Efficient isothermal amplification of the entire genome from single cells.
Preimplantation genetic diagnosis for single gene disorders is usually performed using polymerase chain reaction (PCR)-based methodologies modified for use in single cells. At present, single cell PCR tests require costly and time-consuming development and validation of highly sensitive amplification strategies to cover a growing number of mutations responsible for genetic disease. Whole-genome amplification (WGA) provides an opportunity to amplify the genome from a single blastomere to a level at which multiple tests can be performed on the same cell. Early WGA methods (primer extension preamplification and degenerate oligonucleotide-primed PCR) have not proved sufficiently accurate and reliable for routine clinical use. However, WGA using multiple displacement amplification (MDA) offers approx 5 million-fold amplification with fidelity, apparently sidestepping the limitation of a single cell, and is sufficient for use in most off-the-shelf molecular tests. This chapter describes an optimized MDA protocol for the preparation of genomic DNA from single fibroblasts.
Schowalter
Karen V
KV
Departmeng of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA.
Fredrickson
Jolene
J
Thornhill
Alan R
AR
D002477:Cells / Q000737:chemistry; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods*; D019836:Preimplantation Diagnosis / Q000379:methods*
D019005:Cystic Fibrosis Transmembrane Conductance Regulator