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Chapter: Biochemistry : Biotechnology and Human Disease

Cleavage of large DNA molecules by restriction enzymes produces a bewildering array of fragments.


Cleavage of large DNA molecules by restriction enzymes produces a bewildering array of fragments. How can the DNA sequence of interest be picked out of a mixture of thousands or even millions of irrelevant DNA fragments? The answer lies in the use of a probe, a short piece of ssDNA or RNA, labeled with a radioisotope, such as 32P, or with a nonradioactive molecule, such as biotin or a fluorescent dye. The sequence of a probe is complementary to a sequence in the DNA of interest, called the target DNA. Probes are used to identify which band on a gel or which clone in a library contains the target DNA, a process called screening.


A. Hybridization of a probe to DNA fragments

The utility of probes hinges on the phenomenon of hybridization (or annealing) in which a probe containing a complementary sequence binds a single-stranded sequence of a target DNA. ssDNA, produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane. The immobilized DNA strands are prevented from self-annealing but are available for hybridization to the exogenous, radiolabeled, ssDNA probe. The extent of hybridization is measured by the retention of radioactivity on the membrane. Excess probe molecules that do not hybridize are removed by washing the membrane.


B. Synthetic oligonucleotide probes

If the sequence of all or part of the target DNA is known, short, single-stranded 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 (Figure 33.10). DNA, isolated from leukocytes 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 bS gene, is applied to the membrane. DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell disease) contains a sequence that is complementary to the probe, and a double-stranded hybrid forms that can be detected by electrophoresis. In contrast, DNA obtained from normal individuals is not complementary at this positon and, therefore, does not form a hybrid (see Figure 33.10). 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 (Figure 33.11). [Note: ASO probes are useful only if the mutation and its location are known.]

Figure 33.10 Allele-specific oligonucleotide probe detects hemoglobin (Hb) S allele.

[Note: * indicates 32P radiolabel.] A = adenine; C = cytosine; G = guanine; T = thymine; Pro = proline.

Figure 33.11 Allele-specific oligonucleotide (ASO) probes used to detect the sickle cell mutation and differentiate between sickle cell trait and disease.


C. Biotinylated probes

Because the disposal of radioactive waste is becoming increasingly expensive, nonradiolabeled probes have been developed. One of the most successful is based on the vitamin biotin, which can be chemically linked to the nucleotides used to synthesize the probe. Biotin was chosen because it binds very tenaciously to avidin, a readily available protein contained in chicken egg whites. Avidin can be attached to a fluorescent dye detectable optically with great sensitivity. Thus, a DNA fragment (displayed, for example, by gel electrophoresis) that hybridizes with the biotinylated probe can be made visible by immersing the gel in a solution of dye-coupled avidin. After washing away the excess avidin, the DNA fragment that binds the probe is fluorescent. [Note: Labeled probes can allow detection and localization of DNA or RNA sequences in cell or tissue preparations, a process called in situ hybridization (ISH). If the probe is fluorescent, the technique is called FISH.]


D. Antibodies

If no amino acid sequence information is available to guide the synthesis of a probe for direct detection of the DNA of interest, a gene can be identified indirectly by cloning cDNA in an expression vector that allows the cloned cDNA to translated. A labeled antibody is used to identify which bacterial protein and, therefore, contains the cDNA of interest.

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