Technology development for genome and polymorphism analysis

Abstract

Single nucleotide polymorphism (SNP) is widely expected to provide a multitude of benefits in areas of medical research. There is, however, a growing disparity between the rate of SNP discovery and the development of effective technologies for large scale SNP scoring. This thesis outlines the development of a SNP scoring technology termed dynamic allele-specific hybridization (DASH).

The first publication describes the basic concept of DASH. The strategy involves a short PCR spanning the SNP position, with one PCR primer biotinylated. Via biotin-streptavidine affinity capture the PCR product is bound to the surface of a microtiter plate well. Rinsing in alkali removes the nonbiotinylated strand and then hybridization of an allele-specific oligonucleotide probe to the bound target is done. In the presence of a double strand specific fluorescent dye the probe- target duplex is slowly heated whilst fluorescence is monitored. Denaturation of a duplex where the probe is complementary to the SNP allele will cause a drop in fluorescence at a higher temperature than will a probe mismatching at the polymorphic position. Genotypes are obtained by interpretation of the denaturation profiles.

The second publication is an investigation of how truncated oligonucleotide impurities on a surface affect hybridization. It is well known that DNA chips created by photolithography suffer from carrying such impurities, but the effect of these contaminants upon hybridization has not previously been investigated. Impurities were modeled (oligonucleotides of different lengths) in ratios reported from in situ array creation by photolithography. Subsequent hybridization experiments where denaturation profiles were generated gave insight in how impurities influence hybridization.

The third publication describes a simple routine for array creation on different surfaces by centrifugation. The centrifugation array concept was later implemented in the second generation of DASH (see below).

The fourth manuscript included in the thesis describes a tool for automated DASH assay design. Secondary structure formation in the hybridization target molecule may compete with the allele-specific probe and can cause an assay to fail. However, by careful assay design it is possible to essentially eliminate secondary structure problems and DASH assay failures Strategies for efficient assay design has been developed and implemented in a DASH-assay design software.

The fifth manuscript describes high throughput implementation of DASH. The new system, which we have termed DASH-2, involves small volume multiplex PCR and multiplex melting temperature analysis on arrays created by centrifugation. A new system of generating hybridization signals, termed FRET, offers possibilities for multiplexed analysis by separating the assays spectrally. The system is highly flexible (any plate format, PCR multiplexing, serial and parallel array processing, spectral-multiplexing of hybridization probes), thus supporting a wide range of applications, scales and objectives.