MPD^TM Enhanced Detection of Genetic Mutations

Abstract: BioTraces, Inc. has developed new high sensitivity assay protocols for the detection and quantitation of nucleic acids. These new protocols provide a few hundreds fold higher sensitivity for direct (non-amplified) detection and analysis of DNA and can be used in detection of genetic mutations. More specifically, BioTraces' proprietary Multi Photon Detection^TM (MPD^TM) technology permits the MPD enhanced Single Strand Conformation Polymorphism (SSCP) and Denaturating Gradient Gel Electrophoresis (DGGE), which enables more robust and sensitive detection of genetic mutations.

Introduction: An increasing number of genes involved in human disease are being isolated and the nature of the defects causing disease characterized. In some diseases there are a few causal mutations, but in others up to a few tens of mutations are responsable for organism malfunction. The improvement of assay systems for cancer genes is increasingly important, e.g., there is an urgent need for better genetic diagnostic tests for prostate and breast cancers. Better understanding of BRCA1 and BRCA2 genes polymorpisms may permit efficient diagnostics of breast cancer risk but it is necessary that a screening procedure be developed that can detect both known mutations and possible new candidate mutations as subtle as a single base pair. SSCP and DGGE are good approaches for the detection of candidate defects prior to more expensive sequencing of the affecting gene region.

The key technical bottleneck both in SCCP and DGGE is the low sensitivity of previously used detectors. This bottleneck can be removed by the use of ultra-sensitive MPD. We showed single base resolution for ¹²⁵I labeled DNA sequencing ladders for as little as a few zeptomoles/band. We also studied applications of the MPD Imager to SSCP and DGGE, and demonstrated the possibility for improved sensitivity and throughput.

MPD enhanced DGGE and SSCP: MPD permits efficient rejection of background events through sophisticated signal processing and analysis. MPD is able to reduce the number of background events to less than one per day and has achieved 10^-22 mole sensitivity. Thus, DNA can be reliably detected at much lower level. MPD Imagers permit the sub-millimetric spatial resolution and are well suited for analysis of electrophoretic gels, including 2-D gels at sub-attomoles level. Electrophoretic outputs with as little as 0.05 picoCurie activity/band can be reliably quantitated. We improved methods for detection of mutations using methodologies based on 2D gels, e.g. SSCP and DGGE.

The MPD Imager enables the SSCP and DGGE analysis of DNA at sub-attomole/sample level (1 attomole = 10^-18 mole) and permits robust, ultrasensitive methods to detect polymorphisms in human genomic DNA. SSCP and DDGE reaveal mutations which confer mobililty differences. Current limitations are due to gel overloading and temperature variations. Due to its superior sensitivity, MPD instrumentation eliminates overloading artifacts. It also enables the use of internal mobility markers and consequently an increased ability to recognize mutations under SSCP and DDGE. We utilize the high sensitivity of the MPD to increase throughput of perpendicular DGGE by multiplexing the assay, i.e., concurrent screening of dozens of samples in the same DGGE gel.

There are many targets for detection of unknown mutations. For example, there is evidence for genetic predisposition for schizophrenia and many other neurological diseases. Genetic predisposition to breast and ovarian cancer, i.e. BRCA1 and BRCA2 gene mutations are highly polymorphic. Point mutations are found throughout the entire coding sequence thereby making genetic screening quite difficult and laborious. The development of a robust and efficient mutation scanning method for the BCRA1 gene is hampered by the large size of the gene. We developed an improved electrophoresis method for scanning large regions of the gene in single experiments. This is possible because the increased sensitivity provided by MPD allows increased gel resolution enabling the examination of the migration characteristics of many pieces of DNA in a single experiment.

Short of sequencing the entire gene, the human genetics community presently employs SSCP and DGGE methodologies to detect point mutations within a population. Currently, the most popular method of detecting unknown mutations is SSCP. Single stranded DNA undergoes intrastrand bindings between bases which affect the 3D conformation. The majority of mutations will affect DNA conformation, and can consequently be made apparent by the induced changes in mobility under electrophoresis.

