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    Structured Review

    Roche pcr reactions
    Characterization of a novel 3'UTR extended CaMK4 mRNA transcript expressed in rat cutaneous nociceptors A: Summary table of alternate polyadenylation signal characterisation experiments: The POLYAR algorithm was used to predict the position of polyadenylation signals on the final exon of CaMK4 +12kb downstream. Integral (W) scores, cleavage site (CS) and polyadenylation specificity signal (PAS) positions are relative to the start codon. Rapid amplification of <t>cDNA</t> ends (RACE) was performed using 7 distinct primers designed 200–900bp upstream of the predicated cleavage site. B: <t>PCR</t> products were separated by electrophoresis and any observable bands excised for sequencing. Bands with sequences that mapped to CaMK4 were validated polyadenylation sites (arrows). C: To determine if the extended UTR region was contiguous with the coding sequence (CDS), we designed a gene specific primer (GSP) to prime the cDNA synthesis reaction. PCR probing the kinase domain of CaMK4 demonstrated that the extended UTR was part of the same transcript as the CDS. cDNA synthesised without primer (-P) was employed as a negative control. D: UCSC genome browser track centered on the validated polyadenylation site discovered using primer RACE_7. A cluster of expressed sequence tags (ESTs - black lines) terminate at this highly conserved CS which is ≈ 10kb downstream of the CaMKIV stop codon. The CS is flanked by conserved putative PAS and U-/GU-rich down-stream element (DSE) sequence motifs.
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    Images

    1) Product Images from "IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons"

    Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons

    Journal: The Journal of comparative neurology

    doi: 10.1002/cne.23398

    Characterization of a novel 3'UTR extended CaMK4 mRNA transcript expressed in rat cutaneous nociceptors A: Summary table of alternate polyadenylation signal characterisation experiments: The POLYAR algorithm was used to predict the position of polyadenylation signals on the final exon of CaMK4 +12kb downstream. Integral (W) scores, cleavage site (CS) and polyadenylation specificity signal (PAS) positions are relative to the start codon. Rapid amplification of cDNA ends (RACE) was performed using 7 distinct primers designed 200–900bp upstream of the predicated cleavage site. B: PCR products were separated by electrophoresis and any observable bands excised for sequencing. Bands with sequences that mapped to CaMK4 were validated polyadenylation sites (arrows). C: To determine if the extended UTR region was contiguous with the coding sequence (CDS), we designed a gene specific primer (GSP) to prime the cDNA synthesis reaction. PCR probing the kinase domain of CaMK4 demonstrated that the extended UTR was part of the same transcript as the CDS. cDNA synthesised without primer (-P) was employed as a negative control. D: UCSC genome browser track centered on the validated polyadenylation site discovered using primer RACE_7. A cluster of expressed sequence tags (ESTs - black lines) terminate at this highly conserved CS which is ≈ 10kb downstream of the CaMKIV stop codon. The CS is flanked by conserved putative PAS and U-/GU-rich down-stream element (DSE) sequence motifs.
    Figure Legend Snippet: Characterization of a novel 3'UTR extended CaMK4 mRNA transcript expressed in rat cutaneous nociceptors A: Summary table of alternate polyadenylation signal characterisation experiments: The POLYAR algorithm was used to predict the position of polyadenylation signals on the final exon of CaMK4 +12kb downstream. Integral (W) scores, cleavage site (CS) and polyadenylation specificity signal (PAS) positions are relative to the start codon. Rapid amplification of cDNA ends (RACE) was performed using 7 distinct primers designed 200–900bp upstream of the predicated cleavage site. B: PCR products were separated by electrophoresis and any observable bands excised for sequencing. Bands with sequences that mapped to CaMK4 were validated polyadenylation sites (arrows). C: To determine if the extended UTR region was contiguous with the coding sequence (CDS), we designed a gene specific primer (GSP) to prime the cDNA synthesis reaction. PCR probing the kinase domain of CaMK4 demonstrated that the extended UTR was part of the same transcript as the CDS. cDNA synthesised without primer (-P) was employed as a negative control. D: UCSC genome browser track centered on the validated polyadenylation site discovered using primer RACE_7. A cluster of expressed sequence tags (ESTs - black lines) terminate at this highly conserved CS which is ≈ 10kb downstream of the CaMKIV stop codon. The CS is flanked by conserved putative PAS and U-/GU-rich down-stream element (DSE) sequence motifs.

    Techniques Used: Rapid Amplification of cDNA Ends, Polymerase Chain Reaction, Electrophoresis, Sequencing, Negative Control

    2) Product Images from "Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages"

    Article Title: Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20121187

    Zfp521 favors bone formation in mature OBs. (A) Generation of null and conditional Zfp521 alleles. Zfp521 genomic region encoding exon 4 is shown. Restriction fragment sizes are indicated as well as the positions of the internal and the two flanking probes used for genotyping analysis (Roman numerals). The shaded area indicates the part of the genomic region included in the targeting vector, and the three different alleles are shown. “neo” is the result of the gene-targeting event. “cko” is the conditional knockout allele derived from the neo allele after Flpe-mediated excision of the PGK-neo cassette. One Frt site and two loxP sites remain in the locus. “ko” is the null allele derived from the “neo” or from the “cko” allele by Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. Splicing of exon 3 to exon 5 generates a frameshift. loxP and Frt sites are indicated as closed and open triangles, respectively. Neo, PGK-em7-neomycin dual selection cassette for bacteria and embryonic stem cells; TK, thymidine kinase cassette for counter-selection in embryonic stem cells; X, XbaI; B, BamHI; N, NotI. The genomic region is not drawn to scale. (B) Results of a Southern blot analysis of BamHI-digested tail DNA, probed with the internal probe (III). (C) Northern blot analysis of whole-brain RNA from 3-wk-old mice using a full-length Zfp521 cDNA probe. The blot was rehybridized with a Gapdh probe as a control for RNA quality. wt, wild type; Δexon4, position of the residual mRNA after removal of exon 4. (D) Genotyping PCR showing deletion of Zfp521 allele in genomic DNA extracted from Zfp521 hOC −/− long bones cleaned of soft tissues and BM. (E) Von Kossa staining of tibia sections in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls. (F) Histomorphometric analysis of samples in E ( n = 5). (G) Trabecular BV (BV/tissue volume [TV]) at distal femoral metaphysis and in second lumbar vertebra in 3-wk-old Zfp521 −/− and control mice measured by μCT ( n = 5). (H) Von Kossa staining of tibia sections in 6-wk-old Zfp521 hOC −/− mice and littermate controls. (I) Histomorphometric analysis of samples in H ( n = 6). (J) Trabecular BV (BV/TV) at distal femoral metaphysis and in second lumbar vertebra in 12-wk-old Zfp521 hOC −/− and control mice measured by μCT ( n = 5–6). (K) Von Kossa staining of tibia sections in 12-wk-old Zfp521 hOC −/− mice and littermate controls. (L) Histomorphometric analysis of samples in K ( n = 6). (M) Serum PINP and CTX levels in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls ( n = 6–9). (N) Serum PINP and CTX levels in 6-wk-old Zfp521 hOC −/− and control mice ( n = 5–6). N.Ob, number of OBs; N.Oc, number of OCs. All data are means ± SD. *, P
    Figure Legend Snippet: Zfp521 favors bone formation in mature OBs. (A) Generation of null and conditional Zfp521 alleles. Zfp521 genomic region encoding exon 4 is shown. Restriction fragment sizes are indicated as well as the positions of the internal and the two flanking probes used for genotyping analysis (Roman numerals). The shaded area indicates the part of the genomic region included in the targeting vector, and the three different alleles are shown. “neo” is the result of the gene-targeting event. “cko” is the conditional knockout allele derived from the neo allele after Flpe-mediated excision of the PGK-neo cassette. One Frt site and two loxP sites remain in the locus. “ko” is the null allele derived from the “neo” or from the “cko” allele by Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. Splicing of exon 3 to exon 5 generates a frameshift. loxP and Frt sites are indicated as closed and open triangles, respectively. Neo, PGK-em7-neomycin dual selection cassette for bacteria and embryonic stem cells; TK, thymidine kinase cassette for counter-selection in embryonic stem cells; X, XbaI; B, BamHI; N, NotI. The genomic region is not drawn to scale. (B) Results of a Southern blot analysis of BamHI-digested tail DNA, probed with the internal probe (III). (C) Northern blot analysis of whole-brain RNA from 3-wk-old mice using a full-length Zfp521 cDNA probe. The blot was rehybridized with a Gapdh probe as a control for RNA quality. wt, wild type; Δexon4, position of the residual mRNA after removal of exon 4. (D) Genotyping PCR showing deletion of Zfp521 allele in genomic DNA extracted from Zfp521 hOC −/− long bones cleaned of soft tissues and BM. (E) Von Kossa staining of tibia sections in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls. (F) Histomorphometric analysis of samples in E ( n = 5). (G) Trabecular BV (BV/tissue volume [TV]) at distal femoral metaphysis and in second lumbar vertebra in 3-wk-old Zfp521 −/− and control mice measured by μCT ( n = 5). (H) Von Kossa staining of tibia sections in 6-wk-old Zfp521 hOC −/− mice and littermate controls. (I) Histomorphometric analysis of samples in H ( n = 6). (J) Trabecular BV (BV/TV) at distal femoral metaphysis and in second lumbar vertebra in 12-wk-old Zfp521 hOC −/− and control mice measured by μCT ( n = 5–6). (K) Von Kossa staining of tibia sections in 12-wk-old Zfp521 hOC −/− mice and littermate controls. (L) Histomorphometric analysis of samples in K ( n = 6). (M) Serum PINP and CTX levels in 3-wk-old global Zfp521 −/− mice and Zfp521 +/+ littermate controls ( n = 6–9). (N) Serum PINP and CTX levels in 6-wk-old Zfp521 hOC −/− and control mice ( n = 5–6). N.Ob, number of OBs; N.Oc, number of OCs. All data are means ± SD. *, P

