phi29 dna polymerase mutants (New England Biolabs)

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New England Biolabs phi29 dna polymerase mutants

Impaired rolling circle amplification (RCA) activity and reduced fidelity <t>Phi29</t> DNAP P562del. ( A ) The RCA assay used 10 ng of plasmid template and 3 nM of each enzyme in a 3-hour reaction. A sample without enzyme (-ve control) was used to confirm the <t>polymerase-dependent</t> amplification. Restriction of the RCA yields full-length products. ( B ) Relative fidelity of the D12A-THR and p562del <t>mutants</t> to Exo+THR. The corrected total error rates from Table 2 of each mutant were divided by that of the Exo+THR, resulting in the relative fidelity scores.

Phi29 Dna Polymerase Mutants, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Directed evolution of mesophilic HNA polymerases providing insight into DNA polymerase mechanisms"

Article Title: Directed evolution of mesophilic HNA polymerases providing insight into DNA polymerase mechanisms

Journal: bioRxiv

doi: 10.1101/2022.11.08.515583

Impaired rolling circle amplification (RCA) activity and reduced fidelity Phi29 DNAP P562del. ( A ) The RCA assay used 10 ng of plasmid template and 3 nM of each enzyme in a 3-hour reaction. A sample without enzyme (-ve control) was used to confirm the polymerase-dependent amplification. Restriction of the RCA yields full-length products. ( B ) Relative fidelity of the D12A-THR and p562del mutants to Exo+THR. The corrected total error rates from Table 2 of each mutant were divided by that of the Exo+THR, resulting in the relative fidelity scores.

Figure Legend Snippet: Impaired rolling circle amplification (RCA) activity and reduced fidelity Phi29 DNAP P562del. ( A ) The RCA assay used 10 ng of plasmid template and 3 nM of each enzyme in a 3-hour reaction. A sample without enzyme (-ve control) was used to confirm the polymerase-dependent amplification. Restriction of the RCA yields full-length products. ( B ) Relative fidelity of the D12A-THR and p562del mutants to Exo+THR. The corrected total error rates from Table 2 of each mutant were divided by that of the Exo+THR, resulting in the relative fidelity scores.

Techniques Used: Amplification, Activity Assay, Plasmid Preparation, Mutagenesis

$Phi29 DNAP P562del reduced DNA binding capacity. For the EMSA, 60 pM commercial phi29 DNAP (control), D12A-THR or P562del were allowed to bind a fluorescently labelled primer pre-annealed to a ssDNA template ( A ) Reactions with commercial phi29 DNAP show no binding, probably as a result of storage buffer components interfering with quantification. ( B ) Fraction of intermediate Pol-DNA complex, and ( C ) the fraction of stable Pol-DNA complex by D12A-THR (orange) and P562del (blue) over time.$
Figure Legend Snippet: Phi29 DNAP P562del reduced DNA binding capacity. For the EMSA, 60 pM commercial phi29 DNAP (control), D12A-THR or P562del were allowed to bind a fluorescently labelled primer pre-annealed to a ssDNA template ( A ) Reactions with commercial phi29 DNAP show no binding, probably as a result of storage buffer components interfering with quantification. ( B ) Fraction of intermediate Pol-DNA complex, and ( C ) the fraction of stable Pol-DNA complex by D12A-THR (orange) and P562del (blue) over time.

Techniques Used: Binding Assay

Structure of the closed ternary complex of Phi29 DNA Polymerase and InDel library design. ( A ) There domains in the phi29 DNA polymerase (PDB: 2PYJ) are involved in creating a clamp around the nascent DNA duplex: the polymerase thumb (green), the exonuclease domain (yellow) and the TPR2 domain (purple). The DNA duplex is shown in cyan. (B) InDel mutagenesis of the thumb loop through inverse PCR (iPCR). A single reverse primer in combination with forward primers harbouring 1-3 NNS codons or with priming sites that skipped 1-4 codons were used to generate focused libraries or deletion mutants respectively. The same approach was used to target the TPR2 and exonuclease loops.

Figure Legend Snippet: Structure of the closed ternary complex of Phi29 DNA Polymerase and InDel library design. ( A ) There domains in the phi29 DNA polymerase (PDB: 2PYJ) are involved in creating a clamp around the nascent DNA duplex: the polymerase thumb (green), the exonuclease domain (yellow) and the TPR2 domain (purple). The DNA duplex is shown in cyan. (B) InDel mutagenesis of the thumb loop through inverse PCR (iPCR). A single reverse primer in combination with forward primers harbouring 1-3 NNS codons or with priming sites that skipped 1-4 codons were used to generate focused libraries or deletion mutants respectively. The same approach was used to target the TPR2 and exonuclease loops.

Techniques Used: Mutagenesis, Inverse PCR

P562 deletion in phi29 DNAP enables faster initiation of HNA synthesis. Primer extension assays for HNA synthesis were carried out by incubating 10 pmol of single stranded DNA template pre-annealed to 1 pmol of fluorescently labelled DNA primer with 12 nM D12A-THR or P562del phi29 DNAP over different amounts of time. ( A ) HNA primer extension products synthesized by D12A-THR and P562 mutants separated by denaturing PAGE. Unextended primer (U) and extension products of ~37 incorporations (E) are highlighted. ( B ) Average depletion of unextended primer (U) by D12A-THR (orange) and P562del (blue) normalized to the earliest time point (0 min). ( C ) Average extended products with ~37 incorporations (E) by D12A-THR and P562del normalized to the earliest time point (0 min). P562del shows a faster start to its hNTP incorporations than D12A-THR.

