Whole Genome Amplification & Multiple Displacement Amplification

Whole Genome Amplification

Whole Genome Amplification (WGA) is a general term denoting methods that aim to amplify an entire genome, typically starting with low (picogram to nanogram) quantities of DNA and producing up to tens of microgram quantities of amplified products. WGA has become an invaluable approach for utilizing limited samples of precious stock material or to enable sequencing of single-cell genomic DNA. Several methods have been developed for high-fidelity whole genome amplification, including PCR-based methods such as Degenerate Oligonucleotide PCR (DOP-PCR) and Primer Extension Preamplification (PEP), but the most commonly used method is the Multiple Displacement Amplification (MDA) method that uses the strand-displacement activity of DNA polymerases such as phi29 or Bst.

NEB has engineered a novel variant of phi29 DNA polymerase that has improved thermostability and sensitivity: phi29-XT DNA Polymerase. phi29-XT can be found in kits optimized for WGA and Rolling Circle Amplification (RCA): phi29-XT WGA Kit and phi29-XT RCA Kit.

WGA relies upon the MDA mechanism. Random primers (yellow) anneal to the DNA template (gray) and are extended by a DNA polymerase with strand-displacement activity (arrows). This causes the polymerase to kick up any sequence ahead of the polymerase, which can act as substrate for the random primers.

Rolling Circle Amplification

In RCA, phi29 DNA polymerase and exonuclease-resistant primers are combined with circular template material and incubated for 1–12 hours depending on the level of amplification needed. The polymerase extends the nascent DNA around the circular template over and over, producing extremely long (>30 kb) products with varying degrees of branching (based upon primer(s) used) through the multiple displacement mechanism; a single specific primer produces a long, concatemeric single-stranded DNA, use of bidirectional primers results in discrete double-stranded DNA with varying lengths of concatemeric sequence, and random primers result in highly branched double-stranded DNA.

The proofreading (3' → 5') exonuclease activity of phi29 ensures high-fidelity replication of template DNA, but also requires that the 3' end of primers are protected by phosphorothioate linkages.

For next-generation sequencing applications, acoustic shearing or the NEBNext^® Fragmentation System can resolve these products into readable fragments as part of the library prep workflow. The branches can be resolved using T7 Endonuclease 1 when using these products for certain downstream applications, such as nanopore sequencing.

For high yield reactions, pyrophosphatase can be included to hydrolyze the high concentration of pyrophosphate byproduct produced, pushing the reaction forward.

Learn more about our newly released phi29-XT RCA Kit that includes our engineered phi29-XT DNA Polymerase.