asei  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Mutations in the MutS? interaction interface of MLH1 can abolish DNA mismatch repair

doi: 10.1093/nar/gkl944

Figure Lengend Snippet: Analysis of MLH1 mutants for MMR activity and interaction with MutSα–DNA. ( A ) Nineteen MLH1 missense mutants were co-expressed with PMS2 in parallel to wild-type MutLα in HEK293T cells and in vitro mismatch repair activity was assessed as described in Figure 1 and Materials and Methods. AseI digestion of the mismatched test plasmid yielded linearized vector (L), and successful repair additionally produced two fragments of lower molecular weight (D, digestion fragments). Repair activity of each MutLα mutant was assessed in direct comparison to wild-type MutLα which was expressed, processed and analyzed in parallel, and repair levels were quantified in relation to these wild-type preparations. These levels (wildtype = 1.0) are shown; n.d., no detectable repair activity. ( B–F ) The interaction of MLH1 H112D , MLH1 H112A , MLH1 R127E and MLH1 Y130H/A/F with MutSα–DNA was assessed in comparison to MLH1 wt identically as described in Figure 1. ( G ) The ATPase activity of purified GST–MutLα mutants was assessed in comparison to wildtype as detailed in Materials and Methods. Three individual experiments were performed for each protein preparation and for a mock sample without protein. Values of the mock samples were subtracted from the protein samples. The block diagrams show the average ATPase activity, with error bars giving the standard deviations.

Article Snippet: Restriction enzymes N.BstNBI, N.AlwI and AseI as well as T4 DNA ligase were from New England Biolabs (Ipswich, MA, USA).

Techniques: Activity Assay, In Vitro, Plasmid Preparation, Produced, Molecular Weight, Mutagenesis, Purification, Blocking Assay

Journal: Nucleic Acids Research

Article Title: The BCL2 gene is regulated by a special AT-rich sequence binding protein 1-mediated long range chromosomal interaction between the promoter and the distal element located within the 3?-UTR

doi: 10.1093/nar/gkr023

Figure Lengend Snippet: 3C Analysis of the interaction between the mbr and the BCL2 promoter region in vivo . The scheme of BCL2 gene with the positions of restriction sites and positions of primers was indicated ( A ). The 3C experiments were performed as described in the ‘Materials and Methods’ section. Briefly, formaldehyde was used to crosslink protein–DNA interactions in intact nuclei. The crosslinked chromatin was then digested by AseI ( B ) or HindIII ( C ), followed by ligation. Physical interaction between mbr and SBS1 or SBS2 was then determined by specific PCRs that detected hybrid fragments containing either mbr/SBS1 sequences or mbr/SBS2 sequences. 3C data demonstrated that the mbr specifically interacted with SBS1 (B, up panel), but not with SBS2 (C, up panel). PCR products from AseI (B) or HindIII (C) digested crosslinked chromatin without ligation and non-crosslinked genomic DNA with or without ligation were used as negative controls. The bands shown in the bottom panels of B and C represented the PCR products from genomic DNA that was not cut by any restriction enzyme, which were used as the loading control.

Article Snippet: Hundred units of the restriction enzyme (NEB) (AseI for SBS1-mbr, HindIII for SBS2/mbr) were added for a 22-h digestion.

Techniques: In Vivo, Ligation, Polymerase Chain Reaction

Journal: Journal of Clinical Microbiology

Article Title: Detection of Cryptosporidium and Identification to the Species Level by Nested PCR and Restriction Fragment Length Polymorphism

doi: 10.1128/JCM.43.3.1017-1023.2005

Figure Lengend Snippet: Comparative restriction patterns of C. hominis (A), C. parvum (B), C. felis (C), and C. meleagridis (D) obtained with the following enzymes: TaqI (lanes 1), AseI (lanes 2), MseI (lanes 3), BstUI (lanes 4), and SspI (lanes 5). Lanes 6, undigested C. hominis

Article Snippet: The enzymes used were TaqI (Roche-Bohringer, Mannheim, Germany), AseI, MseI, BstUI, and SspI (New England Biolabs, Beverly, Mass.)

Techniques:

Journal: Pathogens

Article Title: High Frequency of Cryptosporidium hominis Infecting Infants Points to A Potential Anthroponotic Transmission in Maputo, Mozambique

doi: 10.3390/pathogens10030293

Figure Lengend Snippet: Genotyping of the Cryptosporidium parasites by PCR-RFLP targeting 18S rRNA gene. M, molecular size makers (100 bp). Lane 1: C. parvum ; Lanes 2, 3 and 4: C. hominis and Lane 5: mixed infection with C. hominis and C. parvum . The upper lanes show SspI digestion products showing a molecular size from 111 bp to 449 bp, and the lower lanes show AseI digestion products with molecular size of approximately 104 bp to 628 bp.

Article Snippet: Genotype identification was made through the analysis of pattern of the secondary product after restriction digestion with the enzymes SspI and AseI (New England BioLabs Inc., Beverly, MA, USA) as described by Xiao et al. [ ].

Techniques: Polymerase Chain Reaction, Infection