What underlies DNA hybridization? Although the double-stranded DNA sequence is generally stable under physiological conditions, changing these conditions in the laboratory (typically by raising the ambient temperature) will cause the molecules to separate into individual strands. The latter are complementary to each other, but may also complement other sequences present in their environment. Lowering the ambient temperature allows the single-stranded molecules to anneal or "hybridize" with each other. This is the DNA hybridization method.
The concept from the point of view of molecular biology
Scientists involved in both DNA replication and the transcription of DNA into RNA rely on nucleotide crossovers and molecular biology techniques. This includes Southern and Northern blots, polymerase chain reaction (PCR), and most DNA-RNA hybridization and sequencing approaches.
Application
Hybridization is the main property of nucleotidesequences and is used in numerous methods of molecular biology. The overall genetic relationship of two species can be determined by hybridizing segments of their DNA (DNA-DNA hybridization). Due to sequence similarities between closely related organisms, a higher temperature is required to melt such DNA hybrids compared to more distant organisms. Various methods use hybridization to determine the origin of a DNA sample, including the polymerase chain reaction (PCR). In another method, short DNA sequences are hybridized to cellular mRNA to identify expressed genes. Pharmaceutical companies are exploring the use of antisense RNA to bind to unwanted mRNA, preventing the ribosome from translating mRNA into protein.
DNA-DNA hybridization generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences. It is commonly used to determine the genetic distance between two organisms. It has been widely used in phylogeny and taxonomy.
Methodology
DNA from one organism was labeled, then mixed with unlabeled DNA that could be compared to it. The mixture is incubated to allow the DNA strands to dissociate and then cool to form a regenerated hybrid double stranded DNA. Hybridized sequences with a high degree of similarity will bind more tightly and require more energy to separate them: i.e., they separate when heated at a highertemperature than dissimilar sequences, a process known as "DNA melting".
DNA melting
Evaluating the melting profile of the hybridized DNA, the double-stranded DNA is bound to a so-called "column" and the resulting mixture is heated. At each step, the column is washed and the DNA sequences that melt become single stranded and wash off the column. The temperatures at which labeled DNA exits the column reflects the amount of similarity between sequences (and the self-folding pattern serves as a control). These results are combined to determine the degree of genetic similarity between organisms. According to modern microbiology, DNA hybridization is impossible without understanding these things.
When multiple ribonucleic acid (or deoxyribonucleic) acid species are compared in this way, the similarity values allow the species to be placed in the phylogenetic tree. Therefore, this is one of the possible approaches to conduct molecular systematics. Charles Sibley and John Ahlquist, the pioneers of this technique, used DNA-DNA hybridization to study the phylogenetic relationships of birds (Sibley-Ahlquist taxonomy) and primates.
Importance for biology
DNA-DNA hybridization is the gold standard for distinguishing bacterial species, with a similarity value of more than 70%, indicating that the compared strains belong to different species. In 2014, a threshold of 79% similarity was proposed for separating a bacterial subspecies.
Critics contend that the technique is inaccurate for comparing closely related species, as any attempt to measure differences between orthologous sequences between organisms is overwhelmed by the hybridization of paralogous counterparts in an organism's genome. DNA sequencing and computational sequence comparisons are currently the commonly used method for determining genetic distance, although this approach is still used in microbiology to help identify bacteria.
The current way is to conduct DNA-DNA hybridization in silicone using fully or partially sequenced genomes. The GGDC developed by the DSMZ is the most accurate known tool for calculating DDH-like values. Among other algorithmic improvements, it solves the problem with paralogous sequences by carefully filtering them out of matches between two genome sequences.
FISH method
Fluorescence In Situ Hybridization (FISH) is a laboratory technique used to detect and sequence DNA, often on a specific chromosome.
In 1969, Joseph Gall and Mary Lou Pardu published a paper demonstrating that radioactive copies of a ribosomal DNA sequence could be used to detect complementary DNA sequences in the nucleus of a frog egg. Since these original observations, many refinements have increased the versatility andthe sensitivity of the procedure to such an extent that in situ hybridization ("in place", Latin) is now considered an important tool in cytogenetics. (The term in situ is now also used to refer to the initial stage of carcinoma growth, when only epithelial tissue is involved in the pathological process.)
Fluorescent hybridization sequence
RNA probes can be designed for any gene or any sequence within a gene to visualize lncRNA and miRNA mRNA in tissues and cells. FISH is used by studying the cycle of cell reproduction, in particular nuclear interphase for any chromosomal abnormalities. FISH allows you to analyze a large series of archival cases, it is much easier to identify the identified chromosome by creating a probe with an artificial chromosome base that will attract similar chromosomes.
Hybridization signals for each probe when a nuclear abnormality is detected: each mRNA and lncRNA detection probe consists of 20 pairs of oligonucleotides, each pair covers a space of 40-50 bp. p. Probes use proprietary chemistry to detect mRNA.
Hybridization with DNA probes
Probes are often made from DNA fragments that have been isolated, purified and amplified for use in the design of the human genome. The size of the human genome is so large compared to the length that can be directly sequenced that it is necessary to divide it intofragments. Ultimately, these fragments were ordered by digesting a copy of each fragment into even smaller units using sequence-specific endonucleases to measure the size of each small fragment using size exclusion chromatography using this information to determine where large fragments overlapped with each other..
To preserve the elements with their individual DNA sequences, the fragments were added to a system of ever-repeating bacterial populations. Clonal populations of bacteria, each population maintaining a single artificial chromosome, are stored in various laboratories around the world. Artificial chromosomes (BACs) can be grown, extracted and labeled in any laboratory containing the library. Genomic libraries are often named after the institutions where they were developed. An example is the RPCI-11 library, named after the Roswell Cancer Institute in Buffalo (New York, USA). These fragments make up about 100 thousand base pairs and are the basis of most FISH probes.
