Over 200 recessive X chromosome-linked diseases, typically affecting only hemizygous males, have been identified. In many of these, prenatal diagnosis is possible by chorion villus sampling (CVS) or amniocentesis, followed by cytogenetic, biochemical or molecular analysis of the cells recovered from the conceptus. In others, the only alternative is to determine the sex of the fetus. If the fetus is affected by the defect or is male, abortion can be offered. Diagnosis of genetic defects in preimplantation embryos would allow those unaffected to be identified and transferred to the uterus. Here we report the first established pregnancies using this procedure, in two couples known to be at risk of transmitting adrenoleukodystrophy and X-linked mental retardation. Two female embryos were transferred after in vitro fertilization (IVF), biopsy of a single cell at the six- to eight-cell stage, and sexing by DNA amplification of a Y chromosome-specific repeat sequence. Both women are confirmed as carrying normal female twins.

Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification

THE development of molecular genetics, both as a self-contained field and as a body of techniques broadly useful in biologic investigation, has had a profound influence on medical research. The beneficiaries include every discipline in basic science and, at least indirectly, most clinical and applied medical disciplines. Certain technical milestones can be identified over the past several decades that have been particularly important in the progress of the field. One is the discovery of restriction endonucleases, which together with the development of DNA ligation and transformation procedures, led to the ability to clone and thus propagate genes of any organism . . .

https://www.nejm.org/doi/pdf/10.1056/NEJM199001183220307

The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis.

The polymerase chain reaction (PCR) has dramatically altered how molecular studies are conducted as well as what questions can be asked. In addition to simplifying molecular tasks typically carried out with the use of recombinant DNA technology, PCR has allowed a spectrum of advances ranging from the identification of novel genes and pathogens to the quantitation of characterized nucleotide sequences. PCR can provide insights into the intricacies of single cells as well as the evolution of species. Some recent developments in instrumentation, methodology, and applications of the PCR are presented in this review.

https://science.sciencemag.org/content/sci/252/5013/1643.full.pdf

Recent advances in the polymerase chain reaction

We have developed an in vitro method for amplifying a large fraction of the DNA sequences present in a single haploid cell by repeated primer extensions using a mixture of 15-base random oligonucleotides. We studied 12 genetic loci and estimate that the probability of amplifying any sequence in the genome to a minimum of 30 copies is not less than 0.78 (95% confidence). Whole genome amplification beginning with a single cell, or other samples with very small amounts of DNA, has significant implications for multipoint mapping by sperm or oocyte typing and possibly for genetic disease diagnosis, forensics, and the analysis of ancient DNA samples.

https://www.pnas.org/content/pnas/89/13/5847.full.pdf

Whole genome amplification from a single cell: implications for genetic analysis

A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).

J.K. Pattinson

Papers

Biopsy of human preimplantation embryos and sexing by DNA amplification.