Restriction endonuclease fragment analysis

The study of catabolic plasmids involves analysis of both structural and regulatory genes and the enzymatic activities of the gene products. Catabolic plasmids that specify similar enzymatic activities often have similar restriction endonuclease fragmentation patterns, and regions of homologous DNA. 

FIGURE 11.33 Restricdon mapping of a DNA molecule as determined by an analysis of the electrophoretic pattern obtained for different restriction endonuclease digests. (Keep in mind that a dsDNA molecule has a unique nucleotide sequence and therefore a definite polarity thus, fragments from one end are distinctly different from fragments derived from the other end.)... 

This activity is intended to be performed in conjunction with Experiment 66. Restriction endonucleases, or restriction enzymes, cleave DNA at specific base sequences, fragmenting the DNA into smaller pieces. The two strands of a DNA double helix are cleaved at different places, resulting in uneven fragments called sticky ends. Cleavage of DNA by restriction enzymes is a required first step in various types of DNA analysis, including DNA fingerprinting and recombinant DNA technology. 

One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded DNA into smaller, more manageable fragments, has opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments. 

Southern blotting is a technique that can be used to detect specific genes present in DNA. The DNA is cleaved using a restriction endonuclease, the pieces are separated by gel electrophoresis and then transferred to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. 

Analysis of DNA Fragments after Digestion by Restriction Endonucleases... 

Restriction endonucleases catalyze the hydrolysis of specific phosphodi-ester bonds in double-stranded DNA. These enzymes are often used to linearize a circular plasmid for hybrid DNA construction. They have also found use in the analysis of DNA and the construction of restriction maps. In this experiment, students will incubate various restriction enzymes with plasmid or viral DNA and analyze the product DNA fragments by agarose gel electrophoresis. 

Galibert et al. (1974) to deduce the sequence of a 48 nucleotide long fragment from X174. While its use for routine analysis has now been superceded problems can still arise for which the newer procedures are not entirely satisfactory. Confirming the sequence of chemically synthesized oligonucleotides for use as primers and the analysis of short sequences adjacent to restriction endonuclease cleavage sites are conveniently carried out by these methods. 

The DNA content of the smallest genome such as that of the DNA virus SV40 is of the order of thousands of base pairs and the analysis of even such small genomes would be impossible if they could not be reproducibly fragmented into smaller pieces. The discovery of restriction endonucleases from several bacterial species provided the tool for the specific cleavage of DNA. Once cleaved, the fragments of different sizes could be separated electrophoretically, isolated, and analyzed. 

Methodologically, both techniques are based on the digestion of the genomic DNA with restriction endonucleases, separation of the fragments by electrophoresis, followed by X transfer to a nylon membrane and finally, detection by hybridization with either a radioactive or chemiluminescent probe. This technique is known as Southern blot analysis. 

DNA extracted from a cell is extremely long, and is usually cut into shorter fragments before electrophoresis to aid the analysis. Restriction endonucleases cut double-stranded... 

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. 

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