mmei  (New England Biolabs)


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    Name:
    MmeI
    Description:
    MmeI 500 units
    Catalog Number:
    r0637l
    Price:
    282
    Size:
    500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs mmei
    MmeI
    MmeI 500 units
    https://www.bioz.com/result/mmei/product/New England Biolabs
    Average 97 stars, based on 113 article reviews
    Price from $9.99 to $1999.99
    mmei - by Bioz Stars, 2020-07
    97/100 stars

    Images

    1) Product Images from "Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens"

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl391

    Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The DNA is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by MmeI to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.
    Figure Legend Snippet: Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The DNA is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by MmeI to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.

    Techniques Used: Amplification, Blocking Assay, Generated, Magnetic Beads, Polymerase Chain Reaction

    2) Product Images from "Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system"

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1173

    Proof-of-principle experiment of RLL-Y2H system. ( A ) Positive control interacting protein pair, murine p53 and SV40 T-antigen were inserted into mBD and mAD of RLL-Y2H system and were transformed into yeast respectively. Only the yeasts containing both p53 and SV40 T-antigen but not the controls can grow on SD/-AHL T selection plates. ( B ) After plasmid extraction from yeast, p53, SV40 T-antigen and their recombinated fragments were amplified by PCR. ( C ) Sanger sequencing of the recombinated p53 and SV40 T-antigen fragments. The linker ATTL and MmeI sites were highlighted. ( D ) The PCR products of the recombinated p53 and SV40 T-antigen fragments before (left panel) and after (right panel) MmeI digestion.
    Figure Legend Snippet: Proof-of-principle experiment of RLL-Y2H system. ( A ) Positive control interacting protein pair, murine p53 and SV40 T-antigen were inserted into mBD and mAD of RLL-Y2H system and were transformed into yeast respectively. Only the yeasts containing both p53 and SV40 T-antigen but not the controls can grow on SD/-AHL T selection plates. ( B ) After plasmid extraction from yeast, p53, SV40 T-antigen and their recombinated fragments were amplified by PCR. ( C ) Sanger sequencing of the recombinated p53 and SV40 T-antigen fragments. The linker ATTL and MmeI sites were highlighted. ( D ) The PCR products of the recombinated p53 and SV40 T-antigen fragments before (left panel) and after (right panel) MmeI digestion.

    Techniques Used: Positive Control, Transformation Assay, Selection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Sequencing

    3) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    Schematic of DALEC . Brown and green represent priming regions for Solexa bridge amplification primers. Blue indicates the primer binding region for the Solexa sequencing reaction. TCCGAC is the Mme I recognition sequence and the sequence immediately upstream of it (black) is the variable region. The inset gives detailed information for linkers A and B.
    Figure Legend Snippet: Schematic of DALEC . Brown and green represent priming regions for Solexa bridge amplification primers. Blue indicates the primer binding region for the Solexa sequencing reaction. TCCGAC is the Mme I recognition sequence and the sequence immediately upstream of it (black) is the variable region. The inset gives detailed information for linkers A and B.

    Techniques Used: Amplification, Binding Assay, Sequencing

    4) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    Schematic of DALEC . Brown and green represent priming regions for Solexa bridge amplification primers. Blue indicates the primer binding region for the Solexa sequencing reaction. TCCGAC is the Mme I recognition sequence and the sequence immediately upstream of it (black) is the variable region. The inset gives detailed information for linkers A and B.
    Figure Legend Snippet: Schematic of DALEC . Brown and green represent priming regions for Solexa bridge amplification primers. Blue indicates the primer binding region for the Solexa sequencing reaction. TCCGAC is the Mme I recognition sequence and the sequence immediately upstream of it (black) is the variable region. The inset gives detailed information for linkers A and B.

    Techniques Used: Amplification, Binding Assay, Sequencing

    5) Product Images from "MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection"

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn711

    MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.
    Figure Legend Snippet: MmeI cleaves the two DNA strands at one site simultaneously. ( A ) Time course of MmeI digestion of supercoiled pUC19 DNA (2 U/μg) for 10 s, 20 s, 30 s, 1, 3 10, 20, 30 and 60 min. Supercoiled plasmid (sc) is converted directly to linear (lin) DNA, with no accumulation of open circular DNA (oc). ‘A+B cut’ indicates the 184-bp product of MmeI cleavage at both pUC19 sites. ( B ) MmeI digestion of linear pUC19 DNA (previously cut with PstI), in a 2-fold serial dilution from 8 to 0.03 U/μg. MmeI cuts at a one site, forming products from either site A or site B, before forming product from cleavage at both sites (A+B). ‘lin’ indicates linear pUC19, ‘A-R’ and ‘A-L’ indicate the cleavage products from MmeI cutting at the 996 site, ‘B-R’ and ‘B-L’ indicate the cleavage products from MmeI cutting at the 1180 site, while ‘A+B’ indicates the cleavage product from MmeI cutting at both sites.

