ahdi  (New England Biolabs)


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    Name:
    AhdI
    Description:
    AhdI 5 000 units
    Catalog Number:
    r0584l
    Price:
    302
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    Structured Review

    New England Biolabs ahdi
    AhdI
    AhdI 5 000 units
    https://www.bioz.com/result/ahdi/product/New England Biolabs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ahdi - by Bioz Stars, 2021-03
    92/100 stars

    Images

    1) Product Images from "Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia"

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2017.107

    Restriction enzyme digest analysis of rs77355432 SNP in cDNA from family 1; expected fragment sizes reference allele 249 bp (WT), alternative allele 215+34 bp (dupC). Lane 1, uncut DNA. Lane 2, cDNA from the proband is not digested with AhdI. Lane 3, cDNA of the father is cut almost to completion with AhdI. Lane 4, cDNA of the mother is uncut with AhdI. Lane 5, negative control.
    Figure Legend Snippet: Restriction enzyme digest analysis of rs77355432 SNP in cDNA from family 1; expected fragment sizes reference allele 249 bp (WT), alternative allele 215+34 bp (dupC). Lane 1, uncut DNA. Lane 2, cDNA from the proband is not digested with AhdI. Lane 3, cDNA of the father is cut almost to completion with AhdI. Lane 4, cDNA of the mother is uncut with AhdI. Lane 5, negative control.

    Techniques Used: Negative Control

    2) Product Images from "DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells"

    Article Title: DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt804

    Human DHX9 recognizes DNA secondary structures in plasmids. ( A ), representative agarose gel of CC pMEXr (lanes 1–5) and pCEX (lanes 6–10) incubated with 30 nM purified recombinant human DHX9 protein (lanes 3–5 and 8–10) in the presence of 5 mM ATP (lanes 3 and 4, and 8 and 9) or 5 mM AMP-PNP (lanes 5 and 10) followed by MBN cleavage. Lane M, 1 kb DNA marker. ( B ), plot of net percentage of OC and linear ( L ) DNA released from total DNA ( OC + L + CC ) ( CC = closed circular DNA) at the end of the reaction from two experiments, as described in Panel (A). The net percentage was defined as F = f s – f c , were f s was the %[( OC + L )/( OC + L + CC )] for each sample, and f c was the average %[( OC + L )/( OC + L + CC )] for the two samples without MBN (i.e. lanes 1, 3 and 6, 8, respectively). ( C ) , schematic of the diagnostic restriction sites (AhdI and EcoO109I) used to map MBN-specific cleavage in pMEXr. Grey arrows, lengths of restriction fragments released from pCEX and pMEXr when cleaved by AhdI, EcoO109I and EcoRI, which are located several bp from the cloned inserts; C/H, position of the control (C) and H-DNA-forming (H) inserts in pCEX and pMEXr, respectively. ( D ), MBN cleavage mapping. PhosphorImager scan of an agarose gel after electrophoresis of pCEX (lanes 1–4) and pMEXr (lanes 5–8) pre-incubated with 30 nM DHX9 (lanes 2 and 3, and 6 and 7) in the presence of 5 mM ATP, treated with 40 units MBN (lanes 3 and 4, and 7 and 8), end-labeled with T4 DNA polymerase and cleaved with Eco0109I and AhdI. Lane E, control lane containing pMEXr cleaved with EcoRI, labeled with T4 DNA polymerase, and then cleaved with AhdI and EcoO109I; E* , ethidium bromide staining of lane E.
    Figure Legend Snippet: Human DHX9 recognizes DNA secondary structures in plasmids. ( A ), representative agarose gel of CC pMEXr (lanes 1–5) and pCEX (lanes 6–10) incubated with 30 nM purified recombinant human DHX9 protein (lanes 3–5 and 8–10) in the presence of 5 mM ATP (lanes 3 and 4, and 8 and 9) or 5 mM AMP-PNP (lanes 5 and 10) followed by MBN cleavage. Lane M, 1 kb DNA marker. ( B ), plot of net percentage of OC and linear ( L ) DNA released from total DNA ( OC + L + CC ) ( CC = closed circular DNA) at the end of the reaction from two experiments, as described in Panel (A). The net percentage was defined as F = f s – f c , were f s was the %[( OC + L )/( OC + L + CC )] for each sample, and f c was the average %[( OC + L )/( OC + L + CC )] for the two samples without MBN (i.e. lanes 1, 3 and 6, 8, respectively). ( C ) , schematic of the diagnostic restriction sites (AhdI and EcoO109I) used to map MBN-specific cleavage in pMEXr. Grey arrows, lengths of restriction fragments released from pCEX and pMEXr when cleaved by AhdI, EcoO109I and EcoRI, which are located several bp from the cloned inserts; C/H, position of the control (C) and H-DNA-forming (H) inserts in pCEX and pMEXr, respectively. ( D ), MBN cleavage mapping. PhosphorImager scan of an agarose gel after electrophoresis of pCEX (lanes 1–4) and pMEXr (lanes 5–8) pre-incubated with 30 nM DHX9 (lanes 2 and 3, and 6 and 7) in the presence of 5 mM ATP, treated with 40 units MBN (lanes 3 and 4, and 7 and 8), end-labeled with T4 DNA polymerase and cleaved with Eco0109I and AhdI. Lane E, control lane containing pMEXr cleaved with EcoRI, labeled with T4 DNA polymerase, and then cleaved with AhdI and EcoO109I; E* , ethidium bromide staining of lane E.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Purification, Recombinant, Marker, Diagnostic Assay, Clone Assay, Electrophoresis, Labeling, Staining

