Let us make an in-depth study of the molecular diagnosis and treatment of diseases.
A disease, in molecular sense, can be defined as any abnormality in the living system. The abnormality can be caused due to infection by virus, bacteria, fungi, parasites, proteins or small molecules in/from humans, animals, plants, water and soil. The abnormality can also arise due to changes in the molecular structure within the cells. As an example, a change in the DNA sequence known as mutation can cause various disorders/diseases.
The prevention and treatment of these diseases is possible only if the causative agent of the disease can be diagnosed at the appropriate time. Hitherto many costly and laborious clinical procedures were used in the diagnosis and treatment of these diseases. With the advancement of Molecular Biotechnology, various molecular diagnostic methods are now applied in the diagnosis and also treatment of these diseases.
Molecular Diagnosis:
A diagnostic test can be effective only if it is:
(a) Specific for the target molecule
(b) Sensitive to detect even minute levels of the target and
(c) Technically simple.
There are two classes of molecular diagnostic techniques:
(1) DNA detection methods—which uses nucleic acid hybridization or the polymerase chain reaction to detect a specific nucleic acid sequence.
(2) Immunological methods—are based upon the specificity of an antibody for a particular antigen.
1. DNA Detection Methods:
Various methods have been devised for the detection of various diseases, based on the sequence of DNA, built in a specific manner.
Some methods discussed here are:
(a) Detection of a pathogenic organism by nucleic acid hybridization
(b) Diagnosis of genetic disease using restriction endonuclease
(c) Diagnosis of genetic disease by P.C.R./oligonucleotide ligation assay (PCR/OLA) and
(d) detection of mutants at different sites within one gene.
(a) Detection of a pathogenic organism by nucleic acid hybridization:
The disease causing (pathogenic) organism can be detected very specifically in biological samples by nucleic acid hybridization i.e. if the nucleic acid sequence of a disease causing organism is present in the blood, urine, faeces, etc., then it can be hybridized with a nucleic acid probe complementary to the sequence of this target nucleic acid. If the pathogenic organism is present in the biological sample, hybridization occurs and if not, there is no hybridization.
The parasite Plasmodium falciparum causes malaria in man. A specific gene (thereby its product) is the causative agent in this parasite. A complementary DNA probe to this gene is synthesized chemically with radiolabelled 32P. This probe is bonded to a membrane support. Then the biological sample to be analysed is added, under appropriate conditions of temperature and ionic strength to promote base pairing between the probe and the target DNA in the sample.
It is then washed to remove the excess of the sample and then the hybridized double stranded DNA is extracted and the hybrid sequences are detected by autoradiography. The specific DNA probe chosen will hybridize only with P. falciparum but not with P. vivax, P. cyanomolgi or human DNA. This probe can detect as little as 10 picogram of purified P. falciparum DNA or 1 Nano gram of P. falciparum DNA in blood samples. If hybridization has occurred then the pathogenic organism is present and if no hybridization occurs (i.e. no radiations) then the pathogenic organism is absent.
The above procedure is adopted for detection of all pathogenic organisms in any biological sample. Here the disadvantage of using the radioactive phosphorus is that it is hazardous, hence now-a-days nonradioactive hybridization procedures are used. In this method all the DNA from the sample is extracted and is bonded to a support, then the biotin-labelled DNA probe complementary to the pathogen DNA is hybridized to the target DNA.
Then, either avidin or streptavidin is added, which will bind to the biotin on the hybridized probe-target DNA. Then a biotin labelled enzyme like alkaline phosphatase is added which binds to the avidin bonded on the probe. Then the substrate specific for this enzyme is added, which will convert the colourless substrate into a coloured product. Appearance of the colour indicates the presence of the pathogenic DNA and non-development of the colour is an indication of the absence of the organism.
(b) Diagnosis of genetic disease using restriction endonuclease:
Sickle-cell anemia is a genetic disease due to the change in a single nucleotide in the codon for the sixth amino acid of the beta-chain of the hemoglobin molecule. The gene for beta-globin in normal persons is designated as A/A, in heterozygous individuals as A/a, and in homozygous individuals as S/s. Individuals containing the sickle gene are screened before the expression of the symptoms and for screening the carrier, who are at risk of transmitting this gene to their offspring.