Figure Legend Snippet: P562 deletion in phi29 DNAP enables faster initiation of HNA synthesis. Primer extension assays for HNA synthesis were carried out by incubating 10 pmol of single stranded DNA template pre-annealed to 1 pmol of fluorescently labelled DNA primer with 12 nM D12A-THR or P562del phi29 DNAP over different amounts of time. ( A ) HNA primer extension products synthesized by D12A-THR and P562 mutants separated by denaturing PAGE. Unextended primer (U) and extension products of ~37 incorporations (E) are highlighted. ( B ) Average depletion of unextended primer (U) by D12A-THR (orange) and P562del (blue) normalized to the earliest time point (0 min). ( C ) Average extended products with ~37 incorporations (E) by D12A-THR and P562del normalized to the earliest time point (0 min). P562del shows a faster start to its hNTP incorporations than D12A-THR.

Techniques Used: Synthesized, Polyacrylamide Gel Electrophoresis

2) Product Images from "POT-3 preferentially binds the terminal DNA-repeat on the telomeric G-overhang"

Article Title: POT-3 preferentially binds the terminal DNA-repeat on the telomeric G-overhang

Journal: bioRxiv

doi: 10.1101/2022.07.01.497317

Mutation of pot-3 increases telomere length and recombination in a manner that is epistatic with pot-2 . A . POT-3 is 215aa long, the syb2415 allele contains a 500bp deletion spanning the entire OB-fold region. B . Southern blot of terminal restriction fragments from genomic DNA show that mutation of pot-3 results in an increase in telomere length almost to that of pot-2 . Interestingly pot-2; pot-3 double mutants do not have longer telomeres than either single mutant. Membrane was probed with a DIG-labelled (TTAGGC) 4 oligo. C . Telomeric C-circle assays were carried out using phi29 polymerase, spotted onto a nitrocellulose membrane and probed with a DIG-labelled (TTAGGC) 4 oligo. Signal intensity was quantified using ImageJ and results plotted relative to pot-2 (set to 100%). The bar graph shows the average of six independent experiments with individual results displayed as open circles. Mutation of pot-3 increases C-circle levels above that of wildtype but supressed the levels of C-circles in a pot-2 background, ns = not significant, * p =

Figure Legend Snippet: Mutation of pot-3 increases telomere length and recombination in a manner that is epistatic with pot-2 . A . POT-3 is 215aa long, the syb2415 allele contains a 500bp deletion spanning the entire OB-fold region. B . Southern blot of terminal restriction fragments from genomic DNA show that mutation of pot-3 results in an increase in telomere length almost to that of pot-2 . Interestingly pot-2; pot-3 double mutants do not have longer telomeres than either single mutant. Membrane was probed with a DIG-labelled (TTAGGC) 4 oligo. C . Telomeric C-circle assays were carried out using phi29 polymerase, spotted onto a nitrocellulose membrane and probed with a DIG-labelled (TTAGGC) 4 oligo. Signal intensity was quantified using ImageJ and results plotted relative to pot-2 (set to 100%). The bar graph shows the average of six independent experiments with individual results displayed as open circles. Mutation of pot-3 increases C-circle levels above that of wildtype but supressed the levels of C-circles in a pot-2 background, ns = not significant, * p =

Techniques Used: Mutagenesis, Southern Blot

3) Product Images from "ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats"

Article Title: ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkac113

ALT-FISH predicts the ALT status of cell lines from different cancer entities and resolves cellular heterogeneity. ( A ) Representative images of a cancer cell line panel and primary HUVECs stained by TelC ALT-FISH. The telomere maintenance mechanism (TMM) is indicated above and representative CC assay results (+/– phi29 polymerase) are shown below (dot blot). Outlines represent nucleus segmentation. Corresponding results for the TelG probe are shown in Supplementary Figure S3A, B . CC and telomerase activity assays used to determine the TMM status are displayed in Supplementary Figure S3C, D . ( B ) Nuclear TelC ALT-FISH spot numbers for the cell lines shown in panel A. Count data were pooled from 2–4 independent replicates (cell numbers given below). U2OS data are the same as for the untreated control in Figure 1C . The spot distribution in the no TMM/TERT+ cell lines is magnified for better visualization. All boxplots depict the median with first and third quartile of the distribution. Adjusted P -values were calculated using a pair-wise Wilcoxon rank sum test with BH correction, comparing all ALT+ cell lines (including weakly ALT+) to the other groups (no TMM, TERT+). Cyan lines mark the 95th percentile of the spot number distribution in ALT+ cell lines above which cells were defined as super-ALT. Images of super-ALT phenotypes in Saos2, CAL72, MGBMI, U2OS with spot number n given below the image. The cyan box marks the position within the distribution shown on the left side. ( C ) Selected positions picked from U2OS (ALT+) and HeLa LT (TERT+, ALT-prone) TelC ALT-FISH to visualize ALT activity in a spatial context. Examples of intermediate and high-ALT clusters found in U2OS (left). Rare cells with low ALT activity found within the mostly ALT-negative HeLa LT population (right). Nuclear masks are color-coded by spot number (legend). All scale bars in images are 5 μm.

