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The following points highlight the top seven applications of recombinant DNA technology in molecular analysis of disease. The applications are: 1. Normal Gene Variations 2. Gene Variations Causing Disease 3. Point Mutations 4. Deletions, Insertions and Rearrangements of DNA 5. Pedigree Analysis 6. Restriction Fragment Length Polymorphism (RFLP) 7. Gene Therapy.
Applications of Recombinant DNA Technology:
- Normal Gene Variations
- Gene Variations Causing Disease
- Point Mutations
- Deletions, Insertions and Rearrangements of DNA
- Pedigree Analysis
- Restriction Fragment Length Polymorphism (RFLP)
- Gene Therapy
Recombinant DNA Technology: Application # 1. Normal Gene Variations:
a. Polymorphisms occur once in every 500 nucleotides, or about 107 times per genome.
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b. There are deletions and insertions of DNA as well as single base substitutions.
c. In healthy people, these alterations occur in noncoding regions of DNA or at site that cause no change in function of the encoded protein.
d. The polymorphism of DNA structure can be associated with certain diseases.
Recombinant DNA Technology: Application # 2. Gene Variations Causing Disease:
a. Most genetic diseases were due to point mutations that resulted in an impaired protein.
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b. β-globin gene is located in a cluster on chromosome 11. Defective production of β-globin results in a variety of diseases and is due to many different lesions in and around the β-globin gene.
Recombinant DNA Technology: Application # 3. Point Mutations:
a. Sickle cell disease is caused by mutation of a single base out of the 3 x 109 in the genome.
b. The altered codon specifies a different amino acid (valine rather than glutamic acid) and this causes a structural abnormality of the β-globin molecule. β- thalassemia is the result of these mutations.
Recombinant DNA Technology: Application # 4. Deletions, Insertions and Rearrangements of DNA:
a. Pieces of DNA can move from one place to another within a genome on the study of bacteria, viruses, yeasts, and fruit flies.
b. Disease is caused by the deletion of critical piece of DNA, the rearrangement of DNA within a gene, or the insertion of a piece of DNA within a coding of regulatory region can also cause changes in gene expression.
c. Deletions in the alpha-globin cluster, located on chromosome 16, cause alpha thalassemia.
d. Deletions or insertions of DNA larger than 50bp can often be detected by the southern blotting procedure.
Recombinant DNA Technology: Application # 5. Pedigree Analysis:
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a. Intubation of DNA from normal (AA), heterozygous (AS), and homozygous (SS) individuals results in three different patterns on southern blot transfer.
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b. Pedigree analysis has been applied to a number of genetic diseases and is most useful in those caused by deletions and insertions or the rarer instances in which a restriction endonuclease cleavage site is affected.
Recombinant DNA Technology: Application # 6. Restriction Fragment Length Polymorphism (RFLP):
a. Inherited differences in the pattern of restriction is known as RFLP.
b. This results from single base changes (e.g., sickle cell disease) or from deletions or insertions of DNA into a restriction fragment (e.g., the thalassemia) and are proving to be a useful diagnostic measure.
c. PFLPs may disrupt the function of the gene or may have no biological significance.
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d. RFLPs are inherited and they segregate in a Mendelian fashion.
e. These can be used to establish linkage groups, which in turn, by the process of chromosome walking, will define the disease locus.
f. In chromosome walking, a fragment representing one end of a long piece of DNA is used to isolate another that overlaps but extends the first.
g. 20 p.c. of the defined RFLPs are on the X chromosome.
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h. X-linked disorders such as Duchenne type muscular dystrophy will be defined using RFLPs.
i. The defect that causes polycystic kidney disease is linked to the alpha globin locus on chromosome 16.
Recombinant DNA Technology: Application # 7. Gene Therapy:
a. Bone marrow precursor cells are being investigated because they presumably will resettle in the marrow and replicate there. The introduced gene would begin to direct the expression of its protein product and this would correct the deficiency in the host cell.
b. Some percentage of genes injected into fertilized mouse ovum will be incorporated into the genome and found in both somatic and germ cells. These transgenic animals are useful for analysis of tissue— specific effect on gene expression.
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c. The transgenic approach has recently been used to correct a genetic deficiency in mice.
d. Fertilized ova obtained from mice with genetic hypogonadism were injected with DNA containing the coding sequence for the gonadotropin—releasing hormone (GnRH) precursor protein. This gene was expressed and regulated normally in the hypothalamus of a certain number of the resultant mice, and these animals were in all respects normal. Their offspring also showed no evidence of GnRH deficiency.