    3) Product Images from "Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome"

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

    Journal: Nucleic Acids Research

    doi:

    Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).
    Figure Legend Snippet: Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).

    Techniques Used: Ligation, Polymerase Chain Reaction, Construct

    Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.
    Figure Legend Snippet: Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Techniques Used: Flow Cytometry, Mutagenesis, Polymerase Chain Reaction, Isolation

    4) Product Images from "Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia"

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia

    Journal: European Journal of Human Genetics

    doi: 10.1038/ejhg.2017.107

    Restriction enzyme digest analysis of rs77355432 SNP in cDNA from family 1; expected fragment sizes reference allele 249 bp (WT), alternative allele 215+34 bp (dupC). Lane 1, uncut DNA. Lane 2, cDNA from the proband is not digested with AhdI. Lane 3, cDNA of the father is cut almost to completion with AhdI. Lane 4, cDNA of the mother is uncut with AhdI. Lane 5, negative control.
    Figure Legend Snippet: Restriction enzyme digest analysis of rs77355432 SNP in cDNA from family 1; expected fragment sizes reference allele 249 bp (WT), alternative allele 215+34 bp (dupC). Lane 1, uncut DNA. Lane 2, cDNA from the proband is not digested with AhdI. Lane 3, cDNA of the father is cut almost to completion with AhdI. Lane 4, cDNA of the mother is uncut with AhdI. Lane 5, negative control.

    Techniques Used: Negative Control

    Related Articles

    DNA Synthesis:

    Article Title: DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells
    Article Snippet: In the absence of dNTPs, this protocol activated the endonuclease activity of the polymerase as to degrade ∼1.5–2 kb of DNA from any DSBs induced by MBN. .. Two microliters of solution containing 1 mM each of dATP/dGTP/dTTP and dCTP-[α−32 P] (6000 mCi/mmol, PerkinElmer, Waltham, MA, USA) was added, and DNA synthesis was allowed to proceed at 12°C for 30 min. After ethanol precipitation, plasmid DNA was cleaved with Eco0109I and AhdI (New England Biolabs, Beverly, MA, USA), further purified by additional ethanol precipitations and electrophoresed on 1% agarose gels. .. The gels were first stained with ethidium bromide, dried and then exposed to a PhosphorImager (Typhoon FLA 9000, http://www.ge.com ) for visualization.

