PROVIDES CELL PLOIDY STATUS OF SINGLE CELLS & SIMULTANEOUSLY CELL mtDNA LOAD
DOPlify® Kit & Technology
The DOPlify® kit provides robust and reproducible whole genome amplification allowing for cell ploidy status of single cells and simultaneously cell mitochondrial DNA load using a range of different NGS platforms and workflows. The technology captures exclusive know-how for the lysis and whole genome amplification of single or small numbers of cells providing a solution for multiplying the limited DNA in a single cell for a range of downstream applications.
- NGS compatible advanced DOP-PCR
- Single tube lysis & WGA
- No amplification of NTC
- > 1.25 μg of DNA from one cell
- Reduced contamination risk through using a pre-prepared WGA master mix
- PCR program < 2.5 hrs
Why Use DOPlify® Technology?
The DOPlify® kit is a whole genome amplification kit designed to PCR amplify the genome from single cells and limited DNA templates.
The DOPlify® kit is unique in that it uses advanced DOP-PCR, which;
- uses the high processivity and fidelity of new generation polymerases that are NGS application grade
- uses ready-mixes to minimize the pipetting steps and requiring only two sample tube openings to reduce the risk of contamination
- provides an amplification system that is flexible enough to concurrently whole genome amplify a sample whilst specifically amplifying genes of clinical interest, reducing the risk of allele drop-out.
|Cell Lysis||A gentle but effective enzyme-based lysis procedure ensures robust cell lysis and a readily accessible DNA template for whole genome amplification||15 mins|
|Whole Genome Amplification||Whole genome amplification is performed using advanced DOP-PCR, which has been optimised for limited DNA input. DOP-PCR uses degenerate primers to initiate DNA amplification, binding across a broad range of different sequences scattered genome wide||2.5 hrs|
|Agarose Gel Assessment||Following amplification, the use of agarose gel electrophoresis is recommended to ensure that cell amplification has been successful||30 mins|
- PCR-grade Water
- Cell Lysis Enzyme
- Cell Lysis Buffer
- WGA Polymerase
- WGA PCR Buffer
REQUIRED MATERIALS NOT PROVIDED
- 0.2 or 0.5 mL PCR tubes
- Single blastomeres, polar bodies, trophoblastic cells, amniocytes, lymphocytes, or cultured cells
- PCR thermocycler
How many reagents are included in the DOPlify® kit?
Cell lysis buffer, Cell lysis enzyme, PCR-grade H20, Primer, Polymerase and PCR buffer.
How many reactions do I get in a single DOPlify® kit?
How long is the protocol?
~3 hours total (including hands on time and incubations).
What thermocycler do you recommend for PCR?
Any thermocycler that has a programmable ramp rate will be fine.
At what temperature do I store the kit components?
DOPlify® kit should be stored at -20°C in a clean/pre-PCR lab.
What buffer should I use amplify my samples in?
Recommended cell transfer buffers include 10 mM Tris-HCl (pH 8.0) (no EDTA) and PBS (Mg2+, Ca2+ free and BSA free) in 0.2 ml or 0.5 ml PCR tubes.
Publications that Cite Using the DOPlify® Kit for Whole Genome Amplification
Deleye, L., Tilleman, L., Plaetsen, A. V., Cornelis, S., Deforce, D., & Nieuwerburgh, F. V. (2017). Performance of four modern whole genome amplification methods for copy number variant detection in single cells. Scientific Reports, 7(1). doi:10.1038/s41598-017-03711-y.
Deleye, L., Plaetsen, A. V., Weymaere, J., Deforce, D., & Nieuwerburgh, F. V. (2018). Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells. Scientific Reports,8(1). doi:10.1038/s41598-018-19509-5
Plaetsen, A. V., Deleye, L., Cornelis, S., Tilleman, L., Nieuwerburgh, F. V., & Deforce, D. (2017). STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods. Scientific Reports, 7(1). doi:10.1038/s41598-017-17525-5.
The DOPlify® Kit contains reagents for amplifying 50 samples. Ships on on ice packs and remain stable even if the ice packs have thawed and the reagents reach room temperature. Upon receipt, store the kit at -20°C in a constant temperature freezer (not frost free).