Agilent Technologies, Inc.
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Mutation Detection For The K-ras And P16 Genes
Kristen M. Pike1, Theresa M. Plona, Shirley Tsang, David Sun, Nicole L. Lum, Will Ferguson, David J. Munroe
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Figure 1. Wild-type (lanes 10 and 11) versus mutated samples (lanes 4-7), as well as a heterozygous mutation (lanes 2 and 3). PCR products are in even-numbered lanes, and PCR products subjected to BstNI digests are in odd-numbered lanes. The ladder is in lane 1 and water negative controls are in lanes 8 and 9. Figure 2 displays the corresponding DNA sequence chromatograms for these samples.
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Abstract
Mutations in the K-ras gene codon 12 region can lead to colon, pancreas, liver, spleen, stomach, lung and other cancers. The CDKN2A/P16 gene is a familial melanoma gene. Routine PCR and DNA sequencing methods can identify exactly which point mutation is present in patient tissue samples. Freshly frozen tumor sections direct from surgeries can be utilized, as well as archived paraffin-embedded specimens. Prior to DNA sequencing of K-ras, nested PCR products are digested with a restriction enzyme and electrophoresed for quality and sizing purposes. A sample can be determined to be either wild-type or mutated simply by comparing the size of the PCR band to the size of the digested PCR band on a DNA chip. This analysis demonstrates the separation of PCR fragments from 135 bp to 106 bp. DNA sequencing is then utilized to verify the chip results. If a sample is shown to be mutated, sequencing can pinpoint the exact mutation. For P16 exon 3, PCR fragment's are electrophoresed on the 2100 bioanalyzer (Agilent Technologies, Foster City, Calif.), purified, and sequenced. Heterozygous mutations can be resolved on the instrument, accurately within 10-15% of base pair length. Lab-on-a-chip technology (Agilent Technologies) is also incorporated in these diagnostic and quality control assays. Here we demonstrate how extra bands on the chip image correlate to mutated DNA sequences.
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Figure 2. DNA sequence chromatograms from K-ras codon 12 PCR's. A) AAC reverse sequence is GTT forward sequence=valine mutant at codon 12. B) ACC reverse sequence is GGT forward sequence=glycine normal at codon 12. C) ATC/ACC reverse sequence is GAT/GGT forward sequence= aspartic acid mutant and glycine normal at codon 12.
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Introduction
The human K-ras gene is a member of the Ras family of GTPases.(1) Mutant, activated forms of Ras proteins, which are frequently observed in cancer, have an impaired GTPase activity rendering the protein resistant to inactivation by regulatory GAP proteins.(2) The ability to detect changes in the region of this gene that codes for activation is essential. This test was used to determine a cancer patient's eligibility into a clinical trial for a peptide vaccine. The normal form of codon 12 encodes glycine. Known mutations observed at codon 12 are: aspartic acid, valine, serine, cysteine, alanine, arginine, and asparagines. The PCR primers chosen amplified an initial product of 157 base pairs. The forward primer had a mismatch incorporated into it in order to create a BstNI site. This fragment was cut with BstNI, amplified (with an internal reverse primer), and cut again with BstNI.(3) The restriction enzyme's purpose was to trim away excess normal DNA sequences and enrich for any mutant sequences. In mutated samples, a BstNI site was not created, and therefore not recognized. Tumors will inherently contain normal tissue infiltrated throughout. Unless laser capture microdissection is incorporated, normal tissue cannot be removed by simple microtomy, hence the use of BstNI to assist in normal DNA sequence removal. The second nested PCR amplification produced a 135 base pair band. If the sample was wild-type, BstNI recognized its site and trimmed the product to a 106 bp size. If the sample was mutant, the digested product remained at 135 bp.
For P16, a 198 base pair fragment was generated. For normal samples, a single band appeared on the gel image. For mutated samples, a doublet band and sometimes a triplet band were observed. This corresponded 100% with DNA sequence data. In instances of degraded or low amounts of initial DNA template, quality of the PCR product was also viewed on the chip before the reagents and time to sequence it were wasted. Often formalin-fixed tissues or lymph node metastases do not present good quality DNA template for PCR reactions. Chip images provided a qualitative and quantitative tool that indicated if a reaction needed to be repeated to increase its yield. Resolution was better on the 2100 bioanalyzer than on ethidium-stained agarose gels. Chips were safer to work with and preparation time was reduced dramatically.
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Figure 3. Chip image of P16 gene exon 3 PCRs displaying the presence of extra bands in mutated samples.
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Materials and methods
Tissues
Sample blocks were obtained through the National Cancer Institute's Naval Medical Oncology Branch. Ten 5 µm sections were de-paraffinized and extracted using the Series III kit from Xtrana (Xtrana, Inc., Broomfield, Calif.). For P16, DNA was received already extracted in barcoded 96-well plates. DNA was quantitated via spectrophotometry and diluted to 10 ng for starting material.
PCR
Nested PCR was performed for K-ras with final reaction concentrations of 0.25 µm each primer,(3) 0.2 mM dNTPs (Roche Applied Science, Indianapolis, Ind.), 1×buffer (Roche), 1.25 U Taq polymerase (Roche), HPLC-grade water (Fisher Scientific, Hampton, N.H.), and Amp Enhancer (Xtrana). 50 µl were added to the bound DNA on the Xtrana tubes. For samples not extracted using the Xtrana kit, the Amp Enhance solution was eliminated and solubilized template was incorporated. This reaction was cycled 18 times using the 9700 thermal cycler (Applied Biosystems, Foster City, Calif.) under the following conditions: 94 C for 45 seconds, 55 C for 1 minute, and 72 C for 90 seconds. This was purified using the Wizard kit (Promega Corp., Madison, Wis.). The eluant was used as template in a BstNI reaction with BSA (New England BioLabs, Inc., Beverly, Mass.). This reaction was purified using the MinElute kit (Qiagen Inc., Valencia, Calif.). Then an aliquot was used in the second round of PCR; the same conditions as above, but for 40 cycles. Again the amplicon was purified and subjected to the same restriction enzyme digest/purification. At this point the samples were run on the Agilent 2100 bioanalyzer and submitted for DNA sequencing.
