fpg  (New England Biolabs)


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    Name:
    Fpg
    Description:
    Fpg 2 500 units
    Catalog Number:
    m0240l
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    301
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    2 500 units
    Category:
    Other Enzymes
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    New England Biolabs fpg
    Fpg
    Fpg 2 500 units
    https://www.bioz.com/result/fpg/product/New England Biolabs
    Average 99 stars, based on 446 article reviews
    Price from $9.99 to $1999.99
    fpg - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair"

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr463

    Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.
    Figure Legend Snippet: Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.

    Techniques Used: Incubation, Irradiation

    2) Product Images from "Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair"

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr463

    Generation of excess DSBs after in vitro incubation of DNA from irradiated cells at different temperatures. LIG4 − / − MEFs were embedded in agarose, exposed to 20 Gy X-rays and subjected to LTL as described under ‘Materials and Methods’ section. Subsequently, agarose blocks were incubated at different temperatures for different periods of time in TEN-buffer before analysis for DSBs by PFGE. ( A ) Shows typical ethidium bromide stained gels of irradiated and non-irradiated samples at the different treatment conditions. ( B ) Shows change in FDR as a function of time at the different incubation temperatures for samples exposed to IR, as well as for the corresponding non-irradiated controls. The observed increase in FDR reflects an increase in the formation of DSBs during the period of in vitro incubation caused by the conversion of labile lesions to DSBs. Results shown represent the mean and standard error calculated from six determinations in two experiments.
    Figure Legend Snippet: Generation of excess DSBs after in vitro incubation of DNA from irradiated cells at different temperatures. LIG4 − / − MEFs were embedded in agarose, exposed to 20 Gy X-rays and subjected to LTL as described under ‘Materials and Methods’ section. Subsequently, agarose blocks were incubated at different temperatures for different periods of time in TEN-buffer before analysis for DSBs by PFGE. ( A ) Shows typical ethidium bromide stained gels of irradiated and non-irradiated samples at the different treatment conditions. ( B ) Shows change in FDR as a function of time at the different incubation temperatures for samples exposed to IR, as well as for the corresponding non-irradiated controls. The observed increase in FDR reflects an increase in the formation of DSBs during the period of in vitro incubation caused by the conversion of labile lesions to DSBs. Results shown represent the mean and standard error calculated from six determinations in two experiments.

    Techniques Used: In Vitro, Incubation, Irradiation, Staining

    The conversion to DSBs of radiation-induced labile DNA lesions is drastically diminished in a reducing environment . LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays, subject to LTL and treated at different temperatures for different periods of time in TEN buffer supplemented with 2 mM DTT. Other details as in Figure 1 . The dotted lines show the response of samples incubated in TEN in the absence of DTT and have been transferred from Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments.
    Figure Legend Snippet: The conversion to DSBs of radiation-induced labile DNA lesions is drastically diminished in a reducing environment . LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays, subject to LTL and treated at different temperatures for different periods of time in TEN buffer supplemented with 2 mM DTT. Other details as in Figure 1 . The dotted lines show the response of samples incubated in TEN in the absence of DTT and have been transferred from Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments.

    Techniques Used: Incubation

    l -lysine enhances the conversion to SSBs of radiation-induced labile DNA lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays, subjected to LTL and treated at different temperatures for different periods of time in TEN-buffer supplemented with 100 mM l -lysine. The different panels show results obtained at the different temperatures after incubation in the presence or absence of l -lysine. The results without l -lysine have been transferred from Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments.
    Figure Legend Snippet: l -lysine enhances the conversion to SSBs of radiation-induced labile DNA lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays, subjected to LTL and treated at different temperatures for different periods of time in TEN-buffer supplemented with 100 mM l -lysine. The different panels show results obtained at the different temperatures after incubation in the presence or absence of l -lysine. The results without l -lysine have been transferred from Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments.