    Techniques Used: Plasmid Preparation, Knock-Out, Derivative Assay, Modification, Selection, Southern Blot, Northern Blot, Mouse Assay, Polymerase Chain Reaction, Staining

    3) Product Images from "Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation"

    Article Title: Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.141222198

    RT-PCR products of error-prone DNA polymerases at 48 h. cDNA from cells that were untreated or treated under various conditions (+ anti-IgM, + Hut 78, or + IgM + HuT 78) for 0, 12, 24, and 48 h were used in RT-PCR. Stock dilutions (1:10, 1:20, and 1:40) of the cDNA were made and used in the amplifications. For each time point and condition, undiluted cDNA and the aforementioned dilutions were used to detect pols ζ, η, and ι, and GAPDH. No PCR products were detected in the no-RT control cDNAs for each condition (data not shown).
    Figure Legend Snippet: RT-PCR products of error-prone DNA polymerases at 48 h. cDNA from cells that were untreated or treated under various conditions (+ anti-IgM, + Hut 78, or + IgM + HuT 78) for 0, 12, 24, and 48 h were used in RT-PCR. Stock dilutions (1:10, 1:20, and 1:40) of the cDNA were made and used in the amplifications. For each time point and condition, undiluted cDNA and the aforementioned dilutions were used to detect pols ζ, η, and ι, and GAPDH. No PCR products were detected in the no-RT control cDNAs for each condition (data not shown).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Semiquantitation of RT-PCR products of error-prone DNA polymerases in BL2. RT-PCR was performed for pols ζ, η, and ι at 0, 12, 24, and 48 h after various conditions. For each time point and condition, RT-PCR was performed for the housekeeping gene, GAPDH . The OD of each band for the polymerases was measured and normalized against the OD of GAPDH at the respective time point and condition. The PCR products from the 1:10 and 1:20 dilutions of the template cDNA were normalized individually to GAPDH. The normalized values of the PCR products of the 1:20 dilution were doubled and then averaged with the 1:10 PCR products. The average is shown in the bar graph, and the error between the two determinations is depicted by the error bars.
    Figure Legend Snippet: Semiquantitation of RT-PCR products of error-prone DNA polymerases in BL2. RT-PCR was performed for pols ζ, η, and ι at 0, 12, 24, and 48 h after various conditions. For each time point and condition, RT-PCR was performed for the housekeeping gene, GAPDH . The OD of each band for the polymerases was measured and normalized against the OD of GAPDH at the respective time point and condition. The PCR products from the 1:10 and 1:20 dilutions of the template cDNA were normalized individually to GAPDH. The normalized values of the PCR products of the 1:20 dilution were doubled and then averaged with the 1:10 PCR products. The average is shown in the bar graph, and the error between the two determinations is depicted by the error bars.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    4) Product Images from "Duplex Scorpion primers in SNP analysis and FRET applications"

    Article Title: Duplex Scorpion primers in SNP analysis and FRET applications

    Journal: Nucleic Acids Research

    doi:

    ( A ) PCR amplification with FRET duplex Scorpions W-006/W-007, W-009/W-010 and W-011/W-012 (proportional mode). ROX fluorescence was monitored. ( B ) Allelic discrimination with FRET duplex Scorpion W-006/W-007. ROX fluorescence was monitored in channel 2 of the LightCycler (arithmetic mode).
    Figure Legend Snippet: ( A ) PCR amplification with FRET duplex Scorpions W-006/W-007, W-009/W-010 and W-011/W-012 (proportional mode). ROX fluorescence was monitored. ( B ) Allelic discrimination with FRET duplex Scorpion W-006/W-007. ROX fluorescence was monitored in channel 2 of the LightCycler (arithmetic mode).

