Description
DNA sequence analysis and genotyping of biological samples using next-generation sequencing (NGS) platforms is often limited by the amount of available sample. The REPLI-g Advanced DNA Single Cell Kit has been specifically optimized for use in sensitive microarray and NGS applications, providing high uniformity, with minimal allelic dropout, when starting with single cells or purified genomic DNA. Buffers and reagents undergo a unique, controlled decontamination procedure to minimize contaminating DNA and ensure highly reliable results. Accurate amplification of genomes with negligible sequence bias and minimal genomic dropouts is achieved with innovative Multiple Displacement Amplification (MDA) technology.
Genotyping and DNA sequence analysis of biological samples can be limited by a small amount of available sample. The REPLI-g Advanced DNA Single Cell Kit allows uniform amplification of whole genomic DNA from limited sample amounts and enables a greater variety and number of analyses to be performed. The average product length of amplified DNA is typically more than 10 kb, with a range between 2 kb and 100 kb, enabling all downstream applications such as complex genetic analysis, including long-range copy number variations, to be performed (1). DNA amplified with the REPLI-g Advanced DNA Single Cell Kit is highly suited for next-generation sequencing (NGS), array CGH genotyping applications or qPCR analysis. Typical DNA yields from REPLI-g Advanced DNA Single Cell Kit amplifications are approximately 25–35 µg per 50 µl reaction. Depending on the quality of the input cell and its DNA, the resulting amount of amplified DNA may be less (fragmented or damaged gDNA should not be used). For best amplification results, we recommend collecting or storing cells in REPLI-g Advanced sc Storage Buffer (included with kit or available separately).
The REPLI-g Advanced DNA Single Cell Kit contains an optimized Phi 29 polymerase formulation, as well as buffers and reagents, for whole genome amplification (WGA) from single eukaryotic cells, very small amounts of sample or purified genomic DNA using Multiple Displacement Amplification (MDA). Phi 29 polymerase, a phage-derived enzyme, is a DNA polymerase with 3'→5' exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to Taq DNA polymerase. Supported by the unique, optimized REPLI-g Advanced DNA Single Cell buffer system, Phi 29 polymerase easily solves secondary structures such as hairpin loops, thereby preventing slipping, stoppage and dissociation of the polymerase during amplification. The REPLI-g Advanced DNA Single Cell Kit is an improved version of the REPLI-g Single Cell Kit and includes an optimized reaction chemistry, a new single cell storage buffer and an improved protocol to increase the uniformity of amplification and reduce potential amplification bias. In addition, the MDA reaction time has been shortened to 2 hours.
The REPLI-g Advanced DNA Single Cell Kit provides highly uniform amplification across the entire genome, with negligible sequence bias. The method is based on Multiple Displacement Amplification (MDA) technology, which carries out isothermal genome amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 100 kb without dissociating from the genomic DNA template. In contrast to PCR-based methods, Phi 29 polymerase has a 3'–5' exonuclease proofreading activity to maintain 1000-fold higher fidelity than Taq Polymerase during replication. MDA technology is used in the presence of exonuclease-resistant primers to achieve high yields of DNA product from all kinds of eukaryotic tissues. Genetic analyses often require substantial amounts of genomic DNA. Whole genome amplification solves the problem of low DNA quantity when starting with 1–1000 cells. The REPLI-g Advanced DNA Single Cell Kit overcomes these limitations by using a simple and reliable method to achieve accurate genome amplification. In addition, all components provided with the REPLI-g Advanced DNA Single Cell kit are exposed to UV radiation in a standardized procedure to minimize contaminating DNA. Reaction setup takes only 20 minutes and 3 steps, making the REPLI-g Advanced DNA Single Cell Kit protocol easy and reliable. In the first step of the procedure, the cell sample is lysed under isothermal alkaline conditions and the cell is denatured. Cell lysis at room temperature reduces the number of possible DNA breaks and therefore improves uniformity of whole genome amplification. After denaturation has been stopped by the addition of neutralization buffer, a master mix containing buffer and DNA polymerase is added. The isothermal amplification reaction proceeds for 2 hours at 30°C. Because of the shortened MDA reaction time, the REPLI-g Advanced DNA Single Cell Kit enables single cell collection, WGA and even downstream analysis to be performed in a single day.
