r3150  (New England Biolabs)


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    New England Biolabs r3150
    R3150, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    New England Biolabs pvui hf
    Replisome skipping resolves CO collisions if mRNA takeover is blocked. ( A ) Schematic representation of the replication template with relevant features, restriction sites and leading-strand product lengths. ( B ) Sequence of the T7 A1 promoter non-template sequences for both replication templates with relevant transcript lengths and restriction sites. ( C ) Native gel electrophoretic analysis of the products of a replication time course of CO collisions with a single RNAP with a 19mer mRNA transcript either with a 3′-OH group (G19) or a 20mer transcript without a 3′-OH group (3′dC20) on the CO 19 template ( n = 3). Replication efficiency averaged 11 ± 6% (1 min)—13 ± 7% (8 min) for G19 and 10 ± 5% (1 min)—15 ± 7% (8 min) for 3′dC20. Lanes 1-6, reaction products digested with <t>EcoRI</t> and <t>PvuI</t> only; lanes 7–12, replication products were additionally digested with NcoI. ( D ) Gel-filtered [α- 32 P]GMP-labeled 19mer-RNAP CO 19 templates with either a free 3′-OH end (G19; lanes 1–4) or terminated with 3′-dCTP (3′dC20; lanes 5–8) were used in standard replication reactions and the products analyzed by electrophoresis through a composite 5%/20% 7 M urea–polyacrylamide gel. ( E ) Fraction of the labeled RNA extended by mRNA takeover on the gel shown in panel D ( n = 3, mean ± standard deviation). ( F ) Schematic representation of possible replication outcomes with respect to NcoI digestion. E, S and P, EcoRI, ScaI and PvuI restriction sites, respectively; rep. pro., replication proteins; SF, stalled fork; FL, full-length product; BF, broken fork; UC, uncoupled product ( 14 ); skip, NcoI-resistant material resulting from replisome skipping; TO, mRNA takeover product; dark grey oval, replisome; light grey ovals, RNAP.
    Pvui Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    Replisome skipping resolves CO collisions if mRNA takeover is blocked. ( A ) Schematic representation of the replication template with relevant features, restriction sites and leading-strand product lengths. ( B ) Sequence of the T7 A1 promoter non-template sequences for both replication templates with relevant transcript lengths and restriction sites. ( C ) Native gel electrophoretic analysis of the products of a replication time course of CO collisions with a single RNAP with a 19mer mRNA transcript either with a 3′-OH group (G19) or a 20mer transcript without a 3′-OH group (3′dC20) on the CO 19 template ( n = 3). Replication efficiency averaged 11 ± 6% (1 min)—13 ± 7% (8 min) for G19 and 10 ± 5% (1 min)—15 ± 7% (8 min) for 3′dC20. Lanes 1-6, reaction products digested with EcoRI and PvuI only; lanes 7–12, replication products were additionally digested with NcoI. ( D ) Gel-filtered [α- 32 P]GMP-labeled 19mer-RNAP CO 19 templates with either a free 3′-OH end (G19; lanes 1–4) or terminated with 3′-dCTP (3′dC20; lanes 5–8) were used in standard replication reactions and the products analyzed by electrophoresis through a composite 5%/20% 7 M urea–polyacrylamide gel. ( E ) Fraction of the labeled RNA extended by mRNA takeover on the gel shown in panel D ( n = 3, mean ± standard deviation). ( F ) Schematic representation of possible replication outcomes with respect to NcoI digestion. E, S and P, EcoRI, ScaI and PvuI restriction sites, respectively; rep. pro., replication proteins; SF, stalled fork; FL, full-length product; BF, broken fork; UC, uncoupled product ( 14 ); skip, NcoI-resistant material resulting from replisome skipping; TO, mRNA takeover product; dark grey oval, replisome; light grey ovals, RNAP.