    Techniques Used: Polymerase Chain Reaction, Amplification, Fluorescence

    5) Product Images from "A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors"

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.117960

    Novel biosensors from DRIVER exhibit high affinities and selectivities. a. Schematic of overall selection workflow via DRIVER. A high-diversity DNA library of potential RNA biosensors and desired ligands are used as inputs to DRIVER. For each round of selection, the library is transcribed to RNA with or without a ligand mixture present. For rounds with ligands present (yellow) the cDNA corresponding to uncleaved product are amplified with a PCR primer specific to the prefix attached to the input to the current round. For selection rounds without ligand present (blue), the cleaved product is prepended with a new prefix using the regeneration method shown in Fig. 2 and is amplified using a PCR primer specific to this prefix. b. Comparison of cleavage fractions in the presence and absence of the ligand mixture determined via CleaveSeq for a library enriched by DRIVER. Top panel, the enriched library of the S3 selection at round 74 was constricted and CleaveSeq applied in the presence and absence of ligand mixture T1b (N=4,328; at least 30 reads/sequence in each condition). Bottom panel, the same data plotted with the ratio of the cleavage fractions in the presence and absence of the ligand mixture on the x-axis and the standard error of the ratio on the y-axis. Dotted diagonal line, delineates the region where a multiple-hypothesis test would reject the null hypothesis of non-switching, with alpha=1/N. In both panels: dashed line, boundary where the fold change of cleavage is at least 3x; red dots, indicate sequences with strong ( > 3.0), significant (as defined for the diagonal line) switching; green crosses, indicate validated aciclovir biosensors that were first identified from this analysis. c. Binding curves of ligands to select biosensors obtained via SPR analysis. Data points correspond to individual measurements across three experiments. Diamonds mark the equilibrium dissociation constants K D , which are numerically shown in the legend as mean of at least 3 experiments (except for Gard-547, as noted in Methods), and error bars indicate s.e.m. d. Comparison of SPR-derived equilibrium K D and CleaveSeq-derived EC 50 ligand concentrations for select biosensors. K D are shown as mean and error bars in s.e.m of at least 3 independent experiments. CleaveSeq-derived EC 50 and error bars are based on triplicate assays over the ligand and concentrations shown in panel e. e. Fold change of cleavage fractions between the +ligand and −ligand conditions for select biosensors and controls (Dataset S3) under 29 different ligand conditions measured via the CleaveSeq assay. Each column represents a single biosensor sequence (indicated by ID number), grouped by the cognate ligand. Each row represents the ligand concentration (μM) at which the CleaveSeq assay was performed. The color of each cell represents the fold change of cleavage between the −ligand and +ligand conditions. These data viewed as individual gradients for select biosensors to individual ligands are provided in Fig. S6. f. Enrichment of select biosensors. Relative abundance, as determined by NGS sequencing, of select biosensors in the selection libraries during selection rounds immediately prior to their discovery. The legend includes the average enrichment/round and extrapolated round 0 fractions based on an exponential fit to these data.
    Figure Legend Snippet: Novel biosensors from DRIVER exhibit high affinities and selectivities. a. Schematic of overall selection workflow via DRIVER. A high-diversity DNA library of potential RNA biosensors and desired ligands are used as inputs to DRIVER. For each round of selection, the library is transcribed to RNA with or without a ligand mixture present. For rounds with ligands present (yellow) the cDNA corresponding to uncleaved product are amplified with a PCR primer specific to the prefix attached to the input to the current round. For selection rounds without ligand present (blue), the cleaved product is prepended with a new prefix using the regeneration method shown in Fig. 2 and is amplified using a PCR primer specific to this prefix. b. Comparison of cleavage fractions in the presence and absence of the ligand mixture determined via CleaveSeq for a library enriched by DRIVER. Top panel, the enriched library of the S3 selection at round 74 was constricted and CleaveSeq applied in the presence and absence of ligand mixture T1b (N=4,328; at least 30 reads/sequence in each condition). Bottom panel, the same data plotted with the ratio of the cleavage fractions in the presence and absence of the ligand mixture on the x-axis and the standard error of the ratio on the y-axis. Dotted diagonal line, delineates the region where a multiple-hypothesis test would reject the null hypothesis of non-switching, with alpha=1/N. In both panels: dashed line, boundary where the fold change of cleavage is at least 3x; red dots, indicate sequences with strong ( > 3.0), significant (as defined for the diagonal line) switching; green crosses, indicate validated aciclovir biosensors that were first identified from this analysis. c. Binding curves of ligands to select biosensors obtained via SPR analysis. Data points correspond to individual measurements across three experiments. Diamonds mark the equilibrium dissociation constants K D , which are numerically shown in the legend as mean of at least 3 experiments (except for Gard-547, as noted in Methods), and error bars indicate s.e.m. d. Comparison of SPR-derived equilibrium K D and CleaveSeq-derived EC 50 ligand concentrations for select biosensors. K D are shown as mean and error bars in s.e.m of at least 3 independent experiments. CleaveSeq-derived EC 50 and error bars are based on triplicate assays over the ligand and concentrations shown in panel e. e. Fold change of cleavage fractions between the +ligand and −ligand conditions for select biosensors and controls (Dataset S3) under 29 different ligand conditions measured via the CleaveSeq assay. Each column represents a single biosensor sequence (indicated by ID number), grouped by the cognate ligand. Each row represents the ligand concentration (μM) at which the CleaveSeq assay was performed. The color of each cell represents the fold change of cleavage between the −ligand and +ligand conditions. These data viewed as individual gradients for select biosensors to individual ligands are provided in Fig. S6. f. Enrichment of select biosensors. Relative abundance, as determined by NGS sequencing, of select biosensors in the selection libraries during selection rounds immediately prior to their discovery. The legend includes the average enrichment/round and extrapolated round 0 fractions based on an exponential fit to these data.

    Techniques Used: Selection, Amplification, Polymerase Chain Reaction, Sequencing, Binding Assay, SPR Assay, Derivative Assay, Concentration Assay, Next-Generation Sequencing

    D e novo R apid I n V itro E volution of R NA biosensors (DRIVER) is a scalable platform that allows automated, parallelized selection of novel biosensors to diverse ligands. a(i) . Conventional selection starts with an RNA library with randomized regions in an unstructured context and chemical conjugation of a single ligand to a solid phase support such as columns or beads. a(ii) . Scalable DRIVER selection begins with an RNA biosensor library with randomized loop regions in a self-cleaving ribozyme framework and conjugation-free ligands as complex mixtures in solution. b(i) . Conventional SELEX (systematic evolution of ligands by exponential enrichment) involves manually iterating through 1. library incubation with a single immobilized ligand, 2. washing away unbound sequences, 3. eluting enriched sequences, 4. regenerating the DNA library template and transcribing the RNA library. b(ii) . DRIVER selection is fully automated with liquid handling, alternately enriching for sequences that co-transcriptionally self-cleave in the absence of ligands, and remain uncleaved in the presence of ligands. Liquid handling enables selection against complex mixtures in parallel, continuously generating hits across different rounds. c(i) . An enriched library from conventional SELEX can be transformed into bacteria to isolate sequences from single colonies, or deeply sequenced with next generation sequencing (NGS) to identify enriched sequences. c(ii) . DRIVER evolved libraries are screened using the fully automated CleaveSeq assay, which uses NGS to identify sequences that are cleaved and uncleaved in the absence and presence of ligand, respectively. d(i) . Hits from conventional SELEX require further orthogonal assays to verify ligand-specific binding versus PCR amplicons or non-specific binders to the column or bead. Once verified, extensive optimization is usually required to convert ligand-specific aptamers into biosensors, with no guarantee of success. d(ii) . DRIVER hits are directly functional in live cells, requiring no further optimization. Time on individual panels indicates an estimated duration for each set of experimental procedures, and ∞ indicates that the desired outcome is not achieved.
    Figure Legend Snippet: D e novo R apid I n V itro E volution of R NA biosensors (DRIVER) is a scalable platform that allows automated, parallelized selection of novel biosensors to diverse ligands. a(i) . Conventional selection starts with an RNA library with randomized regions in an unstructured context and chemical conjugation of a single ligand to a solid phase support such as columns or beads. a(ii) . Scalable DRIVER selection begins with an RNA biosensor library with randomized loop regions in a self-cleaving ribozyme framework and conjugation-free ligands as complex mixtures in solution. b(i) . Conventional SELEX (systematic evolution of ligands by exponential enrichment) involves manually iterating through 1. library incubation with a single immobilized ligand, 2. washing away unbound sequences, 3. eluting enriched sequences, 4. regenerating the DNA library template and transcribing the RNA library. b(ii) . DRIVER selection is fully automated with liquid handling, alternately enriching for sequences that co-transcriptionally self-cleave in the absence of ligands, and remain uncleaved in the presence of ligands. Liquid handling enables selection against complex mixtures in parallel, continuously generating hits across different rounds. c(i) . An enriched library from conventional SELEX can be transformed into bacteria to isolate sequences from single colonies, or deeply sequenced with next generation sequencing (NGS) to identify enriched sequences. c(ii) . DRIVER evolved libraries are screened using the fully automated CleaveSeq assay, which uses NGS to identify sequences that are cleaved and uncleaved in the absence and presence of ligand, respectively. d(i) . Hits from conventional SELEX require further orthogonal assays to verify ligand-specific binding versus PCR amplicons or non-specific binders to the column or bead. Once verified, extensive optimization is usually required to convert ligand-specific aptamers into biosensors, with no guarantee of success. d(ii) . DRIVER hits are directly functional in live cells, requiring no further optimization. Time on individual panels indicates an estimated duration for each set of experimental procedures, and ∞ indicates that the desired outcome is not achieved.