    Techniques Used: Incubation

    ( A ) Generation of excess DSBs after incubation at different temperatures and for different periods of time of DNA prepared by LTL from non-irradiated cells. LIG4 − / − MEFs were embedded in agarose blocks, subjected to LTL as described under ‘Material and Methods’ section and exposed to 10 Gy X-rays. Subsequently, agarose blocks were incubated in TEN-buffer at different temperatures and for different periods of time as indicated. Other details as in Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments. ( B ) Generation of excess DSBs after incubation at different temperatures and for different periods of time chromatin organized DNA of irradiated cells. LIG4 − / − MEFs were embedded in agarose, exposed to 20 Gy X-rays and transferred immediately in TEN-buffer supplemented with 0.2% Triton X-100. This treatment stops all cellular metabolic activities, including DSB repair, but retains cellular integrity including global nuclear architecture. Agarose blocks were then incubated in the same buffer at different temperatures for different periods of time and were, subsequently, lysed by LTL. Other details as in Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments.
    Figure Legend Snippet: ( A ) Generation of excess DSBs after incubation at different temperatures and for different periods of time of DNA prepared by LTL from non-irradiated cells. LIG4 − / − MEFs were embedded in agarose blocks, subjected to LTL as described under ‘Material and Methods’ section and exposed to 10 Gy X-rays. Subsequently, agarose blocks were incubated in TEN-buffer at different temperatures and for different periods of time as indicated. Other details as in Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments. ( B ) Generation of excess DSBs after incubation at different temperatures and for different periods of time chromatin organized DNA of irradiated cells. LIG4 − / − MEFs were embedded in agarose, exposed to 20 Gy X-rays and transferred immediately in TEN-buffer supplemented with 0.2% Triton X-100. This treatment stops all cellular metabolic activities, including DSB repair, but retains cellular integrity including global nuclear architecture. Agarose blocks were then incubated in the same buffer at different temperatures for different periods of time and were, subsequently, lysed by LTL. Other details as in Figure 1 . Results shown represent the mean and standard error calculated from six determinations in two experiments.

    Techniques Used: Incubation, Irradiation

    3) Product Images from "Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities"

    Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv116

    R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.
    Figure Legend Snippet: R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

    Techniques Used: Incubation, Concentration Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    4) Product Images from "Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase"

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157270

    TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.
    Figure Legend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Techniques Used: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection

    5) Product Images from "MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes"

    Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

    Journal: bioRxiv

    doi: 10.1101/2020.02.13.947598

    Extent of leaf variegation observed for different msh1 mutant alleles. A. An example of an msh1 mutant (CS3372) individual with a leaf-variegation phenotype. B. Values represent the percent of individuals in an F3 family from a homozygous mutant F2 parent that showed visible leaf variegation at time of harvest for mitochondrial and plastid DNA extraction. Means and standard errors are from three replicate F3 families from each mutant line (see Fig. 2 ). Between 45 and 66 individuals were scored for each family. Lowercase letters indicate significant differences between alleles based on a Tukey’s HSD test. Consistent with its lower rate of observed sequence and structural variation in cytoplasmic genomes, the SALK_046763 msh1 mutant line exhibited less severe phenotypic effects.
    Figure Legend Snippet: Extent of leaf variegation observed for different msh1 mutant alleles. A. An example of an msh1 mutant (CS3372) individual with a leaf-variegation phenotype. B. Values represent the percent of individuals in an F3 family from a homozygous mutant F2 parent that showed visible leaf variegation at time of harvest for mitochondrial and plastid DNA extraction. Means and standard errors are from three replicate F3 families from each mutant line (see Fig. 2 ). Between 45 and 66 individuals were scored for each family. Lowercase letters indicate significant differences between alleles based on a Tukey’s HSD test. Consistent with its lower rate of observed sequence and structural variation in cytoplasmic genomes, the SALK_046763 msh1 mutant line exhibited less severe phenotypic effects.

