PCR products are often used as sequencing templates. It is important that the amplicons to be used as sequencing templates are free of residual components of the PCR reaction, especially primers and nucleotides. These reactants can interfere with the sequencing reaction and lower the quality of the sequencing ladder. PCR amplicons can be cleaned by adherence and washing on solid phase (column or bead) matrices, alcohol precipitation, or enzymatic digestion with alkaline phosphatase. Alternatively, amplicons can be run on an agarose gel and the bands eluted. The latter method provides not only a clean template but also confirmation of the product being sequenced. It is especially useful when the PCR reactions are not completely free of misprimed bands or primer dimers (see Chapter 7).
Sequencing buffer is usually provided with the sequencing enzyme and contains ingredients necessary for the polymerase activity. Mixtures of all four dNTPs and one of the four ddNTPs are then added to each tube, with a different ddNTP in each of the four tubes.
The ratio of ddNTPs:dNTPs is critical for generation of a readable sequence. If the concentration of ddNTPs is too high, polymerization will terminate too frequently early along the template. If the ddNTP concentration is too low, infrequent or no termination will occur. In the beginning days of sequencing, optimal ddNTP:dNTP ratios were determined empirically (by experimenting with various ratios). Modern sequencing reagent mixes have preoptimized nucleotide mixes.
With the addition of DNA polymerase enzyme to the four tubes, the reaction begins. After about 20 minutes, the reactions are terminated by addition of a stop buffer. The stop buffer consists of 20 mM EDTA to chelate cations and stop enzyme activity, formamide to denature the products of the synthesis reaction, and gel loading dyes (bromophenol blue and/or xylene cyanol). It is important that all four reactions be carried out for equal time. Maintaining equal reaction times will provide consistent band intensities in all four lanes of the gel sequence, which facilitates final reading of the sequence.
ddATP + four dNTPs ddCTP + four dNTPs ddGTP + four dNTPs ddTTP + four dNTPs ddA
dAdGddC dAdGdCdTdGddC dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC
■ Figure 10-7 Components required for DNA synthesis (template, primer, enzyme, buffers, dNTPs) are mixed with a different ddNTP in each of four tubes (left). With the proper ratio of ddNTPs:dNTPs, the newly synthesized strands of DNA will terminate at each opportunity to incorporate a ddNTP. The resulting synthesis products are a series of fragments ending in either A (ddATP), C (ddCTP), G (ddGTP) or T (ddTTP). This collection of fragments is the sequencing ladder.
Manganese (Mn++) may be added to the sequencing reaction to promote equal incorporation of all dNTPs by the polymerase enzyme.31,32 Equal incorporation of the dNTPs makes for uniform band intensities on the sequencing gel, which eases interpretation of the sequence. Manganese increases the relative incorporation of ddNTPs as well, which will enhance the reading of the first part of the sequence by increasing intensity of the smaller bands on the gel. Modified nucleotides, deaza-dGTP and deoxy-inosine triphosphate (dITP), are also added to sequencing reaction mixes to deter secondary structure in the synthesized fragments. Additives such as Mn++, deaza-dGTP, and dITP are supplied in preop-timized concentrations in commercial sequencing buffers.
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