    Techniques Used: Selection, Conjugation Assay, Incubation, Transformation Assay, Next-Generation Sequencing, Binding Assay, Polymerase Chain Reaction, Functional Assay

    Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p
    Figure Legend Snippet: Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p

    Techniques Used: Selection, Next-Generation Sequencing, In Vivo, Activity Assay, Sequencing, Derivative Assay, Ligation, Polymerase Chain Reaction, Amplification

    6) Product Images from "A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors"

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors

    Journal: bioRxiv

    doi: 10.1101/2020.05.29.117960

    Novel biosensors from DRIVER exhibit high affinities and selectivities. a. Schematic of overall selection workflow via DRIVER. A high-diversity DNA library of potential RNA biosensors and desired ligands are used as inputs to DRIVER. For each round of selection, the library is transcribed to RNA with or without a ligand mixture present. For rounds with ligands present (yellow) the cDNA corresponding to uncleaved product are amplified with a PCR primer specific to the prefix attached to the input to the current round. For selection rounds without ligand present (blue), the cleaved product is prepended with a new prefix using the regeneration method shown in Fig. 2 and is amplified using a PCR primer specific to this prefix. b. Comparison of cleavage fractions in the presence and absence of the ligand mixture determined via CleaveSeq for a library enriched by DRIVER. Top panel, the enriched library of the S3 selection at round 74 was constricted and CleaveSeq applied in the presence and absence of ligand mixture T1b (N=4,328; at least 30 reads/sequence in each condition). Bottom panel, the same data plotted with the ratio of the cleavage fractions in the presence and absence of the ligand mixture on the x-axis and the standard error of the ratio on the y-axis. Dotted diagonal line, delineates the region where a multiple-hypothesis test would reject the null hypothesis of non-switching, with alpha=1/N. In both panels: dashed line, boundary where the fold change of cleavage is at least 3x; red dots, indicate sequences with strong ( > 3.0), significant (as defined for the diagonal line) switching; green crosses, indicate validated aciclovir biosensors that were first identified from this analysis. c. Binding curves of ligands to select biosensors obtained via SPR analysis. Data points correspond to individual measurements across three experiments. Diamonds mark the equilibrium dissociation constants K D , which are numerically shown in the legend as mean of at least 3 experiments (except for Gard-547, as noted in Methods), and error bars indicate s.e.m. d. Comparison of SPR-derived equilibrium K D and CleaveSeq-derived EC 50 ligand concentrations for select biosensors. K D are shown as mean and error bars in s.e.m of at least 3 independent experiments. CleaveSeq-derived EC 50 and error bars are based on triplicate assays over the ligand and concentrations shown in panel e. e. Fold change of cleavage fractions between the +ligand and −ligand conditions for select biosensors and controls (Dataset S3) under 29 different ligand conditions measured via the CleaveSeq assay. Each column represents a single biosensor sequence (indicated by ID number), grouped by the cognate ligand. Each row represents the ligand concentration (μM) at which the CleaveSeq assay was performed. The color of each cell represents the fold change of cleavage between the −ligand and +ligand conditions. These data viewed as individual gradients for select biosensors to individual ligands are provided in Fig. S6. f. Enrichment of select biosensors. Relative abundance, as determined by NGS sequencing, of select biosensors in the selection libraries during selection rounds immediately prior to their discovery. The legend includes the average enrichment/round and extrapolated round 0 fractions based on an exponential fit to these data.
    Figure Legend Snippet: Novel biosensors from DRIVER exhibit high affinities and selectivities. a. Schematic of overall selection workflow via DRIVER. A high-diversity DNA library of potential RNA biosensors and desired ligands are used as inputs to DRIVER. For each round of selection, the library is transcribed to RNA with or without a ligand mixture present. For rounds with ligands present (yellow) the cDNA corresponding to uncleaved product are amplified with a PCR primer specific to the prefix attached to the input to the current round. For selection rounds without ligand present (blue), the cleaved product is prepended with a new prefix using the regeneration method shown in Fig. 2 and is amplified using a PCR primer specific to this prefix. b. Comparison of cleavage fractions in the presence and absence of the ligand mixture determined via CleaveSeq for a library enriched by DRIVER. Top panel, the enriched library of the S3 selection at round 74 was constricted and CleaveSeq applied in the presence and absence of ligand mixture T1b (N=4,328; at least 30 reads/sequence in each condition). Bottom panel, the same data plotted with the ratio of the cleavage fractions in the presence and absence of the ligand mixture on the x-axis and the standard error of the ratio on the y-axis. Dotted diagonal line, delineates the region where a multiple-hypothesis test would reject the null hypothesis of non-switching, with alpha=1/N. In both panels: dashed line, boundary where the fold change of cleavage is at least 3x; red dots, indicate sequences with strong ( > 3.0), significant (as defined for the diagonal line) switching; green crosses, indicate validated aciclovir biosensors that were first identified from this analysis. c. Binding curves of ligands to select biosensors obtained via SPR analysis. Data points correspond to individual measurements across three experiments. Diamonds mark the equilibrium dissociation constants K D , which are numerically shown in the legend as mean of at least 3 experiments (except for Gard-547, as noted in Methods), and error bars indicate s.e.m. d. Comparison of SPR-derived equilibrium K D and CleaveSeq-derived EC 50 ligand concentrations for select biosensors. K D are shown as mean and error bars in s.e.m of at least 3 independent experiments. CleaveSeq-derived EC 50 and error bars are based on triplicate assays over the ligand and concentrations shown in panel e. e. Fold change of cleavage fractions between the +ligand and −ligand conditions for select biosensors and controls (Dataset S3) under 29 different ligand conditions measured via the CleaveSeq assay. Each column represents a single biosensor sequence (indicated by ID number), grouped by the cognate ligand. Each row represents the ligand concentration (μM) at which the CleaveSeq assay was performed. The color of each cell represents the fold change of cleavage between the −ligand and +ligand conditions. These data viewed as individual gradients for select biosensors to individual ligands are provided in Fig. S6. f. Enrichment of select biosensors. Relative abundance, as determined by NGS sequencing, of select biosensors in the selection libraries during selection rounds immediately prior to their discovery. The legend includes the average enrichment/round and extrapolated round 0 fractions based on an exponential fit to these data.

