A useful class of enzymes are site-specific endonucleases, also known as restriction enzymes, that recognize and cut DNA molecules at or near short nucleotide sequences (typically four to eight base pairs in length). Here is a listing of some restriction endonucleases (FIGURE). The recognition sites typically show two-fold rotational symmetry, sometimes called a palindrome. Specific methylation (shown as asterisks in the Figure) protects DNA from cleavage by the restriction endonuclease. Depending on the pattern of endonuclease cleavage, blunt cuts (= blunt or flush ends) or staggered cuts (= cohesive ends) can be created (FIGURE). Cleavage of phosphodiester bonds by restriction endonucleases creates 5'-phosphates and 3'-hydroxyls.
Note: Restriction endonuclease sites are really genetic markers; that is, a point mutation changing any given base pair in the site prevents cleavage of the site. For example, GAATTC is cut by EcoRI, but GTATTC is not (bold shows the nucleotide change; only the sequence of one strand is shown).
Restriction endonucleases cut DNA molecules into smaller fragments at specific nucleotide sequences. Gel electrophoresis is a laboratory process for separating fragments of DNA on the basis of size (FIGURE). The process uses a support medium (agarose or polyacrylamide) called a gel in a buffer solution (liquid containing salts). DNA fragments are "driven" through the medium by an electric current (DNA molecules are negatively charged and move towards the positive electrode). Large molecules have difficulty moving through the gel and travel the least distance. Small molecules travel through the gel more easily and go the farthest. The actual distance a DNA fragment travels is controlled by the size of a DNA molecule. The number of fragments and the sizes (typically expressed in base pairs, bp, or kilobase pairs, kb) of the fragments can be determined by gel electrophoresis (FIGURE). (Note: DNA molecules of known sizes are used as "markers" on the gel). The order of the fragments gives a physical map of the DNA molecule (also known as a restriction map). Here is one way to determine a physical map (FIGURE). The combined physical maps of several restriction endonucleases on a small circular DNA molecule is shown here (FIGURE).
Double-stranded DNA is the native (or normal) secondary structure. The conversion of double-stranded DNA into single strands is called denaturation. The conversion of single strands back to the double-stranded DNA is called renaturation.
Note: DNA can be denatured by heat or alkali (for example, sodium hydroxide). However, RNA phosphodiester bonds are broken by alkali).
How can you find a specific piece of DNA in a mixture containing many DNA molecules? First we separate DNA molecules by size (variations of gel electrophoresis can separate very large chromosomal sized DNA molecules as well as DNA molecules that differ by a single nucleotide). Moreover, gel electrophoresis can be done under conditions that preserve native DNA structure or denature DNA molecules.
DNA molecules separated by size using gel electrophoresis can be transferred to a support matrix (for example, a nitrocellulose membrane) and hybridized to a radioactive "probe" (for example, 32P-labeled DNA). This technique is called Southern blotting (FIGURE).
Northern Blotting is similar to Southern blotting, except that one examines RNA instead of DNA. Probes can be RNA or DNA. The conditions for RNA-RNA or RNA-DNA hybridization are somewhat different than the conditions for DNA-DNA hybridization.
A third type of blotting is called Western Blotting. Western blotting analyzes proteins instead of RNA or DNA. The basic process is the same as above, but the probing is different. In Western blotting, one uses an antibody specific to a particular protein to identify it on the blot.
One place where Southern blotting is useful, is the characterization of differences in DNA sequence between individuals. One type of study is the Restriction Fragment Length Polymorphisms (RFLPs) (FIGURE). Remember that restriction enzymes cut DNA at specific sequences. Remember also that though our genes may be very closely related, there are differences in the sequence of all of our DNAs (except for identical twins). As our DNAs vary, it naturally follows that the restriction maps of pieces of our DNA will vary as well. Many of these differences are inconsequential for gene function. Others may be linked to genetic diseases or disorders.
A special kind of RFLP analysis is called DNA Fingerprinting (FIGURE). It relies on the analysis of what is called VNTR's (or Variable Number of Tandem Repeats). VNTR's consists of many tandem repeats of a short sequence. A particular person's VNTR's come from the genetic information donated by the parents; the VNTR's can be inherited from the mother or father, or a combination, but never a VNTR either of the parents do not have. The repeats vary from individual to individual depending on the amount of crossing over (GENETIC RECOMBINATION) that occurs between the related sequences. Since the sequences are repeated, there are many places where crossing over can occur. If it is "unequal", two VNTR sequences of the same size will give rise to a bigger VNTR sequence and a smaller VNTR sequence, the sum of whose sizes is the same as twice the original size of the original VNTR.
An example of the use of a gene chip is shown (FIGURE).