The REPLI-g Single Cell Kit includes REPLI-g sc Polymerase, an optimized formulation of the innovative, high-fidelity enzyme Phi 29 polymerase, to amplify complex genomic DNA using Multiple Displacement Amplification (MDA) technology, along with gentle alkaline incubation to ensure very low DNA fragmentation or generation of abasic sites. It is specifically designed to provide high yields of amplified DNA from single cells, such as isolated tumor cells or bacteria (see Table 1). In addition, it can be used with various clinical and non-clinical research samples and with purified genomic DNA, while additional protocols are available for use with fresh or dried blood and fresh or frozen tissue. Typical DNA yields consistently reach 40 µg, regardless of the starting quantity of template, meaning subsequent genetic analyses can proceed without additional measurement of DNA concentration. The average product length of over 10 kb and complete genome coverage ensures that DNA amplified with the REPLI-g Single Cell Kit is highly suited for numerous downstream applications, including next-generation sequencing (NGS), array-based comparative genomic hybridization (array CGH), Pyrosequencing, and real-time PCR analysis (Table 2).
Table 2. Downstream applications and instrumentation
|Application ||Instrumentation |
|Whole genome sequencing ||Next-generation sequencing platforms† |
|Exome sequencing |
|SNP genotyping arrays ||Array platforms† |
|Array CGH |
|qPCR/PCR technologies ||Real-time PCR/PCR cyclers† |
|Sanger sequencing ||Capillary sequencers† |
|Pyrosequencing ||PyroMark (QIAGEN) |
The REPLI-g Single Cell Kit uses isothermal genome amplification, termed Multiple Displacement Amplification (MDA), which involves the binding of random hexamers to denatured DNA, followed by strand displacement synthesis at a constant temperature with an optimized form of the enzyme Phi 29 polymerase, which has exceptionally strong strand displacement properties. Additional priming events occur on each displaced strand that serve as a template, enabling generation of high yields of amplified DNA (see figure “ Multiple Displacement Amplification (MDA) technology”). Phi 29 polymerase, a phage derived enzyme, is a DNA polymerase with 3'→5' prime exonuclease activity (proofreading activity) that delivers up to 1000-fold higher fidelity compared to Taq DNA polymerase. Supported by the unique, optimized REPLI-g 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. This enables the generation of DNA fragments up to 100 kb without sequence bias (see figure " Unbiased amplification with Phi 29 polymerase”).
Cell lysis and alkaline denaturation of DNA
Genomic DNA must be denatured before use in enzymatic amplification procedures, which is often accomplished using harsh methods such as incubation at elevated temperatures (heat incubation) or increased pH (chemical alkaline incubation). The REPLI-g Single Cell Kit uses gentle alkaline incubation, allowing cell lysis and uniform DNA denaturation of gDNA with very low DNA fragmentation or generation of abasic sites. This results in amplified DNA with very high integrity, and maximizes the length of amplified fragments so that genomic loci and sequences are uniformly represented (see figure " Effect of heat and alkaline denaturation on loci representation").
Effective elimination of detectable DNA contamination
All REPLI-g Single Cell Kit components undergo a unique, controlled decontamination procedure to ensure elimination of all REPLI-g amplifiable contaminating DNA. Buffers and reagents are exposed to UV radiation by an innovative and standardized procedure to ensure the absence of any detectable residual contaminating DNA (see figure “ Innovative decontamination procedure”). Following UV treatment, the kits undergo stringent quality control to ensure complete functionality.