    Ethanol Precipitation:

    Article Title: DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells
    Article Snippet: In the absence of dNTPs, this protocol activated the endonuclease activity of the polymerase as to degrade ∼1.5–2 kb of DNA from any DSBs induced by MBN. .. Two microliters of solution containing 1 mM each of dATP/dGTP/dTTP and dCTP-[α−32 P] (6000 mCi/mmol, PerkinElmer, Waltham, MA, USA) was added, and DNA synthesis was allowed to proceed at 12°C for 30 min. After ethanol precipitation, plasmid DNA was cleaved with Eco0109I and AhdI (New England Biolabs, Beverly, MA, USA), further purified by additional ethanol precipitations and electrophoresed on 1% agarose gels. .. The gels were first stained with ethidium bromide, dried and then exposed to a PhosphorImager (Typhoon FLA 9000, http://www.ge.com ) for visualization.

    Plasmid Preparation:

    Article Title: DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells
    Article Snippet: In the absence of dNTPs, this protocol activated the endonuclease activity of the polymerase as to degrade ∼1.5–2 kb of DNA from any DSBs induced by MBN. .. Two microliters of solution containing 1 mM each of dATP/dGTP/dTTP and dCTP-[α−32 P] (6000 mCi/mmol, PerkinElmer, Waltham, MA, USA) was added, and DNA synthesis was allowed to proceed at 12°C for 30 min. After ethanol precipitation, plasmid DNA was cleaved with Eco0109I and AhdI (New England Biolabs, Beverly, MA, USA), further purified by additional ethanol precipitations and electrophoresed on 1% agarose gels. .. The gels were first stained with ethidium bromide, dried and then exposed to a PhosphorImager (Typhoon FLA 9000, http://www.ge.com ) for visualization.

    Article Title: Systematic Assembly and Genetic Manipulation of the Mouse Hepatitis Virus A59 Genome
    Article Snippet: Incubate at 37°C for 1 h. Add 1 μl Bsm BI and incubate at 55°C for an additional 1 h. pSMART-MHV-D: 10 μl plasmid, 1 μl Bsm BI, 1 μl BSA, 2 μl NEB Buffer 4, 5 μl water. .. Incubate at 55°C for 1 h. Add 1 μl Ahd I and incubate at 37°C for an additional 1 h. pSMART-MHV-E: 10 μl plasmid, 1 μl Bsm BI, 2 μl NEB Buffer 3, 7 μl water. .. Incubate at 55°C for 1 h. pSMART-MHV-F: 10 μl plasmid, 1 μl Bsm BI, 2 μl NEB Buffer 3, 7 μl water.

    Article Title: Systematic Assembly and Genetic Manipulation of the Mouse Hepatitis Virus A59 Genome
    Article Snippet: Incubate at 37°C for 1.5 h. Add 10 μl Bsm BI and incubate at 55°C for an additional 2 h. pSMART-MHV-D: 150 μl plasmid, 10 μl Bsm BI, 2 μl BSA, 20 μl NEB Buffer 4, 8 μl water. .. Incubate at 55°C for 2 h. Add 10 μl Ahd I and incubate at 37°C for an additional 1.5 h. Ahd I cuts the vector into two fragments, which allows the MHV-D fragment to be the largest band. pSMART-MHV-E: 150 μl plasmid, 10 μl BsmBI, 20 μl NEB Buffer 3, 20 μl water. .. Incubate at 55°C for 2 h. pSMART-MHV-F: 150 μl plasmid, 10 μl BsmBI, 20 μl NEB Buffer 3, 20 μl water.

