Ligation-Mediated Single-Sided PCR
This method involves single-sided PCR that initially requires specification of only one primer by modification site; the second primer is defined by the ligation-based addition of a unique DNA linker. This linker, together with the flanking gene-specific primer, allows exponential amplification of any fragment of DNA.
・ 0.4 μg/μl Cleaved genomic DNA in TE buffer (pH 7.5)
・ 4dNTP mix (pH 7.0), 25 mM (25 mM dATP, 25 mM dCTP, 25 mM dATP and 25 mM dTTP, together, yield the 25 mM 4dNTP mix, which is stored at – 20 °C)
5x Amplification Buffer
・ 200 mM NaCl
・ 100 mM Tris-HCl (pH 8.9), at room temperature
・ 25 mM MgSO4
・ 0.05% Gelatin (from bovine skin; Sigma-Aldrich Co.; cat. no. G9382)
・ 0.5% Triton X-100 (Sigma-Aldrich Japan; cat. no. T8787)
Stored at -20 °C.
5x First-Strand Buffer
・ 200 mM NaCl
・ 50 mM Tris-HCl (pH 8.9), at room temperature.
・ 25 mM MgSO4
・ 0.05% Gelatin
Stored at -20 °C.
First-strand synthesis mix, per reaction (25 ml):
・ 6.0 μl of 5x first-strand buffer
・ 0.3 μ1 of 1 pmol/ml oligonucleotide primer 1
・ 0.25 μ1 of 25 mM 4dNTP mix (pH 7.0)
・ 23.2 μ1 of ddH2O
・ 0.25 μl of 2 U/ml DNA polymerase from Thermococcus litoralis (Vent DNA polymerase; New England Biolabs; cat. no. 254L)
Note: Prepare solutions immediately prior to use. Mix all components except Vent DNA polymerase and chill on ice. Then add the polymerase and keep the mixture on ice.
・ Oligonucleotide forward primer 1: 5’-GTCCGCGGACGACCAGGTT
AGCCGAGT-3’.
20 mM Unidirectional linker mix.
・ 20 mM oligonucleotide LMPCR-1 (see below)
・ 20 mM oligonucleotide LMPCR-2 (see below)
・ 250 mM Tris-HCl (pH 7.7)
LMPCR-1: 5’-GCGGTGACCCGGGAGATCTGAATTC-3’ (top strand)
LMPCR-2: 5’-P-GAATTCAGATCT-3’ (bottom strand).
Prepare this mix in advance as follows: (1) purify the oligonucleotides on denaturing polyacrylamide gels; (2) combine the two oligonucleotides in 250 mM Tris-HCl (pH 7.7) and heat for 5 min at 95 °C; (3) transfer the mixture to 70 °C and cool gradually for approximately 1 h to room temperature; (4) allow the mixture to stand for 1 h at room temperature and then cool it gradually for approximately 1 h to 4 °C; and (5) allow mixture to stand for approximately 12 h at 4 °C and store in aliquots at –20°C. Thaw linker mix on ice before use.
Ligase dilution solution, per reaction (20 μl):
・ 2.2 μl of 1 M Tris-HCl (pH 7.5), at room temperature
・ 0.35 μl of 1 M MgCl2 (final, 17.5 mM)
・ 1.0 μl of 1 M DTT (final, 50 mM)
・ 0.25 μl of 10 mg/ml BSA (DNase-free; GE Healthcare Bio-Science AB, Uppsala, Sweden)
・ 16.2 μl ddH2O
Prepare immediately before use and chill on ice.
Ligase mix, per reaction (25 ml):
・ 0.25 μl of 1 M MgCl2 (final, 10 mM)
・ 0.5 μl of 1 M DTT (final, 20 mM)
・ 0.75 μl of 100 mM ATP (pH 7.0; GE Healthcare Bio-Science AB; final, 3 mM)
・ 17.5 μl of ddH2O
・ 5.0 μl of 20 mM unidirectional linker mix [final, 4 μM linker and 50 mM Tris-HCl (pH 7.7)]
・ 1.0 μl of 3 Weiss units/μl T4 DNA ligase (final, 30 units)
Prepare immediately before use. First mix the solutions of MgCl2, DDT, ATP, BSA, and H2O and chill on ice. Next, add ice-cold unidirectional linker mix and T4 DNA ligase.
・ 2,000 to 3,000 Weiss units/ml T4 DNA ligase (GE Healthcare BioScience AB)
・ 100% Ethanol, ice-cold at room temperature
・ 75% Ethanol at room temperature
・ Amplification mix containing unidirectional linker primer mix and primer 1
・ 2 U/μl Vent DNA polymerase mix per reaction (3.0 ml)
Mix 0.6 μl of 5x amplification buffer with 19 ml of H2O and chill for several minutes in an ice-water bath. Add 0.5 μl Vent DNA polymerase (see above), mix gently and return to ice-water bath. Prepare immediately prior to use.
・ Mineral oil (Sigma-Aldrich Co.; cat. no. M5904)
・ Precipitation mix per reaction (9.4 μl):
・ 8.4 μl of 3 M sodium acetate (pH 7.0) (final, 2.7 M)
・ 1.0 μl of 10 mg/ml yeast tRNA (final, ~1 mg/ml)
Prepare immediately before use.
End-labeling mix. containing end-labeled primer 3, per reaction (5 ml):
・ 1.0 μl of 5x amplification buffer
・ 2.3 μl of 1 pmol/ml end-labeled primer 3
・ 0.4 μl of 25 mM 4dNTP mix (pH 7.0)
・ 0.8 μl of ddH2O
・ 0.5 μl of 2 U/ml Vent DNA polymerase (see above)
Prepare immediately prior to use. Mix the first four components and chill on ice before adding Vent DNA polymerase. Then chill and keep on ice.
・Vent DNA polymerase stop solution (295 μl ):
・ 25 μl of 3 M sodium acetate (pH 7.0; final, 260 mM)
・ 266 μl of TE buffer (pH 7.5; final, 10 mM Tris-HCl)
・ 2 μl of 0.5 M EDTA (pH 8.0; final, 4 mM)
・ 2 μl of 10 mg/ml yeast tRNA (final, 68 μg/ml)
Prepare immediately prior to use.
・ Mixture of phenol, chloroform and isoamyl alcohol (25: 24:1; v/v)
・ Automated thermal cycler
Ligation-Mediated Polymerase Chain Reaction (LM-PCR)
Ligation to the extension product is allowed to proceed for 16 h at 16 ˚C and is followed by PCR with gene-specific primers (primer 2, 5’-GGTTGGCCAAGTCCGTCCGTCTGTCTGTCT-3’; and primer 3, 5’-TGGCCAAGTCCGTCCGTCTGTCTGTCTGTC-3’) and the top-strand adaptor primer (LMPCR-1).