Using the tools of recombinant DNA technology to compare the DNA sequences of homologous chromosomal regions carried by different members of a species, researchers have detected enormous variation in nearly all animals and plants.
The ability to distinguish genotypic differences of all kinds extends our concept of a locus and the alleles that define it. A locus is a designated location anywhere on a chromosome; it can range in size from a single base pair to more than a megabase pair. A gene is a coding locus. A DNA locus without any apparent function is an anonymous locus.
When two or more alleles exist at a DNA locus, the locus is polymorphic and the variations themselves are DNA polymorphisms. Polymorphic DNA loci that are useful for genetic studies are known as DNA markers.
There are four classes of DNA polymorphisms: single nucleotide polymorphisms, or SNPs; microsatellites; minisatellites; and deletions, duplications, and insertions in nonrepeat loci.
Once a DNA polymorphism at a particular locus has been uncovered, researchers can use a variety of methods to determine the genotype at that locus in any individual.
There are three ways to detect SNPs.
A small proportion of SNPs, by chance, eliminate or create a restriction site. PCR provides a rapid, cost-effective method for their detection.
A general method for detecting SNPs exploits differences in hybridization between a probe and two target sequences in the center of the region of hybridization that differ by a single nucleotide. Probes developed for this purpose are called allele-specific oligonucleotides, or ASOs.
Primer extension analysis takes advantage of the high fidelity with which DNA polymerase attaches complementary nucleotides to a growing DNA strand during DNA replication.
Polymorphisms in microsatellite and minisatellite loci, and small deletions, duplications, and insertions in nonrepeat loci, change the size of a locus and are easy to detect by PCR.
Microsatellite variants are detectable in this way as are microsatellite-related disease loci like the one responsible for HD.
Minisatellite alleles can also be detected by PCR. Simultaneous Southern blot analysis of cross-hybridizing minisatellite loci can identify multiple unlinked, polymorphic loci around the genome. The result of this analysis is called a DNA fingerprint. DNA fingerprinting provides a quick snapshot of genomic features that can identify an individual or reveal the relationship between two samples from the same population. DNA fingerprints are a powerful tool for forensic analysis.
Positional cloning identifies the genes responsible for traits whose molecular cause is unknown.
To localize a trait-affecting gene to a specific region of chromosomal DNA, researchers combine formal linkage analysis with the use of DNA markers.
Once they have narrowed the location of the gene to a small enough genomic region, researchers catalogue all possible candidate genes that map to that region, using computational tools that search for coding sequences within genome databases and sequences that are conserved between distantly related species. They can also search for EST sequences in clones derived from cDNA libraries, and they can use Northern blots to identify gene sequences that are transcribed into RNAs in tissues affected by the trait.
To identify the one candidate gene that is responsible for the trait, researchers compare groups of phenotypically normal and abnormal individuals. A finding that the DNA sequence or transcription of the candidate gene is altered in all individuals exhibiting the mutant trait is strong evidence that the candidate gene is responsible for the trait.
Incontrovertible evidence that a candidate gene causes a particular phenotype is best obtained in an experimental animal model where it is possible to induce a specific genetic change and demonstrate the appearance of a predicted phenotype. Two technologies can accomplish this goal: nuclear injection of DNA into embryos to produce transgenic mice, and targeted mutagenesis.
Most genetically determined trait variation among individuals of a species is inherited in a complex manner.
With incomplete penetrance, a mutant genotype does not always cause a mutant phenotype.
Mutant traits that arise in the absence of a mutant genotype are considered phenocopies.
Mutant traits caused by mutations at any one of two or more alternative loci show genetic heterogeneity.
A phenotype controlled by alleles at multiple loci is a polygenic trait.
If different combinations of alleles cause quantifiable differences in a trait, the trait is a quantitative trait, and the loci involved are quantitative trait loci, or QTLs.
The contemporary method of dissecting complex traits is basically the same in all sexually reproducing experimental organisms.
Haplotype association analysis is a novel genetic approach to mapping disease loci that is not dependent on the analysis of genetic transmission of alleles from parent to child.
The term haplotype refers to a specific combination of two or more DNA markers situated close together on the same chromosomal homolog. All individuals who carry a particular haplotype must have inherited it from the same ancestor.
Expression of a specific disease phenotype in many people from the same population is often the result of an ancient mutational event in a haplotype of very closely linked SNPs that has been passed intact from generation to generation.
When alleles at separate loci are associated with each other at a significantly higher frequency than is expected by chance, they are in linkage disequilibrium. A finding of linkage disequilibrium is strong evidence that a locus lies in a small, defined chromosomal region.
