Whole-genome multiple displacement amplification from single cells.
Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.
Spits
Claudia
C
Research Centre Reproduction and Genetics, Academisch Ziekenhuis, Vrije Universiteit Brussel, Brussels, 1090, Belgium.
Le Caignec
Cédric
C
De Rycke
Martine
M
Van Haute
Lindsey
L
Van Steirteghem
André
A
Liebaers
Inge
I
Sermon
Karen
K
D002469:Cell Separation / Q000379:methods; D015894:Genome, Human; D006801:Humans; D021141:Nucleic Acid Amplification Techniques