An intact eukaryotic genome is too complex for most types of analysis. Geneticists have appropriated the enzymes that normally operate on the DNA molecule inside a bacterial cell and used them in the test tube to create the tools of recombinant DNA technology. Restriction enzymes cut DNA into pieces of a defined size, ligase splices the pieces together, DNA polymerase makes DNA copies, and reverse transcriptase copies RNA into DNA.
Restriction enzymes cut DNA into restriction fragments at specific sites that are 4-8 bp in length. Enzymes that recognize a 4 bp site digest DNA into fragments that average 256 bp in length; enzymes that recognize 6 bp digest DNA into fragments that average 4 kb in length; enzymes that recognize 8 bp digest DNA into fragments that average 64 kb in length. Complete digestion at every site recognized by a particular enzyme can occur under optimal reaction conditions. Under less than optimal conditions (of temperature, enzyme concentration, or duration of reaction), a partial digestion occurs in which only a random fraction of restriction sites are digested. Partial digestion enables investigators to obtain a sample of DNA fragments having an average size in-between those obtainable by complete digestion with enzymes that recognize 4, 6, or 8 bp sites. Most restriction enzymes produce DNA molecules with sticky ends, but some produce DNA molecules with blunt ends.
Gel electrophoresis provides a method for separating DNA fragments according to their size. When biologists subject a viral genome, plasmid, or small chromosome to restriction digestion and gel electrophoresis, they can observe the resulting DNA fragments by ethidium bromide staining and size the fragments by comparing their migration within the gel with the migration of known marker fragments.
Cloning DNA fragments:
Restriction fragments and cloning vectors with matching sticky ends can be spliced together to produce recombinant DNA molecules. A cloning vector is a DNA sequence that can enter a host cell, make its presence known, and provide a means of replicating and purifying both itself and any DNA to which it is spliced. Different types of vectors carry different-size inserts, which can serve as the basis for different levels of analysis.
Once inside a living cell, vector-insert recombinants are replicated during each cell cycle (just as the cell's own chromosomes are) in a process known as cloning. The millions of cells arising from a single cell by consecutive divisions make up a cellular clone. The vector-insert recombinant molecules inside the cells of a clone, often referred to as DNA clones, can be purified by procedures that separate recombinant molecules from host DNA. The insert can then be cut away from the vector by restriction enzymes and purified.
Genomic libraries are random collections of vector-insert recombinants containing the DNA fragments of a given species. The most useful libraries carry four to five genomic equivalents. cDNA libraries carry DNA copies of the RNA transcripts produced in a particular tissue. The clones in a cDNA library represent only that part of the genome transcribed and spliced into mRNA in the cells of the specific tissue, organ, or organism.
Hybridization is the natural propensity of complementary DNA strands to form stable double helixes. Hybridization makes it possible to use previously purified DNA fragments as labeled probes. Biologists use such probes to identify clones containing identical or similar sequences within genomic or cDNA libraries. Hybridization can also be used in combination with gel electrophoresis as part of an analytic technique called Southern blotting. Southern blot hybridization allows an investigator to determine the numbers and positions of complementary sequences within isolated DNA fragments or whole genomes of any complexity.
The polymerase chain reaction (or PCR) is a method for the rapid purification and amplification of a single DNA fragment from a complex mixture such as the whole human genome. The DNA fragment to be amplified is defined by a pair of oligonucleotide primers complementary to either end on opposite strands. The PCR procedure operates through a reiterative loop that amplifies the sequence between the primers in an exponential manner. PCR is used in place of cloning to purify DNA fragments whenever sequence information for primers is already available.
Sequencing provides the ultimate description of a cloned fragment. Automation has increased the speed and scope of sequencing.
