The below mentioned article provides a study note on the Prenatal Diagnosis of Genetic Diseases.
It is required to analyse DNA of family members of the affected individual to set up a diagnostic tool for a genetic disease. The markers (RFLPs) that are tightly linked to the disease is needed to be identified first.
The physicians are required to have a basic understanding of the application of DNA technology to diagnosis of genetic diseases in the interest of medical and legal reasons. In case of families with a history of severe genetic diseases, it is essential to determine the presence of the disorder in the developing fetus.
Methods Available for Prenatal Diagnosis:
1. The fetus should be visualized by ultrasound if the genetic abnormality is caused in gross anatomic defects.
2. The chemical composition of the amniotic fluid can also give diagnostic clues.
3. Molecular analysis of fetal DNA can provide the most detailed genetic picture permitting reproductive information.
Sources of DNA:
DNA is obtained from white blood cells, amniotic fluid or chorionic villi. The time for DNA analysis has been shortened by the development of PCR.
Diagnosis of Sickle Cell Diseases Using Recombinant DNA Technology:
1. In the past, prenatal diagnosis of hemoglobinopathies was involved in the determination of the amount and kinds of hemoglobin synthesized in red cells obtained from fetal blood.
2. Sickle cell anemia is a genetic disease caused by a point mutation. At present, diagnostic techniques for analysing fetal DNA has become valuable since they provide safe, early detection of genetic diseases.
Phenylketonuria:
1. The phenylalanine hydroxylase (PAH) gene has been located on chromosome 12. The presence of abnormal phenylalanine hydroxylase genes has been detected by the use of DNA polymorphism as markers to distinguish between normal and mutant genes.
2. If an unborn fetus is affected, DNA-based screening is useful not only in determining but also in detecting carriers of the PKU gene. PKU is inherited as an autosomal recessive trait. It is also important to identify heterozygotes for future family planning.
Probes:
1. Probes are generally pieces of DNA or RN A labelled with a p32-containing nucleotide.
2. The probe must know a complementary sequence to be effective.
3. A cDNA synthesized from a specific mRNA can be used to screen either a cDN A library for a longer cDNA or a genomic library for a complementary sequence in the coding region of a gene.
4. cDNA probes are used to detect DNA fragments on southern blot transfers and to detect and quantitate RNA on Northern blot transfers.
Blotting and Hybridization Techniques:
1. Observation of a specific DNA or RNA fragment in many “Contaminating” molecules requires a number of techniques which are collectively termed blot transfer.
2. Sometimes, if a specific base is altered and a restriction site is changed, these procedures can detect a point mutation.
3. The Northern and Western blot transfer techniques are used to size and quantitate specific RNA and protein molecules, respectively.
4. Colony or plaque hybridization is the method by which specific clones are identified and purified. Bacteria are grown on colonies on an agar plate and overlaid with a nitrocellulose filter paper.
5. Perfect matches hybridize readily and withstand high temperatures in the hybridization and washing reactions. These complexes also form in the presence of low salt concentrations.
6. Less than perfect matches do not tolerate these stringent conditions.
7. Gene families can be detected by varying the stringency of the hybridization and washing steps.
DNA Sequencing:
1. The segments of specific DNA molecules can be analysed for their nucleotide sequence. This method depends upon having a large number of identical DNA molecules.
2. This requirement can be satisfied by cloning the fragment of interest by the above techniques.
3. Enzymatic method uses specific deoxy-nucleotide analogs that terminate DNA strand synthesis at specific nucleotides as the strand is synthesized on purified template nucleic acid.
Manual and Automatic Techniques for the Determination of the Sequence of DNA:
1. The segments of specific DNA molecules obtained by recombinant DNA technology can be analysed for their nucleotide sequence. The manual, emymatie method (Sanger’s) employs specific dideoxynucleotides that terminate DNA strand synthesis at specific nucleotides.
2. Polyaerylamitte gel electrophoresis can be used to separate the fragments according to size. Each of the fragments produces an image (band) on an x-ray film.
3. Another manual method, that of Maxam and Gilbert employs chemical methods to cleave the DNA molecules where they contain the specific nucleotides.
4. The most commonly procedure is the four different fluorescent labels, each representing one nucleotide. Each emits a specific signal by a laser beam and this can be recorded by a computer.