Genotyping by Single Base Extension/Fluorescence
Polarization
1. The PCR should be strong and consist of only one band. Faint bands and primer dimers
may interfere with the SBE.
2. Amplify using one of the following cycles:
Cold start Taq polymerases:
95 x 5 Min
95 x 30 Sec
Y x 30 Sec
72 x 30 Sec
72 x 10 min
15 x infinite
Hotstar Taq Polymerase:
95 x 15 Min
95 x 30 Sec
Y x 30 Sec
72 x 30 Sec
72 x 10 min
15 x infinite
These programs should result in consistent PCR band strengths throughout the samples.
If the samples are of consistent intensity, it will help to eliminate lagging samples that
will fall outside your genotyping cluster.
3. Once you have a good, clean, strong PCR amplify the samples that will be genotyped
and proceed on with the rest of the protocol.
Acycloprimer-FP SNP Detection Kit components
(Perkin Elmer Life Sciences, Cat # ACP113B)
PCR Clean-Up Reagent, 10X
PCR Clean-Up Dilution Buffer
Acyclopol
Acycloterminator Mix (G/A, G/C, A/T, C/A. G/T, and C/T)
10 Reaction Buffer
Some notes before you begin the single base extension genotyping step:
-The SNP primer is an oligo complementary to the sequence adjacent to the SNP Site.
For example, consider the following sequence (SNP marked in red):
5’-AGGGCCTGAGCAGGGGAGCCCCTTCTCAGCCCAAATGCCCTAGGGAACCCCCTTGACAT-3’
3’-TCCCGGACTCGTCCCCTCGGGGAAGAGTCGGGTTTACGGGATCCCTTGGGGGAACTGTA-5’
5’-ATGTCAAGGGGGTTCCCTAGGGCATTTGGGCGGAGAAGGGGCTCCCCTGCTCAGGCCCT-3’
3’-TACAGTTCCCCCAAGGGATCCCGTAAACCCGCCTCTTCCCCGAGGGGACGAGTCCGGGA-5’
The primer(underlined) anneals just prior to the SNP of interest.
-Choosing the correct Acycloterminator mix:
The acycloterminator mix chosen should be complementary to the SNP on the target
strand.
On the upper strands in the above example, the next base to be added to the primer will
either be a G or an A so use the G/A acycloterminator mix.
PCR Clean Up
1. Transfer 2.5ul of PCR product to a black PCR plate (MJ Research Cat# MSP9661)
2. Just prior to cleaning your PCR prepare 1X PCR clean up reagent from the 10x
PCR clean-Up reagent and dilution buffer.
3. Add 1ul 1X PCR Clean up to each well, pipet up and down to mix.
4. Add 1 drop of oil to each well using a fine tip transfer pipet.
a. Take care to ensure that only 1 drop is placed on each well. As oil
magnifies the FP reading, differences in oil will affect the calling of the
genotypes.
5. Incubate at 37 for 1 hour to clean the PCR.
6. Heat to 85 for 15 min to inactivate the enzyme.
7. Store at 4 until ready for SBE
Single Base Extension (SBE)
1. Prepare the following mix:
Acyclopol
10X Buffer
(X/Y)Terminator Mix
SNP Primer (10uM)
dH2O
Total
0.025ul
1ul
0.5ul
0.25ul
4.725ul
6.5ul
*Terminator Mix is
light sensitive. Limit
exposure to light.*
2. Add 6.5ul of SBE extension mix to each well, pipet up and down to mix.
3. Quick spin in the centrifuge (up to ~2000rpm)
4. Cycle:
95 x 2 min
95 x 15 sec
55 x 30 sec
04 x Infinite
xN
N = number of cycles
This will vary with each SNPs
Generally between 5-60
5. Quick spin the plate in the centrifuge (up to ~2000rpm)
6. Allow the samples to warm up to RT and read in a fluorescence polarization
reader (e.g., Perkin Elmer Victor2).