    Techniques Used: Plasmid Preparation, Serial Dilution

    DNA sequence containing MmeI and predicted flanking genes from M. methylotrophus genomic locus. ( A–D ) are uncharacterized putative ORFs. RE sites are numbered from the 5′-EcoRI site (in bp). The four MmeI sites are listed with the arrow indicating the orientation of the site: 5′-TCCRACN20/N18-3′.
    Figure Legend Snippet: DNA sequence containing MmeI and predicted flanking genes from M. methylotrophus genomic locus. ( A–D ) are uncharacterized putative ORFs. RE sites are numbered from the 5′-EcoRI site (in bp). The four MmeI sites are listed with the arrow indicating the orientation of the site: 5′-TCCRACN20/N18-3′.

    Techniques Used: Sequencing

    ( A ) In vivo MmeI Modification. MmeI endonuclease digestion of purified plasmid DNA from the construct expressing active or inactive MmeI. ‘Active’ is the MmeI expression plasmid, pTBMmeI.1. ‘Inactive’ is a plasmid DNA derived from pTBMmeI.1 that carries a single point mutation (N 773 D) that renders MmeI inactive. Addition of PhiX174 DNA verifies that the added MmeI endonuclease is active in the reaction wherein pTBMmeI.1 DNA is not cut. ( B ) In vitro MmeI modification. pMMH1 DNA from a dam-deficient host, either modified in vitro by MmeI or not modified, digested with MmeI, HinfI or MboI. ( C ) pMMH1 DNA from a dam-proficient host digested with MmeI, MboI and DpnI. M: Size standards: Lambda-HindIII, PhiX174-HaeIII.
    Figure Legend Snippet: ( A ) In vivo MmeI Modification. MmeI endonuclease digestion of purified plasmid DNA from the construct expressing active or inactive MmeI. ‘Active’ is the MmeI expression plasmid, pTBMmeI.1. ‘Inactive’ is a plasmid DNA derived from pTBMmeI.1 that carries a single point mutation (N 773 D) that renders MmeI inactive. Addition of PhiX174 DNA verifies that the added MmeI endonuclease is active in the reaction wherein pTBMmeI.1 DNA is not cut. ( B ) In vitro MmeI modification. pMMH1 DNA from a dam-deficient host, either modified in vitro by MmeI or not modified, digested with MmeI, HinfI or MboI. ( C ) pMMH1 DNA from a dam-proficient host digested with MmeI, MboI and DpnI. M: Size standards: Lambda-HindIII, PhiX174-HaeIII.

    Techniques Used: In Vivo, Modification, Purification, Plasmid Preparation, Construct, Expressing, Derivative Assay, Mutagenesis, In Vitro

    Digestion of synthetic DNAs containing modified or unmodified adenines in the MmeI recognition sequence. ( A ) UU, fully unmethylated DNA. ( B ) UM, containing N6-methyl adenine in the bottom strand, 5′-GTYGG(m6A)-3′, of the MmeI recognition sequence. ( C ) MU containing N6-methyl adenine in the top strand, 5′-TCCR(m6A)C-3′, of the MmeI recognition sequence. ( D ) MM containing N6-methyl adenine in the both strands of the MmeI recognition sequence. Lanes: 1 = no enzyme; 2 = MmeI (TCCRAC); 3 = Hpy188I (TCNGA); 4 = HinfI (GANTC); 5 = MboI (GATC); 6 = BfuCI (GATC). M: pBR322-MspI size standard. DNA recognition sites: 6 = m6-adenine, MmeI site 5′- TCCGAC -3′ in ‘ bold’, HinfI site 5′-GANTC-3′ top strand underlined, MboI and BfuCI site 5′-GATC-3′ bottom strand underlined.
    Figure Legend Snippet: Digestion of synthetic DNAs containing modified or unmodified adenines in the MmeI recognition sequence. ( A ) UU, fully unmethylated DNA. ( B ) UM, containing N6-methyl adenine in the bottom strand, 5′-GTYGG(m6A)-3′, of the MmeI recognition sequence. ( C ) MU containing N6-methyl adenine in the top strand, 5′-TCCR(m6A)C-3′, of the MmeI recognition sequence. ( D ) MM containing N6-methyl adenine in the both strands of the MmeI recognition sequence. Lanes: 1 = no enzyme; 2 = MmeI (TCCRAC); 3 = Hpy188I (TCNGA); 4 = HinfI (GANTC); 5 = MboI (GATC); 6 = BfuCI (GATC). M: pBR322-MspI size standard. DNA recognition sites: 6 = m6-adenine, MmeI site 5′- TCCGAC -3′ in ‘ bold’, HinfI site 5′-GANTC-3′ top strand underlined, MboI and BfuCI site 5′-GATC-3′ bottom strand underlined.