    Techniques Used: Selection, Amplification, Polymerase Chain Reaction, Sequencing, Binding Assay, SPR Assay, Derivative Assay, Concentration Assay, Next-Generation Sequencing

    D e novo R apid I n V itro E volution of R NA biosensors (DRIVER) is a scalable platform that allows automated, parallelized selection of novel biosensors to diverse ligands. a(i) . Conventional selection starts with an RNA library with randomized regions in an unstructured context and chemical conjugation of a single ligand to a solid phase support such as columns or beads. a(ii) . Scalable DRIVER selection begins with an RNA biosensor library with randomized loop regions in a self-cleaving ribozyme framework and conjugation-free ligands as complex mixtures in solution. b(i) . Conventional SELEX (systematic evolution of ligands by exponential enrichment) involves manually iterating through 1. library incubation with a single immobilized ligand, 2. washing away unbound sequences, 3. eluting enriched sequences, 4. regenerating the DNA library template and transcribing the RNA library. b(ii) . DRIVER selection is fully automated with liquid handling, alternately enriching for sequences that co-transcriptionally self-cleave in the absence of ligands, and remain uncleaved in the presence of ligands. Liquid handling enables selection against complex mixtures in parallel, continuously generating hits across different rounds. c(i) . An enriched library from conventional SELEX can be transformed into bacteria to isolate sequences from single colonies, or deeply sequenced with next generation sequencing (NGS) to identify enriched sequences. c(ii) . DRIVER evolved libraries are screened using the fully automated CleaveSeq assay, which uses NGS to identify sequences that are cleaved and uncleaved in the absence and presence of ligand, respectively. d(i) . Hits from conventional SELEX require further orthogonal assays to verify ligand-specific binding versus PCR amplicons or non-specific binders to the column or bead. Once verified, extensive optimization is usually required to convert ligand-specific aptamers into biosensors, with no guarantee of success. d(ii) . DRIVER hits are directly functional in live cells, requiring no further optimization. Time on individual panels indicates an estimated duration for each set of experimental procedures, and ∞ indicates that the desired outcome is not achieved.
    Figure Legend Snippet: D e novo R apid I n V itro E volution of R NA biosensors (DRIVER) is a scalable platform that allows automated, parallelized selection of novel biosensors to diverse ligands. a(i) . Conventional selection starts with an RNA library with randomized regions in an unstructured context and chemical conjugation of a single ligand to a solid phase support such as columns or beads. a(ii) . Scalable DRIVER selection begins with an RNA biosensor library with randomized loop regions in a self-cleaving ribozyme framework and conjugation-free ligands as complex mixtures in solution. b(i) . Conventional SELEX (systematic evolution of ligands by exponential enrichment) involves manually iterating through 1. library incubation with a single immobilized ligand, 2. washing away unbound sequences, 3. eluting enriched sequences, 4. regenerating the DNA library template and transcribing the RNA library. b(ii) . DRIVER selection is fully automated with liquid handling, alternately enriching for sequences that co-transcriptionally self-cleave in the absence of ligands, and remain uncleaved in the presence of ligands. Liquid handling enables selection against complex mixtures in parallel, continuously generating hits across different rounds. c(i) . An enriched library from conventional SELEX can be transformed into bacteria to isolate sequences from single colonies, or deeply sequenced with next generation sequencing (NGS) to identify enriched sequences. c(ii) . DRIVER evolved libraries are screened using the fully automated CleaveSeq assay, which uses NGS to identify sequences that are cleaved and uncleaved in the absence and presence of ligand, respectively. d(i) . Hits from conventional SELEX require further orthogonal assays to verify ligand-specific binding versus PCR amplicons or non-specific binders to the column or bead. Once verified, extensive optimization is usually required to convert ligand-specific aptamers into biosensors, with no guarantee of success. d(ii) . DRIVER hits are directly functional in live cells, requiring no further optimization. Time on individual panels indicates an estimated duration for each set of experimental procedures, and ∞ indicates that the desired outcome is not achieved.

    Techniques Used: Selection, Conjugation Assay, Incubation, Transformation Assay, Next-Generation Sequencing, Binding Assay, Polymerase Chain Reaction, Functional Assay

    Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p
    Figure Legend Snippet: Regeneration of ribozymes after cleavage enables selection and an NGS-based assay that are correlated with in vivo activity. a. Both CleaveSeq and DRIVER use the same core transcription (in the presence or absence of ligand(s)) and regeneration method of the 5’ cleaved product after cleavage. b . The regeneration method selectively restores the 5’ cleaved portion of the ribozyme and replaces the prefix sequence in the input library with a new prefix (e.g., “W” prefix is replaced with “Z” prefix for cleaved RNA molecules). The process starts with co-transcriptional cleavage of a DNA template library (i.e., simultaneous RNA transcription and cleavage of the product RNA in the presence or absence of ligand(s) to form a population of RNA molecules some of which have undergone self-cleavage thereby removing part of the ribozyme and the fixed prefix sequence from their 5’-end. The resulting pool of RNA is mixed with an oligonucleotide ( Z-Splint ; Dataset S2) that anneals to the 3’-end of the RNA for reverse transcription. The oligonucleotide subsequently hybridizes to the nascent cDNA, forming a partially self-annealing double-stranded hairpin structure that brings together the ends of molecules derived from the cleaved RNA, which is self-ligated with high efficiency. The resulting circularized ligation product is cut at two uracil locations included in the oligonucleotide by Uracil-Specific Excision Reagent (USER), to release a linear DNA strand harboring the desired sequence with a new prefix sequence. Two distinct populations of DNA molecules result: those corresponding to RNA that did not cleave and retain the template-determined prefix sequence and those corresponding to cleaved RNA that have the new prefix. The resulting DNA is selectively PCR-amplified with primers that extend the product with either the T7 promoter (for DRIVER) or NGS adapters (for CleaveSeq). c. Using the regeneration method, the CleaveSeq assay measures the relative abundance of molecules that underwent cleavage or not to provide estimates of cleavage fractions and switching (fold change of cleavage with the addition of ligand) for each sequence in an input library. d. Top panel, representative comparison of computed cleavage fractions for two replicates independently carried through the CleaveSeq assay from transcription through NGS analysis (N=12,025, at least 100 reads/sequence in each analysis). Bottom panel, the same data plotted with the ratio of the two replicate cleavage fraction measurements on the x-axis and the standard error of the ratio on the y-axis. Significant (p

    Techniques Used: Selection, Next-Generation Sequencing, In Vivo, Activity Assay, Sequencing, Derivative Assay, Ligation, Polymerase Chain Reaction, Amplification

    Related Articles

    SYBR Green Assay:

    Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons
    Article Snippet: .. PCR reactions were performed with 5ng input cDNA in triplicate using 2X SYBR green master mix (Roche). qPCR was performed with SYBR green detection using a Corbett Rotorgene instrument with the following cycle parameters:1) hotstart for 10 mins at 95C, 2) cycle: 95C for 30 seconds, anneal/extend for 60s at 60C. .. SYBR green fluorescence was determined at the end of each extension.