Figure Legend Snippet: ALT-FISH predicts the ALT status of cell lines from different cancer entities and resolves cellular heterogeneity. ( A ) Representative images of a cancer cell line panel and primary HUVECs stained by TelC ALT-FISH. The telomere maintenance mechanism (TMM) is indicated above and representative CC assay results (+/– phi29 polymerase) are shown below (dot blot). Outlines represent nucleus segmentation. Corresponding results for the TelG probe are shown in Supplementary Figure S3A, B . CC and telomerase activity assays used to determine the TMM status are displayed in Supplementary Figure S3C, D . ( B ) Nuclear TelC ALT-FISH spot numbers for the cell lines shown in panel A. Count data were pooled from 2–4 independent replicates (cell numbers given below). U2OS data are the same as for the untreated control in Figure 1C . The spot distribution in the no TMM/TERT+ cell lines is magnified for better visualization. All boxplots depict the median with first and third quartile of the distribution. Adjusted P -values were calculated using a pair-wise Wilcoxon rank sum test with BH correction, comparing all ALT+ cell lines (including weakly ALT+) to the other groups (no TMM, TERT+). Cyan lines mark the 95th percentile of the spot number distribution in ALT+ cell lines above which cells were defined as super-ALT. Images of super-ALT phenotypes in Saos2, CAL72, MGBMI, U2OS with spot number n given below the image. The cyan box marks the position within the distribution shown on the left side. ( C ) Selected positions picked from U2OS (ALT+) and HeLa LT (TERT+, ALT-prone) TelC ALT-FISH to visualize ALT activity in a spatial context. Examples of intermediate and high-ALT clusters found in U2OS (left). Rare cells with low ALT activity found within the mostly ALT-negative HeLa LT population (right). Nuclear masks are color-coded by spot number (legend). All scale bars in images are 5 μm.

Techniques Used: Fluorescence In Situ Hybridization, Staining, Dot Blot, Activity Assay

4) Product Images from "Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification"

Article Title: Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification

Journal: Analytical and Bioanalytical Chemistry

doi: 10.1007/s00216-022-04156-7

a Selectivity of RHthio-CuSO 4 . Absorbance spectra of RHthio and RHthio-CuSO 4 recorded in the presence of various anions and biomaterials. All reaction mixtures included 25 mM RHthio or RHthio-CuSO 4 (1 μL) and a reactive material (200 mM, 1 μL) in water (1 mL). b Agarose gel electrophoresis of the RAM reaction. Lane M: DNA marker; lane 1: RAM reaction without phi29 DNA polymerase; lane 2: RAM reaction without Splint R ligase; lane 3: RAM reaction without SARS-CoV-2 target; lane 4: RAM reaction without RAM template; lane 5: RAM reaction without RAM pri-1; lane 6: RAM reaction without pri-2; lane 7: RAM reaction. The gel was stained with EtBr

Figure Legend Snippet: a Selectivity of RHthio-CuSO 4 . Absorbance spectra of RHthio and RHthio-CuSO 4 recorded in the presence of various anions and biomaterials. All reaction mixtures included 25 mM RHthio or RHthio-CuSO 4 (1 μL) and a reactive material (200 mM, 1 μL) in water (1 mL). b Agarose gel electrophoresis of the RAM reaction. Lane M: DNA marker; lane 1: RAM reaction without phi29 DNA polymerase; lane 2: RAM reaction without Splint R ligase; lane 3: RAM reaction without SARS-CoV-2 target; lane 4: RAM reaction without RAM template; lane 5: RAM reaction without RAM pri-1; lane 6: RAM reaction without pri-2; lane 7: RAM reaction. The gel was stained with EtBr

Techniques Used: Agarose Gel Electrophoresis, Marker, Staining

5) Product Images from "The TERT promoter is polycomb-repressed in neuroblastoma cells with long telomeres"

Article Title: The TERT promoter is polycomb-repressed in neuroblastoma cells with long telomeres

Journal: bioRxiv

doi: 10.1101/2022.05.18.492493

Molecular characterization of telomere maintenance in neuroblastoma cell lines. Telomere maintenance mechanism was assessed in eight neuroblastoma cell lines used in this study: MYCN-amplified (NB5, SKNBE2, IMR32), moderate level of MYCN without amplification (NBL-S), ATRX-mutant (SKNMM, CHLA90), low-DAXX (SKNFI) and ever-shorter telomere (LAN6) neuroblastoma cells. A) Representative images of Telomere FISH (green) showing brighter signal in cell lines with long telomeres. B) Telomeric repeats in neuroblastoma cell line relative to those in the telomerase positive 293T cells using qPCR. Telomerase negative, ALT-positive U2OS cells were used as a positive control for long telomeres. C) Representative blot of C-circle assay. U2OS cells were used as a positive control and PC3 cells were used as a negative control. The amount of DNA in nanograms (ng) are indicated on the blot. All reactions were done in the presence or absence of the Phi 29 (Phi) DNA polymerase. RT-qPCR was performed in neuroblastoma cell lines to measure D ) TERT and E ) TERC expression relative to the telomerase-positive 293T cell line. Gene expression was normalized to HPRT expression. The U2OS cell line was used as an ALT-positive control.