    Techniques Used: Modification, Sequencing

    Endonuclease digestion of MmeI in vitro modified or unmodified DNA (150-bp PCR product across pMMHI polylinker region) containing a HinfI site overlapping the top strand of the MmeI site: 5′-TCC GACTC -3′ and an MboI site overlapping the bottom strand of the MmeI site: 5′-GTCG GATC -3′. The restriction endonucleases were mixed with buffer, aliquoted into three reactions to which were added MmeI modified DNA, unmodified DNA or both DNAs. Size standard: pBR322-MspI.
    Figure Legend Snippet: Endonuclease digestion of MmeI in vitro modified or unmodified DNA (150-bp PCR product across pMMHI polylinker region) containing a HinfI site overlapping the top strand of the MmeI site: 5′-TCC GACTC -3′ and an MboI site overlapping the bottom strand of the MmeI site: 5′-GTCG GATC -3′. The restriction endonucleases were mixed with buffer, aliquoted into three reactions to which were added MmeI modified DNA, unmodified DNA or both DNAs. Size standard: pBR322-MspI.

    Techniques Used: In Vitro, Modification, Polymerase Chain Reaction

    Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).
    Figure Legend Snippet: Detection of MmeI modification using antibodies specific for N6-methyladenine (m6A) and N4-methylcytosine (m4C). (Top row) unmethylated DNA; (second row) MmeI in vitro modified DNA; (third row) M.EcoRI in vitro modified DNA; (fourth row) M.BamHI in vitro modified DNA. Positive controls: m6A antibody panel; (fifth row) dam (m6A) in vivo methylated pUC19 DNA (400–25 ng dilution), m4C antibody panel; (fifth row) M.EsaBC3I (m4C) in vivo methylated plasmid DNA (400–25 ng dilution).

    Techniques Used: Modification, In Vitro, In Vivo, Methylation, Plasmid Preparation

    Relative rates of MmeI endonuclease cleavage and DNA methyltransferase activities. MmeI (2 Us/μg) digestion of Phix174 DNA with 1 μM [methyl-3H]-AdoMet over a time course of 0, 0.25, 1, 5, 15, 60 and 120 min. 3 H counts are reported for an equal amount of DNA to that on the gel. The extent of endonuclease cleavage was estimated from the MmeI fragment pattern. The extent of methylation was calculated as a percent of the maximal counts reached at 60 min, subtracting the background counts from each count.
    Figure Legend Snippet: Relative rates of MmeI endonuclease cleavage and DNA methyltransferase activities. MmeI (2 Us/μg) digestion of Phix174 DNA with 1 μM [methyl-3H]-AdoMet over a time course of 0, 0.25, 1, 5, 15, 60 and 120 min. 3 H counts are reported for an equal amount of DNA to that on the gel. The extent of endonuclease cleavage was estimated from the MmeI fragment pattern. The extent of methylation was calculated as a percent of the maximal counts reached at 60 min, subtracting the background counts from each count.

