| Restriction fragment length polymorphisms (RFLPs)
|
| Restriction enzymes cleave DNA at specific recognition sites; if these sites are altered by mutation or polymorphism, the size of DNA fragments on a blot will differ
|
| If the recognition sequence is disrupted, either by a pathologic change in the DNA sequence resulting in a disease (a mutation), or a naturally occurring variation in the DNA sequence unaccompanied by disease (a polymorphism), the results of probing a Southern blot of DNA digested by a restriction enzyme may differ. Such differences in DNA sequence may lead to the creation of new restriction sites or the abolition of existing sites, and result in DNA fragments of different lengths - pattern differences known as restriction fragment length polymorphisms (RFLPs) (Fig. 34.12). Such RFLPs can be used either to identify disease-causing mutations, because of a single point mutation creating or abolishing a restriction site, or to study variation in noncoding DNA for use in the study of genetic linkage.
|
| RFLPs can also help detect larger pathologic changes in the DNA sequence, either deletions or duplications. Large deletions of a gene may abolish restriction sites; this leads to the disappearance of a fragment on a Southern blot in homozygous individuals. Alternatively, if a DNA duplication event occurs, a new gene may be formed, which has a different pattern of restriction sites that allow detection of the new gene. This type of hybridization is performed using large probes (0.5-5.0 kb) and is performed under moderate stringency, i.e. it is sufficiently rigid to allow hybridization of probe and target but also to tolerate minor differences, e.g. in introns or flanking, noncoding DNA.
|
| Low stringency hybridization of a probe to a Southern blot of digested DNA may allow genes related to, but not identical to, the starting gene to be identified. Many genes exist in families, or have nonfunctional, nearly identical copies elsewhere in the genome (pseudogenes), and thus hybridization of a probe may identify one or more restriction fragments, corresponding to related genes, which can be identified by other means. Similarly, related genes in different species may be identified by using a single probe that can hybridize at low stringency to complementary sequences in blots of DNA from mouse, rat or other species.
|
|
