Molecular inversion probes, or MIPs, are a tool for targeted genetic analysis, allowing researchers to focus on specific DNA sequences within a genome. These specialized, single-stranded DNA molecules are engineered to find and capture particular segments of genetic code. This capability enables the detailed examination of genes or regions of interest without sequencing an entire genome, which is useful for identifying small genetic variations between individuals.
MIPs function like a sophisticated search tool for DNA. They are capable of locating a specific genetic sequence within the immensely complex background of a genome. By isolating and copying the sequence of interest, researchers can analyze it in great detail.
Anatomy of a Molecular Inversion Probe
A molecular inversion probe is a single strand of DNA, around 100 to 120 nucleotides long, with several functional parts. The ends of the probe, known as the extension and ligation arms, are designed with sequences complementary to the DNA on either side of a target genetic location. This design ensures the probe binds precisely where intended.
Between these two arms is a central linker region that does not bind to the target DNA. Instead, it contains universal sequences that are the same for every probe in an experiment. These shared sequences act as docking sites for enzymes and primers during later stages of analysis.
The design of the arms and backbone allows the probe to form a circular structure once it finds its target. The arms bind to the genomic DNA so they end up next to each other, with the target nucleotide(s) filling a small gap between them. The unattached linker region then forms a loop, creating a shape similar to a padlock closed around its target.
The Inversion Probe Workflow
The process begins with hybridization, where thousands of different probes are mixed with a sample of genomic DNA. The two arms of a probe bind to its target DNA strand at adjacent locations, leaving a small gap directly over the specific site of interest.
Once the probe is attached, two enzymes are used. First, a DNA polymerase fills the gap between the arms, using the genomic DNA as a template to add the corresponding nucleotides. Following this, a DNA ligase enzyme seals the connection, transforming the linear probe into a closed, circular molecule that has captured a copy of the target sequence.
To isolate these copies, an exonuclease enzyme is added to the mixture. This enzyme digests linear DNA, which eliminates the original genomic DNA and any probes that failed to circularize. This purification step ensures that only the circularized probes carrying the captured genetic information remain.
The final step is to amplify the captured information. The circular probes are linearized, exposing the universal backbone sequences. PCR primers designed to match these universal sequences are then used to amplify all the captured targets in the sample simultaneously, creating a library of DNA molecules ready for sequencing.
Applications in Genetic Analysis
One primary use of MIP technology is for targeted sequencing of specific genes or genomic regions. Researchers can design a panel of thousands of probes to simultaneously target exons of genes associated with a particular condition, like hereditary cancers. This approach allows for a deep and focused analysis of these areas to find potential disease-causing mutations.
The technology is well-suited for SNP genotyping, which is the process of identifying single nucleotide polymorphisms (SNPs). During the MIP workflow, the gap-filling step captures the specific nucleotide present at a target SNP location. Sequencing the amplified probes allows scientists to accurately determine which version of the SNP an individual has.
MIPs can also analyze copy number variations (CNVs), which are differences in the number of copies of a gene from one person to the next. By measuring the quantity of amplified probes for each targeted region, researchers can determine if a segment of DNA has been deleted or duplicated. This analysis is relevant for studying a range of genetic disorders and cancers.
Distinguishing Features of MIP Technology
A defining characteristic of MIPs is their high multiplexing capability. Scientists can pool tens of thousands of unique probes into a single reaction to investigate an equal number of DNA targets simultaneously. This efficiency reduces the amount of sample DNA required and the overall laboratory workload.
The probe design leads to a high degree of specificity. For a probe to be amplified, its arms must bind correctly, and the gap between them must be successfully filled and ligated. This two-step verification, followed by the removal of all non-circularized molecules, minimizes the capture of incorrect sequences and ensures that only accurately captured targets are analyzed.
This technology provides a cost-effective solution for the deep analysis of specific genomic regions. While whole-genome sequencing is comprehensive, it can be expensive and generate unnecessary data for certain research questions. MIPs offer an economical alternative when the goal is to sequence a pre-defined set of targets at great depth, making it a practical choice for large-scale studies.