Products electrophoresis, sequencing

Capillary electrophoresis technology has become an indispensable tool for forensic scientists in the biology field since it is able to provide valuable information to aid in the process of law enforcement. The primary application of the technique is in the qualitative analysis of STRs. Isolation of STR mixtures is also possible using relative peak heights. Other applications of CE include quantitative analysis of PCR products, mtDNA sequencing, and mutation detection for the analysis of plant and bacterial DNA. Based on the performance of the methods illustrated above, it is reasonable to expect future researchers and practitioners to continue working to exploit the capabilities of this robust scientific technique and its application to criminal investigations. 

When reaction is complete, the product consists of a mixture of DNA fragments of all possible lengths, each terminated by one of the four dye-labeled dideoxyribonucleotides. This product mixture is then separated according to the size of the pieces by gel electrophoresis (Section 26-2), and the identity of the terminal dideoxyribonucleotide in each piece—and thus the sequence of the restriction fragment—is identified simply by noting the color with which the attached dye fluoresces. Figure 28.8 shows a typical result. 

The extraordinary complexity of human genes and their products has encouraged the development of extremely high-resolution analytical methods.75 Capillary electrophoresis is competitive with slab gel methods, with resolution up to the order of about 1,000 base pairs for sequencing, sizing, and detection of mutation. Reversed phase HPLC is useful for restriction digest mapping and MALDI-MS up to about 1000 base pairs. 

Resolution in capillary gel electrophoresis of DNA sequencing was shown to be directly proportional to the product of the number of bases and the relative peak distance, i.e., to the mean separation of peaks.43 Reformulation of the treatment of the capacity factor has been used to simplify and clarify the interpretation of the separation factor in electrophoresis.44 Peak... 

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. 

Gel electrophoresis and, more recently, CGE have been employed principally in molecular biology and biochemical science for the separation of macromolecules such as proteins and nucleic acids. And GCE has been successfully used in oligonucleotide purity analysis, antisense gene therapy, DNA sequencing, PCR product analysis, and DNA forensics. 

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