    Techniques Used: Mutagenesis, DNA Extraction, Sequencing

    Crossing design to test candidate nuclear genes involved in RRR of cytoplasmic genomes. Using a wild-type maternal plant (black) and either a homozygous mutant (red) or heterozygous pollen donor, we generated a heterozygous F1 individual that carried cytoplasmic genomes inherited from a wild-type lineage (as indicated by the black mitochondrion). After selfing the F1, we genotyped the resulting F2 progeny to identify three homozygous mutants and three homozygous wild-type individuals. Given that the mutations in candidate RRR genes are expected to be recessive, the F2 generation would be the first in which the sampled cytoplasmic genomes were exposed to the effects (red asterisks) of these mutants. The identified F2 individuals were each allowed to self-fertilize and set seed to produce multiple F3 families that all inherited their cytoplasmic genomes from the same F1 grandparent. The F3 families were used for purification of mitochondrial and plastid DNA for duplex sequencing. Sequencing was performed on three replicate families for each genotype. Arabidopsis silhouette image is from PhyloPic (Mason McNair).
    Figure Legend Snippet: Crossing design to test candidate nuclear genes involved in RRR of cytoplasmic genomes. Using a wild-type maternal plant (black) and either a homozygous mutant (red) or heterozygous pollen donor, we generated a heterozygous F1 individual that carried cytoplasmic genomes inherited from a wild-type lineage (as indicated by the black mitochondrion). After selfing the F1, we genotyped the resulting F2 progeny to identify three homozygous mutants and three homozygous wild-type individuals. Given that the mutations in candidate RRR genes are expected to be recessive, the F2 generation would be the first in which the sampled cytoplasmic genomes were exposed to the effects (red asterisks) of these mutants. The identified F2 individuals were each allowed to self-fertilize and set seed to produce multiple F3 families that all inherited their cytoplasmic genomes from the same F1 grandparent. The F3 families were used for purification of mitochondrial and plastid DNA for duplex sequencing. Sequencing was performed on three replicate families for each genotype. Arabidopsis silhouette image is from PhyloPic (Mason McNair).

    Techniques Used: Mutagenesis, Generated, Purification, Sequencing

    Intact MSH1 transcripts but reduced expression level in homozygous SALK_046763 msh1 mutants. A. Sanger trace from cDNA sequencing confirms that properly spliced transcripts are present in SALK_046763 msh1 mutants despite the large T-DNA insertion in intron 8. The vertical line below the trace indicates the location of the expected splice junction between exons 8 and 9. B. ΔC T values are calculated based on the difference in quantitative reverse-transcriptase PCR (qRT-PCR) threshold cycle value for each indicated MSH1 marker and the geometric mean of the threshold cycle values from two reference genes ( UBC and UBC9 ). Means and standard errors are from four biological replicates (F4 plants derived from crossing design described in Fig. 2 ), each of which is based on the mean of three technical replicates. The SALK_046763 mutants exhibit higher ΔC T (indicating lower MSH1 expression). Both MSH1 markers indicate a similar shift in ΔC T values (2.3 cycles for exons 8/9 and 2.5 cycles for exon 16), corresponding to an approximately 5-fold difference in transcript abundance. Significant differences between mutant and wild type genotypes at a level of P
    Figure Legend Snippet: Intact MSH1 transcripts but reduced expression level in homozygous SALK_046763 msh1 mutants. A. Sanger trace from cDNA sequencing confirms that properly spliced transcripts are present in SALK_046763 msh1 mutants despite the large T-DNA insertion in intron 8. The vertical line below the trace indicates the location of the expected splice junction between exons 8 and 9. B. ΔC T values are calculated based on the difference in quantitative reverse-transcriptase PCR (qRT-PCR) threshold cycle value for each indicated MSH1 marker and the geometric mean of the threshold cycle values from two reference genes ( UBC and UBC9 ). Means and standard errors are from four biological replicates (F4 plants derived from crossing design described in Fig. 2 ), each of which is based on the mean of three technical replicates. The SALK_046763 mutants exhibit higher ΔC T (indicating lower MSH1 expression). Both MSH1 markers indicate a similar shift in ΔC T values (2.3 cycles for exons 8/9 and 2.5 cycles for exon 16), corresponding to an approximately 5-fold difference in transcript abundance. Significant differences between mutant and wild type genotypes at a level of P

    Techniques Used: Expressing, Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Derivative Assay, Mutagenesis

    6) Product Images from "Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase"

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157270

    TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.
    Figure Legend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Techniques Used: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection

    7) Product Images from "Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities"

    Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv116

    R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.
    Figure Legend Snippet: R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

    Techniques Used: Incubation, Concentration Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    Related Articles

    Alkaline Southern Blotting:

    Article Title: The DNA Glycosylase, Ogg1, Defends Against Oxidant-induced mtDNA Damage and Apoptosis in Pulmonary Artery Endothelial Cells
    Article Snippet: .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase, Fpg (New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks detectable on alkaline Southern blot. .. Samples containing 500 ng DNA were treated with 8 units of Fpg in 20 µl of reaction volume at 37°C for 1 h. Subsequently, samples were treated with NaOH (final concentration 0.1 N) for 15 min at 37°C, mixed with loading dye and resolved in 0.6% agarose alkaline gel.