    Journal: Nucleic Acids Research

    Article Title: Bypass of complex co-directional replication-transcription collisions by replisome skipping

    doi: 10.1093/nar/gkab760

    Figure Lengend Snippet: Replisome skipping resolves CO collisions if mRNA takeover is blocked. ( A ) Schematic representation of the replication template with relevant features, restriction sites and leading-strand product lengths. ( B ) Sequence of the T7 A1 promoter non-template sequences for both replication templates with relevant transcript lengths and restriction sites. ( C ) Native gel electrophoretic analysis of the products of a replication time course of CO collisions with a single RNAP with a 19mer mRNA transcript either with a 3′-OH group (G19) or a 20mer transcript without a 3′-OH group (3′dC20) on the CO 19 template ( n = 3). Replication efficiency averaged 11 ± 6% (1 min)—13 ± 7% (8 min) for G19 and 10 ± 5% (1 min)—15 ± 7% (8 min) for 3′dC20. Lanes 1-6, reaction products digested with EcoRI and PvuI only; lanes 7–12, replication products were additionally digested with NcoI. ( D ) Gel-filtered [α- 32 P]GMP-labeled 19mer-RNAP CO 19 templates with either a free 3′-OH end (G19; lanes 1–4) or terminated with 3′-dCTP (3′dC20; lanes 5–8) were used in standard replication reactions and the products analyzed by electrophoresis through a composite 5%/20% 7 M urea–polyacrylamide gel. ( E ) Fraction of the labeled RNA extended by mRNA takeover on the gel shown in panel D ( n = 3, mean ± standard deviation). ( F ) Schematic representation of possible replication outcomes with respect to NcoI digestion. E, S and P, EcoRI, ScaI and PvuI restriction sites, respectively; rep. pro., replication proteins; SF, stalled fork; FL, full-length product; BF, broken fork; UC, uncoupled product ( 14 ); skip, NcoI-resistant material resulting from replisome skipping; TO, mRNA takeover product; dark grey oval, replisome; light grey ovals, RNAP.

    Article Snippet: Aliquots (8 μl) were taken at the indicated times and incubated for 10 min with 0.2 U/μl EcoRI-HF (NEB), 0.2 U/μl PvuI-HF (NEB), 2 mM AMP-PNP (Roche), and 133 μM ddNTPs (GE Healthcare) (‘stop buffer’) before further analysis by gel electrophoresis.

    Techniques: Sequencing, Labeling, Electrophoresis, Standard Deviation

    Bypass of CO RNAP arrays can be promoted by additional factors. Native ( A ) and denaturing ( B ) agarose gels of products in replication time courses of CO replication collisions with RNAP arrays (on template CO100) after the indicated factors were added for 10 min prior to the initiation of replication. Final concentrations were 100 nM Rep (lanes 4–6), 100 nM UvrD (lanes 7–9), 500 nM Mfd (lanes 10–12), 0.1 U/μl RNase H (lanes 13–15), or 20 μM RNase A (lanes 16–18). Quantification of ( C ) stalled forks or ( D ) full length products ( n = 3, mean ± standard deviation). Native agarose gel of replication reaction products ( E ) stained with ethidium bromide or ( F ) visualized by autoradiography of [α- 32 P]GMP-labeled mRNA. Pooled gel filtered DNA-RNAP complexes (lane 1) were incubated for 10 min with the indicated factors (same concentrations as in panel A). Incubation was continued for another 8 min with the omission of any replication proteins (–) or with either the full complement of replication proteins (+) or with DnaA omitted (–A). Reactions were terminated by the addition of 30 mM EDTA without any restriction enzyme digestion. ( G ) Quantification of the fraction of displaced mRNA products ( n = 3, mean ± standard deviation). SF, stalled fork; FL, full length, BF, broken fork; S, leading-strand stall product; RS, leading-strand restart products; OF, Okazaki fragments; R, replicated; N, nicked; L, linear; s.c., supercoiled; D, displaced mRNA; G, [α-32P]GTP; Rn H, RNase H; Rn A, RNase A; rep. pro.; replication proteins. Note that the position of the oriC-independent mRNA extension product denoted by ‘•’ is different on the gels shown in panels E and F compared to the gels shown in Figure 4A – C because in the latter case the DNA products were digested with PvuI and EcoRI, whereas in the former case they were not.

