Restriction enzyme analysis

Mutation screening methods involve testing for specific (previously selected) mutations in a gene. This approach is relatively inexpensive and may be useful for disorders that are caused by one or few common mutations. It is important to take the origin of the patient into consideration, since the frequency of mutations differs markedly between populations. As an example for such a method we discuss restriction enzyme analysis in more detail in this chapter. 

Restriction enzyme analysis (the reverse of the process of insertion and ligation) is suitable, although this requires some knowledge of the size and the restriction pattern of the foreign DNA. (See discussion of restriction fragment length polymorphisms in Section 15.6.3.)... 

For one patient, a new mutation was suspected but the regular screening methods (IEF and restriction enzyme analysis of most of exons 2, 3, and 4) failed to identify the mutation. ESI and MALDI MS were utilized to characterize the novel variant. 

Restriction-enzyme analysis Sections 6.1.1 and 6.1.2 Southern and Northern blotting techniques Section 6.1.2... 

Restriction-enzyme analysis. Restriction enzymes are precise, molecular scalpels that allow the investigator to manipulate DNA segments. 

A PCR restriction enzyme analysis takes advantage of the occurrence of approximately 40 thalassemia mutations that either introduce or remove a restriction endonuclease site. The PCR amplified target sequence is digested using restriction enzymes (enzymes that cleave DNA at particular nucleotide sequences) and the pattern of fragmentation on an agarose gel defines the presence or absence of a particular mutation. 

A PCR restriction enzyme analysis takes advantage of the occurrence of approximately 40 P-thalassemia mutations that either introduce or remove a restriction endonuclease site. 

Manzano M, CocoUn L, Cantoni C, Comi G (1998) A rapid method for the identification and partial serotyping of Listeria monocytogenes in food by PCR and restriction enzyme analysis. Int J Food Microbiol 42 207-212... 

Kopecka H., Macaya G., Cortadas J., Thiery J.P., Bernard G. (1978). Restriction enzyme analysis of satellite DNA components from the bovine genome. Eur. J. Biochem. 84 189-195. 

Lewin A., Morimoto R., Rabinowitz M. (1978). Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast classification of petites, and deletion mapping of mitochondrial genes. Mol. Gen. Genet. 163 257-275. 

Harmon KS, McKay LL (1987) Restriction enzyme analysis of lactose and bacteriocin plasmids from Streptococcus lactis subsp. diacetylactis WM4 and cloning of Belli fragments coding for bacteriocin production. Appl Environ Microbiol 53 1171-1174... 

Two clones, carrying a 5.2 kb insert in inverse orientations were isolated and mapped by restriction enzyme analysis. The two clones were used to construct deletion clones with the exonuclease Ill/mung bean nuclease system. Deletion clones containing the psbG homologons region were sequenced. 

Comi, G., Maifreni, M., Manzano, M., Lagazio, C., CocoUn, L. (2000). Mitochondrial DNA restriction enzyme analysis and evaluation of the enological characteristics of Saccharomyces cerevisiae strains isolated from grapes of the wine-producing area of CoUio (Italy). International Journal of Food Microbiology, 58, 117-121. 

Fig. 2. Restriction enzyme analysis and Southern-blot hybridization of human tumor DNA. Human tumor DNA (20 p-g) was isolated (16) and digested with coRI restriction enzyme, electrophoresed on a 0.8% agarose gel (A shows the ethidium-bromide stain) and after Southern transfer, probed with P-labeled pcD-p(ADP-ribose) polymerase cDNA, (B). Lanes 1, Human placental DNA 2, Laryngeal squamous ceU carcinoma (SQ-20B) 3, Lung adenocarcinoma (A549) 4, Cervical carcinoma (HeLaSs) Ewing s sarcoma (A4573) 6, normal human fibroblasts (NHF). Marker-lambda-DNA.

Rees A, Stocks J, Shoulders C, Carlson LA, Baralle FE, Galton DJ (1984) Restriction enzyme analysis of the apolipoprotein A-I gene in Fish Eye Disease and Tangier Disease. Acta Med Scand 215 235-237... 

Dlauchy, D., j. Tornai-Lehoczli, and G. Peter. 1999. Restriction enzyme analysis of PGR amplified rDNA as a taxonomic tool in yeast identification. Sys. Appl. Microbiol. 22 445-453. 

Because the use of PCR very often results in the introduction of unwanted mutations, it was decided to construct die whole P putida pepK gene from these two clones without PCR. First, the insert fragments of both recombinant clones were isolated and fused in vector pGEM5Zf(+) (Promega). A plasmid with the two insert fragments in correct order and orientation was then identified by restriction enzyme analysis. This plasmid contains the complete P. putida pepA gene without any mutation. 

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