Selective DNA isolation<unk>  allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of<unk>  augments many ways, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region.<unk>  supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material. Other applications of<unk>  include DNA sequencing to determine unknown<unk> -amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid, phage, or cosmid (depending on size) or the genetic material of another organism. Bacterial colonies (such as E. coli) can be rapidly screened by<unk>  for correct DNA vector constructs.<unk>  may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different<unk> -based methods. Electrophoresis of<unk> -amplified DNA fragments: The child has inherited some, but not all of the fingerprints of each of its parents, giving it a new, unique fingerprint. Some<unk>  fingerprint methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms when certain molecular clocks are used (i.e. the 16S rRNA and recA genes of microorganisms). Amplification and quantification of DNA Because<unk>  amplifies the regions of DNA that it targets,<unk>  can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence.<unk>  may also be used in the analysis of ancient DNA that is tens of thousands of years old. These<unk> -based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar and the body of English king Richard III. Quantitative<unk>  or Real Time<unk>  (qPCR, not to be confused with<unk>  methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression. Quantitative<unk>  is an established tool for DNA quantification that measures the accumulation of DNA product after each round of<unk>  amplification. qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method consists of using fluorescent dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA takes place. An interesting technique combination is real-time<unk>  and reverse transcription. This sophisticated technique, called<unk> , allows for the quantification of a small quantity of RNA. Through this combined technique, mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of<unk> , increasing<unk> ces for detection of genes associated with genetic diseases such as cancer. Laboratories use<unk>  for the purpose of sensitively measuring gene regulation. The mathematical foundations for the reliable quantification of the<unk>  and<unk>  facilitate the implementation of accurate fitting procedures of experimental data in research, medical, diagnostic and infectious disease applications. Medical and diagnostic applications Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease. DNA samples for prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in the mother's bloodstream.<unk>  analysis is also essential to preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations. *<unk>  can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. there is even a proposal to replace the traditional antibody-based tests for blood type with<unk> -based tests. * Many forms of cancer involve alterations to oncogenes. By using<unk> -based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient.<unk>  permits early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest-developed in cancer research and is already being used routinely.<unk>  assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods.<unk>  is very useful in the medical field since it allows for the isolation and amplification of tumor suppressors. Quantitative<unk>  for example, can be used to quantify and analyze single cells, as well as recognize DNA, mRNA and protein confirmations and combinations. Infectious disease applications<unk>  allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.<unk>  also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for<unk>  diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes. Characterization and detection of infectious disease organisms have been revolutionized by<unk>  in the following ways: * The human immunodeficiency virus (or HIV), is a difficult target to find and eradicate. The earliest tests for infection relied on the presence of antibodies to the virus circulating in the bloodstream. However, antibodies don't appear until many weeks after infection, maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the infection don't affect the antibodies.<unk>  tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells. Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified. * Some disease organisms, such as that for tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory.<unk> -based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy. The effects of therapy can also be immediately evaluated. * The spread of a disease organism through populations of domestic or wild animals can be monitored by<unk>  testing. In many cases, the appearance of new virulent sub-types can be detected and monitored. The sub-types of an organism that were responsible for earlier epidemics can also be determined by<unk>  analysis. *Viral DNA can be detected by<unk> . The primers used must be specific to the targeted sequences in the DNA of a virus, and<unk>  can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of<unk>  permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead time in treatment. The amount of virus ("viral load") in a patient can also be quantified by<unk> -based DNA quantitation techniques (see below). A variant of<unk>  <unk>  is used for detecting viral RNA rather than DNA: in this test the enzyme reverse transcriptase is used to generate a DNA sequence which matches the viral RNA; this DNA is then amplified as per the usual<unk>  method.<unk>  is widely used to detect the SARS-CoV-2 viral genome. *Diseases such as pertussis (or whooping cough) are caused by the bacteria Bordetella pertussis. This bacteria is marked by a serious acute respiratory infection that affects various animals and humans and has led to the deaths of many young children. The pertussis toxin is a protein exotoxin that binds to cell receptors by two dimers and reacts with different cell types such as T lymphocytes which play a role in cell immunity.<unk>  is an important testing tool that can detect sequences within the gene for the pertussis toxin. Because<unk>  has a high sensitivity for the toxin and a rapid turnaround time, it is very efficient for diagnosing pertussis when compared to culture. Forensic applications The development of<unk> -based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics: * DNA samples are often taken at crime scenes and analyzed by<unk> .In its most discriminating form, genetic fingerprinting can uniquely discriminate any one person from the entire population of the world. Minute samples of DNA can be isolated from a crime scene, and compared to that from suspects, or from a DNA
Polymerase chain reaction
Reverse transcription polymerase chain reaction
Reverse complement polymerase chain reaction
Fluorescence in situ hybridization
Ink blot test
Immunoassay
Broth microdilution
Magnetic immunoassay