Two experiments repeated independently with similar results. As observed in Fig. 2b , while HUSH KO significantly increases L1-GFP transcription, MORC2 KO leads to only a modest increase. This is probably because the L1-GFP reporter does not contain the native L1 5’ UTR sequence, where MORC2 intensively binds (See Extended Data Fig. 7f,g ). The 5 kb and 1.9 kb marks on the membrane refer to the 28S rRNA and 18S rRNA bands respectively. j. Northern blots showing that disruption of MORC2, MPP8 and TASOR increases the expression level of endogenous L1Hs in hESCs, same cell lines as in panel h). Size marker indicated as in panel i). Two experiments repeated independently with similar results. k. Western blots showing protein abundance of L1_ORF1p and HSP90 in the mutant pools of K562 cells and hESCs (same cell line as shown in panel h). Two experiments repeated independently with similar results. Experiments were performed without dox-induction of the transgenic L1 reporter. Due to the strong signal of bands from the KO samples, the blots were exposed for a very short time and the band signal in the Ctrl samples were relatively very weak compared to the KO samples; same case for panels i, j).

    Article Snippet: 300 ng genomic DNA per sample was digested with 50 units PspGI (R0611S, New England Biolabs) in 1× smart buffer (NEB) at 75 °C for 1hr, to cut uniquely at the intron of the GFP cassette.

    Techniques: Mutagenesis, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Western Blot, Knock-Out, Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Quantitation Assay, Expressing, Quantitative RT-PCR, Transgenic Assay, Northern Blot, Sequencing, Marker

    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Article Snippet: Enzymes and Oligonucleotides Taq DNA Polymerase, PspGI and Hpy99I, were purchased from New England Biolabs (NEB), Inc. A target oligonucleotide and two antisense probes targeting human microRNA miR-375 were custom synthesized and HPLC-purified by Invitrogen Life technologies.

    Techniques:

    Double digestion of the PEAR product by PspGI and Hpy99I. The sense and the antisense strand are indicated respectively by (+) and ( − ). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“ ^ ”). The antisense strands ( A ) are black boxed, the sense strands ( B and C ) are boxed, and the by-products ( D and E ) are grey boxed. The expected length for each strand is indicated in parenthesis.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: Double digestion of the PEAR product by PspGI and Hpy99I. The sense and the antisense strand are indicated respectively by (+) and ( − ). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“ ^ ”). The antisense strands ( A ) are black boxed, the sense strands ( B and C ) are boxed, and the by-products ( D and E ) are grey boxed. The expected length for each strand is indicated in parenthesis.

    Article Snippet: Enzymes and Oligonucleotides Taq DNA Polymerase, PspGI and Hpy99I, were purchased from New England Biolabs (NEB), Inc. A target oligonucleotide and two antisense probes targeting human microRNA miR-375 were custom synthesized and HPLC-purified by Invitrogen Life technologies.

    Techniques:

    Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Article Snippet: Enzymes and Oligonucleotides Taq DNA Polymerase, PspGI and Hpy99I, were purchased from New England Biolabs (NEB), Inc. A target oligonucleotide and two antisense probes targeting human microRNA miR-375 were custom synthesized and HPLC-purified by Invitrogen Life technologies.

    Techniques: Produced

    HPLC separation and purification of antisense oligonucleotide. Fractions are indicated by letter A to E as shown in Fig. 4 . Fraction A, which contains the antisense strands, was collected in the indicated interval. Sample preparation: 10 µg PEAR product double digested by PspGI and Hpy99I; Column: SOURCE Q PE 4.6/100; Flow rate: 1 ml/min; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH+2M NaCl, pH 12; Gradient: 20–35% B in 50 column volume.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: HPLC separation and purification of antisense oligonucleotide. Fractions are indicated by letter A to E as shown in Fig. 4 . Fraction A, which contains the antisense strands, was collected in the indicated interval. Sample preparation: 10 µg PEAR product double digested by PspGI and Hpy99I; Column: SOURCE Q PE 4.6/100; Flow rate: 1 ml/min; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH+2M NaCl, pH 12; Gradient: 20–35% B in 50 column volume.

    Article Snippet: Enzymes and Oligonucleotides Taq DNA Polymerase, PspGI and Hpy99I, were purchased from New England Biolabs (NEB), Inc. A target oligonucleotide and two antisense probes targeting human microRNA miR-375 were custom synthesized and HPLC-purified by Invitrogen Life technologies.

    Techniques: High Performance Liquid Chromatography, Purification, Sample Prep, Flow Cytometry