Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (3)
Articles by Theodore Davis in JoVE
JoVE Neuroscience
High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue
Applications and Product Development, New England Biolabs
The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing β-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.
Other articles by Theodore Davis on PubMed
Efficient Isolation of Targeted Caenorhabditis Elegans Deletion Strains Using Highly Thermostable Restriction Endonucleases and PCR
Reverse genetic approaches to understanding gene function would be greatly facilitated by increasing the efficiency of methods for isolating mutants without the reliance on a predicted phenotype. Established PCR-based methods of isolating deletion mutants are widely used for this purpose in Caenorhabditis elegans. However, these methods are inefficient at isolating small deletions. We report here a novel modification of PCR-based methods, employing thermostable restriction enzymes to block the synthesis of wild-type PCR product, so that only the deletion PCR product is amplified. This modification greatly increases the efficiency of isolating small targeted deletions in C.elegans. Using this method six new deletion strains were isolated from a small screen of approximately 400 000 haploid genomes, most with deletions <1.0 kb. Greater PCR detection sensitivity by this modification permitted approximately 10-fold greater pooling of DNA samples, reducing the effort and reagents required for screens. In addition, effective suppression of non-specific amplification allowed multiplexing with several independent primer pairs. The increased efficiency of this technique makes it more practical for small laboratories to undertake gene knock-out screens.
MmeI: a Minimal Type II Restriction-modification System That Only Modifies One DNA Strand for Host Protection
MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5'-TCCRAC-3'. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5'-GTYGGA-3'. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.
The MspJI Family of Modification-dependent Restriction Endonucleases for Epigenetic Studies
MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N(12)/N(16)) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers (m)CWG but not (m)CpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies.
