Single Cell Genomics
A single cell is the smallest structural and functional unit of organism. The estimated number of single cells in the human body is 3.72 x 1013. Amplification of genomic DNA is a necessary first step for the available sequencing technologies, due to the limited DNA content of a single cell (e.g. human diploid cell contains around 6 picograms) .
Single cell genomics analyses DNA at the most fundamental level of biology. SCG consists of a series of integrated processes.
- Single cell isolation
- Cell lysis & DNA extraction and denaturation
- Whole genome amplification (WGA)
- Library prep
Figure 1: Single Cell Genomics (SCG) workflow
Majority of Single cell genomics (SCG) work today relies on fluorescence-activated cell sorting (FACS) for cell separation. This technology provides automated and rapid delivery of individual cells into tubes or microwell plates. Alternatives to that are manual cell sorting, laser capture microdissection, microfluidics, Serial dilution , Cell Search and Nanofilters etc.
Whole genome amplification (WGA) is required before single cell DNA sequencing. Multiple displacement amplification (MDA) is the most widely used technique which produces long overlapping amplicons suitable for whole genome sequencing.
Phi29 is the ideal polymerase for MDA reactions as it has an error rate of 10-7 . It is more suitable for the detection of point mutations. (Source :Cancer genomics: one cell at a time (PMID-25222669))
Key parameters that determine the quality of the amplification are:
• Absence of contaminations and amplification artefacts
• Coverage breadth
• Nucleotide error rates
• Ability to recover single-nucleotide variants (SNVs)
• Ability to recover copy number variants (CNVs) and structural variants
Advantages of Single Cell Sequencing
By sequencing the genomes of single cells, can learn about mutations in single cell. This technology is particularly important in finding function of cells by looking at the kinds of genes they express. It promises to address key issues in cancer research.
Users usually experience contaminations coming in from the environment after the denaturation step.
Expedeon’s TruePrime offer a unique advantage here: TruePrime does not accept non-denatured DNA strands as template as readily as random primed MDA (Multiple Displacement amplification) reactions.
TruePrime™ technology also provides advantages such as:
• Increased even amplification and genome coverage
• Easy and reliable
• No primer artefacts
• Excellent 1fg sensitivity
• Lower allelic dropout
• Suitable for both SNV and CNV
• Suitable for single-cell or extracted DNA
• Exquisite reproducibility
• Ideally suited for next generation sequencing, tested for Illumina and Ion Torrent workflows
Expedeon’s TruePrime Single Cell Whole Genome Amplification (WGA) Kit uses a revolutionary novel multiple displacement amplification (MDA) method. It uses DNA primase TthPrimPol and high-fidelity Phi29 DNA polymerase to amplify uniformly total genomic DNA either from a single or a few cells.
Technical specifications: TruePrime™ Single Cell WGA Kit version 2.0 uses a novel and reliable method to achieve accurate genome amplification from single cells. Up to 50 cells can be amplified with the same protocol. Typical DNA yields from a TruePrime™ Single Cell WGA kit version 2.0 reaction are about 3-4 μg per 50 μl reaction and 3 hours reaction time when starting from a single mammalian cell.
Benefits & Features
• Primer-free method: TthPrimPol synthesizes the primers for Phi29 DNA pol
• No primer artefacts: The absence of random synthetic primers prevents any amplification artefacts generated by random extension of primer dimers, etc.
• Easy handling in a convenient kit format
• Reliable results
• Insensitive to external DNA contaminations
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