Many techniques exist for testing whether a sample of DNA carries a mutation. In this animation, we explore the use of two different techniques for identifying whether an individual carries the sickle-cell allele of the β-globin gene.
One procedure is called allele-specific oligonucleotide hybridization. In allele-specific oligonucleotide hybridization, the binding of a probe to sample DNA indicates that a particular allele is present in the DNA.
The other procedure, DNA testing by allele-specific cleavage, uses restriction enzymes as diagnostic tools. Restriction enzymes are proteins that recognize and cut DNA at specific sequences. Allele-specific cleavage relies on the mutation in the disease allele either adding or eliminating a recognition site for a restriction enzyme.
Mutations that cause genetic disease, such as sickle-cell anemia, can be identified using a DNA test called allele-specific oligonucleotide hybridization. In this procedure, a short DNA fragment—the oligonucleotide (also called the probe)—is created to match the normal version of the gene, and another oligonucleotide is created to match the sickle allele. The normal and sickle probes differ by a single nucleotide.
In this family, a mother, father, child, and fetus are being tested for the sickle-cell allele. The sickle-cell mutation occurs in the β-globin gene, which codes for a subunit of the hemoglobin protein in red blood cells. Each person has two copies of the β-globin gene. The normal allele is usually designated A, and the sickle allele is designated S. Both copies of the gene from an individual are amplified together by PCR and then dotted onto filters for testing.
The DNA on the filters is denatured to make it single stranded. The probes are either fluorescently or radioactively tagged to be able to visualize them. The probes are added to the filters to allow them to hybridize to complementary sequences in the spots of DNA, and after incubation the excess is washed off.
The probes are very specific. Each hybridizes to just one type of β-globin allele to make a perfect hybridization match.
In this test, the red color indicates that the probe hybridized to DNA on the filter. The blue color indicates lack of hybridization.
In this test, the mother, father, and fetus all show DNA hybridizations with both probes, so they are heterozygous, AS. They are carriers of the sickle-cell allele. The child, in contrast, has DNA that only hybridizes with the probe for the normal allele. The child is therefore homozygous for the normal allele, AA.
Another way that the sickle-cell allele has been identified is by using enzymes that cleave DNA. In sickle-cell anemia, the sickle-cell allele has a mutation that converts an adenine base to a thymine base. Whereas the normal sequence is digested by the restriction enzyme MstII, this enzyme cannot recognize the sequence in the sickle-cell allele and therefore does not cut it.
In this test, you use PCR to amplify the DNA region that includes the mutation site. If you were to digest both samples of DNA with MstII, the normal allele would produce two fragments, and the sickle allele would produce just one.
In performing this test, you would digest DNA from the test sample of interest with MstII, as well as digest DNA from a normal β-globin sample and a sickle-cell sample. The digested DNA is then loaded onto an agarose gel for electrophoresis.
The DNA fragments are negatively charged and migrate toward the positive electrode in an electric field, with the smaller fragments moving faster than the larger fragments.
From the banding pattern on the gel, what is the genotype of the person from which the test sample was taken?
The individual has both alleles of the β-globin gene, and is therefore heterozygous, AS.
Allele-specific oligonucleotide hybridization is a widely used test that allows researchers to distinguish between two alleles that differ by a single nucleotide. The oligonucleotide probes are created so that each type hybridizes to DNA from just one of the alleles. Another probe hybridizes to a second allele. The probes need to be perfectly matched to the alleles, because a single mismatch will prevent hybridization of the probe to the DNA.
DNA testing by allele-specific cleavage uses restriction enzymes as diagnostic tools. In the example of sickle-cell anemia, the mutation that causes the disease also eliminates an MstII recognition site from the mutated β-globin gene. For this reason, the allele-specific cleavage test can be used to determine a person's genotype. However, many mutations that cause disease do not affect the recognition sites of restriction enzymes, thereby limiting the use of this particular technique in diagnosing disease.