    Purification:

    Article Title: DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells
    Article Snippet: In the absence of dNTPs, this protocol activated the endonuclease activity of the polymerase as to degrade ∼1.5–2 kb of DNA from any DSBs induced by MBN. .. Two microliters of solution containing 1 mM each of dATP/dGTP/dTTP and dCTP-[α−32 P] (6000 mCi/mmol, PerkinElmer, Waltham, MA, USA) was added, and DNA synthesis was allowed to proceed at 12°C for 30 min. After ethanol precipitation, plasmid DNA was cleaved with Eco0109I and AhdI (New England Biolabs, Beverly, MA, USA), further purified by additional ethanol precipitations and electrophoresed on 1% agarose gels. .. The gels were first stained with ethidium bromide, dried and then exposed to a PhosphorImager (Typhoon FLA 9000, http://www.ge.com ) for visualization.

    Electrophoresis:

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia
    Article Snippet: Primers were designed for all exons and intron two to screen for truncations and intron retention ( ). cDNA was amplified and products were electrophoresed on agarose gels and DNA was visualized by staining with ethidium bromide (EtBr). .. Digestion with PshAI and AhdI and electrophoresis of digests To assess the effect of the paternal variant on RNA expression in family 1, cDNA amplification products were digested with PshAI or AhdI (New England Biolabs Inc., Ipswich, MA, USA) and analyzed by agarose gel electrophoresis. ..

    Variant Assay:

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia
    Article Snippet: Primers were designed for all exons and intron two to screen for truncations and intron retention ( ). cDNA was amplified and products were electrophoresed on agarose gels and DNA was visualized by staining with ethidium bromide (EtBr). .. Digestion with PshAI and AhdI and electrophoresis of digests To assess the effect of the paternal variant on RNA expression in family 1, cDNA amplification products were digested with PshAI or AhdI (New England Biolabs Inc., Ipswich, MA, USA) and analyzed by agarose gel electrophoresis. ..

    RNA Expression:

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia
    Article Snippet: Primers were designed for all exons and intron two to screen for truncations and intron retention ( ). cDNA was amplified and products were electrophoresed on agarose gels and DNA was visualized by staining with ethidium bromide (EtBr). .. Digestion with PshAI and AhdI and electrophoresis of digests To assess the effect of the paternal variant on RNA expression in family 1, cDNA amplification products were digested with PshAI or AhdI (New England Biolabs Inc., Ipswich, MA, USA) and analyzed by agarose gel electrophoresis. ..

    Amplification:

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia
    Article Snippet: Primers were designed for all exons and intron two to screen for truncations and intron retention ( ). cDNA was amplified and products were electrophoresed on agarose gels and DNA was visualized by staining with ethidium bromide (EtBr). .. Digestion with PshAI and AhdI and electrophoresis of digests To assess the effect of the paternal variant on RNA expression in family 1, cDNA amplification products were digested with PshAI or AhdI (New England Biolabs Inc., Ipswich, MA, USA) and analyzed by agarose gel electrophoresis. ..

    Agarose Gel Electrophoresis:

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia
    Article Snippet: Primers were designed for all exons and intron two to screen for truncations and intron retention ( ). cDNA was amplified and products were electrophoresed on agarose gels and DNA was visualized by staining with ethidium bromide (EtBr). .. Digestion with PshAI and AhdI and electrophoresis of digests To assess the effect of the paternal variant on RNA expression in family 1, cDNA amplification products were digested with PshAI or AhdI (New England Biolabs Inc., Ipswich, MA, USA) and analyzed by agarose gel electrophoresis. ..

    Transfection:

    Article Title: Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2
    Article Snippet: All SETX inserts were fully sequenced after subcloning into pDEST26 to confirm all wild-type and mutant sequences. .. For transfection, pDEST- SETX plasmids were linearized with AhdI (New England BioLabs) and introduced using an Amaxa Nucleofector (Lonza) per the manufacturer's instructions. .. Positive selection was performed using 200 μg/ml G418 (Invitrogen, Life Technologies).

