Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Molecular Diagnostics
DNA Diagnostic Systems

Information about the entire diversity of an Organism's traits is encoded in its genetic material. For instance, bacterial pathogenicity is determined by the presence of a specific Gene or set of genes, while a hereditary genetic disease arises from damage to a particular gene. The DNA segment determining a given biological trait has a strictly defined nucleotide sequence and can serve as a diagnostic marker.

Many rapid and reliable diagnostic Methods are based on nucleic acid Hybridization—the pairing of two complementary segments of different DNA molecules. In general terms, the Procedure consists of the following steps.

1. Immobilization of the single-stranded target DNA on a membrane filter.

2. Application of a labeled single-stranded DNA probe, which under specific conditions (Temperature and Ionic strength) pairs with the target DNA.

3. Washing the filter to remove excess unbound labeled DNA probe.

4. Detection of probe/target hybrid molecules.

In diagnostic tests based on nucleic acid hybridization, three components are key: the DNA probe, the target DNA, and the hybridization signal detection method. The detection system must be highly specific and highly sensitive.

Hybridization probes

To ensure the validity of a diagnostic test, hybridization DNA and RNA probes must be highly specific. In other words, the probe must hybridize only with the target nucleotide sequence. If There is a possibility of obtaining a false-positive (presence of a hybridization signal in the absence of the target sequence) or false-negative (absence of a signal in the presence of the target sequence) result, the utility of the test is significantly reduced. Probe Specificity can manifest at different levels: they can distinguish between two or more species, individual strains within a single species, or different genes. Depending on the situation, probes can be DNA or RNA molecules; they can be long (more than 100 NUCLEOTIDES) or short (fewer than 50 nucleotides), and they can be products of chemical synthesis, cloned intact genes, or fragments thereof.

Probes are obtained in various ways. One method is as follows. DNA from a pathogenic microorganism is digested with a restriction endonuclease and cloned into a plasmid vector. Recombinant Plasmids are then screened using genomic DNA from both pathogenic and non-pathogenic strains. Those plasmids containing sequences that hybridize only with the DNA of the pathogenic strain form The basis of species-specific probes. Subsequently, a series of additional hybridizations are performed with DNA isolated from various organisms to ensure that the potential probes do not cross-hybridize with them. To determine the sensitivity of the method, each probe is also tested on mock samples, including mixed cultures.

It is highly desirable for DNA Diagnostics to be performed directly on the raw material, without additional culturing or Nucleic Acid Extraction, especially when testing clinical specimens. Researchers successfully perform hybridization with target DNA present in stool, urine, Blood, throat swabs, and Tissues without prior purification. If the concentration of the target sequence in the test sample is too low, it can be amplified using the Polymerase Chain Reaction (PCR).

Diagnosis of malaria

As an example of using DNA probes for disease diagnosis, the detection procedure for Plasmodium falciparum can be cited. This parasite causes malaria, a disease that threatens approximately one-third of the world's population. It infects and destroys red Blood Cells, leading to fever and, in severe cases, damage to the Brain, Kidneys, and other Organs. To identify sources of infection, evaluate the effectiveness of eradication measures, and ensure early Diagnosis and Treatment, sufficiently sensitive, simple, and inexpensive methods are required. Currently, malaria is diagnosed by microscopic examination of blood smears—an effective but labor-intensive and time-consuming process. Immunological methods for detecting Plasmodium, such as ELISA, are rapid and easy to automate, but they cannot distinguish active infection from past exposure, as they only detect the presence of Antibodies to Plasmodium in the patient's blood.

For the selective DNA diagnosis of active infection, i.e., for detecting the pathogen's DNA, highly repetitive DNA sequences of P. falciparum are used as a basis. First, a genomic DNA library of the parasite is screened using a DNA probe. Clones yielding the most intense hybridization signal are then selected, as they presumably contain the highly repetitive sequences. The DNA of each selected clone is tested for its ability to hybridize with the DNA of Plasmodium species that do not cause malaria. A sequence that hybridizes with P. falciparum DNA but not with P. vivax, P. cynomolgi, or human DNA is chosen as a specific probe. Using this probe, as little as 10 pg of purified P. falciparum DNA or 1 ng of the same DNA in a patient's blood can be detected.