    Techniques Used: Methylation

    MmeI cleavage of a single site substrate is incomplete but can be stimulated by in trans DNA containing an MmeI site. ( A–E ) MmeI digestion in a 2-fold serial dilution from 16 U/μg DNA to 0.125 U/μg DNA on p996SS1 DNA previously linearized by digestion with PstI. (A) no trans DNA; (B) 0.2 μM M12 trans DNA; (C) 0.02 μM M12 trans DNA; (D) 0.2 μM NmeA30 trans DNA that does not contain an MmeI site; (E) 0.2 μM N6-adenine methylated (5′-TCCG(m6A)C-3′) M12 trans DNA; ( F ) 2-fold serial dilution of the M12 trans DNA from 0.16 μM to 0.001 μM in a digestion reaction containing 4 U (0.026 μM) MmeI/μg substrate DNA. Arrows indicate ‘linear’ DNA (uncut by MmeI) and ‘cut’ DNA that is the MmeI cleavage products.
    Figure Legend Snippet: MmeI cleavage of a single site substrate is incomplete but can be stimulated by in trans DNA containing an MmeI site. ( A–E ) MmeI digestion in a 2-fold serial dilution from 16 U/μg DNA to 0.125 U/μg DNA on p996SS1 DNA previously linearized by digestion with PstI. (A) no trans DNA; (B) 0.2 μM M12 trans DNA; (C) 0.02 μM M12 trans DNA; (D) 0.2 μM NmeA30 trans DNA that does not contain an MmeI site; (E) 0.2 μM N6-adenine methylated (5′-TCCG(m6A)C-3′) M12 trans DNA; ( F ) 2-fold serial dilution of the M12 trans DNA from 0.16 μM to 0.001 μM in a digestion reaction containing 4 U (0.026 μM) MmeI/μg substrate DNA. Arrows indicate ‘linear’ DNA (uncut by MmeI) and ‘cut’ DNA that is the MmeI cleavage products.

    Techniques Used: Serial Dilution, Methylation

    MmeI modification following endonuclease cleavage. ‘Cut’ indicates lambda DNA digested with MmeI (2 U/μg DNA) for 1, 5, 15 and 60 min; ‘Lig’ indicates the cut lambda DNA purified from MmeI and ligated into concatamers; ‘Recut’ indicates subsequent MmeI digestion of the ligated DNA concatamers.
    Figure Legend Snippet: MmeI modification following endonuclease cleavage. ‘Cut’ indicates lambda DNA digested with MmeI (2 U/μg DNA) for 1, 5, 15 and 60 min; ‘Lig’ indicates the cut lambda DNA purified from MmeI and ligated into concatamers; ‘Recut’ indicates subsequent MmeI digestion of the ligated DNA concatamers.

    Techniques Used: Modification, Lambda DNA Preparation, Purification

    MmeI cleaves DNA containing a newly synthesized (unmodified) top strand and a genomic M. methylotrophus bottom strand. MmeI digestion of DNAs from one round of primer extension. Lanes 2–5 are the newly synthesized top strand, while lanes 6–10 are the newly synthesized bottom strand. Lanes 2 = uncut; 3 = 20 U MmeI; 4 = 5 U MmeI; 5 = 10 U BspHI; 6 = uncut; 7 = 20 U MmeI; 8 = 5 U MmeI; 9 = 20 U MmeI digestion of both newly synthesized bottom strand and top strand DNA (as a positive control for MmeI activity); 10 = 10 U BspHI. Lanes 1 and 11: PhiX174-HaeIII size standard. MmeI cuts the DNA containing a genomic M. methylotrophus bottom strand and an unmodified top strand (lanes 3, 4 and 9), but not the DNA containing a genomic M. methylotrophus top strand and an unmodified bottom strand (lanes 7, 8 and 9). The native host DNA from M. methylotrophus is thus modified to prevent MmeI cleavage only in the top DNA strand of the MmeI recognition sequence.
    Figure Legend Snippet: MmeI cleaves DNA containing a newly synthesized (unmodified) top strand and a genomic M. methylotrophus bottom strand. MmeI digestion of DNAs from one round of primer extension. Lanes 2–5 are the newly synthesized top strand, while lanes 6–10 are the newly synthesized bottom strand. Lanes 2 = uncut; 3 = 20 U MmeI; 4 = 5 U MmeI; 5 = 10 U BspHI; 6 = uncut; 7 = 20 U MmeI; 8 = 5 U MmeI; 9 = 20 U MmeI digestion of both newly synthesized bottom strand and top strand DNA (as a positive control for MmeI activity); 10 = 10 U BspHI. Lanes 1 and 11: PhiX174-HaeIII size standard. MmeI cuts the DNA containing a genomic M. methylotrophus bottom strand and an unmodified top strand (lanes 3, 4 and 9), but not the DNA containing a genomic M. methylotrophus top strand and an unmodified bottom strand (lanes 7, 8 and 9). The native host DNA from M. methylotrophus is thus modified to prevent MmeI cleavage only in the top DNA strand of the MmeI recognition sequence.