Figure Legend Snippet: Molecular characterization of telomere maintenance in neuroblastoma cell lines. Telomere maintenance mechanism was assessed in eight neuroblastoma cell lines used in this study: MYCN-amplified (NB5, SKNBE2, IMR32), moderate level of MYCN without amplification (NBL-S), ATRX-mutant (SKNMM, CHLA90), low-DAXX (SKNFI) and ever-shorter telomere (LAN6) neuroblastoma cells. A) Representative images of Telomere FISH (green) showing brighter signal in cell lines with long telomeres. B) Telomeric repeats in neuroblastoma cell line relative to those in the telomerase positive 293T cells using qPCR. Telomerase negative, ALT-positive U2OS cells were used as a positive control for long telomeres. C) Representative blot of C-circle assay. U2OS cells were used as a positive control and PC3 cells were used as a negative control. The amount of DNA in nanograms (ng) are indicated on the blot. All reactions were done in the presence or absence of the Phi 29 (Phi) DNA polymerase. RT-qPCR was performed in neuroblastoma cell lines to measure D ) TERT and E ) TERC expression relative to the telomerase-positive 293T cell line. Gene expression was normalized to HPRT expression. The U2OS cell line was used as an ALT-positive control.

Techniques Used: Amplification, Mutagenesis, Fluorescence In Situ Hybridization, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Quantitative RT-PCR, Expressing

DNA hypomethylation of the TERT locus in neuroblastoma cell lines, tumors and O-PDX. A) Alignments of DNA methylation from whole genome bisulfite sequencing for the TERT locus in neuroblastoma cell lines (black), autopsy (red) and O-PDX (blue). Tumor autopsies and O-PDX that are derived from the same patients are connected by a curved line. The hypomethylated region shaded in gray starts −694 bases from the TERT transcriptional start site (TSS) and ends 758 bases after the TSS.

Figure Legend Snippet: DNA hypomethylation of the TERT locus in neuroblastoma cell lines, tumors and O-PDX. A) Alignments of DNA methylation from whole genome bisulfite sequencing for the TERT locus in neuroblastoma cell lines (black), autopsy (red) and O-PDX (blue). Tumor autopsies and O-PDX that are derived from the same patients are connected by a curved line. The hypomethylated region shaded in gray starts −694 bases from the TERT transcriptional start site (TSS) and ends 758 bases after the TSS.

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Derivative Assay

The DNA hypomethylated region extends for tens of thousands of kilobases in neuroblastoma cell cells with long telomeres. TERT locus is shaded in grey.

Figure Legend Snippet: The DNA hypomethylated region extends for tens of thousands of kilobases in neuroblastoma cell cells with long telomeres. TERT locus is shaded in grey.

Techniques Used:

MYCN-binding sites overlap with the hypomethylated region in the TERT promoter. Alignments of DNA methylation from whole genome bisulfite sequencing (WGBS) and MYCN-ChIP-Seq in three MYCN-amplified neuroblastoma O-PDX models and cell line (SKNBE2) showing that MYCN binding overlaps with the hypomethylated region in the TERT promoter. The core promoter is shaded in red and THOR region is shaded in yellow.

Figure Legend Snippet: MYCN-binding sites overlap with the hypomethylated region in the TERT promoter. Alignments of DNA methylation from whole genome bisulfite sequencing (WGBS) and MYCN-ChIP-Seq in three MYCN-amplified neuroblastoma O-PDX models and cell line (SKNBE2) showing that MYCN binding overlaps with the hypomethylated region in the TERT promoter. The core promoter is shaded in red and THOR region is shaded in yellow.

Techniques Used: Binding Assay, DNA Methylation Assay, Methylation Sequencing, Chromatin Immunoprecipitation, Amplification

6) Product Images from "Single-stranded DNA aptamer-based rolling circle amplification as anti-chicken Salmonella bacteriostatic"

Article Title: Single-stranded DNA aptamer-based rolling circle amplification as anti-chicken Salmonella bacteriostatic

Journal: Veterinary World

doi: 10.14202/vetworld.2022.1171-1176

Rolling circle amplification products on denaturing 15%TBE urea gel. The templates were annealed with a single spanning primer. The ligation process with T4 DNA ligase (NEB, Cat# M0202S) was mediated by the phosphorylated upstream template. Last, ɸ29 DNA polymerase was used to polymerize the oligos (NEB, Cat# M0269L). The rolling circle amplification (RCA) products were hybridized with spacer complementary to produce tandem repeats of ssDNA alternating with dsDNA to increase the chance of interaction with target Salmonella. Few controls have been added to ensure the quality of the RCA products.

Figure Legend Snippet: Rolling circle amplification products on denaturing 15%TBE urea gel. The templates were annealed with a single spanning primer. The ligation process with T4 DNA ligase (NEB, Cat# M0202S) was mediated by the phosphorylated upstream template. Last, ɸ29 DNA polymerase was used to polymerize the oligos (NEB, Cat# M0269L). The rolling circle amplification (RCA) products were hybridized with spacer complementary to produce tandem repeats of ssDNA alternating with dsDNA to increase the chance of interaction with target Salmonella. Few controls have been added to ensure the quality of the RCA products.

