Whole Genome Amplification (WGA) and
Multiple Annealing and Looping-based Amplification Cycles (MALBAC)
Nucleic acid analytical platforms (e.g. qPCR, microarray and high throughput sequencing) often require the sample to meet specific requirements for quantity and quality. Therefore, Whole genome amplification (WGA) is often required for analyzing samples with limited quantity, such as single circulating tumor cells, micro-biopsies, and single blastomere.
WGA methods are in general prone to amplification bias, PCR-based WGA introduces sequence-dependent bias mainly due to the exponential amplification. Multiple Displacement Amplification (MDA), which uses random priming and the strand-displacing phi29 polymerase under isothermal condition, has provided improvements over PCR-based methods but still exhibits considerable bias, again due to nonlinear amplification.
The Single Cell WGA kit generates highly uniform amplification across the entire genome. The method is based on the MALBAC (Multiple Annealing and Looping Based Amplification Cycles) technology (1), which carries out close-to-linear pre-amplification cycles of the entire genome using a mixture of highly-processive DNA polymerases with strand displacement activity, followed by an exponential amplification by PCR to a sufficient amount for various downstream analyses.
Figure 1: MALBAC procedure
The Single Cell WGA kit exhibits significantly improved amplification evenness compared with MDA. With 30x sequencing depth, 93% of the single cell genome can be covered compared with ~50-60% by MDA.
Figure2: Comparison of uniformity of Single Cell Amplification between MALBAC and MDA
Figure 3: MALBAC generates significantly improved amplification evenness compared with MDA
DNA amplified with the Single Cell WGA Kit can be used in a variety of downstream analytical platforms:
• Detection of mutations • SNP genotyping
• CNV profiling and aneuploidy detection • Whole genome and exome sequencing
• Real-time PCR • Molecular cloning
• Microarray CGH