    Journal: Nucleic Acids Research

    Article Title: Bypass of complex co-directional replication-transcription collisions by replisome skipping

    doi: 10.1093/nar/gkab760

    Figure Lengend Snippet: Bypass of CO RNAP arrays can be promoted by additional factors. Native ( A ) and denaturing ( B ) agarose gels of products in replication time courses of CO replication collisions with RNAP arrays (on template CO100) after the indicated factors were added for 10 min prior to the initiation of replication. Final concentrations were 100 nM Rep (lanes 4–6), 100 nM UvrD (lanes 7–9), 500 nM Mfd (lanes 10–12), 0.1 U/μl RNase H (lanes 13–15), or 20 μM RNase A (lanes 16–18). Quantification of ( C ) stalled forks or ( D ) full length products ( n = 3, mean ± standard deviation). Native agarose gel of replication reaction products ( E ) stained with ethidium bromide or ( F ) visualized by autoradiography of [α- 32 P]GMP-labeled mRNA. Pooled gel filtered DNA-RNAP complexes (lane 1) were incubated for 10 min with the indicated factors (same concentrations as in panel A). Incubation was continued for another 8 min with the omission of any replication proteins (–) or with either the full complement of replication proteins (+) or with DnaA omitted (–A). Reactions were terminated by the addition of 30 mM EDTA without any restriction enzyme digestion. ( G ) Quantification of the fraction of displaced mRNA products ( n = 3, mean ± standard deviation). SF, stalled fork; FL, full length, BF, broken fork; S, leading-strand stall product; RS, leading-strand restart products; OF, Okazaki fragments; R, replicated; N, nicked; L, linear; s.c., supercoiled; D, displaced mRNA; G, [α-32P]GTP; Rn H, RNase H; Rn A, RNase A; rep. pro.; replication proteins. Note that the position of the oriC-independent mRNA extension product denoted by ‘•’ is different on the gels shown in panels E and F compared to the gels shown in Figure 4A – C because in the latter case the DNA products were digested with PvuI and EcoRI, whereas in the former case they were not.

    Article Snippet: Aliquots (8 μl) were taken at the indicated times and incubated for 10 min with 0.2 U/μl EcoRI-HF (NEB), 0.2 U/μl PvuI-HF (NEB), 2 mM AMP-PNP (Roche), and 133 μM ddNTPs (GE Healthcare) (‘stop buffer’) before further analysis by gel electrophoresis.

    Techniques: Standard Deviation, Agarose Gel Electrophoresis, Staining, Autoradiography, Labeling, Incubation

    Build of RAREΔ SynHoxA assemblons. (A) Schematic of assembly strategy for 130kb RAREΔ SynHoxA and 166kb Enhancers + RAREΔ SynHoxA . Nature of the RARE mutations is shown on the right. RAR binding data comes from previously published reports. (see Methods) (B) Sanger sequencing traces confirmed precise CRISPR editing of RAREs in yeast. (C) SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Bands correspond to expected fragment lengths. (D) Sequencing data of assemblon BACs purified from E. coli aligned to a custom mm10 reference genome. Positions of the enhancers and protein coding genes are shown in black.

    Journal: bioRxiv

    Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

    doi: 10.1101/2021.07.07.451065

    Figure Lengend Snippet: Build of RAREΔ SynHoxA assemblons. (A) Schematic of assembly strategy for 130kb RAREΔ SynHoxA and 166kb Enhancers + RAREΔ SynHoxA . Nature of the RARE mutations is shown on the right. RAR binding data comes from previously published reports. (see Methods) (B) Sanger sequencing traces confirmed precise CRISPR editing of RAREs in yeast. (C) SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Bands correspond to expected fragment lengths. (D) Sequencing data of assemblon BACs purified from E. coli aligned to a custom mm10 reference genome. Positions of the enhancers and protein coding genes are shown in black.