    Techniques Used: Synthesized, Positive Control, Activity Assay, Modification, Sequencing

    6) Product Images from "Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities"

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002442

    MmeI/DNA/Sinefungin ternary complex. (A) The MmeI helicase spacer (residues 156–300), methyltransferase (residues 301–620), DNA target recognition domain (TRD; residues 621–825), and C-terminal helical region (residues 826–919) are shown in purple, green, cyan, and orange, respectively. DNA is in yellow. The flipped-out adenine (orange) and the bound Sinefungin (red) are labeled. (B) Crystallized DNA and Sinefungin with accompanying 2Fo-Fc map contoured at 1.5 σ. The recognition sequence of TCCGAC is labeled along with the complementary strand.
    Figure Legend Snippet: MmeI/DNA/Sinefungin ternary complex. (A) The MmeI helicase spacer (residues 156–300), methyltransferase (residues 301–620), DNA target recognition domain (TRD; residues 621–825), and C-terminal helical region (residues 826–919) are shown in purple, green, cyan, and orange, respectively. DNA is in yellow. The flipped-out adenine (orange) and the bound Sinefungin (red) are labeled. (B) Crystallized DNA and Sinefungin with accompanying 2Fo-Fc map contoured at 1.5 σ. The recognition sequence of TCCGAC is labeled along with the complementary strand.

    Techniques Used: Labeling, Sequencing

    Change in specificity at position 2. (A) Restriction fragment digestion patterns of lambda, PhiX174, and pBR322 DNAs with wt = wild type MmeI, which cuts at TCCRAC20/18; A = MmeI Lys 645 Met mutant, which cuts at TACRAC20/18; R = MmeI Tyr 642 Lys, Lys 645 Met double mutant, which cuts at TRCRAC20/18; M = size standard, lambda-HindIII digest plus PhiX174-HaeIII digest. (B) Cut site determination for MmeI K 645 M mutant showing cutting at TACRAC20/18. Run-off Sanger sequencing of pUC19 DNA (TACRAC site at 376 to 381), priming from both sides (5' and 3') of the TACRAC recognition site and point of DNA cleavage. (C) Cut site determination for MmeI Y 642 K, K 645 M double mutant showing cutting at both TACRAC20/18 (top panel, pUC19 site at 376 to 381) and TGCRAC (bottom panel, pUC19 site at 1842 to 1847). Run-off Sanger sequencing of the cleaved pUC19 DNA, showing priming from 5' to the TACRAC or TGCRAC recognition site (bottom strand cleavage shown).
    Figure Legend Snippet: Change in specificity at position 2. (A) Restriction fragment digestion patterns of lambda, PhiX174, and pBR322 DNAs with wt = wild type MmeI, which cuts at TCCRAC20/18; A = MmeI Lys 645 Met mutant, which cuts at TACRAC20/18; R = MmeI Tyr 642 Lys, Lys 645 Met double mutant, which cuts at TRCRAC20/18; M = size standard, lambda-HindIII digest plus PhiX174-HaeIII digest. (B) Cut site determination for MmeI K 645 M mutant showing cutting at TACRAC20/18. Run-off Sanger sequencing of pUC19 DNA (TACRAC site at 376 to 381), priming from both sides (5' and 3') of the TACRAC recognition site and point of DNA cleavage. (C) Cut site determination for MmeI Y 642 K, K 645 M double mutant showing cutting at both TACRAC20/18 (top panel, pUC19 site at 376 to 381) and TGCRAC (bottom panel, pUC19 site at 1842 to 1847). Run-off Sanger sequencing of the cleaved pUC19 DNA, showing priming from 5' to the TACRAC or TGCRAC recognition site (bottom strand cleavage shown).

    Techniques Used: Mutagenesis, Sequencing

    Structural comparison between MmeI and BpuSI. (A) MmeI (red) bound to DNA (yellow) superimposed on apo BpuSI (cyan). The helicase connector, methyltransferase, and DNA target recognition domain (TRD) labels correspond to both structures, while the endonuclease domain is only visible in the BpuSI structure. A comparison of the two structures reveals an ~38° rotation in the TRD, which clamps down on the DNA to make specific contacts. The TRD as a whole shifts by ~27 Å between the two structures. (B) A 90° rotation of the view in (a) to show the relative position of the endonuclease domain.
    Figure Legend Snippet: Structural comparison between MmeI and BpuSI. (A) MmeI (red) bound to DNA (yellow) superimposed on apo BpuSI (cyan). The helicase connector, methyltransferase, and DNA target recognition domain (TRD) labels correspond to both structures, while the endonuclease domain is only visible in the BpuSI structure. A comparison of the two structures reveals an ~38° rotation in the TRD, which clamps down on the DNA to make specific contacts. The TRD as a whole shifts by ~27 Å between the two structures. (B) A 90° rotation of the view in (a) to show the relative position of the endonuclease domain.