The principle for the detection is that, within the beta-globin gene of a normal individual, there are three sites for the restriction endonuclease Cvn-1, but in sickle-cell gene one of these sites is lost due to replacement of the single nucleotide.
In the normal gene, the DNA sequence is CCTGAGG whereas in the sickle-cell anemia gene, the sequence is CCTGTGG. Further the recognition sequence and site of cleavage by Cvn-1 is CCTNAGG. Thus, the difference in sequence of normal and sickle-cell gene in the recognition site of Cvn—1 forms the basis of this DNA diagnosis.
Two primers with sequences that can pair within the Cvn-1 sites in the beta-globin gene are added and this part of DNA is amplified using P.C.R. and then digested with Cvn-1. Finally the cleavage products are separated by gel electrophoresis and stained by ethidium bromide.
The results indicate that in the normal gene, four DNA fragments are obtained with 88, 181, 201 and 256 base pairs. But in heterozygous individual five DNA fragments are obtained containing 88, 181, 201, 256 and 382 base pairs and in homozygous individuals only three fragments are obtained with 88, 256 and 382 nucleotide base pairs, indicating the loss of one of the recognition site in the sickle cell gene.
(c) Diagnosis of genetic disease by PCR/OLA procedure:
This procedure is applied for those disorders, due to genetic mutations, which does not affect the restriction endonuclease sites. Let us take an example of a gene, which has undergone mutation at position 98. At this specific site the base pair in the normal gene be C=G and in the mutant gene let it be A=T.
Two oligonucleotides of about 20 nucleotide length each are synthesized with sequence complementary to one of the strands of this gene on either side of position 98. One of these oligonucleotides has biotin at its 5′ end and ‘C’ as the terminal nucleotide at the 3′ end. The other oligonucleotide probe has ‘A’ base nucleotide at the 5′ end and digoxigenin (compound ‘D’) at its 3′ end.
The target DNA is amplified by PCR and then is hybridized with the synthesized probes. The two probes base pair such that the 5′ end of the 2nd probe lies next to the 3′ end of the 1st probe. Then DNA ligase is added, which will ligate only the normal DNA fragment but not the mutated fragment hybridized with the probes.
This is because of the mismatch between the 2nd probe and the mutated gene, which cannot base pair. Further, in order to determine whether ligation has occurred or not, the hybrid probes are taken into a well containing avidin which binds to biotin. Then it is washed, which removes the un-ligated probe.
Then anti-digoxigenin (‘D’ compound) antibody- alkaline phophatase conjugate is added and washed in both the wells (the normal and mutated hybridized probes). It is expected that the antibody enzyme will bind only to the ligated probe well. When substrate is added, the coloured product is produced only in the well where ligation has occurred, whereas no colour is formed where no ligation has taken place.
(d) Detection of mutations at different sites within one gene:
Beta-thalassemia is a genetic disease that is caused due to mutation in beta-globulin at eight or more sites, thus results in low rate of its synthesis. Hence instead of detecting each mutation separately all the eight sites are scanned at the same time.
DNA probes are synthesized to all these eight sites of beta-globin gene where mutations are expected. Each probe is 20 nucleotide in length with a poly T’ tail at the 3′ end. This is used to attach the probe to a membrane. Segments of the sample DNA (beta-gene) that includes each of the possible mutant sites are amplified by PCR, using primers labeled with biotin at the 5′ end.
The amplified target DNA is then hybridized to the membrane bound probes under conditions that allow only perfect matches to hybridize. Then streptavidin with attached alkaline phosphatase is added, the membrane washed and a colourless substrate is added.
A coloured spot on the membrane appears wherever there is a perfect nucleotide match between the amplified target DNA segment and one of the specific oligoneucleotide probes. Where there is no hybridization (mutant DNA segments) no colour appears. In the illustration given below, gene 1 and 2 are mutated but gene 3 is normal. (Represented as probe 1, 2 and 3 respectively in the figure).
2. Immunological Methods:
Antibody molecules consist of four chains, two identical light chains and two identical heavy chains. The fv (fragment variable) region of each antibody molecule binds tightly to a specific site (epitope) on an antigen. This specificity is used to identify the presence of a particular epitope of a disease causing molecule or organism in a biological sample. There are two methods by which the antigen-antibody reaction or binding is detected.
