Optimization and evaluation of single-cell whole-genome multiple displacement amplification.
The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research.
Spits
C
C
Research Centre for Reproduction and Genetics, Academisch Ziekenhuis, Vrije Universiteit Brussel, Brussels, Belgium.
Le Caignec
C
C
De Rycke
M
M
Van Haute
L
L
Van Steirteghem
A
A
Liebaers
I
I
Sermon
K
K
D002021:Buffers; D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015894:Genome, Human; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction
D002021:Buffers; D004247:DNA; D004259:DNA-Directed DNA Polymerase