Ambergen Technology

Cost effective, high throughput and high sensitivity mutation scanning is a critical tool for a variety of molecular diagnostic assays, such as those for inherited disorders as well as sporadic cancers.

At The Core: Cell-Free Protein Synthesis

Ambergen has developed several novel mutation detection/scanning technologies which use cell-free protein synthesis at their core (Figure 1). As depicted in Figure 1, DNA/RNA is extracted from a biological source such as a patient blood sample. Specialized primers are then used to amplify the genes or gene-regions of interest using PCR/RT-PCR. Primers introduce all sequences necessary for cell-free protein expression and well as introducing various detection and affinity tags, e.g. antibody epitope tags. PCR amplicons are directly and rapidly expressed in a cell-free synthesis system in <1 hour. This facilitates a variety of highly innovative mutation scanning techniques which are performed at the protein level, which affords specific advantages over traditional genetic approaches.

ELISA-PTT

Mutations in the open reading frames of specific genes is at the root cause of many inherited genetic disorders. Difficulties in molecularly based diagnosis of these disorders occur in several important cases where the number of possible mutations ranges from several hundred to more than a thousand, the mutations can occur virtually anywhere throughout the gene, the de novo mutation rate is high, and/or the gene is large in size. Examples include the inherited breast cancer susceptibility genes BRCA1/2, the NF gene responsible for neurofibromatosis and the APC gene responsible for an inherited form of colon cancer (FAP). Fortunately, in these cases, the vast majority of mutations fall into one class, truncation mutations, e.g. nonsense or frameshift mutations, which result in a premature termination of translation and hence a shortened gene product (i.e. protein). DNA level detection would require either a mutation scanning method such as full gene sequencing or hundreds to thousands of DNA point probes to detect all the possible known truncation mutations; the later of which is not compatible with detecting previously uncharacterized mutations. An effective and inexpensive alternative, which is a mutation scanning approach, is the protein truncation test (PTT), whereby following the cell-free protein synthesis process depicted in Figure 1, the truncated protein is electrophoretically detected by a change in mass. The electrophoretically based PTT however is inherently low throughput, is limited by the resolution of SDS-PAGE, and is subject to human error in the interpretation of results. To overcome this, Ambergen has developed a patented ELISA based PTT (Figure 2), a version of which we previously reported in Nature Biotechnology (link to our 2002 ELISA-PTT Nature Biotechnology paper). The assay utilizes a series of antibody epitope tags incorporated prior to cell-free protein expression during the PCR stage. Nascent cell-free expressed proteins are captured/isolated onto the wells of the ELISA plate by their N-terminal capture tag. The N-terminal detection tag (N-Tag) provides a normalization signal for total expressed target protein, while the C-terminal detection tag (C-Tag) provides the relative amount truncated target protein. A C-terminal to N-terminal ratio of 50% relative to a wild-type reference indicates a heterozygous truncation mutation is present.

MASSIVE-PRO

Additional strengths of the ELISA-PTT are its ability to screen gene segments as large as 2,500 bp in a single reaction as well as the high throughput, cost effective, automation friendly, industry standard microtiter plate assay format. However, detection is restricted to truncation mutations and sensitivity is limited to a ~25% mutant population in a background of ~75% wild-type (similar to DNA sequencing); adequate for only inherited diseases. However, in the case of sporadic mutations, such as with non-inherited cancers, the mutant species is often present at levels far below 25% in the biological sample being assayed (e.g. very roughly 1% in stool samples from sporadic colon cancer patients). To overcome these limitations, Ambergen has developed a proprietary mass spectrometry based approach (MASSIVE-PRO) for high sensitivity mutation detection/scanning (Figure 3). Small peptides, corresponding to gene regions of interest from the patient, are again produced by PCR coupled with cell-free expression as previously shown in Figure 1. Similar to ELISA-PTT, nascent peptides are then isolated (purified) using an incorporated N-terminal epitope tag. The purified peptides are then eluted and analyzed tandem matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS/MS). Both truncation and missense mutations are detected by a mass shift resulting from the shortened peptide or amino acid substitution (missense). Mutations are confirmed inline by fragmentation based peptide sequencing in the MALDI-TOF MS/MS instrument. Mass shifts as little as 1 Dalton can be detected and sensitivities as low as 1% mutant population have been achieved.

Real Life Application to Diseases

Ambergen is actively applying these aforementioned technologies to the molecular diagnostics of sporadic cancers such as colorectal cancer (CRC), the second leading cause of cancer deaths in the US, as well as to inherited disorders such as neurofibromatosis and familial breast cancer. We are also actively applying these technologies to the early detection of drug-resistance mutations against molecularly targeted therapies used to treat various cancers, such as chronic myeloid leukemia (CML), as well for therapies in other diseases such as HIV.