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More than 100 different DNA probes have been obtained and characterized, allowing the detection of pathogenic strains of various Bacteria, Viruses, and parasitic Protozoa. For example, probes are available for diagnosing human bacterial Infections caused by Legionella pneumophila (respiratory diseases), Salmonella typhi (food poisoning), Campylobacter hyointestinalis (gastritis), as well as for detecting enterotoxigenic strains of Escherichia coli (gastroenteritis). However, this is just the tip of the iceberg; in principle, hybridization can be used to detect virtually any pathogenic microorganism. Detection of Trypanosoma cruzi: The parasitic protozoan Trypanosoma cruzi causes Chagas disease (American trypanosomiasis), which claims approximately 50,000 lives annually. The parasite is widely distributed in Latin America. Transmitted by kissing bugs, it invades the Liver, Spleen, Lymph Nodes, and Central Nervous system, multiplying and destroying the host cells. To diagnose the acute phase of Chagas disease, microscopic examination of a fresh peripheral blood sample is typically performed. Another test, which is longer but more likely to detect the parasites, can also be used: uninfected insects are fed the patient's blood, and after 30–40 days, their hindguts are examined under a Microscope for the presence of parasites. Both methods are highly labor-intensive, expensive, and time-consuming. The disease can also be diagnosed by immunological methods, but these often yield false-positive results. As an alternative to these less-than-satisfactory Procedures, several PCR-based approaches have been developed. Currently, PCR diagnostics for Chagas disease serve as a Complement to traditional, widely used methods.

One PCR assay is based on the detection of a 188-bp DNA fragment that is present in multiple copies in the T. cruzi genome but absent in the genomic DNA of several related parasites. Following Amplification, this fragment is easily detected by Polyacrylamide gel Electrophoresis. By slightly varying the PCR protocol (for example, by changing The nucleotide sequence of the primers), the assay can be used to detect a wide range of bacteria, viruses, and parasites.

Non-radioactive detection methods

In most laboratories, hybridization is performed using probes labeled with a radioactive isotope, most commonly ³²P. Such probes possess high specific activity and provide a good signal-to-noise ratio. The radiolabeled probe is applied to the filter with the immobilized target DNA, hybridization is carried out, unbound DNA probe is washed away, and the label is detected by autoradiography.

However, ³²P is a short-lived isotope that emits high-energy radiation; working with it requires specialized equipment and safe waste disposal. To circumvent these difficulties, non-radioactive detection systems have been developed. In these systems, the hybridization signal is amplified through the enzymatic conversion of a chromogenic or chemiluminescent substrate: the former changes color under the action of the enzyme, while the latter emits light. Most such systems utilize DNA probes containing biotinylated nucleotides. Hybridization and signal detection are carried out in a more or less standard manner.

1. The biotin-labeled probe is hybridized with the target DNA (Fig. 9.4, A).

2. The filter is washed to remove excess unbound probe.

3. Avidin (egg white protein) or streptavidin (a bacterial analog of avidin) is added (Fig. 9.4, B).

4. A biotinylated enzyme—alkaline phosphatase or horseradish peroxidase—is added (Fig. 9.4, C).

5. Depending on the enzyme used, a chromogenic or chemiluminescent substrate is added, and the color change or luminescence accompanying The conversion of the substrate to product is recorded (Fig. 9.4, D).

Alternatively, after DNA hybridization with a biotinylated probe, a pre-formed streptavidin–enzyme complex containing a biotin-binding site can be added.