Techniques Used: Amplification, Ligation

7) Product Images from "Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription"

Article Title: Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription

Journal: Analytica Chimica Acta

doi: 10.1016/j.aca.2022.339909

Evaluation of tandem isothermal gene amplification with ThT fluorescence enhancement. (A) Evaluation of the ternary RCA (scheme shown above) with electrophoretic analysis (urea-PAGE, 10%) of amplified ssDNA and subsequent ThT fluorescence enhancement under various t-RCA conditions: a, without the 1st dumbbell padlock DNA (DP1); b, without target RNA ( RdRp RNA); c, without hairpin primer (HP); d, without phi29 DNA polymerase; e, all included. Fluorescent gel image was visualized under UV light after SYBR Gold staining of amplified DNA (shown by arrow). ThT fluorescence enhancement resulting from the subsequent SDA/GQ-RCA reaction under various t-RCA conditions (a ∼ e) was measured at an excitation wavelength (λ ex ) of 425 nm with a scanning of emission wavelengths. (B) Evaluation of the SDA/GQ-RCA reaction (scheme shown above) using the DNA amplified with the preceding t-RCA under various SDA/GQ-RCA conditions: a, without the t-RCA product (t-RCAP); b, without the 2nd dumbbell padlock DNA (DP2); c, without the Klenow fragment (KF); d, without the nicking endonuclease (NE); e, without phi29 DNA polymerase; f, all included. Fluorescence emission spectra (λ ex = 425 nm) of ThT intercalated into the SDA/GQ-RCA products under various conditions were obtained as described in (A). Each sample used for fluorescence measurements was visualized under UV light (inset image).

Figure Legend Snippet: Evaluation of tandem isothermal gene amplification with ThT fluorescence enhancement. (A) Evaluation of the ternary RCA (scheme shown above) with electrophoretic analysis (urea-PAGE, 10%) of amplified ssDNA and subsequent ThT fluorescence enhancement under various t-RCA conditions: a, without the 1st dumbbell padlock DNA (DP1); b, without target RNA ( RdRp RNA); c, without hairpin primer (HP); d, without phi29 DNA polymerase; e, all included. Fluorescent gel image was visualized under UV light after SYBR Gold staining of amplified DNA (shown by arrow). ThT fluorescence enhancement resulting from the subsequent SDA/GQ-RCA reaction under various t-RCA conditions (a ∼ e) was measured at an excitation wavelength (λ ex ) of 425 nm with a scanning of emission wavelengths. (B) Evaluation of the SDA/GQ-RCA reaction (scheme shown above) using the DNA amplified with the preceding t-RCA under various SDA/GQ-RCA conditions: a, without the t-RCA product (t-RCAP); b, without the 2nd dumbbell padlock DNA (DP2); c, without the Klenow fragment (KF); d, without the nicking endonuclease (NE); e, without phi29 DNA polymerase; f, all included. Fluorescence emission spectra (λ ex = 425 nm) of ThT intercalated into the SDA/GQ-RCA products under various conditions were obtained as described in (A). Each sample used for fluorescence measurements was visualized under UV light (inset image).

Techniques Used: Amplification, Fluorescence, Polyacrylamide Gel Electrophoresis, Staining

8) Product Images from "One-Step Making of DNA Dumbbells Allows Tunneling of Short-Read Libraries into a Long-Read Format"

Article Title: One-Step Making of DNA Dumbbells Allows Tunneling of Short-Read Libraries into a Long-Read Format

Journal: bioRxiv

doi: 10.1101/2022.05.01.490243

Verification of protocol. ( a ) Two different primers capable of hybridizing with a loop paired with a reverse primer matching the input sequence. Only looped products, and not unconverted inputs, yielded positive PCR bands. ( b ) Rolling circle amplifications by Phi29 polymerase proceeded only on dumbbell templates primed with two different loop-hybridizing primers. No high molecular-weight products (arrow) were produced using unconverted inputs as templates.

Figure Legend Snippet: Verification of protocol. ( a ) Two different primers capable of hybridizing with a loop paired with a reverse primer matching the input sequence. Only looped products, and not unconverted inputs, yielded positive PCR bands. ( b ) Rolling circle amplifications by Phi29 polymerase proceeded only on dumbbell templates primed with two different loop-hybridizing primers. No high molecular-weight products (arrow) were produced using unconverted inputs as templates.

Techniques Used: Sequencing, Polymerase Chain Reaction, Molecular Weight, Produced

9) Product Images from "One-Pot Endonucleolytically Exponentiated Rolling Circle Amplification by CRISPR-Cas12a Affords Sensitive, Expedited Isothermal Detection of MicroRNAs"

Article Title: One-Pot Endonucleolytically Exponentiated Rolling Circle Amplification by CRISPR-Cas12a Affords Sensitive, Expedited Isothermal Detection of MicroRNAs

Journal: bioRxiv

doi: 10.1101/2022.05.01.490215

Comparison of the kinetics and detection sensitivity of RCA, one-pot EXTRA-CRISPR, and multi-step CRISPR-assisted RCA assays. ( a ) The RCA-only method involves two sequential reactions of ligation and linear RCA and can only detect 100 pM miR-21. Ligation condition: 100 nM padlock probe and 0.625 U/ μL SplintR ligase. RCA condition: 2 μL ligation product, 0.2 U/μL phi29, and SYBY Green II for detection. ( b ) The assay that connects three tandem steps of ligation, RCA, and Cas12a readout yielded a LOD of ∼100 fM. The conditions for ligation and RCA were the same as in ( a ). After RCA and denaturing of enzymes, 40 nM RNP and 0.8 μM reporter were added into the reaction. ( c ) In the two-step method, ligation and RCA were combined together, followed by fluorogenic detection using Cas12a RNP, conferring similar sensitivity with the three-step assay in ( b ). ( d ) The one-pot EXTRA-CRISPR method improves the sensitivity to detect 10 fM miR-21 prior to full optimization. Assay concentrations used in ( c ) and ( d ): 100 nM Padlock-1, 0.625 U/μL SplintR ligase, 0.1 U/μL phi29 polymerase, 1 μM reporter, and 1 nM Cas12a RNP.

