Many diseases are linked to genetic and molecular diseases which are quite detrimental to the health of infected individuals who might even lead a healthy live, but due to their genetic defects, suffer from abnormalities of varying impacts. Genetic diseases are difficult to cure because scientists are faced with the problem that the disease is caused by a defect in a particular gene and not the habits or any other organism. If it was caused by a pathogen, then proper diagnosis and treatment after the pathogen has been identified is quite effective, however if it is caused by a defective gene, then the treatment to fully get rid of the disease is quite difficult, as it arises naturally inside the body. Our DNA is everywhere and the genes it carries encodes for a number of things and is more than a million-base pair in size. It is quite difficult to pinpoint the gene that has been mutated and is causing this defect and then to cure the gene. However, in recent years with the advent of new technology like DNA recombinant technology, PCR, CRISPER gene editing, things have become significantly easier. I will talk about two of the highly used and major testing tools for genetic disease diagnosis. This has been important in curing millions of people.
Now we talk about some of the powerful testing tools for molecular diagnosis of genetic diseases.
- Southern/Northern-blot hybridization: This method has been a milestone in molecular diagnosis for detection of large gene alterations or point mutations and polymorphisms altering a restriction site. This method can be used at an early stage of gene characterization, i.e., even before complete sequencing is acquired and only a restriction map is available. In this technique, DNA fragments originated by restriction endonuclease digestion of entire DNA are separated electrophoretically, according to their size, on agarose gel. The size separated DNA molecules are then blotted into a nylon filter by capillary action by a high salt buffer that is passed through the gel from a buffer saturated paper sheet. The blotted nucleic acids are then immobilized on the membrane by a fixation step and hybridized to a radiolabeled single stranded DNA molecule, called as probe, which is complementary to the sequence of blot transferred DNA band or bands, that are to be detected.
- Polymerase chain reaction: A crucial advantage of this reaction or technique is its adaptability to automation, by employing a thermostable DNA polymerase, which can be helpful when a large number of tests are to be performed. This method has significantly simplified complex and time-consuming analytical protocols in molecular biology, thereby facilitating the diagnosis of mutations or various kinds of polymorphisms in human genetics. Approaches for detection of mutations can vary greatly even though all the approaches involve using PCR reactions, and furthermore, novel PCR based methods for detection of mutations have been developed. Short deletions or insertions can be detected by simply analyzing the size of PCR product after electrophoretic separation, as compared to a wild type of control sample. An example of this approach is the analysis of the major mutations causing cystic fibrosis, called DF508, which is a three base pair deletion causing the loss of phenylalanine at position 508 of the putative protein product. This deletion can be easily detected by the amplification reaction using specific flanking oligonucleotides that generate a 98 bp fragment in case of a wildtype allele and a 95 bp fragment in case of a mutated allele. Some PCR based diagnostic methods mainly devised to detect defined mutations include, allele specific oligonucleotide (ASO) hybridization, reverse dot blot (RDB), amplification refractory mutation system (ARMS), PCR-mediated site directed mutagenesis (PSDM), and reverse transcriptase polymerase chain reaction (RT-PCR). PCR based scanning methods include, denaturing gradient gel electrophoresis (DGGE), single stranded conformational polymorphism analysis (SSCP), heteroduplex analysis (HA), chemical mismatch cleavage (CMC) and direct sequencing.