Protocol 71 Template preparation


1. Prepare a PCR premix by combining the following reagents in 15 ^l (final concentrations are given):

MilliQ water

1 x PCR buffer (Solis BioDyne, Estonia) 2.5 mM magnesium chloride

0.2 mM dNTP (20% of the dTTP fraction substituted by dUTP) 15 pmol of forward and reverse primer 20 ng of genomic DNA

1 U Taq DNA polymerase (Solis BioDyne, Estonia).

2. Cycling conditions in the thermal cycler: denaturing at 95°C for 12 minutes, followed by 26 cycles of denaturation at 95°C for 15 s, stepdown annealing at 68°C/-0.5°C per cycle for 20 s, and extension at 72°C for 45 s, with final extension at 72°C for 7 min.

3. Check the result by running 1/10 of each PCR reaction on a horizontal 1% agarose gel.


1. Pool tested PCR products for one chip and purify 5-10 |g of the PCR product mix using a PCR product purification column (General Biosystem, South Korea). Elute the products from the column in 24 |l of MilliQ water.

2. Prepare the UNG-SAP reaction in 30 |l:

1 x UNG buffer

2 U thermolabile UNG 1 U SAP

24 |l of purified PCR products Incubate at 37°C for 1 h.

3. Check the efficiency of fragmentation by running 1/10 of UNG-SAP reaction on a horizontal 1% agarose gel after heating at 95°C for 10 min.


1. Place the DNA microarray slide in a slide holder and rinse as follows:

95°C distilled water for 30 s

100 mM sodium hydroxide for 10 min

95°C distilled water for 30 s, twice.

2. Denature and fragment the purified and UNG-SAP-treated PCR product mix at 95°C for 10 min after adding ThermoSequenase DNA Polymerase reaction buffer (1x final concentration).

3. Prepare the APEX reaction in 35 |l:

Denatured and fragmented PCR products with 1x reaction buffer

1.4 |M of each fluorescently labeled ddNTP: Texas Red-ddATP, fluoresein-ddGTP (Amersham Biosciences), Cy3-ddCTP, Cy5-ddUTP (NEN)

4 U ThermoSequenase DNA Polymerase.

4. Apply the reaction mixture to a microarray slide, cover with a coverslip and incubate in a hybidization chamber at 58°C for 15 min.

5. Stop the reaction by washing the slide three times at 95°C in MilliQ water.

6. Read the slide with the Genorama™ QuattroImager and analyze the sequence variants by using Genorama™ Basecaller genotyping software (Asper, Ltd., Figure 7.2).

Multiplexed SNP genotyping using allele-specific primer extension on microarrays

Juha Saharinen, Pekka Ellonen, Janna Saarela and Leena Peltonen

0 0

Post a comment