Both avidin and streptavidin bind to biotin extremely tightly (dissociation constant (K_d = 10^-15); moreover, each of these Proteins has four independent biotin-binding sites, allowing a single avidin or streptavidin molecule to simultaneously bind both a biotin-labeled enzyme and a biotin-labeled probe. Biotinylation and streptavidin binding do not reduce enzymatic activity. In chromogenic detection systems, an insoluble dye is formed by the action of the enzyme at the site of the hybrid DNA, whereas in chemiluminescent systems, a light-emitting product is generated. Non-radioactive detection systems offer other advantages as well: biotinylated DNA remains stable at room temperature for at least a year; chemiluminescent detection methods are as sensitive as radioactive signal detection; and detecting the emitted light using X-ray film or a luminometer, much like recording color changes, takes only a few hours. It appears that chemiluminescent signal detection systems, which are more sensitive than chromogenic ones, will soon replace all other systems used in DNA diagnostics. If PCR is used, the amplified product can be labeled with a fluorescent dye attached to the 5'-end of each primer. Fluorescein and rhodamine, which emit green and red light respectively, are commonly used as Dyes. Following PCR amplification of the target DNA, the fluorescein-labeled primer is separated from the amplification products, and label incorporation is then detected (Fig. 9.5). If the target DNA is absent from the sample, no fluorescent product will be formed.

Fig. 9.4. Chemiluminescent detection of target DNA. B — biotin, AP — alkaline phosphatase. A. Binding of the biotinylated probe to the target DNA. B. Binding of streptavidin to biotin. C. Binding of biotinylated alkaline phosphatase to streptavidin. D. Formation of a luminescent product by alkaline phosphatase.

One recently developed non-radioactive detection method is based on The Use of a molecular beacon probe (Fig. 9.6). Such a probe consists of 25 nucleotides. The central 15 nucleotides are complementary to the target DNA and do not pair with each other, while the 5 terminal nucleotides on each end are mutually complementary and form a hairpin. A fluorescent chromophore (fluorophore) is attached to the 5'-end, and a non-fluorescent chromophore (quencher) is attached to the 3'-end, which accepts the excitation energy of the fluorophore. In solution at room temperature, the beacon adopts a configuration where the fluorophore and quencher are in close proximity, quenching the fluorescence of the fluorophore. However, when the 15 central nucleotides of the probe hybridize to a complementary target DNA or RNA sequence, the fluorophore and quencher become spatially separated, and the probe emits light. The temperature of the reaction mixture must be close to room temperature, because as the temperature rises, the hairpin denatures, the fluorophore and quencher separate, and fluorescence occurs. It is also necessary that all 15 nucleotides of the probe be complementary to the corresponding target DNA or RNA sequence.

Fig. 9.5. Detection of PCR products using a fluorescent dye attached to primers (P1 and P2).

DNA fingerprinting

DNA fingerprinting (DNA typing) is frequently used in forensic medicine to identify biological samples. It can be used to prove that a suspect committed a crime or, conversely, to establish their innocence. To perform DNA typing, a portion of a biological sample (a blood sample, semen, a piece of Skin, Hair) is first collected, and The amount of intact DNA is assessed to determine if it is sufficient for subsequent analysis. The DNA is then digested with Restriction Endonucleases, and the resulting fragments are separated on an agarose gel and transferred to a nylon membrane. Sequential hybridization is carried out with four or five radioactively labeled probes, each of which recognizes a specific DNA sequence (the previous probe is completely stripped from the membrane before hybridization with the next one). After each hybridization, autoradiography is used to visualize the bands corresponding to the hybridization products of the probe with the digested DNA, and a "fragment ladder" is constructed for all samples (Fig. 9.7). Each step (hybridization and autoradiography) takes 10 to 14 days, so the entire procedure can take many weeks or even several months. Minisatellite DNAs, which occur repeatedly in The Human Genome and consist of tandemly repeated regions, are typically used as probes. The repeat length varies from 9 to 40 bp, and their number ranges from 10 to 30; moreover, the same minisatellite sequences can vary in length among different individuals. These differences arise from an increase or decrease in the number of tandem repeats, likely during METABOLISM/36.html">DNA Replication. Such variations have no biological consequences because minisatellite DNAs do not code for proteins. A child inherits one minisatellite sequence from one parent and another from the other parent.

