Health & Research

Molecular Inversion Probe (MIP) Assay

An efficient technology basically for large-scale Single Nucleotide Polymorphisms (SNPs) analysis.

SNP:

SNP is a single base change in DNA sequence. It is a Genetic variation of a single base that is kept through heredity.

Molecular inversion probe:

  • It is a Single stranded DNA
  • Complementaryto the target sequence in the genome.
  • The total length of the MIP is about 120 nucleotides.

MIP structure:

It consists of

  • Complementary sequences at its 5’ and 3’ ends.

2 complementary sequences each of 20-30 nucleotides.

  • Two universal PCR rimer sites.

Each of about 15-20 nucleotides long.

It is about 4-6 nucleotides long.

  • A probe-specific tag sequence.

It is about 20 nucleotides long.

  • A tag-release site.

It is about 4-6 nucleotides long.

Principle:

  • Both complementary sequences of the probe bind to same target DNA molecule.
  • After hybridization, the probe circularizes and the internal linker region forms a free hanging loop.

Enzymes involved in reaction:

  • DNA polymerase
  • Ligase
  • Restriction enzyme
  • Exonuclease

Procedure Steps 

  1. Anneal probe
  2. Gap filling
  3. ligation
  4. Exonuclease selection
  5. Probe release
  6. Captured target enrichment
  7. Captured target identification
  8. Data analysis
  9. Anneal probe
  • Probes are added to the genomic DNA sample.
  • Hybridized to the target DNA.
  • Probes then undergo circularization
  • Anneal a mixture of DNA, up to 10,000 probes, thermostable ligase and polymerase is heat denatured and brought to annealing temperature. Two sequences located at each termini of the probe hybridize to their respective complementary sites on the genome thus forming a circular conformation with a single nucleotide gap between the termini of the probe.
  1. Gap fill – polymerization:
  • Un labelled d ATP, d CTP, d GTP, d TTP is added to each of the 4 reactions respectively. In reactions where the added nucleotide is complementary to the base being studied, DNA polymerase adds the nucleotide.
  • Size of gap:

It is Single nucleotide gap for SNP genotyping and can be

Several Hundred nucleotides long for loci capture.

  1. Ligaton:

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.

  1. Exonuclease selection:

Exonucleases are then added to digest linear probes in reactions where the added nucleotide was not complementary to the gap and excess linear probe in reactions where circular molecules were formed. The reactions are then heated to inactivate the exonucleases.

  1. Probe release:

The probes are then cleaved to release them from the genomic DNA. Probe release site (commonly a restriction site) is cleaved by restriction enzymes so that the probe becomes linearized.

  1. Captured target enrichment/ amplification:

PCR amplification to enrich the captured target using the universal primers of the probe under DNA polymerase.

  1. Captured target identification:
  1. Data analysis:

GeneChip® Targeted Genotyping Analysis Software is used for analysis of the MIP data from the arrays.

Multiplex analysis:

  • Each probe examines one specific genomic locus.
  • Multiple probes in a single tube simultaneously examine multiple loci.
  • Currently, >55,000 loci in a single multiplexed MIP assay.

MIP Optimization Strategies:

  • Ends of the probe should be Complementary and Specific
  • Annealing temperatures should be Same for both complementary sequences
  • Target length Should not be too long (100 to 200 bp long)
  • Concentrations of Probe, Target and enzymes should be properly adjusted
  • Time duration, hybridization and gap fill time is also set properly to optimize the process.

Advantages of MIP

  • High specificity
  • High levels of multiplexing
  • Simple infrastructure (easy to use)
  • Reproducibility
  • scalability

Applications

Limitations of MIP:

  • Low sensitivity
  • Large gap requires longer probes.
  • Increased costs.

References:

  • Molecular inversion probe assay for allelic quantitation. Methods Mol Biol. 2009 ; 556: 67–87. doi:10.1007/978-1-60327-192-9_6. 2010 November 19.
  • Molecular Inversion Probe Array for the Genetic Evaluation of Stillbirth Using Formalin-Fixed, Paraffin-Embedded Tissue, The Journal of Molecular Diagnostics, Vol. 15, No. 4, July 2013
  • Single molecule molecular inversion probes for targeted, high accuracy detection of low frequency variation. Joseph B Hiatt, Colin C Pritchard, Stephen J Salipante, et al.

 

 

 

 

 

 

 

 

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