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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.

Pcr Reactions, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 1596 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

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

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

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

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

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:

Real-time Polymerase Chain Reaction:

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:

Polymerase Chain Reaction:

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: Coordinated transcriptional regulation of bone homeostasis by Ebf1 and Zfp521 in both mesenchymal and hematopoietic lineages
Article Snippet: .. All primers were from Integrated DNA Technologies, and all PCR reactions were performed using Expand High Fidelity (Roche): 5′ retrieval F, 5′-AATAAAGGATCCGTGCTCCAGGCACTATAGAT-3′; 5′ retrieval R, 5′-AATAAAAAGCTTGACCTGGCCAGGTCATTTAA-3′; 3′ retrieval F, 5′-AATAAAAAGCTTGCCTTTCCAAGTTCCTGACA-3′; and 3′ retrieval R, 5′-AATAAAGCGGCCGCGTTCAAGGCCACTGTGGTTT-3′. .. Cloning sites were BamHI, HindIII, and NotI.

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.