Polymerase chain reaction, example

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

Fig. 9.2 Representative examples of the methylation-specific polymerase chain reaction (MSP) analyses for gene promoter regions. Lanes Lf and M indicate the presence of unmethylated and methylated template, respectively. Placental DNA (PDNA) and in vitro methylated DNA (IMD) served as negative and positive controls, respectively. Water (H) was used to detect contamination. Samples 1, 3, 4, and 7 indicate the presence of a methylated promoter DNA with various degrees of methylation, and samples 2, 5, and 6 represent an unmethylated promoter...

Advances in biotechnology have been tremendous. There are many issues in biochemistry and biotechnology that have not yet been completely elucidated however, progress has been made. For example, antibiotics have evolved, the polymerase chain reaction has been developed, and DNA sequencing, genomics, proteomics, combinatorial synthesis, and selective complexation and recognition chemistry have all advanced. 

The traditional microbiological methods are very time consuming and sometimes limited concerning their interpretation. For that reason fast analysis methods as well as automated methods have been developed the latter are often used in specialised microbiological laboratories. During the last few years more and more modern biotechnological methods have been implemented into quality control, for example the enzyme-linked immunosorbent assay or more recently the polymerase chain reaction, which allows the detection of very specific microorganisms. 

DNA (see, for example, 8.23.2 Polymerase Chain Reaction). Some laboratories prefer to perform extra purification of the genomic DNA prior to MLPA [7]. It is not recommended to quantitatively compare DNA from different sources, such as different laboratories. The use of heparinised blood may cause a similar problem. 

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