    Techniques Used:

    Related Articles

    DNA Cleavage Assay:

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities
    Article Snippet: .. DNA Cleavage Assay Endonuclease activity was assayed by incubating various amounts of MmeI (wt or mutant) enzyme for 30 min at 37°C in NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT) supplemented with AdoMet at 80 μM, containing 1 μg substrate DNA per 50 μl. .. Reactions were terminated by the addition of loading dye (NEB B7024) and reaction products were analyzed by gel electrophoresis in 1% LE agarose gels.

    Ligation:

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
    Article Snippet: .. MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: .. Ligation to Linker B Linker B was purchased as two separate oligonucleotides (5'P-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTCGGTGGTCGCCGTATCATT-OH3', 5'OH-TCATCTTTCCCTACACGACGCTCTTCCGATCTNN-OH3') and hybridized using the same procedure as described for Linker A. Mme I products were ligated to Linker B using the following 50 μl reaction mix: 10.0 μl Mme I product, 1.0 μl of 0.05 mM Linker B, 5.0 μl 10× ligase buffer, 3.0 μl T4 DNA ligase (2,000 U/μl; NEB #M0202), 31.0 μl dH2 O. Ligations were performed overnight using a PCR machine. ..

    Mutagenesis:

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities
    Article Snippet: .. DNA Cleavage Assay Endonuclease activity was assayed by incubating various amounts of MmeI (wt or mutant) enzyme for 30 min at 37°C in NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT) supplemented with AdoMet at 80 μM, containing 1 μg substrate DNA per 50 μl. .. Reactions were terminated by the addition of loading dye (NEB B7024) and reaction products were analyzed by gel electrophoresis in 1% LE agarose gels.

    Isolation:

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
    Article Snippet: .. MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

    Purification:

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
    Article Snippet: .. Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification. .. Prepare Illumina P5 and P7 adapters by annealing overhang NN-nucleotides P5: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3′ and 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3′; P7: 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′ and 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNN-3′, respectively.

    Incubation:

    Article Title: Targeted and genome-scale methylomics reveals gene body signatures in human cell lines
    Article Snippet: .. This was incubated at 50°C for 20 min, then 85°C for 20 min. Ethanol precipitation was performed again, and the pellet was resuspended to 50ul with a reaction mixture containing 2 units Mme I (NEB), 50 µM SAM and 1× NEBuffer 4. .. This was incubated at 37°C for 2 hours, then 80°C for 20 min. To this was added 1.66 µl of 10 µM adapter B, 6 µl 10mM ATP, and 3 µl T4 DNA ligase, and the mixture was incubated at 16°C for 4 hours, then 65°C for 15 min.

    other:

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: Mme I digestion Linker A-ligated molecules were subjected to Mme I digestion using the following 200 μl reaction mix: 20 μl Linker A-ligated product, 20.0 μl 10× NEB 4, 0.3 μl 32 mM S-adenosyl methionine, 2.0 μl Mme I (2 U/μl; NEB R0637), 157.7 μl dH2 O.

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: MATERIALS AND METHODS MmeI, AdoMet, all restriction endonucleases, T4 DNA Ligase, Phusion DNA polymerase, DNA size standards and competent cells were obtained from New England Biolabs (Ipswich, MA).

    Activity Assay:

    Article Title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities
    Article Snippet: .. DNA Cleavage Assay Endonuclease activity was assayed by incubating various amounts of MmeI (wt or mutant) enzyme for 30 min at 37°C in NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT) supplemented with AdoMet at 80 μM, containing 1 μg substrate DNA per 50 μl. .. Reactions were terminated by the addition of loading dye (NEB B7024) and reaction products were analyzed by gel electrophoresis in 1% LE agarose gels.

