Multiple displacement amplification WGA

Multiple displacement amplification (MDA) involves the binding of random hexamers to denatured DNA followed by strand displacement synthesis at a constant temperature using the enzyme Phi29 polymerase. Additional priming events can occur on each displaced strand leading to a network of branched DNA structures (see figure Schematic representation of MDA).

Phi29 polymerase does not dissociate from the genomic DNA template allowing the generation of DNA fragments up to 100 kb without sequence bias. The enzyme has a 3'– 5' exonuclease proofreading activity and provides error rates up to1000 times lower than Taq DNA polymerase-based methods (see PCR-based WGA).

PCR-based WGA methods (e.g., PEP and adaptor-ligation PCR) are affected by secondary DNA structures. These structures can cause enzyme slippage (4) or dissociation of the enzyme from the template resulting in nonspecific amplification artifacts, incomplete coverage of loci, and short DNA fragments (less than 1 kb) that cannot be used in many downstream applications (5). In contrast, the strand-displacing enzyme Phi29 polymerase resolves secondary DNA structures (see figure Secondary DNA structures), enabling accurate and uniform amplification of the genome. The long DNA fragment lengths generated using the highly processive Phi29 polymerase ensure that Phi29-amplified DNA covers the whole genome, enabling consistent and unbiased locus representation.

The long DNA fragment lengths generated using the highly processive Phi29 polymerase ensure that MDA-amplified DNA covers the whole genome, enabling consistent and unbiased locus representation.

In a bias-study, various amounts of genomic DNA (0.3–300 ng) were amplified by using MDA, DOP-PCR, and PEP. The relative representation of 8 loci was determined using quantitative real-time PCR (see figure Highly representative amplification using MDA). Locus representation was determined by comparison to 1 µg of unamplified control DNA (5).

Due to unequal amplification of different loci caused by unresolved secondary structures, PCR-based WGA methods like DOP-PCR or PEP exhibit frequent locus dropout. MDA shows highly representative DNA amplification and minimal risk of locus dropout.

Long stretches of intact DNA are the ideal template for successful WGA to ensure the highest sensitivity of downstream assays. Assays performed on amplified, fragmented DNA are not as sensitive or reliable, as the risk of a DNA breakpoint in the locus of interest is higher (see figure Effect of DNA fragmentation on WGA). For optimal results, it is important that the template DNA is completely denatured. However, heat denaturation at 95°C can damage the template DNA, resulting in incomplete and less consistent locus representation. Alkaline DNA denaturation circumvents these problems and enables uniform DNA amplification across the whole genome with minimal sequence bias.

In addition to affecting locus representation, DNA denaturation at 95°C should be avoided, as it also affects the length of DNA products amplified by MDA by prematurely dissociating Phi29 from the DNA template. In general, Phi29 polymerase is capable of replicating up to 100 kb without dissociating from the genomic DNA template. DNA amplified using MDA has an average product length greater than 10 kb, with a range between 2 kb and 100 kb. Thus, the DNA yielded by MDA amplification is ideal for use in downstream applications that require long DNA fragments (e.g., RFLP analysis and Southern blotting).

In order to make sure that the whole genome is used for single-cell WGA, the whole intact cell is added to the WGA reaction. Because every DNA break results in the loss of the sequence information at that site, MDA is a highly suited for amplifying the whole genome. The reaction is used for the WGA since Phi29 polymerase is capable of replicating up to 100 kb without dissociating from the genomic DNA template.

In a study, the genomic DNA of single cells (3 replicates) was amplified using MDA. Approximately 40 µg of DNA was generated during single-cell WGA. Single-cell WGA DNA was compared with WGA DNA from 1000 cells and nonamplified DNA in whole genome sequencing. Whole genome sequencing was performed on the Illumina MiSeq instrument starting with 2 µg of DNA (WGA-DNA or nonamplified genomic DNA). Comparable sequence coverage was observed for gDNA and single-cell amplified DNA. Comparison of nonamplified and REPLI-g amplified DNA revealed error rates in a similar, very low, percentage range (see figure Comparable NGS results using genomic or single-cell amplified DNA).

MDA requires average genomic DNA fragment sizes of approximately 2 kb in order to amplify DNA without introducing any bias. Fragmented or low-quality DNA can be used as long some DNA fragments are above 2 kb in length; although fragments can be ligated to create longer DNA. This is because randomly fragmented DNA should contain multiple intact copes of each locus. However, to ensure accurate locus representation, the starting amount of template DNA should be increased.

Formalin fixation is a commonly used technique for preserving tissue samples for paraffin embedding (FFPE). The fixation ensures the preservation of tissue architecture and cell morphology by cross-linking biomolecules. Different sample types may require a different fixation procedure: tissues with a soft consistency, such as breast tissue samples, usually require a longer fixation step to preserve tissue morphology. Longer fixation times may result in two effects:

• A higher degree of cross-links is generated between biomolecules
• A higher degree of DNA fragmentation occurs — resulting in small DNA fragments of usually several hundred base pairs in length

Fragmentation of DNA causes a reduction of genome equivalents that can be detected by PCR and therefore has a large effect on downstream assays (see Critical factors that influence PCR-based analysis following MDA).

Although no simple method is currently available to determine the degree of cross-linking within a sample, gel electrophoresis of DNA isolated from FFPE samples can give valuable hints about the quality of the DNA.