Molecular Colony Diagnostics: Detection and Quantitation of Viral Nucleic Acids by In-Gel PCR

1,047

973

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.diagnostics | early diagnosis | biomarkers | genetics | cancer

mentioning

confidence: 99%

Detection and quantification of rare mutations with massively parallel sequencing

Kinde

Papadopoulos

et al. 2011

1,047

973

“…In analogous fashion, small numbers of DNA molecules that vary by subtle changes (single base-pair substitutions or small deletions or insertions) can be counted directly by amplifying individual DNA molecules (single-molecule PCR) (7)(8)(9)(10)(11)(12)29). Such digital techniques have been shown to be extremely useful for measuring variation in genes or their transcripts, but digital technologies have been limited thus far to counting tens to thousands of molecules in the wells of microtiter plates, on microscope slides, or after electrophoresis of individual PCR products.…”

mentioning

confidence: 99%

Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

Dressman

Yan

Traverso

et al. 2003

735

479

Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single magnetic particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently labeled particles via flow cytometry. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and used for further experimentation. BEAMing can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.

“…Polony genotyping is an inherently digital process; each DNA or RNA molecule is separately counted and provides one bit of information. The power of a digital genotyping has been demonstrated by Digital PCR (28), as well as other similar technologies (29,30), finding applications in detecting loss of heterozygosity (31), quantifying allelic imbalance (26), and detecting rare somatic mutations (30). The polony genotyping presented here should further extend the utility of the digital genotyping, because millions of polonies (18) can be genotyped in a single reaction.…”

Section: Discussionmentioning

confidence: 99%

Digital genotyping and haplotyping with polymerase colonies

Mitra

Butty

Shendure

et al. 2003

139

Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two singlenucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.T he study of genetic variation in the human population has the potential to greatly improve human health, both by predicting susceptibility to disease and guiding choice of therapy. The most common genetic variations in the human population are single-nucleotide polymorphisms (SNPs). By studying candidate genes and performing genome-wide linkage disequilibrium studies, scientists are trying to uncover the ''causative SNP,'' the SNP that alters gene function and thereby increases the risk of disease. It has been shown that deriving haplotypes increases the efficiency of linkage disequilibrium studies (1). Surprisingly, recent studies (2, 3) suggest that haplotypes will also be critical for candidate gene studies. These studies found that for some diseases, there is not one single SNP that is responsible for altering gene function, but instead, multiple SNPs interact to alter the function or expression of a protein (4). These alterations occur only when specific combinations of SNPs are present on the same chromosome, so one must determine the haplotype to find a correlation to the observed phenotype. In these cases, the focus has shifted from a causative SNP to a causative haplotype.The most common approach for determining the haplotype, or phase, of a set of SNPs is computational inference from unphased data (3,(5)(6)(7)(8). Although this methodology has greatly increased the power of both linkage and candidate gene studies, a recent study estimates the error rate to be between 19 and 48%, depending on the algorithm used (6). This error rate presents challenges for the use of this method as a research tool and makes it an unlikely candidate for use in the clinical setting. Recent findings suggest a way to improve the accuracy of haplotype inference. Daly et al. (9) and others (10, 11) have shown that SNPs tend to be inherited in larger haplotype blocks than previously thought, and that there are relatively few variants of each block. This observation has sparked a public effort to characterize all common haplotypes in the human population (12). ...