    Irradiation:

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair
    Article Snippet: .. Irradiated and non-irradiated DNA obtained by LTL of cells embedded in agarose (~1.2 µg DNA per plug) was treated for 24 h at 20°C with Fpg (400 ng, New England Biolabs, M0240 L) in the buffer provided by the manufacturer, or Nth (1.2 µg, NEB, M0268 L) in a buffer [70 mM HEPES/KOH pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 50 µg/ml bovine serum albumin] reducing non-specific nuclease activity ( ). .. After enzyme treatment agarose blocks were incubated at 20°C for 2 h with 1 mg/ml protease in TEN-buffer and washed once in TEN-buffer before PFGE.

    other:

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site
    Article Snippet: Bbv CI nicking endonuclease, Eco NI, Fpg, hOGG1, UDG, APE1, and T5 exonuclease were purchased from New England Biolabs (Ipswich, MA, USA).

    Activity Assay:

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair
    Article Snippet: .. Irradiated and non-irradiated DNA obtained by LTL of cells embedded in agarose (~1.2 µg DNA per plug) was treated for 24 h at 20°C with Fpg (400 ng, New England Biolabs, M0240 L) in the buffer provided by the manufacturer, or Nth (1.2 µg, NEB, M0268 L) in a buffer [70 mM HEPES/KOH pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 50 µg/ml bovine serum albumin] reducing non-specific nuclease activity ( ). .. After enzyme treatment agarose blocks were incubated at 20°C for 2 h with 1 mg/ml protease in TEN-buffer and washed once in TEN-buffer before PFGE.

    Construct:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: .. Fpg and Nth Nicking Assays 250 ng of each construct were digested with Formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs, Cat. #M0240S) using 1 μL of Fpg (8 units) in the presence of BSA, according to the manufacturer’s instructions, in 1X NEBuffer 1 for 1 hr at 37°C. ..

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    New England Biolabs endo iii
    R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. <t>Endo</t> <t>III</t> (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.
    Endo Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

    Journal: Nucleic Acids Research

    Article Title: Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

    doi: 10.1093/nar/gkv116

    Figure Lengend Snippet: R.PabI forms Schiff-base intermediates. ( A ) Sequences of substrates and markers used for analysis of reaction intermediates. ( B ) Analysis of DNA–R.PabI complexes. R.PabI (0–6 pmol, 0–300 nM) and a double-stranded substrate (GT#C40 (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) were incubated at 70°C for 20 min and then with 0.1 M NaBH 4 (or 0.1 M NaCl) at 25°C for 30 min. The reaction mixture was mixed with a gel loading buffer (final SDS concentration = 3%), denatured at 90°C for 10 min, and separated by 10% SDS-PAGE. Endo III (20 units) was incubated with a substrate (GT#C40EIII (Supplementary Table S2) with a 5′- 32 P label in the top strand, 0.2 pmol, 10 nM) at 37°C for 20 min and subjected to NABH 4 -trapping. ( C ) Analysis of covalently-trapped intermediates. NaBH 4 -trapping reactions of R.PabI and Endo III were performed as in A (5-fold scale relative to A). DNA–enzyme complexes were separated by SDS-APGE, and gel bands containing DNA–enzyme complexes (corresponding to bands marked as DNA–enzyme complexes in B) were excised. DNA–enzyme complexes were electro-eluted from the gel (multiple bands altogether), desalted by a spin column, and treated with the indicated amounts of proteinase K. The resulting products were analyzed by 18% denaturing PAGE. The bands indicated as a DNA–peptide crosslink for R.PabI and Endo III likely correspond to (xi) in Supplementary Figure S4C except that R.PabI and Endo III proteins were digested into short peptides of different sizes.

