Textbook Assignment: Chapter 13, Pages 404-417: Please also read Chapter 26, Pages 749 - 751.
Major concepts:
DNA polymorphisms: Previous lectures have placed emphasis on genetic markers that produce measurably different phenotypes, either at the visual level or at the biochemical level. With the rapid development of molecular techniques that permit examination of precise details of DNA sequence, it has become possible to use other types of changes in DNA sequence, including naturally occurring differences among individuals as genetic markers. Differences among individuals at particular genomic sites that can be used as genetic markers are commonly referred to as polymorphisms. This lecture describes a number of types of easily detected polymorphisms that have come into widespread usage as genetic markers that can be used for a wide variety of purposes, ranging from chromosomal mapping to forensics and pedigree analysis.
Codominant inheritance: The differences that give rise to polymorphic markers are carried on individual chromosomes. The pattern associated with a particular chromosome is referred to as a haplotype. The genome of an individual who is heterozygous for a polymorphic marker will contain two different haplotypes. In most (but not all) cases, the detection methods that are used are capable of detecting both variants in a single assay. Thus, the two haplotypes can usually be observed as codominant "phenotypes". This makes such markers particularly useful in pedigree analysis, where each haplotype can usually be traced back to the parent or forward into the progeny.
Restriction fragment length polymorphism (RFLP): When a specific cloned DNA probe is used to analyze a Southern blot of human (or other) DNA, a limited number of restriction fragments of specific and characteristic lengths will be identified. Because single base mutations can either create additional restriction sites or destroy pre-existing sites, DNA preparations from different individuals frequently exhibit different patterns of size distribution of restriction fragments that hybridize with a particular probe. These differences are called restriction fragment length polymorphisms (RFLPs). In many cases, the genetic polymorphisms that generate RFLPs will have no obvious phenotypic effect because they are located in introns or involve "silent" mutations that convert a codon to different codon specifying the same amino acid.
Annonymous probes: One major advantage of RFLPs as genetic markers is that they do not need to have any special properties other than the availability of a probe that can be used to visualize the alternative patterns of restriction fragments that are obtained depending on whether a particular cut site is present or absent. Any randomly cloned DNA, including sequences located in introns or between genes, that happens to emerge during "shotgun" cloning can potentially be used as a probe, as long as it can detect an RFLP. Probes of this sort that do not correspond to any known genes are referred to as annonymous probes. Many useful human RFLPs are identified with annonymous probes.
Human linkage markers: Prior to widespread use of molecular markers such as RFLPs (and many others that we will learn about in this and future lectures) it was difficult to find suitable markers for human genetic linkage studies. The total number of known genes is still rather small (although it is now growing rapidly because of the human genome project). In addition, to be of maximum usefullness, a genetic marker needs to have alternative alleles that both occur with a reasonably high frequency. Many of the more thoroughly studied human genes were originally identified because relatively rare alleles cause disease phenotypes. In such cases, the vast majority of the population carries only the wild-type alleles that do not differ in significant ways from one individual to another.
Codominant expression of RFLP haplotypes: In cases where an RFLP is carried uniquly on a single chromosome (which is expected for RFLPs in unique sequence DNA), each haploid genome will generate a specific pattern of restriction fragments that can be detected with the probe in question. If there is a polymorphism, the same region of the homologous chromosome will generate an alternative pattern with the same probe and restriction endonuclease. Because they reflect DNA sequences uniquely present on specific chromosomes, RFLP haplotypes are stable genetic markers that are inherited in a codominant manner. In addition, since they are not associated with any pathology or outward phenotypic differences, relatively high frequencies of the alternative alleles (haplotypes) can often be found in populations of healthy individuals. This allows RFLPs to be used in all types of genetic studies, including analysis of their linkage to the genes responsible for human genetic diseases. Because of their usefulness as genetic markers, large numbers of human RFLPs have been studied in detail, including identification of the chromosomal locations of the DNA sequences responsible for the polymorphisms.