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    New England Biolabs ahdi
    Restriction enzyme digest analysis of rs77355432 SNP in <t>cDNA</t> from family 1; expected fragment sizes reference allele 249 bp (WT), alternative allele 215+34 bp (dupC). Lane 1, uncut DNA. Lane 2, cDNA from the proband is not digested with <t>AhdI.</t> Lane 3, cDNA of the father is cut almost to completion with AhdI. Lane 4, cDNA of the mother is uncut with AhdI. Lane 5, negative control.
    Ahdi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ahdi/product/New England Biolabs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ahdi - by Bioz Stars, 2021-03
    92/100 stars
      Buy from Supplier

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    Restriction enzyme digest analysis of rs77355432 SNP in cDNA from family 1; expected fragment sizes reference allele 249 bp (WT), alternative allele 215+34 bp (dupC). Lane 1, uncut DNA. Lane 2, cDNA from the proband is not digested with AhdI. Lane 3, cDNA of the father is cut almost to completion with AhdI. Lane 4, cDNA of the mother is uncut with AhdI. Lane 5, negative control.

    Journal: European Journal of Human Genetics

    Article Title: Identification of causative variants in TXNL4A in Burn-McKeown syndrome and isolated choanal atresia

    doi: 10.1038/ejhg.2017.107

    Figure Lengend Snippet: Restriction enzyme digest analysis of rs77355432 SNP in cDNA from family 1; expected fragment sizes reference allele 249 bp (WT), alternative allele 215+34 bp (dupC). Lane 1, uncut DNA. Lane 2, cDNA from the proband is not digested with AhdI. Lane 3, cDNA of the father is cut almost to completion with AhdI. Lane 4, cDNA of the mother is uncut with AhdI. Lane 5, negative control.

    Article Snippet: Digestion with PshAI and AhdI and electrophoresis of digests To assess the effect of the paternal variant on RNA expression in family 1, cDNA amplification products were digested with PshAI or AhdI (New England Biolabs Inc., Ipswich, MA, USA) and analyzed by agarose gel electrophoresis.

    Techniques: Negative Control

    Human DHX9 recognizes DNA secondary structures in plasmids. ( A ), representative agarose gel of CC pMEXr (lanes 1–5) and pCEX (lanes 6–10) incubated with 30 nM purified recombinant human DHX9 protein (lanes 3–5 and 8–10) in the presence of 5 mM ATP (lanes 3 and 4, and 8 and 9) or 5 mM AMP-PNP (lanes 5 and 10) followed by MBN cleavage. Lane M, 1 kb DNA marker. ( B ), plot of net percentage of OC and linear ( L ) DNA released from total DNA ( OC + L + CC ) ( CC = closed circular DNA) at the end of the reaction from two experiments, as described in Panel (A). The net percentage was defined as F = f s – f c , were f s was the %[( OC + L )/( OC + L + CC )] for each sample, and f c was the average %[( OC + L )/( OC + L + CC )] for the two samples without MBN (i.e. lanes 1, 3 and 6, 8, respectively). ( C ) , schematic of the diagnostic restriction sites (AhdI and EcoO109I) used to map MBN-specific cleavage in pMEXr. Grey arrows, lengths of restriction fragments released from pCEX and pMEXr when cleaved by AhdI, EcoO109I and EcoRI, which are located several bp from the cloned inserts; C/H, position of the control (C) and H-DNA-forming (H) inserts in pCEX and pMEXr, respectively. ( D ), MBN cleavage mapping. PhosphorImager scan of an agarose gel after electrophoresis of pCEX (lanes 1–4) and pMEXr (lanes 5–8) pre-incubated with 30 nM DHX9 (lanes 2 and 3, and 6 and 7) in the presence of 5 mM ATP, treated with 40 units MBN (lanes 3 and 4, and 7 and 8), end-labeled with T4 DNA polymerase and cleaved with Eco0109I and AhdI. Lane E, control lane containing pMEXr cleaved with EcoRI, labeled with T4 DNA polymerase, and then cleaved with AhdI and EcoO109I; E* , ethidium bromide staining of lane E.