DGGE is an alternative methodology and is often more sensitive to point mutations. Here all point mutations contribute to the denaturation profile of a double stranded DNA fragment. The detection probability for mutations is close to 100%, but DGGE is considerably more complicated and manpower intensive than SSCP. Thus, simplified DGGE with higher throughput may become an important method for the study of mutations. MPD enhanced DGGE may permit a factor of 20 fold higher throughput.

SSCP/MPD- pilot studies: The sample preparation protocol uses a "cold" PCR from a genomic template followed by the steps of iodination and PCR product analysis by agarose gel electrophoresis. We developed a two-color SSCP/MPD assay: electrophoresis of radioidinated samples with a ³²P-labeled mobility marker in each lane, and co-electrophoresis of ¹²⁵I labeled sample and ³²P-labeled wild-type DNA in each lane. This allows us to compensate for temperature nonuniformity during the SSCP electrophoresis step. A suitable set of 10 SS DNA mobility markers has been developed for SSCP electrophoresis of fragments 100 to 350 nucleotides long. We developed the software for automated processing of the SSCP assay data, which can be used to normalize the mobilities of the sample strand(s) in each lane. Optionally, one can co-electrophorese distinguishably labeled wild-type and mutant samples. The optimization includes separate SSCP of each strand, restriction endonuclease digestion of some fragments to reduce the length, as well as finding the optimal gel matrix, temperature conditions, glycerol content, etc. To test the ability of the MPD Imager to discriminate between two different radiolabels, we amplified one strand of a 385 bp fragment of E.coli 23S rRNA gene, incorporating ³²P-dCTP. A 20-cycle single-primer amplification was performed. Although in this case we used linear (single-primer) amplification, rather than PCR, the concentrations of dNTPs were lowered and the extension time lengthened to allow efficient label incorporation. At the same time the cycling reaction selectively amplified only one of the two strands. Following the labeling procedures, ¹²⁵I-labeled product from each sample and ³²P-labeled product were mixed and diluted with sequencing stop solution, heat denatured and placed on ice. The samples were electrophoresed for 16 hours at a constant power of 8W in the cold room (4^o) without temperature control in MDE gel. The gel was then dried and the lanes read out with the MPD Imager. The spectra were processed to discriminate between ³²P and ¹²⁵I based on energy and coincident emission of the iodine isotope. The data demonstrate that low-concentration PCR labeling produces readable SSCP band patterns and that the MPD Imager can efficiently discriminate between ³²P and ¹²⁵I.

In summary, we performed preliminary studies of MPD enhanced SSCP. We used two radioiodination techniques to efficiently label the PCR products: an isothermal labeling/extension protocol using Sequenase 2.0 and a second cyclic amplification with lowered concentrations of dNTPs. Both labelling techniques are 100-fold more efficient than the standard internal incorporation of radiolabel during PCR. Signal to background ratio of 100:1 was achieved using only 1 nCi of radiolabel per sample.

DGGE/MPD - pilot studies: In perpendicular DGGE the direction of electrophoresis is perpendicular to direction of denaturant gradient. In principle, perpendicular DGGE can detect mutations in every DNA melting domain. However, only a small number of samples can be analyzed simultaneously, due to the topographical constraints of the system and limited dynamic range. Perpendicular DGGE has a limited dynamic range because of detector sensitivity (lower limit) and the maximum amount of DNA that can be loaded in a gel without affecting background and electrophoretic resolution (upper limit). Increased detection sensitivity improves the dynamic range and thus permits pooling of numerous samples. However, this method may be limited by biological constraints such as Taq polymerase errors. We minimize these artifacts by using less then 20 PCR cycles, i.e. remaining in the exponential phase of PCR yield. Furthermore, the shape of the curve and the branching which occurs in perpendicular DGGE in the presence of mutations is extremely robust to artifacts.

We performed a series of experiments investigating perpendicular DGGE assays at DNA levels much lower than possible with silver staining techniques. We performed perpendicular DGGE of a fragment of human DNA fragment. Comparison with silver stain shows about 5,000-fold higher sensitivity. The higher sensitivity of the perpendicular DGGE might allow pooling, e.g., 50 samples in screening for polymorphisms, bypassing the low throughput of perpendicular DGGE while retaining its mutation detection efficiency.