High-throughput genotyping experiments using the TAQMAN assay

Summary

This unit describes the application of 5'-nuclease allele specific analysis or TaqMan method for single nucleotide polymorphism (SNP) typing.

Operation method

High-throughput genotyping experiments using the TAQMAN assay

Materials and Instruments

Genomic DNA
PCR mixes
96-well transparent plates and lids Horizontal centrifuge ABI PRISM 7700 Sequencing System Thermocycler Statistical Software Package

Move

1. 2ul (30ng) of genomic DNA was added to each well of rows B through H of the 96-well transparent plate, and row A was retained as a control - 4 wells were spiked with only 2ul of buffer as a "no DNA" control, while the remaining 8 wells were spiked with 24 genomic DNA per well as a positive control, and 4 wells were spiked with each of the two pure strains.

2. Add 23ul of PCR mix to each well, cover with a clear cap, and centrifuge briefly in a horizontal centrifuge to obtain a pre-PCR fluorescence read using an ABI PRISM 7700 sequencer. Using the "PlateRead" form, make sure that the Use Spectral Compensation forEndpoint item is checked (under the Advanced Optionsinthe Instrument and Diagnostics menu).

3. Start the PCR on the thermal cycler with the following conditions.

1 cycle: 2 min 50°C

1 cycle: 10 min 95°C (denaturation)

40 cycles: 15s 94°C (denaturation)

1 min 62°C (denaturation/extension)

Final step: infinite 4°C (maintenance)

4. Read the fluorescence again to get a post PCR reaction read. If you use the allele-calling software provided with the ABI PRISM 7700 and choose the "AUelicDiscrimination" mode to read the incandescent light, the algorithm allows you to automatically sort out the genotypes or genotype by visual inspection.

For large-scale genotyping (e.g., >500 samples), it is more accurate to pool sample data from multiple well plates to determine the genotype. In this way, follow the remaining steps in this protocol.

5. Import the raw data (both pre- and post-PCR) from the well plate read form into the statistical package. Calculate normalized fluorescence values as follows: (reported fluorescence value one background)/(reference fluorescence value one background); reported fluorescence is in the FAM and VIC lanes, reference fluorescence is in the ROX lane.

6. Compare the average PCRhli standard extinction for each reporter dye between well plates using a nonparametric test (e.g., Kruskal-WaUis test). If one plate (or group of plates) has a significantly different pre-PCR highlight than the others, adjust the post-PCR fluorescence value accordingly. For example, if the average fluorescence of the FAM reporter dyes in a particular well plate is 1.5 times that of the other well plates, divide the post-PCR FAM fluorescence value for each well of that plate by that factor.

7. Use stepwise cluster analysis (K-meansclustering) to automatically divide the corrected post-PCR data into 4 groups-3 genotypes and 1 "no-DNA" control. Most statistical software packages provide cluster analysis algorithms that can be used directly.

Often, clustering is obvious and requires no further processing. To determine whether genotype assignment is meaningful, it is important to examine the scatterplot of the data. For example, extreme values can corrupt the cluster analysis algorithm and produce obviously incorrect classification results. Removing such extreme values usually results in correct classification results.

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