Real-Time Gap Ligase Chain Reaction

Patients and Sample Collection.

Primary carcinomas of the head and neck and lung were surgically resected at The Johns Hopkins Hospital. After appropriate approval of the Institutional Review Board and patient consent, tumor, surgical margins, and resected lymph nodes were collected during surgical resection at the Johns Hopkins Medical Institutions. Those tissues not used for diagnostic histological analysis were fresh frozen for molecular analysis. Primary tumors with <70% tumor cells were microdissected to remove areas of normal tissue. The p53 gene was analyzed in primary tumors by direct sequencing as described previously (3 , 7) . After resection of the primary tumor, margins were examined by frozen section to confirm adequacy of resection, and then additional normal-appearing tissue was removed from the edges of the surgical defect and fresh frozen for molecular analysis. For this validation study, 10 cases were selected to encompass a range of common p53 mutations in the primary tumor, identified in previous studies (Table 1)⇓ . All tissue samples were cut into 12-μm sections and placed in a mixture of 1% SDS and proteinase K (0.5 mg/ml) at 48°C overnight. In addition, 5-μm sections were taken every 15 slices and stained with H&E for examination under light microscopy by a pathologist in a blinded manner. DNA was then extracted from all samples with phenol/chloroform and precipitated with ethanol. A total of 41 lymph node or tumor margin samples was prepared and tested.

Plaque Hybridization Assay.

The exon containing the known p53 mutation for each DNA sample was amplified by PCR using primers containing EcoRI digestion sites (5/6F 5′-GTAGGAATTCACTTGTGCCCTGACTTTC-3′, 5/6R 5′-CATCGAATTCTTAACCCCTCCTCCCAGAG-3′; 7/9F 5′-GTAGGAATTCTTGGGCCTGTGTTATCTCC-3′, 7/9R 5′-CATCGAATTCAGAAAACGGCATTTTGAGTG-3′). Reactions were carried out in a volume of 50 μl using 500 ng of template, 2 μm of each primer (Invitrogen, Carlsbad, CA), 5 units of TaqDNA polymerase (Invitrogen), 1.5 mm each dATP, dCTP, dGTP, dTTP, 16.6 mm ammonium sulfate, 67 mm Trizma, 6.7 mm magnesium chloride, 10 mm mercaptoethanol, and 0.1% DMSO. The exons were amplified for 35 cycles (95°C for 30 s, 57°C for 1 min and 72°C for 1 min).

The PCR products were then cloned into Lambda ZapII bacteriophage vector and amplified additionally in XL1Blue E. coli cells (Stratagene, La Jolla, CA). The PCR products were initially purified using a Qiagen PCR purification kit (Qiagen, Valencia, MA), digested overnight at 37°C with EcoRI (New England Biolabs, Beverly, MA) and repurified using a Qiagen Gel extraction kit before overnight ligation with the vector at 14°C using T4 ligase (Invitrogen) and packaging into Gigapack III Gold Packaging Extract as per manufacturers instructions (Stratagene). Serial dilutions of phage were plated with 200 μl XL1Blue E. coli cells (A_{600 nm} 0.5) and 3 ml of l-topose onto 100-mm LB agar plates and incubated overnight at 37°C. The resulting clone colonies were transferred to nylon membranes (NEN, Boston, MA) and hybridized with oligonucleotide probes end labeled with γP³² ATP (3 , 6 , 7) . In each case, the probe was specific for the known mutation in the primary tumor (sequences available on request). After hybridization the membranes were washed stringently at 53°C–60°C to detect mutant-specific binding of the probes. The membranes were then exposed to X-ray film (Kodak) and hybridizing plaques counted. In addition, wild-type end-labeled probes were also hybridized to the membranes as a measure of total plaques containing insert present. The percentage of tumor cells in each specimen was estimated by dividing the number of labeled mutant plaques by the total number of plaques present containing p53 insert (hybridizing to a wild-type probe). For each set of margins or lymph nodes examined, positive (primary tumor) and negative (leukocyte) controls were simultaneously analyzed. All positive assays were repeated.

Real-Time QGLCR.

Tumor, lymph node, margin, and normal DNA samples for each case were amplified by PCR using primers for the specific exon containing the mutation in each case. Reaction conditions were as described in the previous section but using 100–200 ng templates. (Exon 5, F 5′-CACTTGTGCCCTGACTTTCA-3′, R 5′-AACCAGCCCTGTCGTCTCT-3′; exon 6, F 5′-AGAGACGACAGGGCTGGTT-3′, R 5′-CTTAACCCCTCCTCCCAGAG-3′; exon 7, F 5′-CCTCATCTTGGGCCTGTGT-3′, R 5′-CCGGAAATGTGATGAGAGGT-3′; and exon 8, F 5′-TTTCCTTACTGCCTCTTGCTTC-3′, R 5′-GCTTCTTGTCCTGCTTGCTT-3′.)

