The following points highlight the three main molecular basis of inherited DNA diseases. The molecular basis are: 1. Genetic Diseases 2. Cleavage of DNA into Fragments 3. DNA Cloning.
Molecular Basis # 1. Genetic Diseases:
The human genome contains DNA with about 3 billion (109) base pairs that encode 50,000 to 100,000 genes located on 23 pairs of chromosomes. The molecular defects of inherited diseases are based on mutation in DNA causing the incorporation of an incorrect amino acid into a protein. Genetic diseases are divided into five principal categories.
i. Single Gene Defects:
(a) Single gene disorders result from a mutation at a single site on a chromosome. These single gene defects indicate new significance to the old genetic terms “recessive” and “dominant”.
(b) Recessive pattern shows the deficiencies in enzymes. In a heterzygote for a recessive disorder the one normal gene often provides sufficient enzyme to prevent clinical symptoms.
(c) The heterozygote with one normal and one defective gene coding for structural proteins exhibit disease symptoms.
ii. Multifactorial Disorders:
(a) These are determined by many genes and are influenced by environmental factors, e.g., diabetes mellitus, hypertensive and manic depression.
(b) These occur more frequently in the immediate family of an affected person.
(c) Twins that are genetically identical show a greater similarity than twins that have half of their genes in common.
iii. Chromosome Disorders:
(a) These are caused by the loss, gain or abnormal arrangement of one or more of the 46 chromosomes composing the human diploid genome (two copies of each of the 22 autosomal chromosomes plus two sex chromosomes).
(b) Chromosomal abnormalities are common.
(c) The most common autosomal defect is trisomy 21 (three copies of chromosome 21) which is responsible for about 95 percent of cases of Down syndrome.
iv. Somatic Cell Gene Defects:
Somatic cell gene mutations are important in tumorigenesis.
v. Mitochondrial Mutations:
(a) Circular molecules of DNA (mt DNA) are present in mitochondria. Each codes for 13 of 100 polypeptides required for the process of oxidative phosphorylation, as well as for 2 species of rRNAs and 22 kinds of tRNA.
(b) mt DNA is maternally inherited because mitochondria from the sperm cell do not enter the fertilized egg.
(c) mt DNA has a high rate of mutation and mitochondrial defects cause a number of human diseases.
Molecular Basis # 2. Cleavage of DNA into Fragments:
The process of DNA analysis has been discovered by a special group of bacterial enzymes called restriction endonucleases (restriction enzymes) that cleave double-stranded DNA into smaller fragments. Restriction enzymes are used experimentally to obtain the defined DNA segments called restriction fragments.
i. Specificity of Restriction Endonucleases:
These enzymes recognize specific nucleotide sequences (generally 4 or 6 base pairs). These sequences exhibit two-fold rotational symmetry which means the nucleotide sequence on the “top” strand read 5′ → 3′ is identical to that of the “bottom” strand also read in the 5′ → 3′ direction.
ii. Nomenclature:
(a) A restriction enzyme is named according to the organism from which it was isolated.
(b) The first letter of the name is from the genus of the bacteria.
(c) The next two letters are from the name of the species.
(d) An additional subscript letter indicates the type or strain.
(e) The final number appears to indicate the order in which the enzyme was discovered in the particular organism.
Example:
Hae 111 is the third restriction endonuclease isolated from the bacterium Haemophilus aegyptius.
iii. “Sticky” arid “Blunt” Ends:
(a) Restriction enzymes cleave DNA to produce a 3′-hydroxyl group on one end and a 5′-phosphate group on the other.
(b) Some restriction endonucleases, such as Taq 1, produce “sticky” or cohesive ends, i.e., the resulting DN A fragments have single stranded sequences that are complementary to each other.
(c) Other restriction nucleases, such as Hae 111, cleave in the middle of their recognition sequence and produce fragments that have “blunt” ends that do not form hydrogen bonds with each other.
(d) Using the enzyme DNA ligase, sticky ends of a DNA fragment can be covalently joined with other DNA fragments that have sticky ends produced by cleavage with the some restriction endonulcease. The hybrid combination of two fragments is called a recombinant DNA molecule.
iv. Restriction Sites:
(a) A DNA sequence recognized by a restriction enzyme is called a restriction site.
(b) Since these sites occur at random, restriction endonucleases cleave DNA into fragments of different sizes.
(c) An enzyme that recognizes a specific four base-pair sequence produces many cuts in the DNA molecule. But an enzyme requiring a sequence of six base pairs produces fewer cuts and hence, longer pieces.
Molecular Basis # 3. DNA Cloning:
If a foreign DNA molecule is introduced into a replicating cell, it permits the amplification (production of many copies) of the DNA. In some cases, a single DNA fragment to be cloned can be isolated and purified. To clone a nucleotide sequence, the total cellular DNA is first cleaved with a specific restriction enzyme forming thousands of fragments.
These “insert” DNA fragments are joined to DNA cloning vectors to form hybrid molecules. Each hybrid recombinant DNA molecule conveys its insert DNA into a single host cell.
As the host cell multiplies, it forms a clone in which every bacterium carries copies of the some insert DNA fragment, hence the name “cloning”. The cloned DNA is released from its vector by cleavage (using the suitable restriction endonuclease) and is isolated.
i. Vectors:
(a) A vector is a molecule of DNA to which the fragment of DNA to be cloned is attached.
(b) The important properties of vector are:
i. It must be capable of autonomous replication.
ii. It must contain at least one specific nucleotide sequence recognized by a restriction endonuclease.
iii. It must carry at least one gene.
iv. The used vectors are commonly plasmids and bacterial and animal viruses.
(1) Prokaryotic plasmids:
(a) Prokaryotic organisms contain single, large, circular chromosomes.
(b) Most species of bacteria also contain small circular, extra-chromosomal DNA molecule called plasmids.
(c) Plasmids may carry genes that convey antibiotic resistance to the host bacterium and may transfer the genetic information from one bacterium to another.
(d) Plasmids can be isolated from bacterial cells and their circular DNA can be cleaved at specific sites by restriction endonucleases and the foreign DNA can be inserted into the circle.
(e) The hybrid plasmid can be reintroduced into a bacterium and large numbers of copies of the plasmid containing the foreign DNA are produced.
(2) Other Vectors:
(a) The development of improved vectors can accommodate large DNA segments showing improved molecular genetic research.
(b) Yeast plasmids, and bacterial and mammalian viruses are widely used as cloning vectors.
ii. DNA Libraries:
A DNA library is a collection of cloned restriction fragments of the DNA of an organism.
Two kinds of libraries are:
(a) Genomic libraries;
(b) cDNA libraries.
(a) Genomic DNA libraries:
(a) A genomic library is the collection of fragments of double-stranded DNA obtained by digestion of the total DNA with a restriction endonuclease.
(2) The amplified DNA fragments represent the entire genome of the organism and are called a genomic library.
(3) Cleavage occurs only at a fraction of the restriction sites on any one DNA molecule producing fragments of about 20,000 base pairs.
(b) Complementary DNA (cDNA) libraries:
(1) cDNA libraries contain only those DNA sequences that appear as mRNA molecules.
(2) This mRNA can be used as a template to make a cDNA using the enzyme reverse transcriptase.
(3) It can be used as a probe to locate the gene that coded for the original mRNA in mixtures containing many unrelated DNA fragments.