Figure Legend Snippet: Comparison of the kinetics and detection sensitivity of RCA, one-pot EXTRA-CRISPR, and multi-step CRISPR-assisted RCA assays. ( a ) The RCA-only method involves two sequential reactions of ligation and linear RCA and can only detect 100 pM miR-21. Ligation condition: 100 nM padlock probe and 0.625 U/ μL SplintR ligase. RCA condition: 2 μL ligation product, 0.2 U/μL phi29, and SYBY Green II for detection. ( b ) The assay that connects three tandem steps of ligation, RCA, and Cas12a readout yielded a LOD of ∼100 fM. The conditions for ligation and RCA were the same as in ( a ). After RCA and denaturing of enzymes, 40 nM RNP and 0.8 μM reporter were added into the reaction. ( c ) In the two-step method, ligation and RCA were combined together, followed by fluorogenic detection using Cas12a RNP, conferring similar sensitivity with the three-step assay in ( b ). ( d ) The one-pot EXTRA-CRISPR method improves the sensitivity to detect 10 fM miR-21 prior to full optimization. Assay concentrations used in ( c ) and ( d ): 100 nM Padlock-1, 0.625 U/μL SplintR ligase, 0.1 U/μL phi29 polymerase, 1 μM reporter, and 1 nM Cas12a RNP.

Techniques Used: CRISPR, Ligation

Optimization of the one-pot EXTRA-CRISPR miR-21 assay. (a) The position of the CRISPR module in a padlock sequence affects the detection signal and background level. The assays were conducted with 1 pM miR-21 and 100 nM padlock. (b) Comparison of T4 ligase and SplintR ligase for the one-pot assay. The assays were conducted with 40 U/μL T4 ligase or 2.5 U/μL SplintR ligase, 100 nM Padlock-1, 0.2 U/μL phi29 polymerase, 50 nM Cas12a RNP, and 1 μM reporter. (c-f) Optimization of the concentration of phi29 ( c ), padlock ( d ), Cas12a RNP ( e ), and reporter ( f ). ΔRFU, unless otherwise specified, was the signal increase from 0 min to 120 min. Error bars represent one standard deviation (S.D., n = 3). The selected optimal conditions were indicated by a color background. ( g ) Representative real-time curves for calibrating the one-pot assay with serial dilutions of synthetic miR-21 standards using the optimized protocol. The inset displays the curves of averaged signal for 0 (NC), 5, and 10 fM miR-21 with the shaded bands indicating one S.D. ( n = 5). ( h ) Titration curves plotted at various time points for the assay calibration in ( g ) show a strong dependence of the assay performance on the reaction time. (i) Diagram of the analytical figures of merit determined by the assay calibration, including LOD, sensitivity (the slope of linear calibration curve), and linear dynamic range defined by the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ). ( j ) Linear calibration obtained with the optimal assay time of 100 min yields a LOD of 1.64 fM miR-21 calculated from 3× S.D. of the background level and a linear range from 5.47 fM to 500 fM. Error bars indicate one S.D. ( n = 5). ( k ) Specificity of the EXTRA-CRISPR for detecting miR-21 against a single-mismatch miR-21 sequence and eight different miRNAs (1 pM each). The color intensity represents the averaged signal level of two replicates.

Figure Legend Snippet: Optimization of the one-pot EXTRA-CRISPR miR-21 assay. (a) The position of the CRISPR module in a padlock sequence affects the detection signal and background level. The assays were conducted with 1 pM miR-21 and 100 nM padlock. (b) Comparison of T4 ligase and SplintR ligase for the one-pot assay. The assays were conducted with 40 U/μL T4 ligase or 2.5 U/μL SplintR ligase, 100 nM Padlock-1, 0.2 U/μL phi29 polymerase, 50 nM Cas12a RNP, and 1 μM reporter. (c-f) Optimization of the concentration of phi29 ( c ), padlock ( d ), Cas12a RNP ( e ), and reporter ( f ). ΔRFU, unless otherwise specified, was the signal increase from 0 min to 120 min. Error bars represent one standard deviation (S.D., n = 3). The selected optimal conditions were indicated by a color background. ( g ) Representative real-time curves for calibrating the one-pot assay with serial dilutions of synthetic miR-21 standards using the optimized protocol. The inset displays the curves of averaged signal for 0 (NC), 5, and 10 fM miR-21 with the shaded bands indicating one S.D. ( n = 5). ( h ) Titration curves plotted at various time points for the assay calibration in ( g ) show a strong dependence of the assay performance on the reaction time. (i) Diagram of the analytical figures of merit determined by the assay calibration, including LOD, sensitivity (the slope of linear calibration curve), and linear dynamic range defined by the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ). ( j ) Linear calibration obtained with the optimal assay time of 100 min yields a LOD of 1.64 fM miR-21 calculated from 3× S.D. of the background level and a linear range from 5.47 fM to 500 fM. Error bars indicate one S.D. ( n = 5). ( k ) Specificity of the EXTRA-CRISPR for detecting miR-21 against a single-mismatch miR-21 sequence and eight different miRNAs (1 pM each). The color intensity represents the averaged signal level of two replicates.