Fig. 9.6. Hybridization of a molecular beacon probe with a target DNA. The single-stranded region of the probe hybridizes to the complementary target DNA sequence, its hairpin Structure is disrupted, the fluorophore and fluorescence quencher are no longer in contact, and fluorescence is observed. This indicates that hybridization has occurred between the probe and the target sequence. Modified from Tyagi, Kramer, Nat. Biotechnol 14: 303–308, 1996.

An individual's DNA fingerprint is a set of fragments of varying lengths that correspond to the minisatellite sequences in their genome. Due to the high diversity of these repeats, the probability of finding two individuals in a population with identical DNA fingerprints is 10^-5 to 10^-8. In other words, the banding pattern of minisatellite DNA is almost as unique as a fingerprint. DNA fingerprinting is also used in paternity testing. Some bands of the child's DNA fingerprint must match the mother's bands, while others must match the father's. If the DNA in the test sample is insufficient but not severely degraded, small regions of minisatellite DNA can be amplified by PCR and then sequenced; this method is more sensitive than determining tandem repeat length polymorphism.

Fig. 9.7. Use of Southern hybridization for forensic analysis. DNA isolated from the victim's blood, from a bloodstain on the suspect's shirt, and from the suspect's blood was digested with the same restriction endonuclease. The fragment ladder for the DNA isolated from the bloodstain on the shirt is identical to that of the victim's DNA and differs from the fragment ladder of the suspect's DNA.

Use of Polymorphic DNA Markers

DNA fingerprinting can also be useful for distinguishing between plant cultivars. One variant of this method is based on random amplified polymorphic DNA (RAPD) markers. For this purpose, arbitrary primers 9–10 nucleotides in length, which do not contain palindromic sequences and have a GC content of 50–80%, are added individually to plant chromosomal DNA preparations. Each PCR is initiated by a single primer, which must be capable of binding to both strands of the target DNA. Although The nucleotide sequences of all oligonucleotides are known, it is unclear which one will serve as an effective PCR initiator. If the primer hybridizes to both strands of the target DNA in the appropriate orientation and the sites are located 100 to 3000 bp apart, the flanked DNA segment will be amplified, and the resulting fragment can be separated by gel electrophoresis and visualized by staining. The number of different DNA fragments produced during amplification depends on the primer and the genomic DNA. For the same primer and target DNA, the amplification products will be identical every time, whereas a single nucleotide change in the primer will completely alter the set of resulting fragments. Thus, using the same set of oligonucleotide primers, one can compare the RAPD DNA fingerprints of different plant cultivars, and consequently, the cultivars themselves. To detect differences between two very closely related plant varieties or cultivars, it is often necessary to use several arbitrary primers with known nucleotide sequences (Fig. 9.8). Like all other molecular markers, RAPD can be used to characterize entire genomes, individual Chromosomes, or genes. Compared to other methods for identifying complex DNA, the RAPD method offers several advantages: 1) the same (universal) set of oligonucleotide primers can be used for all plant species; 2) there is no need to construct Genomic Libraries, use radioactive probes, or perform hybridization, allowing for the rapid and easy characterization of A large number of samples; 3) the process can be automated. Furthermore, while standard PCR requires knowledge of the nucleotide sequence of the target gene or fragment to be amplified, RAPD amplifies any genomic region containing two sequences complementary to the primer that flank a DNA segment 100 to 3000 bp in length.

Fig. 9.8. Polyacrylamide gel electrophoresis of PCR-amplified plant DNA fragments followed by ethidium bromide staining. Three different arbitrary primers were used to amplify fragments from each of the two cultivars. For primers A and C, the banding patterns in the polyacrylamide gel are identical for cultivars 1 and 2, whereas when primer B is used, the band positions differ. Thus, primer B can be used to distinguish between cultivars 1 and 2.

Using the RAPD method, it was possible to distinguish six inbred lines of maize from one another and to show that the PCR products of maize hybrids represent a combination of the PCR products of the parental inbred lines. RAPD markers were also used to screen different strains of the fungus Leptosphaeria maculans, which causes blackleg disease in crucifers. A distinction was established between non-virulent (non-pathogenic) and virulent (pathogenic) strains.