    Article Title: Seasonal expressions of prolactin, prolactin receptor and STAT5 in the scented glands of the male muskrats (Ondatrazibethicus)
    Article Snippet: .. The PCR reactions were carried out in a 10 μL volume using FastStart DNA MasterPlast SYBR green Kit (Roche Molecular System Inc., Basel, Switzerland). .. The PCR conditions were performed in ABI PRISM 7500 Fast Real-Time 7500 System (Applied Biosystems, Foster City, CA, USA) as described below: 10 min at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C.

    Amplification:

    Article Title: Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation
    Article Snippet: .. After quantitation and DNaseI treatment (Roche, Indianapolis), cDNA was synthesized with Superscript preamplification system for first-strand cDNA synthesis with oligo(dT) primers according to the manufacturer's recommendations. cDNA created from 3.5 μg RNA was amplified in PCR reactions with Taq polymerase (Roche). ..

    Synthesized:

    Article Title: Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation
    Article Snippet: .. After quantitation and DNaseI treatment (Roche, Indianapolis), cDNA was synthesized with Superscript preamplification system for first-strand cDNA synthesis with oligo(dT) primers according to the manufacturer's recommendations. cDNA created from 3.5 μg RNA was amplified in PCR reactions with Taq polymerase (Roche). ..

    Real-time Polymerase Chain Reaction:

    Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons
    Article Snippet: .. PCR reactions were performed with 5ng input cDNA in triplicate using 2X SYBR green master mix (Roche). qPCR was performed with SYBR green detection using a Corbett Rotorgene instrument with the following cycle parameters:1) hotstart for 10 mins at 95C, 2) cycle: 95C for 30 seconds, anneal/extend for 60s at 60C. .. SYBR green fluorescence was determined at the end of each extension.

    Concentration Assay:

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors
    Article Snippet: .. Surface plasmon resonance (SPR) measurement of the binding affinities of RNA biosensorsTo prepare DNA templates for transcription (see Dataset S2 for oligonucleotide sequences), all PCR reactions in this section used the Kapa HiFi HotStart PCR Kit (Roche) and 400 nM each of two primers, performed for 10 cycles with 10 nM starting template concentration, at an annealing temperature of 55 °C, using the GC buffer and 1 M betaine monohydrate, unless otherwise specified. .. To prepare DNA templates of biosensors for SPR binding assays, the previously prepared double stranded templates as described in the CleaveSeq validation assays were amplified using primers BT1285p and JX457, to append the T7 promoter and the poly(A) sequence for hybridizing transcribed RNA molecules to the poly(T) sequence on the sensor chip.

    Quantitation Assay:

    Article Title: Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation
    Article Snippet: .. After quantitation and DNaseI treatment (Roche, Indianapolis), cDNA was synthesized with Superscript preamplification system for first-strand cDNA synthesis with oligo(dT) primers according to the manufacturer's recommendations. cDNA created from 3.5 μg RNA was amplified in PCR reactions with Taq polymerase (Roche). ..

    Polymerase Chain Reaction:

    Article Title: IB4-binding sensory neurons in the adult rat express a novel 3′ UTR-extended isoform of CaMK4 that is associated with its localization to axons
    Article Snippet: .. PCR reactions were performed with 5ng input cDNA in triplicate using 2X SYBR green master mix (Roche). qPCR was performed with SYBR green detection using a Corbett Rotorgene instrument with the following cycle parameters:1) hotstart for 10 mins at 95C, 2) cycle: 95C for 30 seconds, anneal/extend for 60s at 60C. .. SYBR green fluorescence was determined at the end of each extension.

    Article Title: Duplex Scorpion primers in SNP analysis and FRET applications
    Article Snippet: .. All PCR reactions were carried out on a Roche LightCycler. .. Each reaction was made up from 1 µl (5 ng/µl) of DNA (wild-type, heterozygote, homozygote mutant or negative control), 1 µl of 10× buffer [Advanced Biotechnologies Buffer IV: 200 mM (NH4 )2 SO4 , 750 mM Tris–HCl, 0.1% Tween], 1 µl of a 5 µM solution of Scorpion Primer, 1 µl of a 25 µM solution of quencher oligonucleotide when required, 1 µl of 5 µM solution of lower unlabelled primer, 1 µl of a 2 mM solution of dNTPs (each of the four nucleotides), 250 ng/µl of BSA, 0.1 µl (0.5 U) of BioTaq polymerase (Bioline), 1.6 µl of a 25 mM solution of MgCl2 and water to a final volume of 10 µl.

    Article Title: Seasonal expressions of prolactin, prolactin receptor and STAT5 in the scented glands of the male muskrats (Ondatrazibethicus)
    Article Snippet: .. The PCR reactions were carried out in a 10 μL volume using FastStart DNA MasterPlast SYBR green Kit (Roche Molecular System Inc., Basel, Switzerland). .. The PCR conditions were performed in ABI PRISM 7500 Fast Real-Time 7500 System (Applied Biosystems, Foster City, CA, USA) as described below: 10 min at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C.

    Article Title: Expression of error-prone polymerases in BL2 cells activated for Ig somatic hypermutation
    Article Snippet: .. After quantitation and DNaseI treatment (Roche, Indianapolis), cDNA was synthesized with Superscript preamplification system for first-strand cDNA synthesis with oligo(dT) primers according to the manufacturer's recommendations. cDNA created from 3.5 μg RNA was amplified in PCR reactions with Taq polymerase (Roche). ..

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors
    Article Snippet: .. Surface plasmon resonance (SPR) measurement of the binding affinities of RNA biosensorsTo prepare DNA templates for transcription (see Dataset S2 for oligonucleotide sequences), all PCR reactions in this section used the Kapa HiFi HotStart PCR Kit (Roche) and 400 nM each of two primers, performed for 10 cycles with 10 nM starting template concentration, at an annealing temperature of 55 °C, using the GC buffer and 1 M betaine monohydrate, unless otherwise specified. .. To prepare DNA templates of biosensors for SPR binding assays, the previously prepared double stranded templates as described in the CleaveSeq validation assays were amplified using primers BT1285p and JX457, to append the T7 promoter and the poly(A) sequence for hybridizing transcribed RNA molecules to the poly(T) sequence on the sensor chip.

    Article Title: Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages
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    Nested PCR:

    Article Title: Dysregulated FAM215A Stimulates LAMP2 Expression to Confer Drug-Resistant and Malignant in Human Liver Cancer
    Article Snippet: .. Total RNA from Hep3B cells was used as the template for nested-PCR reactions using a 5’/3’-RACE Kit, 2nd Generation (Catalog No. 03353621001; Roche Applied Science, CA, USA, according to the manufacturer’s instructions. .. The products obtained from RACE PCR were cloned into a TA vector and sequenced.

    Binding Assay:

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors
    Article Snippet: .. Surface plasmon resonance (SPR) measurement of the binding affinities of RNA biosensorsTo prepare DNA templates for transcription (see Dataset S2 for oligonucleotide sequences), all PCR reactions in this section used the Kapa HiFi HotStart PCR Kit (Roche) and 400 nM each of two primers, performed for 10 cycles with 10 nM starting template concentration, at an annealing temperature of 55 °C, using the GC buffer and 1 M betaine monohydrate, unless otherwise specified. .. To prepare DNA templates of biosensors for SPR binding assays, the previously prepared double stranded templates as described in the CleaveSeq validation assays were amplified using primers BT1285p and JX457, to append the T7 promoter and the poly(A) sequence for hybridizing transcribed RNA molecules to the poly(T) sequence on the sensor chip.

