Qualitative and Quantitative DNA and RNA Analysis by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry - Clinical Applications of PCR - page 67

Table 1
Preparation of Polymerase Chain Reaction Cocktail

Final Volume for Volume for

reagent concentration one reaction one 384-plate

Water ( see Subheading 2. ) N/A 2.24 µ L 1120 µ L 10X

Hot Start Taq PCR buffer, 1X 0.50 µ L 250 µ L

containing 15 m M MgCl 2 1.5 m M MgCl 2

25 m M MgCl 2 1 m M MgCl 2 0.20 µ L 100 µ L

dNTP mix, 25 m M each 200 µ M each 0.04 µ L 20 µ L

Hot Start Taq Polymerase, 0.1 U/reaction 0.02 µ L 10 µ L

L
Forward and reverse PCR

5 U/ µ

200 n M 1.00 µ L 500 µ L

primers (1 M each)

Oligonucleotide standard

µ

0.50 µ L 250 µ L

( see Note 4 )
DNA/cDNA (5 ng/ µ L; 2.5 ng/reaction 0.50 µ L 250 µ L

see Note 5 )

Total volume 5.00 µ L 2500 µ L

Table 2
Preparation of Shrimp Alkaline Phosphatase Reaction Solution

Volume for Volume for

Reagent one reaction one 384-well plate

Water 1.53 µ L 881.3 µ L

hME buffer 0.17 µ L 97.9 µ L

SAP 0.30 µ L 172.8 µ L

Total volume 2.00 µ L 1152.0 µ L

1. Prepare the SAP reaction solution as described in Table 2 . Make sure the solution

is mixed well because this solution can be viscous as a result of the high concen-
tration of glycerol.

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

reaction. Each well now contains 7

µ

µ L solution.

Fig. 3. ( continued from opposite page ) (filled block arrows). The low abundance of the
fetal allele (filled peak) is overshadowed by the nonmutant allele (unfilled peak) in the
mass spectrum. As SABER involves the extension of only the mutant allele, the latter's
presence (filled peak) can be robustly identified from the mass spectrum. The striped
peaks represent the unextended primer.