Single nucleotide polymorphisms and next generation sequencing

Abstract

Single cell analysis for preimplantation genetic diagnosis (PGD) of single gene defects was first used to identify the sex of embryos in a series of couples at risk of various X-linked conditions, which typically only affect males. The use of PCR at the single cell level was still in its infancy and even doubling the typical number of amplification cycles failed to amplify unique target sequences reliably. For this reason, PCR amplification of a Y-linked repeat sequence, present in thousands of copies per cell, was used for identification of male embryos. However, the extreme sensitivity of this protocol and the rapid build-up of amplified DNA products in the laboratory environment, soon led to the appearance of contamination and false positive results in blank (negative) controls. Furthermore, in a minority of single male cells amplification still failed, resulting in a male embryo being identified as female, which lead to the first clinical misdiagnosis following PGD.