Applied Biosystems

About: Applied Biosystems is a based out in . It is known for research contribution in the topics: Mass spectrometry & Nucleic acid. The organization has 1521 authors who have published 1579 publications receiving 285423 citations.

Haplotypes and informative SNP selection algorithms: don't block out information

Abstract: It is widely hoped that variation in the human genome will provide a means of predicting risk of a variety of complex, chronic diseases. A major stumbling block to the successful identification of association between human DNA polymorphisms (SNPs) and variability in risk of complex diseases is the enormous number of SNPs in the human genome (4,9). The large number of SNPs results in unacceptably high costs for exhaustive genotyping, and so there is a broad effort to determine ways to select SNPs so as to maximize the informativeness of a subset.In this paper we contrast two methods for reducing the complexity of SNP variation: haplotype tagging, i.e. typing a subset of SNPs to identify segments of the genome that appear to be nearly unrecombined (haplotype blocks), and a new block-free model that we develop in this report. We present a statistic for comparing haplotype blocks and show that while the concept of haplotype blocks is reasonably robust there is substantial variability among block partitions. We develop a measure for selecting an informative subset of SNPs in a block free model. We show that the general version of this problem is NP-hard and give efficient algorithms for two important special cases of this problem.

Quantitative PCR Analysis of DNA, RNAs, and Proteins in the Same Single Cell

Abstract: BACKGROUND: The single cell represents the basic unit of all organisms. Most investigations have been performed on large cell populations, but understanding cell dynamics and heterogeneity requires single-cell analysis. Current methods for single-cell analysis generally can detect only one class of analytes. METHODS: Reverse transcription and the proximity ligation assay were coupled with quantitative PCR and used to quantify any combination of DNA, mRNAs, microRNAs (miRNAs), noncoding RNAs (ncRNAs), andproteinsfromthesamesinglecell.Themethodwas used on transiently transfected human cells to determinetheintracellularconcentrationsofplasmids,their transcribed mRNAs, translated proteins, and downstream RNA targets. RESULTS: We developed a whole-cell lysis buffer to release unfractionated DNA, RNA, and proteins that wouldnotdegradeanydetectableanalyteorinhibitthe assay. The dynamic range, analytical sensitivity, and specificity for quantifying DNA, mRNAs, miRNAs, ncRNAs,andproteinswereshowntobeaccuratedown tothesingle-celllevel.Correlationstudiesrevealedthat the intracellular concentrations of plasmids and their transcribed mRNAs were correlated only moderately withtranslatedproteinconcentrations(Spearmancorrelation coefficient, 0.37 and 0.31, respectively; P 0.01). In addition, an ectopically expressed gene affected the correlations between analytes and this gene, which is related to gene regulation. CONCLUSIONS: This method is compatible with most cell-sampling approaches, and generates output for the same parameter for all measured analytes, a feature facilitating comparative data analysis. This approach should open up new avenues in molecular diagnostics for detailed correlation studies of multi

Automated DNA sequencing methods involving polymerase chain reaction.

Abstract: Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.