Restriction digestion

The extraordinary complexity of human genes and their products has encouraged the development of extremely high-resolution analytical methods.75 Capillary electrophoresis is competitive with slab gel methods, with resolution up to the order of about 1,000 base pairs for sequencing, sizing, and detection of mutation. Reversed phase HPLC is useful for restriction digest mapping and MALDI-MS up to about 1000 base pairs. 

Saperstein, D. and Nickerson, J.M. (1991) Restriction fragment length polymorphism analysis using PCR coupled to restriction digests. BioTechniques 10, 488-489. 

The first step in creating a specific mRNA species is to construct a plasmid encoding the mRNA behind a T7, T3, or SP6 RNA polymerase promoter sequence. The S boundary of the mRNA is defined by the position of the promoter transcription start site and the S end is defined by restriction digest of the plasmid prior to in vitro transcription. 

Prepare 1 pg of purified DNA probe, either by restriction digestion or by synthetic means. 

A RESTRICTION MAP is used to identify and locate specific restriction sites on a given piece of DNA. The size of a fragment is determined by running the restriction digest on an agarose gel. Fragments separate by size—the smaller ones move farther toward the bottom of the gel. 

We set up a restriction digest using 10 pg of DNA in a final reaction volume of approximately 10 pL. The reaction is mixed and incubated in a 37°C water bath for 1 h (temperature and incubation time may vary with type of enzyme used check supplier s protocol). 

Perform restriction digests and sequencing to identity chimeras... 

Southern blot analysis is used to confirm the PCR results. The genomic DNA is isolated from PCR-positive ES cell clones. With the choice of restriction digest and probes for hybridization, the wild-type allele can readily be distinguished from the targeted allele since predicted novel restriction fragments are generated by the homologous recombination event. 

The presence of PCR primers in restriction digests, which inhibit the restriction enzyme. 

Layer the DNA solution from the restriction digest on top of the sucrose gradient. Fill the tube with paraffin to approximately 3 mm below the rim. Balance with paraffin. 

Matejusova, I., Koubkova, B. and Cunningham, C.O. (2004) Identification of European diplozoids (Diplozoidae, Monogenea) by restriction digestion of the ribosomal RNA internal transcribed spacer (ITS2). Journal of Parasitology 90, 817-822. 

Hybridization analysis using immobilized DNA includes dot blots and slot blots. Dot and slot blots are named for the circular and slotted well shapes in the templates used to present test samples to a membrane surface. They eliminate the need for restriction digestion and electrophoretic resolution steps by depositing samples of DNA to be tested directly onto a hybridizable membrane surface. Dot blot methodology is faster and easier for hybridization screening than Southern blotting, especially when many samples are to be screened... 

---