Diagnostic and Clinical Applications of DNA

DNA Probes:
                    The ability to isolate specific fragments of DNA containing known sequences of genes gives rise to DNA probes that can be used for a variety of diagnostic, forensic, and therapeutic purposes. DNA probes can be labeled with either radioactive or nonradioactive markers. A DNA probe has a strong interaction with (ideally) a specific DNA target and can be detected after the interaction. DNA probes consisting of 20 bases or fewer usually will have a unique target even in a large set of DNA molecules. The probability that any base will follow any other base in DNA is one in four or 0.25. Therefore, the probability of a specific sequence of 20 bases occurring in a DNA molecule by chance is 0.25 to the power of 20 a vanishingly small number. The use of DNA probes in various aspects of medical diagnostics is increasing rapidly. 

                     DNA probes can be used to identify infectious agents if sequences specific to different pathogens are known. Identification of a pathogen by DNA probes can be done in hours as compared to days or weeks by conventional culturing of microorganisms. DNA probes are now used routinely to detect the presence of mutant alleles in fetal cells obtained by amniocentesis, as well as in cells removed from affected adults or carriers. Many inherited disorders, such as sickle cell disease, cystic fibrosis, Huntington's disease, Duchenne's muscular dystrophy, and dozens of other Mendelian (single-gene) disorders, can now be diagnosed in fetuses and adults. In addition to inherited disorders, DNA probes are used to detect the presence of active oncogenes or inactive tumor suppressor genes in cancerous tissues removed from patients.

Southern Blot Analysis:

                     The Southern blot (named for its inventor, E. M. Southern) is a method for hybridizing one or more labeled DNA probes to a large number of DNA fragments and discriminating among them. The procedure depends on the ability of denatured DNA single strands to bind tightly to nitrocellulose under certain conditions. The DNA to be investigated is digested with several restriction enzymes to generate DNA fragments of varying sizes which are then separated by agarose gel electrophoresis. After the fragments are separated, the gel is immersed in a denaturing solution that converts all DNA to single strands. Then the gel, which is in the form of a broad, flat slab, is placed on top of filter paper supported by a glass plate. 

                      A nitrocellulose filter is placed over the gel and covered with another sheet of filter paper. Finally, paper towels and weights are placed on top of the gel. The glass plate supporting the filter papers and gel is adjusted so that the ends of the filter paper are suspended in buffer and act as wicks. The buffer moves upward through the gel and into the wad of paper towels that act to absorb the buffer. The DNA migrates from the gel to the nitrocellulose filter that binds the single-stranded DNA fragments. The nitrocellulose filter is dried and exposed to high temperature to keep the DNA in a denatured state. A labeled DNA probe containing a known sequence or gene is then incubated with the nitrocellulose filter under a variety of hybridization conditions to identify a particular fragment of DNA from the sample. 

                      Strands of the DNA probe bind to fragments of the separated DNA that contain similar or identical sequences and hybridize together to form double-stranded DNA. After the hybridization reaction is complete, the solution is cooled and the nitrocellulose filter is washed to remove unbound probe DNA and dried. The labeled DNA fragments can be visualized by autoradiography (if the probe was radioactive) or by fluorescence. The DNA fragments of interest also can be eluted from the nitrocellulose filter for further analysis.

Southern blot

Polymorphisms:

                 Approximately 25% of all human genes occur among individuals in a population in multiple allelic forms referred to as polymorphisms. For example, the ABO locus, an important factor in blood transfusions, consists of three different alleles (alternative states of a gene) called A, B, and O. Individuals can be homozygous or heterozygous for any pair of A, B, and O alleles. The HLA family of genes that play a vital role in the rejection of tissue transplants consists of hundreds of different alleles. No two individuals, except for identical twins, share exactly the same set of HLA alleles. For a gene to be polymorphic, alternative alleles must be present in at least 1% of individuals in a population. The relative frequencies of the different alleles at a polymorphic locus usually vary from one human population to another.
                  The remaining 75% of human genes consist of a single allelic form and are said to be monomorphic. For example, all human beings have the same α and β genes for hemoglobin, presumably because the protein structure has been optimized by millions of years of human evolution. All persons, irrespective of race or ethnicity, have hemoglobin genes that are identical in sequence. The only exceptions to the monomorphic state of the hemoglobin genes are the rare individuals who inherit or acquire a mutation in an α or β genes that gives rise to the several hundred characterized human hemoglobinopathies.
                   Restriction enzyme sites in DNA also are highly polymorphic in human populations. The pattern of DNA fragments produced by digesting DNA from different individuals will usually show a difference if a sufficient number of fragment sizes are examined; again, only identical twins have identical restriction enzyme sites. The different fragments produced by digesting DNA with one or more restriction enzymes are called restriction fragment length polymorphisms (RFLPs). RFLPs have been extremely useful in constructing a restriction map of the human genome (which can be correlated with the genetic and physical maps), in screening human populations for the presence of mutant alleles, and for diagnosing hereditary disorders in fetuses and prospective parents.                                        An early success in mapping the human genome was determining the location of more than 10,000 RFLPs on the 23 human chromosomes. Most mutant genes that cause hereditary disorders are linked to specific RFLP differences among affected and unaffected persons that can be detected by using DNA probes carrying the gene in question. For example, the single-base change found in all cases of sickle cell anemia also changes a restriction site located within the/3-globin gene that is recognized by the restriction enzyme MstII. When DNA from unaffected and affected individuals is digested with MstII and a Southern blot analysis is performed with the appropriate probe, affected individuals show a different pattern of DNA fragments as compared with unaffected individuals. Huntington's disease (HD) is an autosomal dominant disorder characterized by progressive chorea, dementia and ultimately death.                     

                    It is a late-onset hereditary disease that usually manifests after age 40. Any son or daughter who had a parent with Huntington's disease has a 50% probability of inheriting the mutant allele from the affected parent. A HindIII restriction site is closely linked to the HD gene in asymptomatic family members who carry the mutant allele but is absent in family members who have not inherited the mutant allele. Southern blot analysis can determine which family members are destined to die of the disease later in life. (Not all families that carry a HD gene mutation have this particular mutation that was characteristic of the original large family investigated.).

                   Genetic testing for susceptibility to Huntington's disease (and many others) raises profound ethical questions for society, for families, and for individuals who may or may not have inherited disease-causing mutant alleles, and who may or may not want to know their disease status. Persons diagnosed with a gene for an inherited disorder may face discrimination in obtaining health insurance, life insurance, job opportunities, and so path. Hundreds of Mendelian (single-gene) disorders can now be detected with appropriate DNA probes. However, genetic screening generally is not advised, particularly if no medical treatment is available for the particular disease.

Forensic DNA Analysis:

                   RFLPs are also widely used in forensic pathology, criminology, and cases of contested paternity. A particular set of DNA probes is specific for hypervariable sequences in the human genome that are inherited in a Mendelian pattern identical to the inheritance of genes. Hypervariable sequences are highly polymorphic minisatellite loci that are unique to each individual just as each individual has a unique set of genes. These hypervariable sequences can be identified by a special set of DNA probes called Jeffreys probe after the scientist discovered them. A Southern blot analysis of fragments of DNA from an individual using the Jeffreys probe possesses a unique "genetic fingerprint." Only monozygous twins have the same pattern of hypervariable sequences and identical genetic fingerprints. DNA obtained from a blood stain, a human hair cell, or even a few sperm provides enough material to match with the DNA obtained from a suspect. DNA testing of suspects in rape, murder, and other crimes in which a sample of DNA was obtained at the scene of the crime is now a routine procedure. DNA sequences can be amplified by the polymerase chain reaction (PCR) technique. Only an infinitesimal amount of DNA is needed, e.g., a sample of DNA from a single hair follicle or sperm. 

