Whole Genome Amplification (WGA) is a laboratory technique designed to generate large quantities of DNA from very small or degraded samples. WGA addresses this challenge by producing microgram quantities of DNA from nanogram or even picogram starting material, making various downstream genetic analyses possible.
Methods of Whole Genome Amplification
Whole Genome Amplification relies on distinct molecular strategies to achieve broad amplification of the entire genome. These methods can be broadly categorized into PCR-based techniques and isothermal amplification methods, each with unique operational principles and characteristics.
PCR-based Methods
PCR-based WGA methods adapt the foundational Polymerase Chain Reaction (PCR) for whole-genome coverage, typically involving degenerate or random primers.
Degenerate Oligonucleotide-Primed PCR (DOP-PCR)
Degenerate Oligonucleotide-Primed PCR (DOP-PCR) employs primers with a short, specific sequence at their 3′ end, followed by a degenerate (random) sequence, and then another specific sequence at the 5′ end. Initially, a low annealing temperature allows these degenerate primers to bind to numerous sites across the genome. Subsequent cycles use higher annealing temperatures, promoting more specific amplification from the initially primed regions, thereby amplifying many loci simultaneously.
Primer Extension Preamplification (PEP)
Primer Extension Preamplification (PEP) is another PCR-based WGA technique, often applied to single cells. PEP utilizes random 15-base primers that anneal to numerous genomic DNA sites, initiating DNA synthesis. The reaction starts at a lower temperature, around 37°C, and then extends continuously up to 55°C, ensuring that a broad range of genomic sequences are targeted.
Isothermal Methods
Isothermal WGA methods, unlike PCR, operate at a constant temperature, eliminating the need for thermal cycling equipment.
Multiple Displacement Amplification (MDA)
Multiple Displacement Amplification (MDA) is the most widely used isothermal WGA technique. MDA employs a highly processive DNA polymerase, such as phi29 DNA polymerase, which possesses strong strand displacement activity and a 3’→5′ exonuclease proofreading function. Random hexamer primers bind to denatured DNA templates at multiple locations. The phi29 polymerase then extends these primers, continuously synthesizing new DNA strands while displacing any downstream strands, leading to a highly branched, exponential amplification of the entire genome.
The phi29 DNA polymerase’s proofreading capability contributes to high fidelity, minimizing errors during replication. This enzyme also resolves secondary DNA structures, leading to more uniform amplification and longer DNA fragments.
Comparison of Methods
PCR-based methods require repeated temperature changes, typically using a thermal cycler. In contrast, isothermal methods like MDA maintain a constant reaction temperature, often around 30°C, eliminating the need for specialized equipment. MDA, with phi29 DNA polymerase, offers higher accuracy due to its proofreading activity, leading to fewer errors and more uniform amplification. PCR-based methods can be more prone to amplification bias and may produce shorter DNA fragments.
Applications of Whole Genome Amplification
Whole Genome Amplification has revolutionized various scientific and clinical fields by enabling genetic analysis from previously insufficient DNA samples.
Forensic Science
In forensic science, WGA is highly valuable for amplifying trace amounts of DNA often found at crime scenes, such as from a single hair, saliva, or touch DNA. It provides the necessary amplification for standard DNA profiling techniques, like Short Tandem Repeat (STR) analysis, when starting material is limited. This is useful for analyzing degraded samples or DNA mixtures.
Genetic Disease Research and Diagnosis
WGA plays a significant role in genetic disease research and diagnosis, especially when limited biological samples are available. For example, in preimplantation genetic diagnosis (PGD), WGA allows for the genetic analysis of embryos before implantation, often from a single cell (blastomere) or a few cells from the trophectoderm biopsy. This enables the detection of specific inherited disorders or chromosomal abnormalities, such as alpha-thalassemia, allowing for the selection of unaffected embryos for transfer.
Cancer Research
WGA facilitates advanced genomic profiling in cancer research, particularly when dealing with rare tumor cells or circulating tumor DNA (ctDNA). Circulating tumor cells (CTCs) are often present in very low frequencies, making direct DNA analysis challenging. WGA allows researchers to amplify DNA from individual CTCs, providing enough material to investigate genetic mutations and copy number variations, which can guide personalized cancer therapies. WGA also supports the analysis of ctDNA, enabling non-invasive monitoring of tumor burden and treatment response.
Single-Cell Genomics
Single-cell genomics relies on WGA, as individual cells contain only picogram quantities of DNA. WGA enables the amplification of the entire genome from a single cell, allowing for the study of genetic heterogeneity within cell populations. This is particularly relevant in fields like developmental biology, neurobiology, and cancer research, where understanding cell-to-cell variations is paramount.
Next-Generation Sequencing (NGS)
Next-Generation Sequencing platforms typically require specific quantities of input DNA for library preparation and sequencing. When starting material is scarce, WGA provides the necessary amplification to meet these requirements, making NGS feasible. WGA amplified DNA can be used for various NGS applications, including whole-genome sequencing, exome sequencing, and metagenomics research.
Understanding Challenges in Whole Genome Amplification
Despite its utility, Whole Genome Amplification is not without its limitations, which can impact the accuracy and reliability of downstream genetic analyses.
Amplification Bias
Amplification bias is a common issue where certain regions of the genome are over-represented or under-represented in the amplified DNA compared to the original sample. Such bias can lead to “holes” in sequencing data, affecting the completeness and accuracy of genomic profiling.
Allelic Drop-Out (ADO)
Allelic drop-out (ADO) is another significant challenge, occurring when one of the two alleles at a heterozygous genetic locus fails to amplify. This leads to an incorrect interpretation of homozygosity, potentially causing misdiagnosis in genetic testing.
Contamination
Contamination poses a heightened risk in WGA due to the extreme sensitivity of the amplification process. Minute amounts of foreign DNA from reagents, laboratory equipment, or even human skin cells can be inadvertently amplified, overwhelming the target DNA from the precious, low-input sample.
Fidelity and Accuracy
Fidelity and accuracy relate to the potential for polymerases used in WGA to introduce errors during DNA synthesis. While high-fidelity polymerases with proofreading capabilities, like phi29 DNA polymerase, significantly reduce error rates, some errors can still occur. These errors can be propagated during amplification, leading to artificial mutations in the final amplified product.