The PCR products were purified using a Qiagen gel extraction kit (Qiagen). The concentration of all purified products was stringently determined using the TD-360 MiniFluorometer (Turner Designs, Sunnyvale, CA). All samples from a single patient were measured concurrently on two separate occasions with multiple readings taken each time. Accuracy of the fluorometer was also validated with standards of known DNA concentration and by comparison to agarose gel analysis with a Low DNA Mass Ladder (Invitrogen) and real-time quantitative PCR (data not shown). Stock solutions of each PCR product were made in 10 mm Tris (pH 8) to give 10¹⁰ amplicon copies/5 μl calculated from the size of the amplicon for each exon and assuming 1 bp of double-stranded DNA = 660pg/pmol and 1 pmol = 6.02 × 10¹¹copies.

Ligase detection reactions involve the use of two adjacent oligonucleotide primers, which hybridize to a single strand of target DNA, which will be ligated only if there is an exact match to the target sequence. In the presence of a mismatch, ligation will not occur. Point mutations are best detected by designing oligonucleotides so that the mutation site is at the 3′-end of the upstream 5′-primer. This linear reaction can increase exponentially by the addition of two further complementary oligonucleotides that hybridize to the cDNA strand able to ligate only in the presence of an exact match. Using a thermostable ligase in a cycling reaction, ligated products from both reactions are subsequently used as templates, creating exponential amplification, dubbed a LCR. Although highly sensitive, background ligation can still occur at low level with this basic technique. An additional modification to further improve specificity is the use of a single nucleotide gap at the site of the point mutation between the adjacent oligo primers (22) . This gap is filled using a thermostable DNA polymerase in the presence of the appropriate mutant nucleotide in the reaction mix adding substantial additional specificity to the reaction. On the basis of these principles, we developed a modified gap-LCR reaction that could be monitored in real-time using fluorescent-labeled oligos to allow quantitation, QGLCR. In our assay, one oligonucleotide is labeled with a reporter dye at the 5′-end and the adjacent oligonucleotide to which it will ligate is labeled at the 3′-end with a quencher dye. Excitation of the reporter dye results in fluorescence energy transfer to the quencher dye and subsequent fluorescence at the characteristic spectrum of the quencher dye. The onset of rise in fluorescence by cycle number is proportional to the initial amount of target mutation. A schematic representation of QGLCR is shown in Fig. 1⇓ .

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Fig. 1.

Schematic diagram of the quantitative gap ligase chain reaction (QGLCR). F, FAM; T, TAMRA; p, phosphorylation.

The assay was modified and developed over a 2-year period, initially using K-ras- and p53-mutated cell lines, radiolabelled primers, and high percentage polyacrylamide gels before converting to real time using fluorescent labeled primers. The assay was then optimized for each mutation, using varying concentrations of tumor DNA initially diluted in water and subsequently diluted in normal DNA. Different lengths and concentrations of primers were tested at varying temperatures and with varying amounts of the other reagents. Blocking oligonucleotides, extra gaps, differential positioning of the gap, and additional mismatches in the primers were also tested. In general, fluorescent primer lengths of 15 or 16 bp were found to be optimal for producing the best signal, creating a ligated product of 30–32 bp. Some mutations worked best with an additional mismatch nucleotide. All primer sequences are available on request. A two-step cycling reaction was found to work better than an initial three-step reaction, and annealing temperature was found to be critical for maximizing specificity for each particular mutation.

For the QGLCR assay, 10⁹ copies of PCR product template from each sample was used. In addition, serial 10-fold dilutions of tumor mixed into normal template (down to 1:10,000) were constructed for each case and 10⁹ copies from each dilution used to generate standard comparison curves against which to compare the unknown margin and lymph node samples (Fig. 2)⇓ . Each reaction also contained in a 25-μl reaction volume: 2.5 μl of Platinum Taq 10× buffer; 0.625 μl of Taq ligase 10× buffer; 1 mm NADβ, 100 μm mutant-complementary insertion nucleotide; 1 unit of Platinum Taq polymerase (Invitrogen); 8 units TaqDNA ligase (NEB); 400 nm two nonlabeled reverse strand mutation specific oligomers; 400 nm one FAM 5′-labeled forward strand mutation specific oligomer and 600 nm one TAMRA 3′-labeled forward strand mutation-specific oligomer. All oligos 3′ of the ligation site were phosphorylated at their 5′-end. Reactions were run for 40 cycles (94°C for 2 min initiation, then 50°C 30 s, 94°C 30 s) on a 96-well plate using an Applied Biosystems 7700 Sequence detector (Applied Biosystems, Foster City, CA). All samples were run in duplicate and each plate was run twice. All samples from each patient were run concurrently on the same plate. Each plate also included multiple water blanks, containing everything except DNA, as an additional negative control. Threshold values for the margin and lymph node amplification curves were quantitated relative to the thresholds for the corresponding serial dilutions of tumor in normal for each case.

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Fig. 2.

Standard curve for quantitative gap ligase chain reaction, generated from dilutions of tumor DNA in normal DNA (case 7). X axis, starting quantity of mutant DNA (copy number)/well; y axis, quantitative gap ligase chain reaction cycle number.