Techniques Used: CRISPR, Sequencing, Concentration Assay, Standard Deviation, Titration

10) Product Images from "Chemiluminescent screening of specific hybridoma cells via a proximity-rolling circle activated enzymatic switch"

Article Title: Chemiluminescent screening of specific hybridoma cells via a proximity-rolling circle activated enzymatic switch

Journal: Communications Biology

doi: 10.1038/s42003-022-03283-2

Design of the enzymatic switch and feasibility of proximity-rolling circle activated enzymatic switch. a Schematic diagram of the pyrophosphate anions-activated enzymatic switch. b Comparison of enzymatic switches constructed with different metal ions. c Chemiluminescent intensity ( I ) of 10 mM pH 7.2 Tris-HCl containing 0.2 mM luminol, 1 mM H 2 O 2 and (I) 0.2 μg mL −1 HRP, (II) 0.2 μg mL −1 HRP-Cu 2+ , (III) 0.2 μg mL −1 HRP and 0.12 mM Cu 2+ or (IV,V,VI) reaction mixture of 0.2 μg mL −1 HRP-Cu 2+ and 2 mM pyrophosphate anions (IV), 0.2 μg mL −1 HRP and 0.12 mM CuPPi 2 (V) and 0.2 μg mL −1 HRP-Cu 2+ and 2 mM dNTPs (VI) after 5-min incubation. d Chemiluminescent intensity of 10 mM pH 7.2 Tris-HCl (50 μL) containing 0.2 mM luminol, 1 mM H 2 O 2 and 0.2 μg mL −1 HRP-Cu 2+ without (I) or with (II,III) reaction mixture (20 μL) of 1.0 μg mL −1 PCSK9-DNA probes, 0.1 μM block-primer, 0.3 μM padlock, 0.625 U μL −1 splint R ligase, 0.025 U μL −1 phi 29 polymerase, 1.0 mM BSA and 2.0 mM dNTPs in absence (II) and presence (III) of 0.5 μg mL −1 PCSK9-Ab. Error bars were estimated from three parallel experiments.

Figure Legend Snippet: Design of the enzymatic switch and feasibility of proximity-rolling circle activated enzymatic switch. a Schematic diagram of the pyrophosphate anions-activated enzymatic switch. b Comparison of enzymatic switches constructed with different metal ions. c Chemiluminescent intensity ( I ) of 10 mM pH 7.2 Tris-HCl containing 0.2 mM luminol, 1 mM H 2 O 2 and (I) 0.2 μg mL −1 HRP, (II) 0.2 μg mL −1 HRP-Cu 2+ , (III) 0.2 μg mL −1 HRP and 0.12 mM Cu 2+ or (IV,V,VI) reaction mixture of 0.2 μg mL −1 HRP-Cu 2+ and 2 mM pyrophosphate anions (IV), 0.2 μg mL −1 HRP and 0.12 mM CuPPi 2 (V) and 0.2 μg mL −1 HRP-Cu 2+ and 2 mM dNTPs (VI) after 5-min incubation. d Chemiluminescent intensity of 10 mM pH 7.2 Tris-HCl (50 μL) containing 0.2 mM luminol, 1 mM H 2 O 2 and 0.2 μg mL −1 HRP-Cu 2+ without (I) or with (II,III) reaction mixture (20 μL) of 1.0 μg mL −1 PCSK9-DNA probes, 0.1 μM block-primer, 0.3 μM padlock, 0.625 U μL −1 splint R ligase, 0.025 U μL −1 phi 29 polymerase, 1.0 mM BSA and 2.0 mM dNTPs in absence (II) and presence (III) of 0.5 μg mL −1 PCSK9-Ab. Error bars were estimated from three parallel experiments.

Techniques Used: Construct, Incubation, Blocking Assay

Chemiluminescent responses to pyrophosphate anions, primer and PCSK9-Ab. a Schematic diagram of activation of enzymatic switch by pyrophosphate anions. b Chemiluminescent intensity ( I ) and c relative chemiluminescent signal ( ΔI ) as function of logarithm of pyrophosphate anions concentration in 10 mM pH 7.2 Tris-HCl containing 200 ng mL −1 HRP-Cu 2+ , 0.2 mM luminol and 1 mM H 2 O 2 . d Schematic diagram of activation of enzymatic switch by a primer-induced rolling circle amplification. e Chemiluminescent intensity and f relative chemiluminescent signal as function of logarithm of primer concentration in 10 mM pH 7.2 Tris-HCl buffer (50 μL) containing 0.2 μg mL −1 HRP-Cu 2+ , 0.2 mM luminol, 1 mM H 2 O 2 and the reaction mixtures (20 μL) of 0.3 μM padlock, 0.625 U μL −1 splint R ligase, 0.025 U μL −1 phi 29 polymerase, 1.0 mM BSA, 2.0 mM dNTPs with different concentrations of primer for 1-h incubation at 37 o C. g Schematic diagram of chemiluminescent assay of PCSK9-Ab via proximity-rolling circle activated enzymatic switch. h Chemiluminescent intensity and i relative chemiluminescent signal as function of logarithm of PCSK9-Ab concentration in 10 mM pH 7.2 Tris-HCl (50 μL) containing 0.2 μg mL −1 HRP-Cu 2+ , 0.2 mM luminol, 1 mM H 2 O 2 and the reaction mixtures (20 μL) of 1.0 μg mL −1 PCSK9-DNA probes, 0.1 μM block-primer, 0.3 μM padlock, 0.625 U μL −1 splint R ligase, 0.025 U μL −1 phi 29 polymerase, 1.0 mM BSA and 2.0 mM dNTPs with different concentrations of PCSK9-Ab for 1-h incubation at 37 o C. Error bars were estimated from three parallel experiments.