    SPR Assay:

    Article Title: A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors
    Article Snippet: .. Surface plasmon resonance (SPR) measurement of the binding affinities of RNA biosensorsTo prepare DNA templates for transcription (see Dataset S2 for oligonucleotide sequences), all PCR reactions in this section used the Kapa HiFi HotStart PCR Kit (Roche) and 400 nM each of two primers, performed for 10 cycles with 10 nM starting template concentration, at an annealing temperature of 55 °C, using the GC buffer and 1 M betaine monohydrate, unless otherwise specified. .. To prepare DNA templates of biosensors for SPR binding assays, the previously prepared double stranded templates as described in the CleaveSeq validation assays were amplified using primers BT1285p and JX457, to append the T7 promoter and the poly(A) sequence for hybridizing transcribed RNA molecules to the poly(T) sequence on the sensor chip.

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    Quantitative Real Time Transcription Polymerase Chain Reaction Pepper, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reduced tolerance of CaDIK1- silenced pepper plants to drought stress. (A) Drought sensitive phenotype of CaDIK1 -silenced pepper plants. Three-week-old TRV2: CaDIK1 and TRV2:00 pepper plants were exposed to drought stress by withholding watering for 10 days and re-watering 2 days. Representative images were taken before (left) and after (middle) drought stress and re-watering (right). (B) Survival rates of plants after re-watering. Data represent the mean ± standard error of three biological replicates, each evaluating 20 plants. (C) Transpirational water loss from the leaves of empty vector control and CaDIK1 -silenced pepper plants at various times after detachment of leaves. Data represent the mean ± standard deviation of three independent biological replicates. (D , E) Decreased leaf temperatures of CaDIK1- silenced pepper plants in response to ABA treatment. Leaf temperatures were measured 6 h after treatment with 100 μM ABA using thermal imaging and representative images were taken. (D) ; the mean leaf temperature was measured using 12 plants of each line (E) . Data represent the mean ± standard deviation of three independent experiments. Data represent the mean ± SD of three independent biological replicates. (F , G) Stomatal apertures in empty vector control and CaDIK1 -silenced pepper plants after ABA treatment. Representative images were taken under a microscope (F) and the stomatal apertures were measured (G) . Leaf peels were harvested from 2-week-old pepper plants and incubated in stomatal opening solution containing 0, 10, and 20 μM ABA; the stomatal apertures were then measured under a microscope. Representative images were taken at 2.5 h after various concentrations of ABA treatment. Data represent the mean ± standard error of three independent experiments, each evaluating 20 plants. (H) Transcript expression of drought-inducible genes using quantitative reverse transcription polymerase chain reaction in control and CaDIK1 -silenced pepper plants exposed to drought stress at 0, 2, and 4 h after detachment. The relative expression levels (δδCT) of each gene were normalized to the geometric mean of CaACT1 as an internal control gene. Data represent the mean ± standard error of three independent biological replicates, each evaluating 20 plants. Asterisks indicate significant differences between the control and the CaDIK1 -silenced pepper plants (Student’s t -test; * P

    Journal: Frontiers in Plant Science

    Article Title: Pepper Novel Serine-Threonine Kinase CaDIK1 Regulates Drought Tolerance via Modulating ABA Sensitivity

    doi: 10.3389/fpls.2020.01133

    Figure Lengend Snippet: Reduced tolerance of CaDIK1- silenced pepper plants to drought stress. (A) Drought sensitive phenotype of CaDIK1 -silenced pepper plants. Three-week-old TRV2: CaDIK1 and TRV2:00 pepper plants were exposed to drought stress by withholding watering for 10 days and re-watering 2 days. Representative images were taken before (left) and after (middle) drought stress and re-watering (right). (B) Survival rates of plants after re-watering. Data represent the mean ± standard error of three biological replicates, each evaluating 20 plants. (C) Transpirational water loss from the leaves of empty vector control and CaDIK1 -silenced pepper plants at various times after detachment of leaves. Data represent the mean ± standard deviation of three independent biological replicates. (D , E) Decreased leaf temperatures of CaDIK1- silenced pepper plants in response to ABA treatment. Leaf temperatures were measured 6 h after treatment with 100 μM ABA using thermal imaging and representative images were taken. (D) ; the mean leaf temperature was measured using 12 plants of each line (E) . Data represent the mean ± standard deviation of three independent experiments. Data represent the mean ± SD of three independent biological replicates. (F , G) Stomatal apertures in empty vector control and CaDIK1 -silenced pepper plants after ABA treatment. Representative images were taken under a microscope (F) and the stomatal apertures were measured (G) . Leaf peels were harvested from 2-week-old pepper plants and incubated in stomatal opening solution containing 0, 10, and 20 μM ABA; the stomatal apertures were then measured under a microscope. Representative images were taken at 2.5 h after various concentrations of ABA treatment. Data represent the mean ± standard error of three independent experiments, each evaluating 20 plants. (H) Transcript expression of drought-inducible genes using quantitative reverse transcription polymerase chain reaction in control and CaDIK1 -silenced pepper plants exposed to drought stress at 0, 2, and 4 h after detachment. The relative expression levels (δδCT) of each gene were normalized to the geometric mean of CaACT1 as an internal control gene. Data represent the mean ± standard error of three independent biological replicates, each evaluating 20 plants. Asterisks indicate significant differences between the control and the CaDIK1 -silenced pepper plants (Student’s t -test; * P

    Article Snippet: Quantitative Real-Time Transcription-Polymerase Chain Reaction Pepper and Arabidopsis cDNAs were synthesized using a Transcript First Strand cDNA Synthesis kit (Roche).

    Techniques: Plasmid Preparation, Standard Deviation, Imaging, Microscopy, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction

    Enhanced drought tolerance of CaDIK1- OX plants. (A) Drought stress phenotype of wild-type (WT) and CaDIK1- OX transgenic plants. Three-week-old wild-type and transgenic plants were subjected to drought stress by withholding watering for 14 days and re-watering for 2 days. (B) Survival rates of plants after re-watering. Data represent the mean ± SE of three independent biological replicates, each evaluating 30 plants. (C) Transpirational water loss of detached rosette leaves in wild-type and transgenic plants leaves at various time points. Data represent the mean ± standard error of three independent biological replicates, each evaluating 50 leaves. (D , E) Enhanced leaf temperatures of CaDIK1 -OX plants in response to abscisic acid (ABA) treatment. Representative thermographic images of wild-type and CaDIK1- OX plants 6 h after treatment with 100 μm ABA (D); the mean leaf temperatures of the three largest leaves were measured using 10 plants of each line (E) . Data represent the mean ± standard deviation of three independent biological replicates, each evaluating 10 plants. (F , G) Stomatal apertures in wild-type and CaDIK1- OX plants treated with ABA. Leaf peels were harvested from three-week-old plants of each line and incubated in stomatal opening solution containing 0, 10, or 20 μM ABA. Representative images were taken under a microscope (F) and stomatal apertures were measured (G) . Data represent the mean ± standard error of three independent biological replicates. (H) Transcript expression of drought-inducible genes using quantitative reverse transcription polymerase chain reaction in CaDIK1 -OX plants exposed to drought stress at 3 h after detachment. The relative expression levels (δδCT) of each gene were normalized to the geometric mean of Actin8 as an internal control gene. Data represent the mean ± standard error of three independent biological replicates. Asterisks indicate significant differences between wild-type and transgenic lines (Student’s t -test; * P