RFLP

The DNA to be amplified is mixed with three other components in an automated PCR procedure:

1. A heat-stable DNA polymerase that is isolated from a thermophilic bacterium,
2. An excess of two short primer DNAs that are complementary to opposite strands of the DNA fragment that is to be amplified, and
3. An excess of deoxyribonucleotide triphosphates.
PCR consists of repeated cycling of three reactions:
denaturation of the DNA by heating, reannealing of the primers with the target DNA by cooling, and synthesis of new DNA strands. The sequence of reactions is automatically repeated at defined intervals to yield an exponential increase in the amount of DNA. Twenty cycles of PCR amplify DNA by about a factor of 10*6 and 30 cycles by about 10*9. PCR amplification of DNA is one of the most widely used techniques in medical research and diagnostics. PCR is used in forensic pathology (to identify human remains), in rapid identification of infectious microorganisms, in diagnosis of inherited diseases, and in archaeology and anthropology where small DNA samples can be recovered.

Sequencing DNA:

Until the development of automated DNA sequencing machines in the 1990s, two techniques were used to sequence the bases in a segment of DNA. Each of the techniques involves the isolation of a restriction fragment containing a few hundred or a few thousand base pairs. The DNA is denatured and each strand is sequenced separately so that the sequences can be compared in order to eliminate
errors. One sequencing technique is the Sanger method (developed by Fred Sanger) which uses dideoxynucleotides that stop chain elongation at the site of their incorporation. The four dideoxynucleotide substrates are labeled with radioactivity and used in four separate reactions in vitro in which a single-stranded DNA template is copied. A series of radioactive DNA fragments are separated according to length by agarose gel electrophoresis (fragments differing by only one nucleotide are separated), and the DNA sequence can be read directly from the pattern of radioactive
bands in the gel. Another sequencing technique is the Maxam-Gilbert method (developed by Alan Maxam and Walter Gilbert). In this method, a single DNA strand is labeled at the 5' end with radioactive phosphorus (p32). The radioactive DNA is divided into four portions and each one is exposed to different chemical reactions. Each reaction causes a 5' cleavage adjacent to either
1. A or T,
2. G alone,
3. C or T, or
4. C alone.
The reactions are carried out for a short time so that, on average, only one cleavage occurs in each DNA molecule. This produces a set of DNA fragments (one set for each reaction), whose length identifies that position of a particular base. For example, a fragment containing 19 nucleotides in the G-only reaction mixture identifies G at position 20 from the 5' end. Similarly, a fragment containing 27 nucleotides present in the C or T reaction but not in the C-only reaction indicate that T is at position 28. The lengths of DNA fragments are determined by polyacrylamide gel electrophoresis, a technique that can separate DNA fragments differing in length by only one nucleotide.

Gene Therapy:

              One expected advance in medical treatment is the use of gene therapy to ameliorate or cure a variety of inherited disorders as well as diseases caused by somatic mutations such as cancer. The goal of gene therapy is the replacement of a defective, disease-causing gene in an individual by normal, functioning copies. In essence, gene therapy is a novel form of drug therapy; it uses the biochemical capacity of a patient's cells to synthesize the therapeutic agent from the introduced gene. The first trails at gene therapy were directed at trying to correct severe combined immunodeficiency (SCID) which is caused, in some cases, by a deficiency of adenosine deaminase (ADA), that is expressed in all tissues. ADA deaminates both adenosine and deoxyadenosine and, in the absence of the enzyme ADA, deoxyadenosine accumulates in cells. 

              Deoxyadenosine can be phosphorylated by the enzyme deoxcytidine kinase to produce deoxyadenosine triphosphate, a toxic substance that kills dividing cells. As a result, individuals with an ADA deficiency are defective in both humoral and cell-mediated immunity. In the mid-1980s, the first clinical gene therapy trials were attempted to correct ADA deficiency in two children with SCID. The ADA gene was cloned into a viral vector and inserted into peripheral T cells removed from the affected children. After a period of growth of the genetically modified cells in vitro, they were reintroduced into the patients. Synthesis of ADA could be detected for a time but the activity disappeared as the introduced cells died. 

