A procedure for highly specific, sensitive, and unbiased whole-genome amplification

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) is a significant challenge in genomic analysis.
• Existing methods may struggle with low DNA input quantities or introduce biases, limiting their utility for single-cell or trace DNA samples.

Purpose of the Study:

• To develop and optimize a DNA amplification method using phi29 DNA polymerase and trehalose for highly specific amplification of complex DNA pools.
• To generate micrograms of specific amplicons without TIPs from subfemtogram amounts of DNA.
• To assess the fidelity and accuracy of the amplified DNA for various genomic analyses.

Main Methods:

• Utilized phi29 DNA polymerase and trehalose with optimized amplification control.
• Tested amplification with varying human genomic DNA (gDNA) inputs, from subfemtograms to nanograms, including intact cells.
• Evaluated amplicon quality using high-resolution chromosome-wide comparative genomic hybridization and 550k Infinium BeadChip SNP typing.

Main Results:

• Generated micrograms of specific amplicons without TIPs from as little as 0.5-2.5 ng of human gDNA or a few cells.
• Achieved high locus representation, comparable to native DNA, and accurately detected small deletions (down to <1 kb) and duplications (down to 18 kb).
• Demonstrated >99.7% accuracy in SNP typing, favorably comparing with unamplified DNA, and improved performance with partially degraded DNA input.

Conclusions:

• The developed method provides highly specific and unbiased DNA amplification from minute DNA quantities, suitable for single-cell genomics.
• The procedure generates amplicons with high fidelity and accuracy, enabling reliable detection of genomic variations.
• The amplified DNA is suitable for downstream applications including massively parallel sequencing and microarray hybridization.