Probes : Synthetic oligonucleotide probes

If the sequence of all or part of the target DNA is known, short, singlestranded oligonucleotide probes can be synthesized that are complementary to a small region of the gene of interest. If the sequence of the gene is unknown, the amino acid sequence of the protein, the final gene product, may be used to construct a nucleic acid probe using the genetic code as a guide. Because of the degeneracy of the genetic code , it is necessary to synthesize several oligonucleotides. [Note: Oligonucleotides can be used to detect single-base changes in the sequence to which they are complementary. In contrast, cDNA probes contain many thousands of bases, and their binding to a target DNA with a single-base change is unaffected.]
1. Detecting the βS-globin mutation: A synthetic allele-specific oligonucleotide (ASO) probe can be used to detect the presence of the sickle cell mutation in the β-globin gene (Fig. 1). DNA, isolated from white blood cells (WBC) and amplified, is denatured and applied to a membrane. A radiolabeled oligonucleotide probe, complementary to the point mutation (GAG → GTG, glutamate → valine) at codon 6 in patients with the βS gene, is applied to the membrane. DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell anemia) contains a sequence that is complementary to the probe and a double-stranded hybrid form can be detected. In contrast, DNA obtained from normal individuals is not complementary at this position and, therefore, does not form a hybrid (see Fig. 1). Use of a pair of such ASO probes (one specific for the normal allele and one specific for the mutant allele) allows all three possible genotypes (homozygous normal, heterozygous, and homozygous mutant) to be distinguished (Fig. 2). [Note: ASO probes are useful only if the mutation and its location are known.]

Figure 1: Allele-specific oligonucleotide probe detects hemoglobin (Hb) S allele. [Note: The * indicates ³²P radiolabel.]

Figure 2: Allele-specific oligonucleotide (ASO) probes used to detect the sickle cell mutation and differentiate between sickle cell trait and disease.