               Several other trials of gene therapy for ADA deficiency showed promise, but it was still necessary to maintain the children with SCID on enzyme replacement therapy. Finally, in 2000, successful treatment of an X-linked form of SCID (SCID-X1) by gene therapy was reported by a French medical team. Almost a year after a normal gene was introduced into their cells, two children were still synthesizing the enzyme that they lacked. This was the first success for gene therapy in curing a disease after years of effort. Since the 1980s, hundreds of clinical trials of gene therapy for cystic fibrosis, osteogenesis imperfecta, Gaucher's disease, Fanconi's anemia, and several forms of cancer have been attempted. The problems that need to be overcome in developing a successful strategy for gene therapy are:
1. Development of safe and effective vectors for cloning the gene and inserting it into palients cells,
2. Regulation of expression of the desired gene product so that it is produced at the right time and in the correct amounts in the appropriate tissues, and
3. Stability of the inserted genetic construct in cells so that the product will continue to be produced.
               Many different viral vectors have been developed to deliver genes to various organs in the body; these include retroviruses that can insert themselves and the genes they carry into the chromosomes of cells; adenovirus, a DNA virus that causes respiratory infections, which has been inactivated by removal of many viral genes; and lentiviruses, slow-growing retroviruses. All of the viral vectors carry with them some risks. In 1999, a gene therapy patient died of complications from the use of adenovirus that was being tested as a therapy for an inherited liver disease caused by a deficiency in the enzyme ornithine transcarbamylase (OTC). Despite the problems that have beset gene therapy trials, it is expected that development of safer vectors and new techniques for delivering genes to cells will eventually make gene therapy a vital part of medical treatment.

DNA Vaccines:

              An extension of gene therapy, and one that may turn out to be of worldwide importance, is the use of naked DNA as a vaccine to prevent viral diseases. For example, plasmid DNA can be injected into tissues; upon entry, the DNA expresses any cloned gene, such as a viral antigen, that is carried by the plasmid. DNA vaccines have the advantage that the viral protein that is expressed in the cells stimulates both humoral and cell-mediated immunity. Fragments of the synthesized viral protein are carried to the cells' surface where they stimulate CD8 + cytotoxic T cells and, thereby, cell mediated-immunity. In experiments with mice, DNA vaccines have proven to be very effective. The gene coding for the core protein of the influenza virus was cloned into a plasmid vector and injected into mice. 

              The mice developed immunity not only to the strain of influenza from which the gene was derived but from other strains of influenza virus as well. Inducing an immune response with a viral core antigen is thought to be more effective than capsid antigens because viral core proteins from related viral strains do not differ much in structure or antigenicity. Viral capsid proteins, on the other hand, evolve rapidly; such changes, for example, account for the different strains of influenza virus that arise each year. Before DNA vaccines can replace conventional vaccines that use inactivated or attenuated viruses, the safety of the plasmid vectors must be rigorously proved. The plasmids might occasionally integrate into the host genome or they might stimulate an immune response to tissues containing the plasmid DNA. Either event might dictate against the widespread use of DNA vaccines.
Antibodies to DNA:

              Both single- and double-stranded DNA are antigenic; antibodies to DNA are normally found in the circulation, but in some individuals overproduction of DNA antibodies causes disease. In particular, patients with systemic lupus erythematosus (SLE) exhibit abnormal levels of antibodies to double-stranded DNA. Antibodies against single stranded DNA also bind to bases, nucleosides, nucleotides, oligonucleotides, and the ribose-phosphate backbone of RNA. Antibodies against double-stranded DNA also bind to base pairs, chromatin, nucleosomes, type IV collagen, and the deoxyribose-phosphate backbone of DNA. Antibodies against DNA consist of both IgM and IgG classes. Healthy individuals generally have low-affinity IgM antibodies to DNA; however, if these undergo an isotype switch to IgG, they may become pathogenic. 

               Tests for DNA antibodies help establish a diagnosis of SLE, although the level of such antibodies is not always predictive of the severity of the disease. Genetic susceptibility also seems to play a role in the pathogenicity of DNA antibodies in some individuals. Antibodies to double-stranded DNA may cause glomerulonephritis by forming antibody-DNA complexes that become trapped in the glomeruli. In some cases, DNA antibodies can be retrieved from damaged tissues in patients with SLE or glomerulonephritis, showing that the antibodies are involved in tissue damage. The production of DNA antibodies is decreased by immunosuppressive drugs, and immunosuppressive therapy is the approved treatment for SLE and related diseases caused by antibodies directed against DNA.