    Ethanol Precipitation:

    Article Title: Targeted and genome-scale methylomics reveals gene body signatures in human cell lines
    Article Snippet: .. This was incubated at 50°C for 20 min, then 85°C for 20 min. Ethanol precipitation was performed again, and the pellet was resuspended to 50ul with a reaction mixture containing 2 units Mme I (NEB), 50 µM SAM and 1× NEBuffer 4. .. This was incubated at 37°C for 2 hours, then 80°C for 20 min. To this was added 1.66 µl of 10 µM adapter B, 6 µl 10mM ATP, and 3 µl T4 DNA ligase, and the mixture was incubated at 16°C for 4 hours, then 65°C for 15 min.

    Polymerase Chain Reaction:

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: .. Ligation to Linker B Linker B was purchased as two separate oligonucleotides (5'P-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTCGGTGGTCGCCGTATCATT-OH3', 5'OH-TCATCTTTCCCTACACGACGCTCTTCCGATCTNN-OH3') and hybridized using the same procedure as described for Linker A. Mme I products were ligated to Linker B using the following 50 μl reaction mix: 10.0 μl Mme I product, 1.0 μl of 0.05 mM Linker B, 5.0 μl 10× ligase buffer, 3.0 μl T4 DNA ligase (2,000 U/μl; NEB #M0202), 31.0 μl dH2 O. Ligations were performed overnight using a PCR machine. ..

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
    Article Snippet: .. Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification. .. Prepare Illumina P5 and P7 adapters by annealing overhang NN-nucleotides P5: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3′ and 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3′; P7: 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′ and 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNN-3′, respectively.

    Gel Extraction:

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens
    Article Snippet: .. MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8). .. Blocking of MmeI-tagged primers : 25 μl Of the fragment is used as primer with 10 μl Driver bio-dsDNA template in the presence of 2.5 mM each ddNTPs-bio (Biotin-11-ddNTPs, NEN® Life Science Products Inc., Boston, MA), 0.025 mM each dNTPs (Roche) and Thermo Sequenase™ (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Plasmid Preparation:

    Article Title: Generating high accuracy peptide binding data in high throughput with yeast surface display and SORTCERY
    Article Snippet: .. Zymoprep Yeast Plasmid Miniprep I (Zymo Research) Isopropanole High-Fidelity DNA Polymerase (e.g. Phusion) Thermocycler Gel equipment PCR purification and gel extraction kits (QiaGen) MmeI (New England Biolabs): MmeI restriction enzyme, NEB Cut Smart Buffer, 1 mM SAM T4 Ligase Primers and oligos .. Dilute cells to OD600 of 0.05 in SD + CAA and grow for 8 h at 30 °C.

    Gel Purification:

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
    Article Snippet: .. Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification. .. Prepare Illumina P5 and P7 adapters by annealing overhang NN-nucleotides P5: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN-3′ and 5′-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3′; P7: 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′ and 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCNN-3′, respectively.

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    New England Biolabs mmei
    Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The <t>DNA</t> is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by <t>MmeI</t> to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.
    Mmei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The DNA is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by MmeI to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.

    Journal: Nucleic Acids Research

    Article Title: Primer Extension Enrichment Reaction (PEER): a new subtraction method for identification of genetic differences between biological specimens

    doi: 10.1093/nar/gkl391

    Figure Lengend Snippet: Primer Extension Enrichment Reaction (PEER). ( A ) Generation of dsDNA from total Nucleic Acid. (1) Tester NA (white and gray rectangle) is split in two aliquots and denatured; Driver NA (white rectangle) is denatured as well. (2) Single strands are reverse transcribed (RT) by Super Script RT with three different primers—AFMmeIN6 * for the first Tester aliquot, T2N6 (diagonal fill rectangle) for the second aliquot and D0N6 (red rectangle) for the Driver. (3) The reverse transcriptase switches templates and copies the annealed SMART primers (SMART technology, Clontech). (4) The RT products are amplified with Advantage2 Polymerase to yield Tester1 dsDNA with primers AMmeIPCR (black rectangle), Tester23 dsDNA with T3PCR (vertical fill rectangle) and T2PCR (diagonal fill rectangle) and Driver bio-dsDNA with D0bioPCR biotinylated at the 5′ end (red rectangle with red circle). ( B ) Processing of Tester1 dsDNA. (1) The DNA is cleaved by a cocktail of restriction enzymes that leave 3′ GC protruding ends. (2) The ends are treated with the Klenow fragment of DNA Polymerase I in the presence of dCTP only and then ligated to AMmeIAdapter. (3) The tagged fragments are cut to uniform size by MmeI to create multiple AMmeIPrimers. ( C ) Blocking reaction. (1) AMmeIPrimers generated from Tester1 dsDNA are extended on Driver bio-dsDNA template in the presence of biotinylated ddNTPs (red circles) and ThermoSequenase™. (2) Biotinylated molecules are captured with streptavidin-coated magnetic beads (white crescent with gray bar) and removed from the reaction. ( D ) Retrieval of targets of interest from the Tester23 dsDNA. (1) Capture PCR—AMmeIPrimers that were not blocked and removed in the preceding steps are added to Tester23 dsDNA and in the presence of regular dNTP are annealed and extended to capture the targets of interest. (2) Regular PCR amplification of the capture products with different primer combinations. Black rectangles, primers AFMmeIN6, AFMmeISMART, AMmeIPCR, AMmeIAdapter.

