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Optimization and evaluation of single‐cell whole‐genome multiple displacement amplification
Author(s) -
Spits C.,
Le Caignec C.,
De Rycke M.,
Van Haute L.,
Van Steirteghem A.,
Liebaers I.,
Sermon K.
Publication year - 2006
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.20324
Subject(s) - multiple displacement amplification , biology , dna polymerase , microbiology and biotechnology , dna , polymerase chain reaction , genomic dna , genetics , polymerase , computational biology , gene , dna extraction
Abstract 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 ϕ29 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 ϕ29 and Bst polymerases on DNA samples and single cells. The ϕ29 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 ϕ29 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. Hum Mutat 27(5), 496–503, 2006. © 2006 Wiley‐Liss, Inc.