For the P16 gene, the final reaction concentrations were 0.1 µm of each primer(4) (Invitrogen Corp., Carlsbad, Calif.), with 0.2 mM dNTPs (Invitrogen), 1 unit of Platinum Taq High Fidelity, 1× PCR buffer (Invitrogen), 3 mM magnesium sulfate, 3%DMSO (Sigma-Aldrich Co., St. Louis, Mo.), 10 ng of DNA template, and HPLC-grade water (Fisher) in a total volume of 50 µl. An initial 3 minute denaturation at 95 C was followed by 40 cycles at the following conditions, again on ABI's 9700 thermal cycler: 95 C for one minute, 58 C for 1 minute, and 72 C for 1 minute. A ten minute final extension at 72 C was also incorporated. Amplicons were freed of excess primers and nucleotides using ExoSap (USB), analyzed on the bioanalyzer, and submitted for DNA sequencing. Sequencing reactions were purified/desalted using Sephadex G50 (Sigma).
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Figure 4. DNA sequence chromatogram data corresponding to Agilent chip data. Heterozygous mutations display an extra upper band.
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Analysis of PCR-products
Amplified products were electrophoresed on the 2100 bioanalyzer. The DNA 1000 LabChip® kit (Agilent Technologies) was used in accordance with manufacturer's instructions. Briefly, 9 µl of the gel dye mixture was added to the chip well labeled "G." This was pressurized for one minute throughout the chip with the syringe attachment provided. Then 9 µl of the gel dye mixture was added to the other two chip wells labeled with "G." 1 µl of ladder was added to the ladder well, followed by 5 µl of the gel dye mixture. This was pipetted up and down several times to mix. 5 µl of the markers were added to each of the twelve sample wells. 1 µl of each sample was added to the corresponding wells on the chip. The chip was run for one minute on the IKA vortex adapter (IKA® Works, Inc., Wilmington, NC) provided at the recommended setting. The chip was placed in the 2100 bioanalyzer and the double-stranded DNA 1000 assay software was run. Twelve samples could be run in a 30-40 minute time frame. Data was saved to a server and analyzed using the Agilent analysis software package provided.
Results and discussion
The established PCR assays enable the analysis of two gene regions relevant to cancer. The significance of these diagnostics relies on accurate interpretation of visual data. First we compared mutant versus wild type samples for K-ras codon 12 mutation. The sizing differences, as well as the intensity of heterozygous bands, would be difficult to discern on a standard agarose gel, and would be time-consuming. By applying the Agilent microfluidic technology, we were able to easily confirm our data with that of DNA sequencing. The sizing and quantitation offered by the software were invaluable to eliminating any guesswork previously involved in this assay.
Second, the analysis of the exon 3 region of the P16 gene was researched. At first, the discovery of multiple bands on the chip image was thought to be primer dimer or an annealing stringency problem. After comparing normal and mutated samples from DNA sequencing data to the Agilent data, the correspondence was perfect. The PCR product at size 198 bp was the main band. Only mutated samples showed these extra bands. The mutated samples were found to be heterozygous at either position 316 or 356. The CG mutation at position 316 produced one extra band. However, when a sample displayed the CG mutation at position 316 and CT mutation at position 356, two extra bands could be seen. The extra bands detected here are due to the slower mobility of the heteroduplex formed by the heterozygote mutant of the samples. Detection on the 2100 bioanalyzer completely matched results obtained using the 3700 sequencer. Figure 3 shows heterozygous mutations and double homozygous mutations.
Figure 4 shows the DNA sequence chromatogram data that corresponds to the Agilent data generated. This size range resolution could not be visualized on a slab gel.
Conclusion
The data obtained from our studies indicate that Agilent's 200 bioanalyzer can tell the difference between mutated and normal DNA samples in particular genes. This is extremely important in making a genetic diagnosis in cancer patients. This makes for a rapid and accurate screening assay, which can be employed by any molecular biology laboratory. These findings suggest that the Agilent Bioanalyzer could be used to discriminate single base changes (i.e. mutations or SNPs).
Acknowledgements
The authors would like to thank Lynn Rasmussen, Claudia Stewart, Melissa Gregory, Robin Stewart, Anne Book, Marianne Subleski, Kelly Martin, and Casey Frankenberger of the Laboratory of Molecular Technology, SAIC-Frederick.
Kristen M. Pike, Theresa M. Plona, Shirley Tsang, David Sun, Nicole L. Lum and David J. Munroe are with the Laboratory of Molecular Technology, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, Md. Will Ferguson is with Agilent Technologies in their Palo Alto, Calif. Offices.
This work was supported by NCI contract: NO1-CO-12400.
Corresponding author Kristen M. Pike can be reached at: Mailing address: Laboratory of Molecular Technology, SAIC-Frederick, Inc., National Cancer Institute-Frederick, 915 Tollhouse Avenue, Suite 211, Frederick, MD 21701. Phone 301-846-6897; email: pike@mail.ncifcrf.gov.
This work was supported by NCI Contract Number NO1-CO-12400.
References
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