Immobilized Mismatch Binding Protein (IMBP) 96-well Plates are a breakthrough in mutation/polymorphism detection. IMBP plates are coated microtiter plates designed to bind DNA molecules containing single base pair mismatches or small (1-4 base) additions or deletions. IMBP plates are suited for single nucleotide polymorphism (SNP) detection, allele identification, mutation scanning, PCR optimization and PCR fidelity monitoring. Typical IMBP assays require less than one hour to complete and do not require gel electrophoresis or radioactivity. IMBP plates are available as individual plates (12 strips of 8 wells) or in kits containing plates, buffers, DNA standards and colorimetric reagents. IMBP 96-well plates provide a robust, economical and easy-to-use assay to detect, identify and even isolate sequences which contain any type of single base alteration. Please contact Gene Check for further examples of this emerging technology.

IMBP plate assays are simple to perform. After DNA amplification and hybridization, results are obtained in less than 60 minutes using standard laboratory equipment and supplies.

Simple Protocol Outline:

1. Rinse to remove storage buffer.
2. Add DNA to wells. Incubate 15-30 minutes.
3. Wash off unbound DNA.
4. Add Streptavidin-HRP. Incubate 15 minutes.
5. Wash.
6. Incubate with TMB.
7. Add stop acid and measure OD (450 nm).

Complete Protocol Outline:

Assay Set Up

1. Dilute 10X Wash Buffer by adding 25 ml to 225 ml molecular grade deionized water (sufficient for one plate). Store unused Wash Buffer at 4°C.
2. Prepare Stop Acid (0.5 M H2SO4). The use of stop acid increases the sensitivity of TMB and is recommended.
3. If necessary, remove any unused strips from the frame and store at -20°C.

IMBP Plate Protocol. Perform all steps at room temperature.

1. Remove storage buffer from the wells by rinsing with 150 µL Wash Buffer and shaking off the liquid. Rinse four more times. After the last rinse, rap the inverted plate on a pile of laboratory tissues to remove any remaining liquid. Examine each well to insure that no Wash Buffer remains. To avoid background signals, do not allow wells to dry once they have been hydrated.
2. Add DNA samples (30 µL per well). Cover the plate and incubate for 20 minutes. While DNA is binding make up Streptavidin-HRP solution by mixing 5 µL conjugate with 10 ml 1X Wash Buffer (sufficient for two plates).
3. Add 150 µL Wash Buffer to DNA containing wells and shake out liquid. Rinse four additional times. After the last rinse, rap the plate on tissues and check for any residual liquid.
4. Add 100 µL diluted Streptavidin-HRP conjugate to each well. Cover the plate and incubate for 15 minutes.
5. Shake off Steptavidin-HRP and rinse the wells 5 X with Wash Buffer (150 µL). Again, be certain that all wash is removed from the wells using the rapping technique.
6. Add 80 µL TMB to each well. Allow 10-15 minutes for blue color development. If desired, read the blue color 370 or 655 nm. To increase sensitivity add 80 µL of Stop Acid (0.5 M H2SO4) to each well and read the resulting yellow color at 450 nm.

Genotyping

High through put genotyping is an ideal application of IMBP Plate technology and completely avoids the use of gels. Utilizing IMBP assays with allele specific probes (3), Gene Check has genotyped more than 40,000 sheep for specific alleles of the prion protein gene:

Scanning

Samples can be screened for SNPs by simply amplifying with biotin labeled primers and denaturing and annealing the PCR product to allow heterozygous SNPs to form mismatches with wild type DNA.

Examples of SNP detection in fragments of BRCA1 and p53 genes:

SNP Enrichment

IMBP plates can be used to enrich SNP and other polymorphisms from mixed samples containing normal and tumor cells or pooled samples.

Enrichment of 1% mutant sequence (GTC) in a wild type (GCC) background. PCR amplicons were denatured, annealed and then exposed to IMBP plates. Bound DNA was released, amplified, and sequenced.

PCR Fidelity Optimization

PCR fidelity is critical to many applications including mutation detection and cloning. PCR errors include both misincorporation and mispriming. Only IMBP assays detect both types of errors and can be used to measure PCR fidelity rapidly and inexpensively for any template.

Comparison of four PCR polymerases. (All PCR products gave single bands on polyacrylamide gels.) High fidelity equals low signal:

For another example of IMBP fidelity testing visit Finnzyme Oy.

Please see the Research Section for a complete list of references.