Detection of sickle cell anemia heterozygotes by RFLP: In special cases, the DNA sequence associated with a genetic disease may generate an RFLP. The example cited in our textbook is sickle cell anemia, which is caused by a one nucleotide substitution in the coding sequence for beta-globin that converts a glutamic acid to a valine. As shown on page 404, the normal allele for beta-globin contains a cut site for the restriction endonuclease DdeI, which is missing from the sickle cell mutant form. When a Southern blot is probed with a partial beta-globin sequence that spans the polymorphic cut site, normal hemoglobin allele yields 2 restriction fragments 175 and 201 nucleotides long, whereas the sickle cell allele that lacks the cut site yields a single fragment that is 376 nucleotides long (figure 13.28). DNA from an individual who is heterozygous exhibits both patterns codominantly. Thus the probe identifies bands corresponding to fragment lengths of 175, 201, and 376 nucleotides (figure 13.29) Thus, in a family where healthy children may or may not be carriers, RFLP analysis can be used to distinguish easily between the two possibilities. .
Variable number tandem repeats (VNTR): As described in chapter 9, the genomes of humans and other complex organisms contain numerous DNA sequences that are present in multiple copies, including some that are highly repetitive and others that are repeated only a few times. Some of the sequences that are repeated only a few times are clustered together as tandem repeats. Such clusters are called minisatellites. The size distributions detected on Southern blots with probes for some of the moderately repeated sequences were found to vary greatly among individuals. Further analysis revealed that the reason for the variation was that the numbers of tandem repeats were different in different individuals. Such sequences are now commonly referred to as variable number tandem repeats (VNTR).
Codominant Mendelian inheritance: VNTRs show up on Southern blots as fragments of different sizes that are inherited as Mendelian markers with strictly codominant expression. In most cases, each individual will exhibit only two bands, one from each parent. Although usually discussed separately because they are caused by the presence of repeated sequences, VNTRs can be viewed as a specialized type of restriction fragment length polymorphism (RFLP) in which the variation in fragment length reflects the number of repeats, rather than the presence or absence of cut sites. However, unlike RFLPs caused by differences in cut sites, VNTRs can also be detected as bands of differing size after PCR amplification with primers that anneal just outside the repeated regions.
Short tandem repeat polymorphism (STRP): STRPs, also called microsatellites, consist of repeats of two, three, or four nucleotides. We have already seen examples of triplet repeats in Huntington disease and the fragile X syndrome (textbook pages 128-131). Like VNTRs, STRP markers can be detected through the use of restriction endonucleases that cut on either side of the repeat, followed by Southern blotting and detection with a probe specific for the repeated sequence. Alternatively, PCR amplification with primers located just outside of the region containing the repeated sequence can be used to generate amplification fragments whose lengths reflect the number of repeats. STRP markers are inherited codominantly. One potential use is to determine whether a child of a person who has developed Huntington disease will develop the disease later in life.
Random-amplified polymorphic DNA (RAPD): RAPD begins with a single primer for PCR that is only about 10 nucleotides in length. Such a primer has about a one in a million chance of binding at any particular site in a human genome, which means there are about 3000 such sites. The chance of a second binding on the complementary strand close enough to support PCR is quite small, such that about 4-8 amplification products are typically obtained, even with reduced stringency during the annealing phase. The patterns that are obtained have substantial individuality, since a one base mutation is likely to be enough to create or destroy a site under the conditions that are used. In this case, the pattern of inheritance is dominant, since the assay only sees the presence or absence of a band, making it impossible to distinguish homozygous positive from heterozygous. However, because of its speed, RAPD is often used in preliminary testing.
DNA fingerprinting: With appropriate combinations of the procedures described above, it is possible to identify sets of DNA markers (RFLP, VNTR, STRP, RAPD, and others) that are highly individualistic. Techniques such as these have made possible a procedure that has come to be known as DNA fingerprinting, which is now widely used in criminal investigations to match blood, hair or semen left at a crime scene to that of suspects. When done properly, such techniques can identify a specific individual with virtual certainty (although defense lawyers are still finding clever ways to challenge the evidence). Another area where DNA fingerprinting is extremely useful is in determining paternity. DNA fingerprinting is briefly discussed on pages 749-751 of the textbook. Non-human applications are also possible.