    Journal: Frontiers in Plant Science

    Article Title: Pepper Novel Serine-Threonine Kinase CaDIK1 Regulates Drought Tolerance via Modulating ABA Sensitivity

    doi: 10.3389/fpls.2020.01133

    Figure Lengend Snippet: Enhanced drought tolerance of CaDIK1- OX plants. (A) Drought stress phenotype of wild-type (WT) and CaDIK1- OX transgenic plants. Three-week-old wild-type and transgenic plants were subjected to drought stress by withholding watering for 14 days and re-watering for 2 days. (B) Survival rates of plants after re-watering. Data represent the mean ± SE of three independent biological replicates, each evaluating 30 plants. (C) Transpirational water loss of detached rosette leaves in wild-type and transgenic plants leaves at various time points. Data represent the mean ± standard error of three independent biological replicates, each evaluating 50 leaves. (D , E) Enhanced leaf temperatures of CaDIK1 -OX plants in response to abscisic acid (ABA) treatment. Representative thermographic images of wild-type and CaDIK1- OX plants 6 h after treatment with 100 μm ABA (D); the mean leaf temperatures of the three largest leaves were measured using 10 plants of each line (E) . Data represent the mean ± standard deviation of three independent biological replicates, each evaluating 10 plants. (F , G) Stomatal apertures in wild-type and CaDIK1- OX plants treated with ABA. Leaf peels were harvested from three-week-old plants of each line and incubated in stomatal opening solution containing 0, 10, or 20 μM ABA. Representative images were taken under a microscope (F) and stomatal apertures were measured (G) . Data represent the mean ± standard error of three independent biological replicates. (H) Transcript expression of drought-inducible genes using quantitative reverse transcription polymerase chain reaction in CaDIK1 -OX plants exposed to drought stress at 3 h after detachment. The relative expression levels (δδCT) of each gene were normalized to the geometric mean of Actin8 as an internal control gene. Data represent the mean ± standard error of three independent biological replicates. Asterisks indicate significant differences between wild-type and transgenic lines (Student’s t -test; * P

    Article Snippet: Quantitative Real-Time Transcription-Polymerase Chain Reaction Pepper and Arabidopsis cDNAs were synthesized using a Transcript First Strand cDNA Synthesis kit (Roche).

    Techniques: Transgenic Assay, Standard Deviation, Incubation, Microscopy, Expressing, Reverse Transcription Polymerase Chain Reaction

    Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).

    Journal: PLoS ONE

    Article Title: Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection

    doi: 10.1371/journal.pone.0150711

    Figure Lengend Snippet: Quantitative RT-PCR analysis of candidate genes. Mock infected plants were used to normalize fold change of early and late samples in leaf and tuber tissues as indicated. White bars represent the RT-qPCR analysis; grey bars show the microarray data. Error bars represent standard error (SE). L23 and PP2A genes were used as internal normalization controls. Asterisk indicates that the means of fold change of the mock and infected samples are significantly different (Student’s t-test).

    Article Snippet: Three technical replicates of each three biological replicates (for infected tuber tissue only two biological replicates) were used in subsequent Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) reactions using FastStart Universal Probe Master with Rox (Roche Diagnostics, Switzerland).

    Techniques: Quantitative RT-PCR, Infection, Microarray

    Comparison of methylation status of PAX1 gene by thiol-labeled AuNPs methods and qMSP. Notes: ( A ) Receiver operating characteristic (ROC) curve analysis by qMSP and ( B and C ) thiol-labeled AuNPs methods. Methylation distribution at various disease stages by histopathology detected by qMSP method. The y-axis is Δ Cp , which represents the DNA methylation level of the PAX1 gene. The dashed line represents the cut-off with a Δ Cp of 10.57. ( D ) Methylated rate distribution at various disease stages detected by thiol-labeled AuNP method. The y-axis is percentage of methylation rate. The dashed line represents the cut-off with a methylated percentage of 31.27% (* P -value

    Journal: International Journal of Nanomedicine

    Article Title: Real-time colorimetric detection of DNA methylation of the PAX1 gene in cervical scrapings for cervical cancer screening with thiol-labeled PCR primers and gold nanoparticles

    doi: 10.2147/IJN.S116288

    Figure Lengend Snippet: Comparison of methylation status of PAX1 gene by thiol-labeled AuNPs methods and qMSP. Notes: ( A ) Receiver operating characteristic (ROC) curve analysis by qMSP and ( B and C ) thiol-labeled AuNPs methods. Methylation distribution at various disease stages by histopathology detected by qMSP method. The y-axis is Δ Cp , which represents the DNA methylation level of the PAX1 gene. The dashed line represents the cut-off with a Δ Cp of 10.57. ( D ) Methylated rate distribution at various disease stages detected by thiol-labeled AuNP method. The y-axis is percentage of methylation rate. The dashed line represents the cut-off with a methylated percentage of 31.27% (* P -value

    Article Snippet: Quantification of DNA methylation through qMSP Quantitative methylation-specific polymerase chain reaction (qMSP) by TaqMan-based technologies was performed in Lightcycler LC480 real-time polymerase chain reaction (PCR) system (Hoffman-La Roche Ltd., Basel, Switzerland) to detect DNA methylation.

    Techniques: Methylation, Labeling, Histopathology, DNA Methylation Assay

    Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Journal:

    Article Title: First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression

    doi: 10.1007/s12192-009-0139-4

    Figure Lengend Snippet: Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Article Snippet: Quantitative real-time PCR analysis HSP70 gene expression data were quantified using quantitative polymerase chain reaction (qPCR) performed on a LightCycler® sequence analysis system (Roche Diagnostics, Basel, Switzerland) using the QuantiTect SYBR® Green PCR kit (Qiagen). qPCR amplification was performed using synthetic DNA-specific primers corresponding to the transcript of SmRNA that allowed us to determine the reverse transcription efficiency for each sample. qPCR amplification of the gene of interest, HSP70, was then performed using specific primers designed from the alignment of the eight sequences of HSP70 from Niphargus obtained in this study, forward primer F307-PCRQ 5′-GCTGCGATTGCTTACGG-3′ and reverse primer R408-PCRQ 5′-CGCCAGCAGTAGATTTCACCTC-3′.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Journal:

    Article Title: First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression

    doi: 10.1007/s12192-009-0139-4

    Figure Lengend Snippet: Graphical representation of quantitative PCR results. Total RNA was extracted from control and heat-shocked crustaceans, and Nrhe_Chalamont_Hsp70 mRNA expression was analysed with specific probes. Control animals ( C ; fold induction = 1)

    Article Snippet: Quantitative real-time PCR analysis HSP70 gene expression data were quantified using quantitative polymerase chain reaction (qPCR) performed on a LightCycler® sequence analysis system (Roche Diagnostics, Basel, Switzerland) using the QuantiTect SYBR® Green PCR kit (Qiagen). qPCR amplification was performed using synthetic DNA-specific primers corresponding to the transcript of SmRNA that allowed us to determine the reverse transcription efficiency for each sample. qPCR amplification of the gene of interest, HSP70, was then performed using specific primers designed from the alignment of the eight sequences of HSP70 from Niphargus obtained in this study, forward primer F307-PCRQ 5′-GCTGCGATTGCTTACGG-3′ and reverse primer R408-PCRQ 5′-CGCCAGCAGTAGATTTCACCTC-3′.

    Techniques: Real-time Polymerase Chain Reaction, Expressing