Automation of diagnostic genetic testing: mutation detection by cyclic minisequencing.

BACKGROUND: The rising role of nucleic acid testing in clinical decision making is creating a need for efficient and automated diagnostic nucleic acid test platforms. Clinical use of nucleic acid testing sets demands for shorter turnaround times (TATs), lower production costs and robust, reliable methods that can easily adopt new test panels and is able to run rare tests in random access principle. Here we present a novel home-brew laboratory automation platform for diagnostic mutation testing. METHOD: This platform is based on the cyclic minisequecing (cMS) and two color near-infrared (NIR) detection. Pipetting is automated using Tecan Freedom EVO pipetting robots and all assays are performed in 384-well micro plate format. The automation platform includes a data processing system, controlling all procedures, and automated patient result reporting to the hospital information system. CONCLUSIONS: We have found automated cMS a reliable, inexpensive and robust method for nucleic acid testing for a wide variety of diagnostic tests. The platform is currently in clinical use for over 80 mutations or polymorphisms. Additionally to tests performed from blood samples, the system performs also epigenetic test for the methylation of the MGMT gene promoter, and companion diagnostic tests for analysis of KRAS and BRAF gene mutations from formalin fixed and paraffin embedded tumor samples. Automation of genetic test reporting is found reliable and efficient decreasing the work load of academic personnel.

Multiple heat pulses during PCR extension enabling amplification of GC-rich sequences and reducing amplification bias.

PCR amplification over GC-rich and/or long repetitive sequences is challenging because of thermo-stable structures resulting from incomplete denaturation, reannealing, and self-annealing of target sequences. These structures block the DNA polymerase during the extension step, leading to formation of incomplete extension products and favoring amplification of nonspecific products rather than specific ones. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to temporarily destabilize such blocking structures, in order to enhance DNA polymerase extension over GC-rich sequences. With this novel type of protocol, we were able to amplify all expansions of CGG repeats in five Fragile X cell lines, as well as extremely GC-rich nonrepetitive segments of the GNAQ and GP1BB genes. The longest Fragile X expansion contained 940 CGG repeats, corresponding to about 2.8 kilo bases of 100% GC content. For the GNAQ and GP1BB genes, different length PCR products in the range of 700 bases to 2 kilobases could be amplified without addition of cosolvents. As this technique improves the balance of amplification efficiencies between GC-rich target sequences of different length, we were able to amplify all of the allelic expansions even in the presence of the unexpanded allele.

Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors.

Detection of recurrent somatic rearrangements routinely allows monitoring of residual disease burden in leukemias, but is not used for most solid tumors. However, next-generation sequencing now allows rapid identification of patient-specific rearrangements in solid tumors. We mapped genomic rearrangements in three cancers and showed that PCR assays for rearrangements could detect a single copy of the tumor genome in plasma without false positives. Disease status, drug responsiveness, and incipient relapse could be serially assessed. In future, this strategy could be readily established in diagnostic laboratories, with major impact on monitoring of disease status and personalizing treatment of solid tumors.

Fluorescence resonance energy transfer (FRET) using ssDNA binding fluorescent dye.

There is a need for simple and inexpensive methods for genotyping single nucleotide polymorphisms (SNPs) and short insertion/deletion variations (InDels). In this work, I demonstrate that a single-stranded DNA (ssDNA) binding dye can be used as a donor fluorophore for fluorescence resonance energy transfer (FRET). The method presented is a homogenous assay in which detection is based on the FRET from the fluorescence of the ssDNA dye bound to the unmodified detection primer to the fluorescent nucleotide analog incorporated into this detection primer during cyclic template directed primer extension reaction. Collection of the FRET emission spectrum with a scanning fluorescence spectrophotometer allows powerful data analysis. The fluorescence emission signal is modified by the optical properties of the assay vessel. This seems to be a completely neglected parameter. By proper selection of the optical properties of the assay plate one can improve the detection of the fluorescence emission signal.

Novel heat pulse extension-PCR-based method for detection of large CTG-repeat expansions in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal-dominant disease caused by an expansion of CTG repeats in the 3' untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene. Detection and accurate sizing of the CTG-repeat expansions is clinically important, because the number of CTG repeats correlates with the disease severity. Because difficulties in PCR amplification over large expansions, molecular diagnosis of DM1 is still primarily based on Southern blotting, which is technically demanding and time consuming and requires large amounts of genomic DNA samples. We have recently discovered that the use of multiple heat pulses during Heat Pulse Extension PCR (HPE-PCR) enables efficient amplification over repetitive and GC-rich sequences. Based on this principle, we have developed an assay for efficient amplification of large CTG-repeat expansions seen in DM1 patients. The HPE-PCR method was able to amplify different DMPK1 repeat expansions of up to 1750 CTG repeats in 78 clinical samples with a varying degree of tissue heterogeneity, even in the presence of the short wild-type allele. The CTG-repeat lengths and fragmentation patterns obtained with HPE-PCR were fully concordant with the original diagnostic Southern blotting results. This novel technique provides a PCR-based platform for molecular diagnosis of DM1, and it has been adopted for routine diagnostic use.