    Article Snippet: For controls, 20 units of Endo III (New England Biolabs) and 0.2 pmol (10 nM) a double-stranded substrate containing an AP site and a 5′-32 P label on the top strand was incubated in 10 mM Tris–HCl (pH 7.5), 100 mM NaCl and 1 mM EDTA (total 20 μl) at 37°C for 20 min followed by 100 mM NaBH4 (or 100 mM NaCl) as above.

    Techniques: Incubation, Concentration Assay, SDS Page, Polyacrylamide Gel Electrophoresis

    TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Journal: PLoS ONE

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    doi: 10.1371/journal.pone.0157270

    Figure Lengend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Article Snippet: As positive control, the oligo AP was incubated with 1U Endo III (E . coli NTH1, New England Biolabs), or with 2U Exonuclease III (Exo III, E . coli AP endonuclease, New England Biolabs) or with 1 μg of purified recombinant TcAP1 T . cruzi AP endonuclease.

    Techniques: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection

    Extent of leaf variegation observed for different msh1 mutant alleles. A. An example of an msh1 mutant (CS3372) individual with a leaf-variegation phenotype. B. Values represent the percent of individuals in an F3 family from a homozygous mutant F2 parent that showed visible leaf variegation at time of harvest for mitochondrial and plastid DNA extraction. Means and standard errors are from three replicate F3 families from each mutant line (see Fig. 2 ). Between 45 and 66 individuals were scored for each family. Lowercase letters indicate significant differences between alleles based on a Tukey’s HSD test. Consistent with its lower rate of observed sequence and structural variation in cytoplasmic genomes, the SALK_046763 msh1 mutant line exhibited less severe phenotypic effects.

    Journal: bioRxiv

    Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

    doi: 10.1101/2020.02.13.947598

    Figure Lengend Snippet: Extent of leaf variegation observed for different msh1 mutant alleles. A. An example of an msh1 mutant (CS3372) individual with a leaf-variegation phenotype. B. Values represent the percent of individuals in an F3 family from a homozygous mutant F2 parent that showed visible leaf variegation at time of harvest for mitochondrial and plastid DNA extraction. Means and standard errors are from three replicate F3 families from each mutant line (see Fig. 2 ). Between 45 and 66 individuals were scored for each family. Lowercase letters indicate significant differences between alleles based on a Tukey’s HSD test. Consistent with its lower rate of observed sequence and structural variation in cytoplasmic genomes, the SALK_046763 msh1 mutant line exhibited less severe phenotypic effects.

    Article Snippet: Half of the cleaned sample was then treated with a cocktail of repair enzymes to remove single-stranded damage in a 50 μl reaction volume containing 1x NEB CutSmart Buffer, 8 U Fpg (New England Biolabs M0240), 5 U Uracil-DNA Glycosylase (New England Biolabs M0280), and 10 U Endonuclease III (New England Biolabs M0268) for 30 min at 37° C. Samples were then cleaned with 1.6 volumes of SPRI beads.

    Techniques: Mutagenesis, DNA Extraction, Sequencing

    Crossing design to test candidate nuclear genes involved in RRR of cytoplasmic genomes. Using a wild-type maternal plant (black) and either a homozygous mutant (red) or heterozygous pollen donor, we generated a heterozygous F1 individual that carried cytoplasmic genomes inherited from a wild-type lineage (as indicated by the black mitochondrion). After selfing the F1, we genotyped the resulting F2 progeny to identify three homozygous mutants and three homozygous wild-type individuals. Given that the mutations in candidate RRR genes are expected to be recessive, the F2 generation would be the first in which the sampled cytoplasmic genomes were exposed to the effects (red asterisks) of these mutants. The identified F2 individuals were each allowed to self-fertilize and set seed to produce multiple F3 families that all inherited their cytoplasmic genomes from the same F1 grandparent. The F3 families were used for purification of mitochondrial and plastid DNA for duplex sequencing. Sequencing was performed on three replicate families for each genotype. Arabidopsis silhouette image is from PhyloPic (Mason McNair).

