Polymorphism Ratio Sequencing: A New Approach for Single Nucleotide Polymorphism Discovery and Genotyping

Abstract

Polymorphism ratio sequencing (PRS) combines the advantages of high-throughput DNA sequencing with new labeling and pooling schemes to produce a powerful assay for sensitive single nucleotide polymorphism (SNP) discovery, rapid genotyping, and accurate, multiplexed allele frequency determination. In the PRS method, dideoxy-terminator extension ladders generated from a sample and reference template are labeled with different energy-transfer fluorescent dyes and coinjected into a separation capillary for comparison of relative signal intensities. We demonstrate the PRS method by screening two human mitochondrial genomes for sequence variations using a microfabricated capillary array electrophoresis device. A titration of multiplexed DNA samples places the limit of minor allele frequency detection at 5%. PRS is a sensitive and robust polymorphism detection method for the analysis of individual or multiplexed samples that is compatible with any four-color fluorescence DNA sequencer.Genetic studies are expanding past the identification of rare Mendelian diseases to encompass common complex disorders such as cancer, neurological diseases, autoimmune diseases, and cardiovascular diseases. The abundance and stability of single nucleotide polymorphisms (SNPs) has made them the DNA variation of choice for such endeavors. Due to the low penetrance of individual alleles, association studies designed to uncover significant genetic risk factors require the scoring of hundreds of thousands of SNPs (Kruglyak 1999) in several thousand samples (Dahlman et al. 2002). Multiplexed highthroughput genotyping methods are essential to accommodate this challenging number of SNPs and samples.Current methods of SNP detection include microarrays (Chee et al. 1996), allele-specific polymerase chain reaction (PCR; Liu et al. 1997), oligonucleotide ligation assays (Grossman et al. 1994;Samiotaki et al. 1994;Tobe et al. 1996), single-base extension (Syvanen et al. 1993;Pastinen et al. 1996;Shumaker et al. 1996), TaqMan (Livak et al. 1995, and molecular beacons (Tyagi et al. 1998). These methods are designed to interrogate SNPs in one sample at a time. Modifications such as kinetic PCR (Germer et al. 2000) and detection using denaturing high-performance liquid chromatography (HPLC; Giordano et al. 2001) can extend these procedures to multiplexed sample analysis. Each method offers a trade-off between sensitivity, throughput, parallel processing, user accessibility, and cost, and they all experience limitations due to local DNA hybridization characteristics. Selection of allelespecific probes is confined to the DNA sequence immediately adjacent to or surrounding the targeted polymorphism. This restriction can lead to nonuniform hybridization characteristics, ambiguous results, or the rejection of otherwise informative SNPs in a significant percentage of the markers. Additionally, novel polymorphisms are not detected by these approaches. These limitations are particularly important when studying functional or rare SNPs.Quality-based resequencing a...

DNA sequencing and genotyping in miniaturized electrophoresis systems

“…Alternative mutation detection techniques have been developed based on electrophoretic size-and/or conformation-based DNA separations, such as SSCP and HA [30][31][32], or tandem SSCP-HA [33,34], SBE [35,36], and polymorphism ratio sequencing (PRS) [37]. Among these techniques, SSCP (which involves a mobility shift assay combining mutated DNA with known wild-type DNA, and creating single-stranded conformers by denaturing and snap-cooling) alone or in conjunction with HA (which involves analyzing the combined hybrid duplexes of both mutant and wild-type DNA), are two very promising approaches to creating a simple, robust, low-cost, and high-speed electrophoretic method to screen for genetic alterations related to disease [38].…”

Miniaturization in Futuristic Clinical Laboratory Medicine

“…8 Likewise, the inclusion of microfabricated thin-film heating elements and platinum resistive temperature detectors in a thermal cycling PCR device coupled to a highspeed microchip capillary electrophoresis system has opened up opportunities for amplification of even a single molecule. 9,10 Control of fluid flow in chip-based microfluidic systems can also be affected by techniques that utilize either pressurecontrolled air bubbles, surface tension, gravity, or by the mere application of electrical potential.…”

Rapid Determination of Monozygous Twinning with a Microfabricated Capillary Array Electrophoresis Genetic-Analysis Device1

“…These advantages include high-throughput DNA sequencing and capabilities for analyzing single base pairs, multicolor fluorescence detection, requirements for only minute quantities of sample and reagents, and a small device footprint. The capabilities of CAE devices have been demonstrated in rapid (approximately 30 min) high-quality genomic and mitochondrial DNA sequencing analyses (1-bp resolution with 99% accuracy) of 96 samples simultaneously (8,9 ). The facile scaling capability of these devices was illustrated in the typing of single-stranded conformation polymorphisms for 384 bladder cancer samples and the mutational screening of 384 samples for hereditary hemochromatosis-related single-nucleotide polymorphisms in Ͻ6 min (Ͼ1 sample/s throughput) (10 ).…”