    Article Snippet: Field Inversion Gel Electrophoresis (FIGE) SynHox assemblon BACs were verified by digesting a ∼250-500ng purified by alkaline lysisfrom small scale (5-10 mL) saturated bacterial culture with PvuI-HF (New England Biolabs R3150S).

    Techniques: Binding Assay, Sequencing, CRISPR, Purification

    Build of 134kb SynHoxA assemblon. (A) Layout of rat HoxA locus in the rn6 genome assembly. The rn6 genome includes an erroneous duplication at the HoxA locus between gaps in the assembly. The SynHoxA assemblon sequence is based on bringing together the two ‘separate’ RnHoxA segments. The sequence was segmented into 28 ∼5kb PCR amplicons with terminal homology of ∼200bp to adjacent amplicons. Conservation to the mouse genome is depicted using the multiz track from the UCSC genome browser. (B) Schematic depicting the assembly workflow for the 134kb SynHoxA assemblon. BACs containing Rat HoxA were used as PCR template to generate 28 segments tiling the entire HoxA locus. These segments were co-transformed into yeast with appropriate linkers and assembly vector to build two ∼65kb half assemblons into centromeric yeast-bacteria shuttle vectors. These half assemblons are recovered to bacteria and amplified. Full 134kb assemblon was built from half assemblons after releasing them from the vector using terminal restriction enzymes ( AsiSI ) and transforming into yeast. Full assemblon was then recovered from yeast into bacteria for amplification and verification. (C) Agarose gel of the 28 PCR amplicons that tile the 134kb SynHoxA assemblon. (D) Strategy to PCR-screen yeast colonies derived from assembly experiments. Primers (red arrows) span assembly junctions and test presence/absence of amplicons in many yeast colonies. Reproduced from ref ( 47 ) with permission from authors. (E) Agarose gel showing one yeast colony carrying the full 134kb SynHoxA assemblon verified manually for the presence of all assembly junctions, using the strategy outlined in panel D. (F) Half and Full 134kb SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using field inversion gel electrophoresis (FIGE). Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

    Journal: bioRxiv

    Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

    doi: 10.1101/2021.07.07.451065

    Figure Lengend Snippet: Build of 134kb SynHoxA assemblon. (A) Layout of rat HoxA locus in the rn6 genome assembly. The rn6 genome includes an erroneous duplication at the HoxA locus between gaps in the assembly. The SynHoxA assemblon sequence is based on bringing together the two ‘separate’ RnHoxA segments. The sequence was segmented into 28 ∼5kb PCR amplicons with terminal homology of ∼200bp to adjacent amplicons. Conservation to the mouse genome is depicted using the multiz track from the UCSC genome browser. (B) Schematic depicting the assembly workflow for the 134kb SynHoxA assemblon. BACs containing Rat HoxA were used as PCR template to generate 28 segments tiling the entire HoxA locus. These segments were co-transformed into yeast with appropriate linkers and assembly vector to build two ∼65kb half assemblons into centromeric yeast-bacteria shuttle vectors. These half assemblons are recovered to bacteria and amplified. Full 134kb assemblon was built from half assemblons after releasing them from the vector using terminal restriction enzymes ( AsiSI ) and transforming into yeast. Full assemblon was then recovered from yeast into bacteria for amplification and verification. (C) Agarose gel of the 28 PCR amplicons that tile the 134kb SynHoxA assemblon. (D) Strategy to PCR-screen yeast colonies derived from assembly experiments. Primers (red arrows) span assembly junctions and test presence/absence of amplicons in many yeast colonies. Reproduced from ref ( 47 ) with permission from authors. (E) Agarose gel showing one yeast colony carrying the full 134kb SynHoxA assemblon verified manually for the presence of all assembly junctions, using the strategy outlined in panel D. (F) Half and Full 134kb SynHoxA assemblon BACs purified from E.coli were digested with PvuI and separated using field inversion gel electrophoresis (FIGE). Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