    Journal: bioRxiv

    Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

    doi: 10.1101/2020.02.13.947598

    Figure Lengend Snippet: Crossing design to test candidate nuclear genes involved in RRR of cytoplasmic genomes. Using a wild-type maternal plant (black) and either a homozygous mutant (red) or heterozygous pollen donor, we generated a heterozygous F1 individual that carried cytoplasmic genomes inherited from a wild-type lineage (as indicated by the black mitochondrion). After selfing the F1, we genotyped the resulting F2 progeny to identify three homozygous mutants and three homozygous wild-type individuals. Given that the mutations in candidate RRR genes are expected to be recessive, the F2 generation would be the first in which the sampled cytoplasmic genomes were exposed to the effects (red asterisks) of these mutants. The identified F2 individuals were each allowed to self-fertilize and set seed to produce multiple F3 families that all inherited their cytoplasmic genomes from the same F1 grandparent. The F3 families were used for purification of mitochondrial and plastid DNA for duplex sequencing. Sequencing was performed on three replicate families for each genotype. Arabidopsis silhouette image is from PhyloPic (Mason McNair).

    Article Snippet: Half of the cleaned sample was then treated with a cocktail of repair enzymes to remove single-stranded damage in a 50 μl reaction volume containing 1x NEB CutSmart Buffer, 8 U Fpg (New England Biolabs M0240), 5 U Uracil-DNA Glycosylase (New England Biolabs M0280), and 10 U Endonuclease III (New England Biolabs M0268) for 30 min at 37° C. Samples were then cleaned with 1.6 volumes of SPRI beads.

    Techniques: Mutagenesis, Generated, Purification, Sequencing

    Intact MSH1 transcripts but reduced expression level in homozygous SALK_046763 msh1 mutants. A. Sanger trace from cDNA sequencing confirms that properly spliced transcripts are present in SALK_046763 msh1 mutants despite the large T-DNA insertion in intron 8. The vertical line below the trace indicates the location of the expected splice junction between exons 8 and 9. B. ΔC T values are calculated based on the difference in quantitative reverse-transcriptase PCR (qRT-PCR) threshold cycle value for each indicated MSH1 marker and the geometric mean of the threshold cycle values from two reference genes ( UBC and UBC9 ). Means and standard errors are from four biological replicates (F4 plants derived from crossing design described in Fig. 2 ), each of which is based on the mean of three technical replicates. The SALK_046763 mutants exhibit higher ΔC T (indicating lower MSH1 expression). Both MSH1 markers indicate a similar shift in ΔC T values (2.3 cycles for exons 8/9 and 2.5 cycles for exon 16), corresponding to an approximately 5-fold difference in transcript abundance. Significant differences between mutant and wild type genotypes at a level of P

    Journal: bioRxiv

    Article Title: MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

    doi: 10.1101/2020.02.13.947598

    Figure Lengend Snippet: Intact MSH1 transcripts but reduced expression level in homozygous SALK_046763 msh1 mutants. A. Sanger trace from cDNA sequencing confirms that properly spliced transcripts are present in SALK_046763 msh1 mutants despite the large T-DNA insertion in intron 8. The vertical line below the trace indicates the location of the expected splice junction between exons 8 and 9. B. ΔC T values are calculated based on the difference in quantitative reverse-transcriptase PCR (qRT-PCR) threshold cycle value for each indicated MSH1 marker and the geometric mean of the threshold cycle values from two reference genes ( UBC and UBC9 ). Means and standard errors are from four biological replicates (F4 plants derived from crossing design described in Fig. 2 ), each of which is based on the mean of three technical replicates. The SALK_046763 mutants exhibit higher ΔC T (indicating lower MSH1 expression). Both MSH1 markers indicate a similar shift in ΔC T values (2.3 cycles for exons 8/9 and 2.5 cycles for exon 16), corresponding to an approximately 5-fold difference in transcript abundance. Significant differences between mutant and wild type genotypes at a level of P

    Article Snippet: Half of the cleaned sample was then treated with a cocktail of repair enzymes to remove single-stranded damage in a 50 μl reaction volume containing 1x NEB CutSmart Buffer, 8 U Fpg (New England Biolabs M0240), 5 U Uracil-DNA Glycosylase (New England Biolabs M0280), and 10 U Endonuclease III (New England Biolabs M0268) for 30 min at 37° C. Samples were then cleaned with 1.6 volumes of SPRI beads.

    Techniques: Expressing, Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Derivative Assay, Mutagenesis