Whole Genome Amplification by Isothermal Multiple Strand Displacement Using Phi29 DNA Polymerase

Area of Science:

• Molecular Biology
• Genomics

Background:

• Minute DNA quantities pose challenges for molecular analyses.
• Whole genome amplification (WGA) is crucial for amplifying limited DNA samples.

Purpose of the Study:

• To describe and evaluate a novel isothermal whole genome amplification (iWGA) method.
• To assess the suitability of iWGA for downstream applications.

Main Methods:

• Utilized a Phi29 DNA polymerase-based kit (Illustra GenomiPhi V2 DNA Amplification Kit).
• Employed random hexamer primer binding and multiple strand displacement for amplification.
• Applied the method to genomic DNA and single cells.

Main Results:

• Achieved amplification of minute DNA quantities, yielding up to 5 μg of product.
• Generated iWGA products with fragment lengths of 10 kb and longer.
• Demonstrated suitability of amplified DNA for downstream applications like sequencing and DNA profiling.

Conclusions:

• Isothermal whole genome amplification (iWGA) is an effective method for amplifying limited DNA samples.
• The generated long DNA fragments are advantageous for various molecular applications.
• The method is not suitable for degraded DNA samples.