Qualitative and Quantitative DNA and RNA Analysis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry - Clinical Applications of PCR - page 68

Table 3
Preparation of Primer Extension Cocktail

Stock Final Volume for Volume for

Reagent concentration concentration one reaction one 384-plate

Water N/A N/A 1.674 µ L 915.70 µ L

ddNTP/dNTP mix 10X Buffer 1X Buffer with 0.200 µ L 105.98 µ L

with 2.25 m M 50 µ M

d/ddNTPs each d/ddNTPs each

Extension primer 100 µ M 1.2 µ M 0.108 µ L 28.62 µ L

ThermoSequenase 32 U/ µ L 0.064 U/ µ L 0.018 µ L 9.54 µ L

Total volume 2.000 µ L 1059.84 µ L

3. Seal the plate and centrifuge it at 600 g for 3 min.

4. Thermocycle the sample plate as follows:

a. 37°C, 20 min.
b. 85°C, 5 min.
c. 4°C, forever.

3.2.3. Primer Extension Reaction
In this step, an extension primer is annealed to the region immediately
upstream of the mutation site. The ThermoSequenase, three different ddNTPs,
and one dNTP are used to extend the primer for one or two bases (terminated
by ddNTP). This step produces two extension products (typically 18 to 25 bases
long) with different molecular weights. In SABER, only one ddNTP specific
for the mutant DNA is used ( 14 ) .
1.

Prepare primer extension cocktail as described in Table 3 . Make sure the solu-
tion is mixed well before aliquoting.

2. Add 2 L of the solution from step 1 to each well of the 384-plate from the SAP

reaction. Each well now contains 9

µ

µ L solution.

3. Seal the plate and centrifuge at 600 g for 3 min.

4. Thermocycle the sample plate as follows:

a. 94°C, 2 min.
b. 94°C, 5 s.
c. 52°C, 5 s.
d. 72°C, 5 s.
e. Perform step b 39 times.
f. 4°C, forever.

3.2.4. Cation Removal
This step is used to remove cations in the solution, because cations might
interfere in mass spectra. For high-throughput analyses, please refer to “Clean-