    Article Snippet: MmeI digestion : The ligation products are digested with 5 U MmeI (NEB) for 2 h. The cleaved DNA is resolved in 10% polyacrylamide gel, the resulting 50 bp fragment is cut out, isolated from the gel with QIAquick gel extraction kit (Qiagen) and resuspended in 50 μl of 10 mM Tris (pH 8).

    Techniques: Amplification, Blocking Assay, Generated, Magnetic Beads, Polymerase Chain Reaction

    Proof-of-principle experiment of RLL-Y2H system. ( A ) Positive control interacting protein pair, murine p53 and SV40 T-antigen were inserted into mBD and mAD of RLL-Y2H system and were transformed into yeast respectively. Only the yeasts containing both p53 and SV40 T-antigen but not the controls can grow on SD/-AHL T selection plates. ( B ) After plasmid extraction from yeast, p53, SV40 T-antigen and their recombinated fragments were amplified by PCR. ( C ) Sanger sequencing of the recombinated p53 and SV40 T-antigen fragments. The linker ATTL and MmeI sites were highlighted. ( D ) The PCR products of the recombinated p53 and SV40 T-antigen fragments before (left panel) and after (right panel) MmeI digestion.

    Journal: Nucleic Acids Research

    Article Title: Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system

    doi: 10.1093/nar/gkx1173

    Figure Lengend Snippet: Proof-of-principle experiment of RLL-Y2H system. ( A ) Positive control interacting protein pair, murine p53 and SV40 T-antigen were inserted into mBD and mAD of RLL-Y2H system and were transformed into yeast respectively. Only the yeasts containing both p53 and SV40 T-antigen but not the controls can grow on SD/-AHL T selection plates. ( B ) After plasmid extraction from yeast, p53, SV40 T-antigen and their recombinated fragments were amplified by PCR. ( C ) Sanger sequencing of the recombinated p53 and SV40 T-antigen fragments. The linker ATTL and MmeI sites were highlighted. ( D ) The PCR products of the recombinated p53 and SV40 T-antigen fragments before (left panel) and after (right panel) MmeI digestion.

    Article Snippet: Digest the purified PCR products with MmeI (NEB, CA) which yields a ∼110 bp target band, followed by low-melting gel purification.

    Techniques: Positive Control, Transformation Assay, Selection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Sequencing

    Schematic of DALEC . Brown and green represent priming regions for Solexa bridge amplification primers. Blue indicates the primer binding region for the Solexa sequencing reaction. TCCGAC is the Mme I recognition sequence and the sequence immediately upstream of it (black) is the variable region. The inset gives detailed information for linkers A and B.

    Journal: BMC Genomics

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    doi: 10.1186/1471-2164-11-465

    Figure Lengend Snippet: Schematic of DALEC . Brown and green represent priming regions for Solexa bridge amplification primers. Blue indicates the primer binding region for the Solexa sequencing reaction. TCCGAC is the Mme I recognition sequence and the sequence immediately upstream of it (black) is the variable region. The inset gives detailed information for linkers A and B.

    Article Snippet: Ligation to Linker B Linker B was purchased as two separate oligonucleotides (5'P-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTCGGTGGTCGCCGTATCATT-OH3', 5'OH-TCATCTTTCCCTACACGACGCTCTTCCGATCTNN-OH3') and hybridized using the same procedure as described for Linker A. Mme I products were ligated to Linker B using the following 50 μl reaction mix: 10.0 μl Mme I product, 1.0 μl of 0.05 mM Linker B, 5.0 μl 10× ligase buffer, 3.0 μl T4 DNA ligase (2,000 U/μl; NEB #M0202), 31.0 μl dH2 O. Ligations were performed overnight using a PCR machine.

    Techniques: Amplification, Binding Assay, Sequencing