(a) Radio-immuno assay (RIA):
The concentration of progesterone in blood (for example) is to be determined by RIA. First of all antibodies to progesterone are raised and taken in a test tube containing glass beads. The antibodies get readily attached to the glass beads. Then progesterone containing sample is added to this test tube which binds the antibodies forming antigen-antibody complex, whose concentration depends upon the amount of progesterone in the blood sample.
Another test tube is taken and the antibodies are labelled with radioactive compounds like 123I or 3H or 14C. This radio labelled antibody is then added to the first test tube containing progesterone attached to un-labelled antibodies. Radio labelled antibodies will now attach to the progesterone and form labelled antigen-antibody complex which is measured using a scintillation counter.
(b) Enzyme linked immunosorbent assay (ELISA):
The sample which is to be tested for the presence of a specific molecule or organism is bound to a solid support such as a plastic plate. Then a marker- specific antibody (primary antibody) is added to the bound material and then the support is washed to remove unbound primary antibody.
Then a second antibody (secondary antibody) is added, which binds specifically to the primary antibody and not to the target molecule. The secondary antibody contains bound enzyme like alkaline phosphatase which catalyses the conversion of a colorless substrate into a colored product. The system is washed again to remove any unbound secondary antibody-enzyme conjugate.
Then a colorless substrate is added which is converted to a colored product only if the specific antigen is present, if not there is no colour. If there is no antigen (or the causative agent) then the primary antibody will not bind to the target site in the sample, hence the first washing step removes it. Consequently, the secondary antibody—enzymes conjugate will have nothing to bind to and is removed during the second washing step, and the final mixture remains colorless.
Conversely, if the antigen (or the causative agent) or the target site is present in the sample, then the primary antibody binds to it, the secondary antibody binds to the primary antibody and the attached enzyme will catalyze the reaction to from a colored product which can be detected colorimetrically.
Molecular Treatment or Gene Therapy:
In order to treat a genetic disease, the normal gene for that disease has to be sequenced and cloned. This cloned normal gene can be used to correct the defect in individuals who have a mutant form of that gene. Here, the objective is to add a normal functioning gene to defective cells, thereby providing the required protein and correcting the genetic disease. In addition, it will be necessary to prevent the over expression of a deregulated normal gene, in some diseases.
There are three methods for the therapy of genetic diseases:
(1) Ex vivo gene therapy
(2) In vivo gene therapy and
(3) Antisense therapy.
(a) Ex vivo Gene Therapy:
Somatic cells from an affected individual are collected. The isolated cells are grown in culture. These cells are then transfected by retroviral cloning vectors containing the remedial gene construct. The cells are further grown and those cells which contain the gene of interest are selected and finally transplanted or transfused back into the patient. These transplanted transfected cells will synthesize the gene product i.e. the protein. Examples for this type of treatment include gauche disease, sickle cell anaemia, thalassemia etc.
(b) In vivo Gene Therapy:
In this type of treatment there is the direct delivery of the remedial gene into the cells of a particular tissue of the patient, using retroviral vectors. Even plasmid DNA constructs
are used. This type of treatment is used in case of muscular dystrophy, neuronal degeneration and brain cancer patients.
(c) Antisense Therapy:
Antisense therapy is designed to prevent or lower the expression of a specific gene. In some type of genetic diseases and cancers, the genes are deregulated or over expressed resulting in the production of excess of the gene product or its continuous presence in the cell will disrupt the normal functioning of the cell.
In such type of diseases the addition of normal gene will not solve the problem; instead blocking the synthesis of the gene product (protein) will be helpful. Thus in anti-sense therapy a nucleic acid sequence is introduced into the target cell which is complementary to complete or a part of that specific mRNA.
Hence the mRNA produced by the normal transcription of the gene will hybridize with the antisense oligonucleotide by base pairing, thereby preventing the translation of this mRNA, resulting in reduced amount of target protein. The antisense therapy is used in treatment of various cancers, AIDS, atherosclerosis, leukemia and sickle cell anaemia.
