Single Base Extension Using CFET Tags and BiotinddNTPs

The scheme for detecting multiplex SNPs using CFET-SBE and biotin-ddNTPs is shown in Fig. 3. The sequences of CFET tags with their nomenclatures are described in Table 1 and Fig. 2 of ref. 15.

1. Combine the following materials in one tube and use a vacuum centrifuge to remove the solvent:

a. 0.5 pmol of FAM-labeled probe.

Fig. 3. Combinatorial fluorescence energy transfer single base extension (CFET-SBE) for multiplex single-nucleotide polymorphism (SNP) detection using biotin-ddNTPs. DNA template containing polymorphic sites is incubated with CFET-labeled probes, Biotin-ddNTPs and DNA polymerase. The nucleotide at the 3'-end of each CFET-labeled probe is complementary to a particular SNP on the template. Only the fully complementary CFET-labeled probe is extended by DNA polymerase of a biotin-ddNTP. After solid-phase capture and isolation of the biotinylated DNA extension fragments, the SBE products are analyzed for their fluorescence signatures and each of which codes for a unique SNP. (This figure also appears in color on the Companion CD.)

Fig. 3. Combinatorial fluorescence energy transfer single base extension (CFET-SBE) for multiplex single-nucleotide polymorphism (SNP) detection using biotin-ddNTPs. DNA template containing polymorphic sites is incubated with CFET-labeled probes, Biotin-ddNTPs and DNA polymerase. The nucleotide at the 3'-end of each CFET-labeled probe is complementary to a particular SNP on the template. Only the fully complementary CFET-labeled probe is extended by DNA polymerase of a biotin-ddNTP. After solid-phase capture and isolation of the biotinylated DNA extension fragments, the SBE products are analyzed for their fluorescence signatures and each of which codes for a unique SNP. (This figure also appears in color on the Companion CD.)

d. 0.68 pmol of PCR product.

2. Dilute the Thermo Sequenase from 32 U/|L to 0.5 U/|L.

3. Dilute the biotin-11-dd(A,C,G,U)TP from 1 mM to 1.33 |M.

4. Add 1.5 |L of each diluted biotin-11-dd(A,C,G,U)TP, 2 |L of Thermo Sequenase reaction buffer, 2 |L of diluted Thermo Sequenase to the CFET-labeled oligonucleotides and PCR product. Vortex the mixture gently.

5. Perform the extension by heating the mixture at 94°C for 30 s and 54°C for 30 s.

3.7. Solid Phase Purification

1. Wash 50 |L of streptavidin-coated magnetic beads with 2X 50 |L B&W buffer and resuspense the beads in the B&W buffer (buffer volume depends on the liga-tion or extension mixture volume).

2. Combine equal volume of each of the streptavidin-coated magnetic beads and the CFET-labeled ligation (or single base extension) products at 25°C for 0.5 h with occasional mixing.

3. Remove the supernatant while immobilizing the magnetic beads with a magnet.

4. Wash the beads with 3X 50 |L of B&W buffer.

5. Add 50 |L of fresh 0.1 M NaOH to the beads and keep it at 25°C for 5 min to denature the DNA template from the ligation or single base extension product.

6. Wash the bead mixture with 2X 50 | L water.

7. Add 20 |L of 98:2 (v/v) formamide/10 mM EDTA to the bead mixture and heat the solution at 94°C for 5 min to release the ligation (or single base extension) products from the magnetic beads.

3.8. Multicolor Fluorescence Detection of CFET-Labeled DNA

by MegaBACE 1000 Capillary Array Electrophoresis DNA Analysis System

1. Dilute the 10X LPA buffer to the 1X buffer by water.

2. Centrifuge the LPA matrix tubes at 956g for 2 min to remove the air bubbles.

3. Inject and equilibrate the gel matrix and perform a prerun on the instrument.

4. Electrokinetically inject the CFET-labeled ligation or extension DNA products at 3 kV for 120 s.

5. Electrophorese at 8 kV for 2 h.

6. Analyze the result with the sequencing analysis tool from the MegaBACE 1000 instrument. The black, green, and blue (default color) of the analyzed data on electropherograms correspond to the FAM, TAMRA, and Cy5 signals, respectively (see Note 9).

3.9. Multicolor Fluorescence Detection of CFET-Labeled DNA

by ABI377 DNA Analysis System

1. Prepare the gel for electrophoresis by mixing 5 mL Long Ranger (50%), 18 g urea, 5 mL 10X TBE buffer, and 26 mL water.

2. Stir the gel mixture until a clear solution is obtained.

3. Degas the gel solution for 5 min.

4. Add 250 pL of 10% (w/v) fresh ammonium persulfate solution followed by 35 pL of TEMED with gentle mixing for 30 s.

5. Introduce the gel mixture immediately between the precasted glass plates and insert a comb (see Note 6).

6. Prepare a sufficient quantity of 1X TBE buffer to fill both anodal and cathodal chambers by diluting a 10X TBE stock.

7. Mount the gel cassette onto the sequencing apparatus and perform "Plate Check A."

8. Pre-electrophorese the gel until a temperature of 51°C is reached.

9. Flush the wells in the gel with the upper reservoir buffer and carefully load the samples.

10. Electrophorese at 91W (3 kV, 40 mA) for at least 2 h.

11. Analyze the result with the sequencing analysis tool from the ABI 377 instrument. The blue, black, and red (default color) of the analyzed data on electro-pherograms correspond to the FAM, TAMRA, and ALEXA signals, respectively.

4. Notes

1. The following phosphoramidites require longer coupling time than the other nucleoside phosphoramidites: FAM-dT (10 min), TAMRA-dT, amino-modifier C6-dT, and Chemical Phosphorylation Reagent II (6 min).

2. Use "Trityl On" in the final coupling step for the synthesis of CFET-labeled and other nondye-labeled oligonucleotides. This is owing to the fact that the dyes on the 5'-end do not have a dimethoxytrityl group, whereas the dimethoxytrityl group is required for OPC purification in the other nondye-labeled oligonucleotides.

3. Use 1 mL of fresh ammonium hydroxide for deprotection and cleavage. Keep the CFET-labeled oligonucleotides away from light during this process.

4. Do not shake the vial vigorously because the CFET-labeled oligonucleotides may come to the inside wall of the vial. On the other hand, insufficient shaking may lead to incomplete reaction.

5. The distinction between the unreacted dye and dye-labeled oligonucleotide may not be clear in the column. Exercise judgment to collect the appropriate fraction.

6. Before the gel polymerizes completely, make sure that no bubbles exist in the gel. Also, allow the gel to polymerize for at least 3 h.

7. If insufficient materials are loaded, the product bands can be visualized using an ultraviolet lamp in a darkroom.

8. Use 1:20 (v/v) ammonium hydroxide for oligonucleotides of 35mer or shorter and 1:10 (v/v) ammonium hydroxide for oligonucleotides longer than 35mer.

9. In the MegaBACE 1000 instrument, three bandpass filters, 520DF20, 585DF20, and 670DF20, are used to detect the fluorescence signals from FAM, TAMRA, and Cy5. The detection requires a replacement of the 610LP (long pass) filter in channel 3 of the system by the 670DF20 filter to detect the fluorescence signal from Cy5.

0 0

Post a comment