John M. S. Bartlett.pdf - Bio-Nica.info

Solid-Phase Minisequencing 363
a microtiter plate or test tube format and the result of the assay is obtained as an
objective numeric value, which is easy to interpret. Furthermore, the solid-phase
minisequencing method allows quantitative detection of a sequence variant present as a
minority of less than 1% in a sample (2,3,5,6). We have used the sensitive quantitative
analysis for detecting point mutations in malignant cells present as a minority in a cell
population (5) and for analyzing heteroplasmic mutations of mitochondrial DNA (3,6).
The high sensitivity is an advantage of the minisequencing method compared with
dideoxynucleotide sequencing, in which a sequence variant must be present as 10 to
20% of a mixed sample to be detectable. A limitation of the solid-phase minisequencing
method is that it is restricted to analyzing variable nucleotides only at positions
predefined by the detection step primers used. The method is based on the use of
equipment and reagents that are available from common suppliers of molecular
biological products, facilitating easy setup. In the future, high-throughput analysis of
nucleotide sequence variation will be performed by rapid, automatic methods based
on homogeneous detection principles or alternatively using methods in microarray
or chip formats. The minisequencing reaction principle is applicable for both types
of assay formats (7,8).
2. Materials
2.1. Equipment
1. Programmable heat block, and facilities to avoid contamination in PCR.
2. Microtiter plates with streptavidin-coated wells (e.g., Combiplate 8, Labsystems, Helsinki,
Finland; see Note 1).
3. Multichannel pipet and microtiter plate washer (optional).
4. Shaker at 37°C.
5. Water bath or incubator at 50°C.
6. Liquid scintillation counter.
2.2. Reagents
All the reagents should be of standard molecular biology grade. Use sterile distilled
or deionized water.
1. Thermostable DNA polymerase. We use Thermus aquaticus (5 U/µL, Promega or Perkin–
Elmer-ABI) or Thermus brockianus (Dynazyme II, 2 U/µL, Finnzymes, Espoo, Finland)
DNA polymerase. Store at –20°C (see Note 2).
2. 10× concentrated DNA polymerase buffer: 500 mM mM MgCl 2 , 1% (v/v) Triton X-100,
and 0.1% (w/v) gelatin or 10× concentrated buffer supplied with the DNA polymerase
enzyme. Store at –20°C.
3. dNTP mixture: 2 mM dATP, 2 mM dCTP, 2 mM dGTP, and 2 mM dTTP stored at –20°C.
4. PBS/Tween: 20 mM sodium phosphate buffer, pH 7.5, and 0.1% (v/v) Tween 20 store at
4°C. 50 mL is enough for several full-plate analyses.
5. TENT (washing solution): 40 mM Tris-HCl, pH 8.8, 1 mM EDTA, 50 mM NaCl, and
0.1% (v/v) Tween 20. Store at 4°C. Prepare 1 to 2 L at a time, which is enough for several
full-plate analyses.
6. NaOH (50 mM; make fresh every 4 wk) stored in a plastic vial at room temperature
~20°C).
7. [ 3 H]-labeled deoxynucleotides (dNTPs): dATP to detect a T at the variant site, dCTP to