Figure Legend Snippet: Chemiluminescent responses to pyrophosphate anions, primer and PCSK9-Ab. a Schematic diagram of activation of enzymatic switch by pyrophosphate anions. b Chemiluminescent intensity ( I ) and c relative chemiluminescent signal ( ΔI ) as function of logarithm of pyrophosphate anions concentration in 10 mM pH 7.2 Tris-HCl containing 200 ng mL −1 HRP-Cu 2+ , 0.2 mM luminol and 1 mM H 2 O 2 . d Schematic diagram of activation of enzymatic switch by a primer-induced rolling circle amplification. e Chemiluminescent intensity and f relative chemiluminescent signal as function of logarithm of primer concentration in 10 mM pH 7.2 Tris-HCl buffer (50 μL) containing 0.2 μg mL −1 HRP-Cu 2+ , 0.2 mM luminol, 1 mM H 2 O 2 and the reaction mixtures (20 μL) of 0.3 μM padlock, 0.625 U μL −1 splint R ligase, 0.025 U μL −1 phi 29 polymerase, 1.0 mM BSA, 2.0 mM dNTPs with different concentrations of primer for 1-h incubation at 37 o C. g Schematic diagram of chemiluminescent assay of PCSK9-Ab via proximity-rolling circle activated enzymatic switch. h Chemiluminescent intensity and i relative chemiluminescent signal as function of logarithm of PCSK9-Ab concentration in 10 mM pH 7.2 Tris-HCl (50 μL) containing 0.2 μg mL −1 HRP-Cu 2+ , 0.2 mM luminol, 1 mM H 2 O 2 and the reaction mixtures (20 μL) of 1.0 μg mL −1 PCSK9-DNA probes, 0.1 μM block-primer, 0.3 μM padlock, 0.625 U μL −1 splint R ligase, 0.025 U μL −1 phi 29 polymerase, 1.0 mM BSA and 2.0 mM dNTPs with different concentrations of PCSK9-Ab for 1-h incubation at 37 o C. Error bars were estimated from three parallel experiments.

Techniques Used: Activation Assay, Concentration Assay, Amplification, Incubation, Blocking Assay

11) Product Images from "HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale"

Article Title: HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale

Journal: Frontiers in Bioengineering and Biotechnology

doi: 10.3389/fbioe.2022.832176

HyperXpress workflow and effect on product formation of the most relevant optimizations. (A) The HyperXpress workflow starts with the assembly of DNA parts using LCR. This assembly includes a circularization of the DNA construct which upon addition of the components required for RCA results in an amplification of circular constructs. In order to provide optimal reaction conditions for CFPS a buffer exchange is achieved through DNA precipitation and resuspension. Upon addition of the components required for CFPS each DNA assembly is assayed for product formation. (B) Results of the sequential optimizations of the workflow compared to the corresponding 0 nM bridging oligo reaction as a negative control (Neg.). EP29: EquiPhi29™ DNA Polymerase; N2(sN)2N2: endonuclease protected random hexamer oligonucleotides; PEG prec.: in-well DNA precipitation using polyethylenglycol; res.: resuspension of the DNA precipitate in ultrapure water; p09005 coexp.: bicistronic operon plasmid with sfGFP and mKate2 genes used instead of p10024 template with monocistronic GFP reporter construct ( n = 6).

Figure Legend Snippet: HyperXpress workflow and effect on product formation of the most relevant optimizations. (A) The HyperXpress workflow starts with the assembly of DNA parts using LCR. This assembly includes a circularization of the DNA construct which upon addition of the components required for RCA results in an amplification of circular constructs. In order to provide optimal reaction conditions for CFPS a buffer exchange is achieved through DNA precipitation and resuspension. Upon addition of the components required for CFPS each DNA assembly is assayed for product formation. (B) Results of the sequential optimizations of the workflow compared to the corresponding 0 nM bridging oligo reaction as a negative control (Neg.). EP29: EquiPhi29™ DNA Polymerase; N2(sN)2N2: endonuclease protected random hexamer oligonucleotides; PEG prec.: in-well DNA precipitation using polyethylenglycol; res.: resuspension of the DNA precipitate in ultrapure water; p09005 coexp.: bicistronic operon plasmid with sfGFP and mKate2 genes used instead of p10024 template with monocistronic GFP reporter construct ( n = 6).

Techniques Used: Construct, Amplification, Buffer Exchange, Negative Control, Random Hexamer Labeling, Plasmid Preparation

phi29 dna polymerase mutants (New England Biolabs)

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11) Product Images from "HyperXpress: Rapid Single Vessel DNA Assembly and Protein Production in Microliterscale"

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