As with all molecular methods, the quality of the DNA/ RNA is of particular importance, and this is highlighted in LD-PCR where obtaining high-quality DNA template is paramount. If the DNA is nicked or fragmented, then the size of amplicon that can be produced will obviously be limited, and if the DNA is impure, then the PCR amplification is going to be inhibited.
Many commercial kits that extract genomic DNA from a variety of starting materials are now available (as discussed in Inverse PCR). They provide DNA of high purity and length (average length 50-100 kb Genomic-tip 100/G columns, Qiagen, Crawley, UK) and remove the need for time-consuming purification procedures e.g., pulsed-field, low-melting point agarose gel electrophoresis.
As for all PCR primer pairs, the quality of their design can only be conclusively demonstrated by experimentation, although if certain strategies are adhered to optimal theoretical design can be achieved. For LD-PCR, it is essential to minimize secondary structures, especially at the 3' ends, as the formation of dimers/polymers will create a competition for reagents and reduce the concentration of the amplicon. Thus, it is important to ensure that the primers are of the highest specificity to minimize nonspecific amplification and prevent the design of a competitive PCR. This can be achieved by design of longer primers, although there is a balance between specificity and optimal amplification, and reports vary as to the optimal length for LD-PCR. In general, optimal primer length is similar to that for normal PCR (18-25 bp),[4'9] although longer primers (27-33 bp) have been shown to reduce the frequency of failed LD-PCR reactions. The concentrations of LD-PCR primers are similar to a normal PCR (0.1-1.0 p.M), although increased concentrations (4 mM) have been used, in an attempt to reduce the annealing time required. However, at higher concentrations this benefit may be negated by the formation of secondary structures and the possibility of mispriming. For further information on primer design, see Foord and Rose.
When LD-PCR was initially developed, the use of many individual polymerases was evaluated and rTth DNA polymerase provided a consistent and reproducible amplification.1-4,7,9-1 As the LD-PCR technique progressed, it was discovered that the use of a polymerase/exonucle-ase mix could benefit the LD-PCR amplification,1-4,5,7,12-1 but had the drawback of determining optimal enzyme concentrations and buffer solutions. Since then, a variety of commercial LD-PCR kits and enzyme mixes are available (Table 1), providing optimal relative enzyme concentrations and buffers. Recently, the use of Escherichia coli exonuclease III was found beneficial when trying to perform LD-PCR. It permitted amplification of moderately damaged DNA templates when added to the polymerase mix, although it had no benefit where extensive damage had occurred.
Typical buffer compositions have been stated above and generally are similar to normal PCR buffers except concentrations are nonlimiting. Certain protocols report the use of Tricine instead of Tris-HCl because its pKa is less temperature dependent, and so provides DNA with a greater protection against nicking and depurination. By adding certain cosolvents, i.e., glycerol (up to 8%), DMSO (1-3%) or a mix of the two (5% of each), the melting and strand-separation temperature can be lowered. This can increase the length of amplification by 10 kb by facilitating denaturation and minimizing the exposure of any heat-labile components (DNA template) to extended high temperatures.
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