Long-Range PCR Overview & Best Practices

Polymerase chain reaction (PCR) has become an indispensable tool used to amplify specific fragments of DNA throughout biology. However, in traditional PCR, the maximum size of amplified fragments is highly limited because of errors that occur in nucleotide polymerization and template deterioration during thermal cycling. To counter these issues, long-range PCR was developed which has enabled the amplification of larger fragments, reported to be around 3-15 kb or more.

Long-range PCR has many advantages, one being that they typically yield PCR products with around tenfold fewer mutations than those observed in products resulting from conventional PCR. In practice, long-range PCR has been frequently combined with sequencing to achieve higher sensitivity and provide a faster, cost effective, tool for the field of molecular genetics. Just a few applications of the technique have been in identifying structural rearrangements of mitochondrial DNA, enabling rapid sequence alignment of clones in gene libraries, mapping of chromosomal translocation breakpoints, and amplifying long stretches of trinucleotide repeat expansions. Long-range PCR has also been used to amplify RNA templates, by converting the RNA into a DNA intermediate and then extending the cDNA, which has aided in identifying deletions in exons prior to subsequent mutation detection.

Long-range PCR does, however, come with challenges:

• When a DNA fragment is too long some DNA polymerases are inefficient for PCR extension which can result in a low quantity of amplicons after PCR is performed. Because of this, long-range DNA polymerases have been developed; little, however, is documented on the advantages and/or disadvantages of which, and their respective performance on various amplicons is largely unreported.
• Studies on the efficacy of some PCR additives (such as dimethyl sulfoxide (DMSO), formamide, and dithiothreitol (DTT), in the synthesis of long DNA fragments remains limited, although the positive effects are well known for shorter segments of DNA used in conventional PCR.

Long-Range PCR Best Practices

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For these reasons, study optimization is paramount for any reliable and effective long-range PCR experiment.

• The method of nucleic acid extraction from a starting sample must accommodate a very high molecular-weight DNA or RNA.
• Large primers must be designed to accommodate the larger DNA or cDNA fragment. Long-range DNA polymerase mixes also typically include either Taq polymerase, a truncated version called Klentaq that has no 3'-exonuclease proofreading activity, or an archaebacterial DNA polymerase with proofreading activity.
• dUTPase is normally included into the mix as it prevents the incorporation of dUTP, the deaminated form of dCTP, into the DNA.
• A high concentration of betaine, compared to other PCR additives, is frequently included as it has been shown to significantly help promote long-range PCR amplification, up to DNA segments of at least 20 kb.

References

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Long and accurate polymerase chain reaction
Long-range PCR in next-generation sequencing: comparison of six enzymes and evaluation on the MiSeq sequencer
PCR enhancers: Types, mechanisms, and applications in long-range PCR
Long-Range PCR
Betaine improves LA-PCR amplification