A polymer most frequently used as a sieving matrix in automatic DNA sequencers is polyacrylamide. It can be used in linear or cross-linked form. The porosity of the gel is defined by the concentration of acrylamide and cross-linking agent (typically N,N-methylene-bis-acryl-amide). The concentration of acrylamide may vary from 3% to 10%. The concentration of a cross-linker is ~3% to 5% of the total acrylamide concentration. An increase in the acrylamide concentration suggests a higher resolution of the DNA bands and allows calling more bases, but this leads to slower runs. Linear polyacrylamide (LPA), which is more fluid, is typically used in capillary electrophoresis (CE) where the sieving matrix is replaced after each run so the capillary can be reused. The last requirement is a result of the high cost of the capillary arrays and complexity of alignment required after their replacement. Other types of polymers used in CE include hydroxyethyl cellulose (HEC), poly(ethylenox-yde) (PEO), polyvinyl pyrrolidone (PVP), and others.
One of the major figures of merits in the DNA sequencers is the resolution of the DNA bands. A reduction in the width of the DNA band improves resolution, allowing base calling of longer DNA chains. Resolution depends also on the distance which DNA molecules migrate in a sieving matrix. Separation lengths of 10 to 50 cm are typical but they may be longer for higher resolution. However, there is a trade-off between increase in resolution and run time: for a given voltage, velocity of DNA molecules in the gel is inversely proportional to the separating length, L, resulting in a migration time proportional to L2.
For analysis of single-stranded DNA, denaturants such as urea (concentration 4-8 M) or formamide (up to 10%) are added to a gel. Additionally, the electrophoretic runs are conducted at elevated temperature (40-70 °C), which helps to prevent self-hybridization.
developed and manually analyzed. Lack of automation in the data analysis and risk associated with using radioactive materials are the most serious drawbacks of this method. Also, the resolution at high base numbers in manual sequencers is lower than that in automatic sequencers because of the much shorter migration distance.
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