Powered by phi29-XT DNA Polymerase, an engineered polymerase with improved thermostability and sensitivity
High sensitivity: amplify from as little as 10 fg of input DNA
Flexible protocol offers compatibility with different types of sample input material
Simple workflow produces high yield in a short reaction time (less than 2 hours)
Whole genome representation for human and microbial DNA
Enables multiple downstream applications without further processing steps
Product Information
The phi29-XT WGA Kit is optimized for sensitive, fast, and robust whole genome amplification (WGA), an isothermal method for amplification of an entire genome, starting from femtogram quantities of DNA and resulting in microgram quantities of amplified products. The kit features phi29-XT DNA Polymerase, an engineered polymerase with improved thermostability, sensitivity, and capable of generating a higher yield in a shorter reaction time than wild-type phi29 DNA Polymerase. Also included are exonuclease-resistant random primers (containing phosphorothioate bonds) to universally amplify any DNA sequence, neutralization buffer, and dNTPs. Input material can be purified genomic DNA, cells, or microbiomes. WGA enables several downstream applications, including single cell whole genome sequencing (WGS), genomic DNA enrichment, and environmental DNA (eDNA) enrichment and sequencing.
Figure 1: Overview of the phi29-XT WGA Kit
The phi29-XT WGA Kit (NEB #E1604) is a fast, easy-to-use, and highly versatile kit containing all the required components for whole genome amplification (WGA) using a random primer mix. The kit delivers high yields of DNA products from a variety of starting materials including purified genomic DNA, cells, or microbiomes. This kit is ideal for various DNA applications such as single cell whole genome sequencing (WGS), genomic DNA enrichment, and environmental DNA (eDNA) enrichment and sequencing.
Figure 2 :phi29-XT DNA Polymerase generates more product in less time than wild-type phi29 DNA Polymerase in WGA applications at elevated temperatures
Triplicate WGA reactions were performed with 100 pg human genomic DNA input at the indicated temperatures for 1.5 hours. The reactions were then heat-inactivated at 65°C for 10 minutes. All reactions contained 1 mM dNTPs and 50 μM Exonuclease-Resistant Random Primers. Wild-type phi29 reactions were carried out with 10 units of phi29 DNA Polymerase (NEB #M0269) in 1X phi29 DNA Polymerase Reaction Buffer and 0.1 mg/mL Recombinant Albumin, and phi29-XT reactions were carried out with 1X phi29-XT DNA Polymerase for WGA and 1X phi29-XT Reaction Buffer for WGA. Reaction yields (dots) were quantified using Quant-iT® PicoGreen® dsDNA Reagent and were averaged (lines) to determine the yield at each reaction temperature. While the working reaction temperature of wild-type phi29 DNA Polymerase is typically between 30°C and 38°C, phi29-XT DNA Polymerase generates robust product yields around 42°C. The optimal temperature range for wild-type phi29 increased by approximately three degrees for WGA using human genomic DNA when compared to RCA using pUC19 plasmid as input.
Figure 3: phi29-XT DNA Polymerase generates high yields of product in a short period of time, even from 10 fg genomic DNA input
Duplicate WGA reactions were carried out using various amounts of human genomic DNA as input and were incubated for 0.5 to 8 hours at 42°C, followed by heat inactivation at 65°C for 10 minutes. All reactions contained 1 mM dNTPs and 50 μM Exonuclease-Resistant Random Primers. The resulting reaction yields (dots) were quantified using Quant-iT® PicoGreen® dsDNA Reagent and were averaged (line) to determine the yield at each reaction temperature. While nonspecific amplification will eventually occur in the absence of template from primer-dimer amplification, there is no NTC amplification within the recommended incubation period of 1.5 h. Nonspecific, primer-dimer amplification is not expected to significantly contribute to the WGA yield when DNA input is included in the reaction. NTC = no template control.
Figure 4: phi29-XT WGA Kit can be used with a variety of input sources, consistently yielding more than 10 μg of DNA
Bar chart showing the average yield (μg) of triplicate reactions from various input sources. Samples were incubated at 42°C for 1.5 hours, then heat-inactivated at 65°C for 10 minutes. All WGA reactions contained 1 mM dNTPs and 50 μM Exonuclease-Resistant Random Primers. DNA yield is quantified using Quant-iT® PicoGreen® dsDNA Reagent (Invitrogen®, #P11496). Input amounts are listed below each sample type. With the exception of the human cell sample, DNA used as input was extracted from the source prior to WGA.
Figure 5: phi29-XT WGA Kit ensures whole genome coverage for human genomic DNA
Integrative Genome Viewer (IGV) visualization of read coverage across the human genome (human NA19240 gDNA). Libraries were constructed using the NEBNext® Ultra II FS DNA Library Prep Kit for Illumina (NEB #E7805) and sequenced on a NovaSeq® instrument (2 x 150 bp). Coverage depth per base was determined, reads were down-sampled with seqtk and aligned to the human reference genome (NCBI, GRCh38) with Bowtie2. As previously reported, drops in coverage occurred in centromeric regions, which are known for their highly repetitive nature and pose an amplification challenge for DNA polymerases.
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