ahdi  (New England Biolabs)

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

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

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

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