Restriction fragments are discrete pieces of double-stranded DNA having unique termini that result from the cleavage of larger DNA by restriction endonucleases, enzymes that recognize specific sequences (usually 4-8 bp in length, in duplex DNA and catalyze the cleavage of both strands within or adjacent to the sequence to generate cohesive ends or blunt ends (see Restriction Enzymes and Staggered Cut). The discovery of restriction endonucleases allowed the manageable analysis and manipulation of an otherwise huge, experimentally inaccessible macromolecule. The use of restriction endonucleases also led to the development of engineered DNA vectors (plasmids, cosmids, bacteriophages, and yeast artificial chromosomes (YACs)), which are self-replicating DNAs that are used as carriers to clone and characterize restriction fragments. Important features of these vectors include selectable phenotypic markers (such as antibiotic resistance genes), unique restriction sites grouped in multiple cloning sites, where restriction fragments can be introduced, promoters for the expression of the inserted DNA, and mechanisms for DNA replication. Vector or chromosomal DNA can be digested into restriction fragments with one or more restriction endonucleases and analyzed for size and fragment distribution using gel electrophoresis (see Restriction Map). Restriction-fragment size is dependent on the type of restriction endonuclease used and the particular sequence. For instance, assuming a random distribution bases in DNA with a 50% ( G + C ) composition, a given tetranucleotide sequence will occur randomly approximately every 256 base pairs, whereas a hexanucleotide sequence occurs approximately every 4096 base pairs. Large restriction fragments can be generated by (1) rare-cutting enzymes, like the octanucleotide ( GCA GGCCGC , where "a" indicates the hydrolysis site in the DNA recognition sequence) cutter R*AotI (where "R*" designates restriction endonuclease), (2) partial R*endonuclease digestion resulting from short digestion duration or dilute enzyme concentrations, or (3) limited methylation of R*endonuclease sites with the cognate methyltransferase enzyme. The properly sized spectrum of generated restriction fragments can be covalently ligated into cloning vectors to form a collection of chimeric DNA molecules that can replicate as individual clones once introduced individually into a host organism, such as Escherichia coli, by transformation, transfection, or transduction. Such a population of clone-containing fragments encompassing all of the original DNA is called a "gene library." The genetic information encoded on an individual restriction fragment or library of fragments is easily amplified and manipulated.
1. Methods for Analyzing Restriction Fragments
Several methods are used to analyze restriction fragments separately or in a collection of clones. In Southern blotting, restriction fragments separated on an agarose gel by electrophoresis are processed by transferring the DNA from the gel to a nitrocellulose filter, which provides a stable support for hybridization with single-strand labeled probes containing known sequences of interest. If a restriction fragment contains the complementary sequence, the probe will hybridize, and subsequent analysis of the filter for the label will identify the fragment. It can then be isolated and further characterized by sequencing. Another related method, called colony blotting, involves hybridizing DNA probes to colonies containing recombinant DNA that have been fixed to nitrocellulose filters and lysed . Analysis of the filter can then identify clones carrying sequences of interest. Chromosome walking uses a genomic library of clones to determine consecutive DNA fragments for mapping or locating a particular region in the genome. In this technique, a cloned DNA fragment is isolated, and the ends of the insert fragment are subcloned and used as probes to find clones containing the same DNA sequences, specifically, DNA adjacent to that generating the probes. Variations in DNA sequences between individual organisms are revealed by digesting DNA with various restriction endonucleases to reveal restriction fragment length polymorphisms (RFLP).
RFLPs result from changes in the fragmentation pattern of a restriction digest due to DNA sequence differences, which can be as small as single-base-pair substitutions. RFLP mapping provides a "genetic fingerprint" of the DNA of organisms. DNA fragments containing regulatory sequences, such as, promoters or operators, can be analyzed by placing them in vectors containing adjacent reporter genes that encode a protein with a measurable activity. Expression of the reporter gene is thereby placed under the control of the regulatory sequence. For instance, an antibiotic resistance gene can be used to report promoter activity. Only clones carrying the expressed antibiotic resistance gene will grow on media containing the antibiotic. Another approach uses enzymatic or functional assays of cell lysates of clones to identify those carrying a DNA fragment containing a gene that encodes the protein of interest.
The generation of restriction fragments by restriction endonucleases has contributed greatly to biotechnology. Fragmentation and sequencing of DNA allows characterization of genes, as well as probing for similar genes in different organisms using hybridization techniques. Sequencing of restriction fragments also reveals the open reading frames (ORF) of new genes, and their spatial organization with respect to other genes. RFLP mapping is important in forensic and medical analysis. Restriction fragments provide a starting point for biochemists and molecular biologists to mutagenize genes to determine how resultant changes are reflected in protein structure or function. Specially designed cloning vectors containing powerful promoters can overexpress cloned genes of interest to increase protein production. Molecular cloning of restriction fragments is an invaluable tool that has yielded a wealth of information with widespread applications.
