Digital PCR and the QuantStudio™ 3D Digital PCR System.

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for detection and quantification of genetic material. This chapter walks the reader through the fundamentals of digital PCR technology including digital PCR modeling using Poisson statistics. It describes a highly successful implementation of digital PCR technology using the chip-based nanofluidic Applied Biosystems™ QuantStudio™ 3D digital PCR system.

Tumor burden monitoring using cell-free tumor DNA could be limited by tumor heterogeneity in advanced breast cancer and should be evaluated together with radiographic imaging.

Background: Accurate measurement of tumor burden in breast cancer disease is essential to improve the clinical management of patients. In this study, we evaluate whether the fluctuations in the fraction of PIK3CA mutant allele correlates with tumor response according to RECIST criteria and tumor markers quantification.

Methods: Eighty six plasma samples were analyzed by digital PCR using Rare Mutation Assays for E542K, E545K and H1047R.

Mediterranean types of beta-thalassemia in the German population.

Forty beta-thalassemia genes from unrelated German heterozygotes with no known foreign ancestry were examined using the oligonucleotide technique and DNA restriction analysis, with the aim of determining the contribution of Mediterranean beta-thalassemia mutations to the prevalence of this trait in the German population. Of the 40 beta-thalassemia genes, 26 were identified as Mediterranean types (20 beta 39 nonsense, 3 IVS2 nt 110, 2 IVS2 nt1, 1 IVS1 ntl G----A). The geographic distribution of the birthplaces of the probands' grandparents revealed no difference in the proportion of Mediterranean and unidentified beta-thalassemia genes in the west and the north of Germany.

Direct demonstration of the HB Suan-Dok mutation in the alpha 2-globin gene by restriction analysis with Sma I.

Hb Suan-Dok [alpha 2(109)(G16)Leu-greater than Arg beta 2] has an alpha-thalassemia-like effect due to low production and instability of the altered alpha-globin chain. Since the Hb Suan-Dok mutation (CTG-greater than CGG) creates a new Sma I restriction site, it was possible to diagnose the mutation by restriction analysis. The location in the alpha 2-globin gene was confirmed.

Study of alpha-thalassemia in northeastern Thailand at the DNA level.

The frequency of alpha-thalassemias in a general population sample from northeastern Thailand and in an Austroasiatic group with high frequencies of hemoglobin E and beta-thalassemia, the So, was estimated using DNA techniques. Among 64 healthy adult subjects from the Khonkaen and Ubol areas, the following haplotype frequencies were determined: alpha alpha, 0.742; -alpha 3.

The distribution of the Hb constant spring gene in Southeast Asian populations.

The distribution of the hemoglobin Constant Spring (Hb CS) gene in eight populations in Southeast Asia (including Assam) was determined using oligonucleotide hybridization. Hb CS was absent in two Assamese populations with a high prevalence of Hb E. The Hb CS gene frequency was 0.

Beta zero-thalassemia in a Thai family is caused by a 3.4 kb deletion including the entire beta-globin gene.

DNA analysis of a Northern Thai family with a child affected with beta-thalassemia major revealed a novel deletion of 3.4 kb removing the entire beta-globin gene in the proposita and her mother. Detailed mapping of the deletion located the 5' breakpoint in the region between nucleotides -810 and -128 of the beta-globin locus, and the 3' breakpoint between the Ava II and Xmn I sites located downstream of the beta-globin gene.

The spectrum of beta-thalassemia mutations in northern and northeastern Thailand.

A total of 123 beta-thalassemia genes from northern (n = 113) and northeastern (n = 10) Thailand were examined. Using five oligonucleotide probes, the mutation in 108 genes (88%) was identified: 50 nonsense 17, 49 frameshift 41-42, 4-28(A----G), 2 IV1 nt5(G----C), 2IVS2 nt654, and 1 deletion removing the entire beta-globin gene. The nonsense 17 mutation (n = 39) was linked to a single haplotype, whereas the frameshift 41-42 mutation occurred with several haplotypes.

DNA haplotypes and frameworks linked to the beta-globin locus in an Austro-Asiatic population with a high prevalence of hemoglobin E.

DNA haplotypes (HT) and frameworks (FW) linked to the beta-globin locus were determined by restriction fragment analysis using eight restriction enzymes on chromosomes bearing the Hb A gene (HBB*A) or the HbE gene (HBB*E) in the So, an Austro-Asiatic population of northeast Thailand with an HBB*E frequency near 0.5. All HBB*E genes were present with FW2, and only two haplotypes were observed (25 HT 27-2, -+- +-; 10 HT 41-2, +----++-).

Beta-globin gene linked DNA haplotypes and frameworks in three South-East Asian populations.

DNA haplotypes and frameworks (numbers in parenthesis) linked to the beta-globin gene were determined by restriction fragment analysis using eight restriction endonucleases on 86 (97) chromosomes bearing the normal beta-globin gene (HBB*A) and 108 (118) chromosomes bearing HBB*E in subjects homozygous for HBB*A or HBB*E from three South-East Asian populations with high HBB*E frequencies (northern Thailand, north-eastern Thailand and Cambodia). A systematic nomenclature for beta-globin gene-linked haplotype characterized by six polymorphic sites is introduced. In all populations, HBB*A occurred preferentially (greater than 80%) in linkage with the haplotype 41 (+----+) and all three frameworks described by Antonarakis et al.