Molecular Inversion Probe

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration (as suggested by the name of the technique) and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterationsand characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously. Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration (as suggested by the name of the technique) and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterationsand characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously. The probes are designed with sequences that are complementary to the genomic target at its 5’ and 3’ ends.The internal region contains two universal PCR primer sites that are common to all MIPs as well as a probe-release site, which is usually a restriction site. If the identification of the captured genomic target is performed using array-based hybridization approaches, the internal region may optionally contain a probe-specific tag sequence that uniquely identifies the given probe as well as a tag-release site, which, similar to the probe-release site, is also a restriction site. Probes are added to the genomic DNA sample. After a denaturation followed by an annealing step, the target-complementary ends of the probe are hybridized to the target DNA. The probes then undergo circularization in this process. These probes, however, are designed such that a gap delimited by the hybridized ends of the probes remains over the target region. The size of the gap ranges from a single nucleotide for SNP genotyping to several hundred nucleotides for loci capture (e.g. exome capture). The gap is filled by DNA polymerase using free nucleotides and the ends of the probe are ligated by ligase, resulting in a fully circularized probe. Since gap filling is not performed for non-reacted probes, they remain linear. Exonuclease treatment removes these non-reacted probes as well as any remaining linear DNA in the reaction. In some versions of the protocol, the probe-release site (commonly a restriction site) is cleaved by restriction enzymes such that the probe becomes linearized. In this linearized probe the universal PCR primer sequences are located at the 5’ and 3’ ends and the captured genomic target becomes part of the internal segment of the probe. Other protocols leave the probe as a circularized molecule. If the probe is linearized, traditional PCR amplification is performed to enrich the captured target using the universal primers of the probe. Otherwise, rolling circle amplification is performed for the circular probe. The captured target can be identified either via array-based hybridization approaches or by sequencing of the target. If array-based approach is used, the probe may optionally contain a probe-specific tag that uniquely identifies the probe as well as the genomic region targeted by it. The tags from each probe are released by cleaving the tag release site with restriction enzymes. These tags are then hybridized to the sequences that are placed on the array and are complementary to them. The captured target can also be identified by sequencing the probe, now also containing the target. Traditional Sanger sequencing or cheaper, more high-throughput technologies such as SOLiD, Illumina or Roche 454 can be used for this purpose. Multiplex analysisAlthough each probe examines one specific genomic locus, multiple probes can be combined into a single tube for multiplexed assay that simultaneously examines multiple loci. Currently, multiplexed MIP analysis can examine more than 55,000 loci in a single assay.