    Article Snippet: Field Inversion Gel Electrophoresis (FIGE) SynHox assemblon BACs were verified by digesting a ∼250-500ng purified by alkaline lysisfrom small scale (5-10 mL) saturated bacterial culture with PvuI-HF (New England Biolabs R3150S).

    Techniques: Sequencing, Polymerase Chain Reaction, Transformation Assay, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Purification, Nucleic Acid Electrophoresis

    Build of 170kb Enhancers+SynHoxA assemblon. (A) Layout of rat HoxA locus from the rn6 genome assembly depicting genes, Rn HoxA cluster segments in black and previously identified distal enhancers in purple. The Enhancers+SynHoxA assemblon sequence is made by stringing all the enhancers directly upstream of the SynHoxA assemblon sequence. Conservation to mouse genome is depicted using multiz track from the UCSC genome browser. (B) PCR amplicons tiling enhancer sequences were generated from Rat HoxA BACs and co-transformed into a yeast strain containing the 134kb SynHoxA assemblon with a gRNA vector targeting the left terminus of the 134kb assemblon. The enhancer PCR amplicons were used to repair this break, resulting in the construction of the 170kb Enhancers+SynHoxA assemblon. Assemblon was recovered into bacteria for amplification and verification. (C) Agarose gel of the 8 PCR amplicons containing enhancer sequences. (D) Agarose gel showing one yeast colony tested for the presence of novel enhancer assembly junctions and with primers spanning 134kb SynHoxA . (E) 134kb and 170kb assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

    Journal: bioRxiv

    Article Title: Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

    doi: 10.1101/2021.07.07.451065

    Figure Lengend Snippet: Build of 170kb Enhancers+SynHoxA assemblon. (A) Layout of rat HoxA locus from the rn6 genome assembly depicting genes, Rn HoxA cluster segments in black and previously identified distal enhancers in purple. The Enhancers+SynHoxA assemblon sequence is made by stringing all the enhancers directly upstream of the SynHoxA assemblon sequence. Conservation to mouse genome is depicted using multiz track from the UCSC genome browser. (B) PCR amplicons tiling enhancer sequences were generated from Rat HoxA BACs and co-transformed into a yeast strain containing the 134kb SynHoxA assemblon with a gRNA vector targeting the left terminus of the 134kb assemblon. The enhancer PCR amplicons were used to repair this break, resulting in the construction of the 170kb Enhancers+SynHoxA assemblon. Assemblon was recovered into bacteria for amplification and verification. (C) Agarose gel of the 8 PCR amplicons containing enhancer sequences. (D) Agarose gel showing one yeast colony tested for the presence of novel enhancer assembly junctions and with primers spanning 134kb SynHoxA . (E) 134kb and 170kb assemblon BACs purified from E.coli were digested with PvuI and separated using FIGE. Lambda monocut ladder sizes are indicated in kb. Band sizes correspond to expected fragments.

    Article Snippet: Field Inversion Gel Electrophoresis (FIGE) SynHox assemblon BACs were verified by digesting a ∼250-500ng purified by alkaline lysisfrom small scale (5-10 mL) saturated bacterial culture with PvuI-HF (New England Biolabs R3150S).

    Techniques: Sequencing, Polymerase Chain Reaction, Generated, Transformation Assay, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Purification

    DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Journal: BMC Molecular Biology

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells

    doi: 10.1186/1471-2199-15-24

    Figure Lengend Snippet: DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Article Snippet: Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

    Techniques: DNA Methylation Assay, Amplification, Generated, Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Western Blot, Autoradiography