DNA chips: DNA “chips” or microarrays have been used as possible tests for multiple mutations. In this technology, individual DNA strands, including sequences from different targets, are attached to a fixed medium in a network format. On the other hand, the DNA of the sample or the cDNA labeled with fluorescent dyes is hybridized into a chip (Fig. 4E)[32]. Then the presence of fluorescence is checked with a laser system; The sequences and their quantities in the sample are determined. In X-linked disease (Fig. 1C3 and Table 1), the mutated gene is located on the X chromosome. A recessive mutation can lead to the disease. The X chromosome gene must be mutated to cause disease; Therefore, X-linked recessive disorder is carried by women, while men are usually affected.
Some of the most important methods are described below. The production of a visual karyotype (Fig. 2C) is carried out by keeping dividing cells in the metaphasic stage with a microtubule polymerization inhibitor such as colchicine; The cells are then distributed on a glass slide and stained with an Giemsa spot (G-band). Chromosomes are examined by taking a photo or digital imaging and then assembling the chromosomes. Human chromomas are classified according to the position of the centromere; In metacentric chromosomes, centromeres are located in the middle (chromosomes 1, 3, 16, 19 and 20), chromosomes 13, 14, 15, 21, 22 and Y are acrocentric (the centromere near one end), and the other chromosomes are submetacentric. Chromosomal arms are defined by region number (from centromere), band, subband and sub-subband numbers, e.g. 12q13.12 refers to chromosome 12, long arm, region 1, volume 3, subband 1, subband 2 (see chromosome 12, q, 1, 3, period, 1, 2). High-resolution bands must be fixed before the chromosomes are fully compacted. Practical methods of chromosomal banding are G-(Giemsa), R-(inverted), C-(centromere) and Q-(quinacrine). In addition to the genetic causes of disorders, determining DNA variations could also reveal predisposition to a disease or treatment options. Molecular diagnostics offer a way to assess the genetic makeup of humans; He combines laboratory medicine with molecular genetics to develop DNA/RNA-based analytical methods to monitor human pathologies. A wide range of methods have been used to detect mutations.
Molecular methods for identifying pathogenic mutations could be classified as methods for known mutations and methods for unknown mutations. However, several criteria must be met in order to choose an appropriate method; For example, the following points should be taken into account: type of nucleic acid (DNA or RNA), type of sample (blood, tissue, etc.), number of mutations and reliability of the method. Pediatricians should be considered when prescribing these tests in order to provide patients with an accurate diagnosis. L.J. Stadler of the University of Missouri and Ralph Singleton of the University of Virginia worked on methods for detecting mutations in corn and barley. Here, models of Mendelian heredity and chromosomal disorders are examined and a brief summary of genetic methods in genetic disorders is presented in order to familiarize pediatricians with the basics of cytogenetics and molecular methods of detecting mutations. Various methods are available for the detection of mutations, both in plants and animals. The seeds of the offspring would normally show dominant characters. If a mutation were induced on a dominant gene, the recessive trait would appear in the offspring. Polymerase chain reaction (PCR) and its versions: In the 1980s, Dr.
Mullis introduced a method of amplifying DNA fragments into a large number of fragments in just a few hours; This method, called polymerase chain reaction (PCR), has been a critical point in molecular biology [29, 30]. The essential components of the polymerase chain reaction are model DNA, primers (a pair of synthetic oligonucleotides complementary to the two target DNA strands), thermostable DNA polymerase enzyme (e.g., Taq), divalent cations (usually Mg2+), deoxynucleoside triphosphates (dNTP), and buffer solution. PCR, consisting of 25 to 40 repeated cycles, consists of three discrete stages of temperature change (Fig. 3A); After a single high temperature step (>90 ° C), a series of denaturation, annealing of primers and extension cycles is performed, followed by a single temperature step called final product expansion or short storage. These steps are as follows: The significant aspect of this crossing is to bring together the same irradiated chromosome in the homozygous state to detect all recessive mutations. In Generation X (generation F1; called X, because individuals X 1 are irradiated, the man`s X chromosome goes to daughters who have another X from the mother; therefore, no induced mutations can be detected in this generation.