The MDA amplification used in this kit to amplify DNA is catalyzed by Phi29 DNA polymerase, which has strong strand displacement activity. This enzyme was cloned from phage and can achieve in vitro thermal-cycle-independent constant temperature DNA polymerization reaction. Single polymerization reaction can achieve up to 100 kb of continuous polymerization. Phi29 DNA Polymerase has strong 3’→5’ exonuclease (proofreading) activity which ensures high fidelity of DNA synthesis, and the fidelity is 1000-fold higher than that of Taq and higher than current most high fidelity polymerases. This kit uses optimized Phi29 DNA polymerase reaction system to achieve constant temperature amplification with high sensitivity of single cell whole genome. This method is suitable for whole genome amplification from single cell, small amount of tissue or minor purified genomic DNA, to obtain massive genomic DNA of high coverage rate.
Discover-sc Single Cell Kit
- High sensitivity: It can simplify whole genome from single cell or very minor amount of genome sample.
- High coverage rate: The coverage rate of single cell genome can be more than 95% after amplification.
- High uniformity: Non-preference based DNA amplification can ensure uniform amplification of whole genome.
- High fidelity: Phi29 DNA polymerase has higher fidelity than current major polymerases.
- High yield: 6-8 hr reaction can produce 30-40 µg genome DNA.
- Easy operation: Only 3 steps reactions are needed, and whole operation process costs less than 10 min.
- Low requirements for facilities: Only water-bath is needed for the whole reaction.
Wide applicability of amplification products
The amplification products of Discover-sc Single Cell Kit were distributed at 2-100 kb, with average product size more than 20 KB, could be widely used in two generation sequencing, large segment copy number variation analysis, microsatellite analysis, qPCR analysis, gene chip analysis, Array CGH and other downstream analysis. The left picture shows electrophoresis results of amplification products of Discover-sc single cell kit.
M: marker,
C: amplification products of single cell,
D: amplification products of genome,
G: un-amplified genome DNA.
High sensitivity of reaction system! High coverage! High uniformity!
Discover-sc Single Cell Kit employs optimized reaction system, able to achieve single cell whole genome amplification. When ensuring coverage and uniformity, amplification sensitivity of purified eukaryotic genomic DNA could be 1 ng, and 10 pg for the bacterial genome. The right picture shows uniformity and coverage test results of Discover-sc Single Cell Kit genome amplification products. Using 8 pairs of primers distributed on different chromosomes and multiplex PCR to detect the uniformity and coverage of Discover-sc Single Cell Kit whole genome amplification products, the amplification products showed the same uniformity and coverage as the non-amplified genome.
M: marker, C: amplification products of single cell, D: amplification products of genome, G: un-amplified genome DNA. NC: Negative control.
High amplification yield
A single amplification reaction of Discover-sc Single Cell Kit can produce 30-40 µg whole genome DNA, and can be used for massive parallel experiments analysis. The following picture shows cumulative curves of amplification products using single cell and 10 ng purified genomic DNA as initial template.
Stable reaction system! Uniform product accumulation!
Discover-sc Single Cell Kit can achieve uniform product accumulation during amplification of trace sample, and amplification can be ended according to time and yield at different time points and get whole genome DNA of high coverage and higher uniformity. 24-hour reaction at 30°C will not affect the quality of product. The right picture shows the genome coverage and uniformity detection result of products for single cell after amplification at different times.
M: Marker;
G: un-amplified genomic DNA;
NC: negative control.
The super exponential amplification principle
Super simple operation flow
|
Components |
N601-01 (24 rxn) |
N601-02 (96 rxn) |
|
Discover-sc DNA Polymerse |
48 μl |
192 μl |
|
Discover-sc Reaction Buffer |
750 μl |
3×1 ml |
|
Buffer D |
1 ml |
2×1 ml |
|
Buffer N |
1 ml |
2×1 ml |
|
DTT, 1 M |
1 ml |
1 ml |
|
PBS |
1 ml |
2×1 ml |
|
H2O |
1 ml |
2×1 ml |
Discover-sc Single Cell Kit is shippe in dry ice package, after receiving the kit please immediately store all the components in -20°C constant temperature refrigerator, avoid automatic defrost refrigerator. With correct storage and operation, the quality assurance of this kit is 6 months. For long-term storage, please store the kit at -70°C.