    Journal: Nucleic Acids Research

    Article Title: DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

    doi: 10.1093/nar/gkt804

    Figure Lengend Snippet: Human DHX9 recognizes DNA secondary structures in plasmids. ( A ), representative agarose gel of CC pMEXr (lanes 1–5) and pCEX (lanes 6–10) incubated with 30 nM purified recombinant human DHX9 protein (lanes 3–5 and 8–10) in the presence of 5 mM ATP (lanes 3 and 4, and 8 and 9) or 5 mM AMP-PNP (lanes 5 and 10) followed by MBN cleavage. Lane M, 1 kb DNA marker. ( B ), plot of net percentage of OC and linear ( L ) DNA released from total DNA ( OC + L + CC ) ( CC = closed circular DNA) at the end of the reaction from two experiments, as described in Panel (A). The net percentage was defined as F = f s – f c , were f s was the %[( OC + L )/( OC + L + CC )] for each sample, and f c was the average %[( OC + L )/( OC + L + CC )] for the two samples without MBN (i.e. lanes 1, 3 and 6, 8, respectively). ( C ) , schematic of the diagnostic restriction sites (AhdI and EcoO109I) used to map MBN-specific cleavage in pMEXr. Grey arrows, lengths of restriction fragments released from pCEX and pMEXr when cleaved by AhdI, EcoO109I and EcoRI, which are located several bp from the cloned inserts; C/H, position of the control (C) and H-DNA-forming (H) inserts in pCEX and pMEXr, respectively. ( D ), MBN cleavage mapping. PhosphorImager scan of an agarose gel after electrophoresis of pCEX (lanes 1–4) and pMEXr (lanes 5–8) pre-incubated with 30 nM DHX9 (lanes 2 and 3, and 6 and 7) in the presence of 5 mM ATP, treated with 40 units MBN (lanes 3 and 4, and 7 and 8), end-labeled with T4 DNA polymerase and cleaved with Eco0109I and AhdI. Lane E, control lane containing pMEXr cleaved with EcoRI, labeled with T4 DNA polymerase, and then cleaved with AhdI and EcoO109I; E* , ethidium bromide staining of lane E.

    Article Snippet: Two microliters of solution containing 1 mM each of dATP/dGTP/dTTP and dCTP-[α−32 P] (6000 mCi/mmol, PerkinElmer, Waltham, MA, USA) was added, and DNA synthesis was allowed to proceed at 12°C for 30 min. After ethanol precipitation, plasmid DNA was cleaved with Eco0109I and AhdI (New England Biolabs, Beverly, MA, USA), further purified by additional ethanol precipitations and electrophoresed on 1% agarose gels.

    Techniques: Agarose Gel Electrophoresis, Incubation, Purification, Recombinant, Marker, Diagnostic Assay, Clone Assay, Electrophoresis, Labeling, Staining

    Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).

    Journal: Nucleic Acids Research

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

    doi:

    Figure Lengend Snippet: Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).

    Article Snippet: To generate the HPRT target with the natural clamp suitable for CDCE analysis (Fig. ), restriction digestion by AhdI and HinfI (New England Biolabs) was performed with 1/20 of the target-renatured samples (i.e. ∼1.5 × 107 target copies).

    Techniques: Ligation, Polymerase Chain Reaction, Construct

    Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Journal: Nucleic Acids Research

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

    doi:

    Figure Lengend Snippet: Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Article Snippet: To generate the HPRT target with the natural clamp suitable for CDCE analysis (Fig. ), restriction digestion by AhdI and HinfI (New England Biolabs) was performed with 1/20 of the target-renatured samples (i.e. ∼1.5 × 107 target copies).

    Techniques: Flow Cytometry, Mutagenesis, Polymerase Chain Reaction, Isolation