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Detection of Rare Mutant Alleles by Restriction Endonuclease-mediated Selective-PCR: Assay Design and Optimization

¹ Johnson & Johnson Research Pty Limited, Australian Technology Park, Level 4, 1 Central Ave., Eveleigh NSW 1430, Australia

² Department of Medical Oncology, St. Vincent’s Hospital, Darlinghurst, Sydney 2010, Australia.

³ School of Pathology, University of New South Wales, Sydney 2052, Australia.
^a Author for correspondence. Fax 61-2-8396-5811; e-mail cfuery{at}medau.jnj.com

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site.

Methods: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR.

Results: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status.

Conclusion: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.

	Introduction

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Restriction endonucleases (REs)¹ in combination with PCR have been used extensively for detecting point mutations (1)(2)(3)(4)(5)(6)(7)(8). Detection of rare alleles can be achieved using enriched PCR, which requires multiple rounds of PCR (5)(6), or using restriction endonuclease-mediated selective (REMS)-PCR, which achieves the same result in a single round of PCR (7)(8). The recognition sites for REs used in these assays must span the bases to be screened for mutations and must be present in one allele but not the variant allele. The recognition site can be either natural or induced during PCR by the use of mismatched primers. Rules for designing PCR primers that contain mismatched bases near the 3' terminus have been established (9). The thermostable RE present in the REMS-PCR mixture inhibits amplification of sequences that contain its recognition site, thus producing selective amplification of amplicons that lack its recognition site. REMS-PCR systems contain primers for amplification of both diagnostic and control amplicons, the presence or absence of which confirms the specific sequences at the diagnostic locus and efficient assay function, respectively.

Development of REMS-PCR protocols requires identification of enzymes and specific buffer conditions that are capable of sustaining enzymatic activity despite repeated cycles of thermal denaturation. This report describes assays used to identify suitable conditions. The utility of these assays in defining conditions for REMS-PCR is demonstrated using the combination of Taq DNA polymerase and the RE BstNI.

	Materials and Methods

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re thermostability assay
Mock PCR mixtures, excluding genomic DNA but including standard PCR components and RE, were thermocycled and then incubated with plasmid DNA at the optimal temperature (as recommended by the manufacturer) to assess the residual RE cleavage activity. We studied BstNI, BslI, Tsp509I, Tru9I, TaqI, TaiI, TthHB8I, and MwoI and report experiments with BstNI. Reactions for assessment of BstNI activity contained 7.5 pmol of each of the primers 5BKIT (5'-TATAAACTTGTGGTAGTTGGACCT-3') and 3KiE (5'-CTCATGAAAATGGTCAGAGAAAC-3'), each dNTP at 100 µmol/L (Amersham Pharmacia Biotech), 0.5 U of AmpliTaq^® DNA polymerase (PE Biosystems), and 20 U of BstNI (10 U/µL; New England Biolabs). We assessed the effect on thermostability of buffers, MgCl₂ (PE Biosystems), dithiothreitol (DTT; Promega), bovine serum albumin (BSA; New England Biolabs), and glycerol (AnalaR grade; BDH Laboratory Supplies). Reactions were made up in thin-walled tubes to a total volume of 25 µL.

The reactions were placed in a GeneAmp^® PCR system 9600 Thermal Cycler (PE Biosystems), heated to 94 °C for 2 min, and then subjected to 15 or 30 cycles of 60 °C for 1 min followed by 92 °C for 20 s. After thermocycling, 8 µg of plasmid DNA (pGFP-C1; Clontech) was added in 5 µL of Tris-EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 7.4 at 25 °C), and reactions were incubated at 60 °C for 1 h. Cleavage of plasmid DNA was assessed by electrophoresis on a 3% NuSieve^® GTG^® gel (FMC BioProducts). The RE was scored as inactive or as having low, moderate, or high activity based on the number of restriction fragments produced. Reactions for assessment of other REs were performed similarly.

pcr efficiency assay
We assessed the compatibility of the buffers and additives with the PCR by amplifying a 185-bp region of exon 1 of the K-ras gene. Reactions contained 800 ng of DNA from the cell line K562 (CCL 243; ATCC), 30 pmol each of 5BKIT and 3KiE, and 100 µmol/L of each dNTP (Amersham Pharmacia). AmpliTaq DNA polymerase (4 U; PE Biosystems) was mixed with TaqStart^TM antibody (Clontech Laboratories) to give a final molar ratio of 1:5. The DNA polymerase:antibody mixture was incubated for 15 min at room temperature before addition to the PCR mixtures. Reactions were made up in thin-walled tubes to a total reaction volume of 100 µL and thermocycled as above for 30 cycles. A 28-µL aliquot of each reaction was analyzed by electrophoresis on a 5% NuSieve GTG gel; and the efficiency of PCR was rated as low, moderate, or high according to the intensity (by eye) of the specific amplicon produced. Mispriming events reduced the intensity of the specific amplicons. PCR was assessed using Stoffel fragment DNA polymerase similarly.

assessment of the limit of detection of rems-pcr
Genomic DNA was extracted from human cancer cell lines K562 and Calu 1 (HTB 54; ATCC). K562 is wild type at K-ras codon 12, whereas Calu 1 has wild-type (GGT) and mutant (TGT) alleles. Calu 1 DNA was diluted with K562 DNA at ratios (by mass) of Calu 1:K562 of 1:10, 1:10², and 1:10³. The REMS-PCR mixtures (50 µL) contained 500 ng of genomic DNA; 50 pmol of 5BKIT [5'-TATAAACTTGTGGTAGTTGGACCT-3' (underlined base is mismatched with respect to K-ras)]; 50 pmol of 3K2 (5'-CGTCCACAAAATGATTCTGA-3'); 2 pmol of 5BK36 [5'-CTAGAACAGTAGACACAAACCA-3' (underlined base is mismatched with respect to K-ras)]; 2 pmol of 3K37 (5'-GATTTTGCAGAAAACAGATC-3'); 2 pmol of 5BK38 (5'-GTACACATGAAGCCATCGTATA-3'); 2 pmol of 3K39 (5'-CCACTTGTACTAGTATGCCTTAAG-3'); 1 mmol/L DTT; each dNTP at 50 µmol/L; and 40 U of BstNI in 100 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.3), 4 mmol/L MgCl₂. A control reaction contained K562 DNA without BstNI. AmpliTaq DNA polymerase (6 U) was mixed with TaqStart antibody (molar ratio of 1:5) and incubated for 15 min at room temperature before addition to the mixtures. Reactions were placed in the thermal cycler, denatured at 94 °C for 2 min, and then subjected to 30 cycles of 58 °C for 1 min followed by 92 °C for 20 s. A 25-µL aliquot of each reaction was analyzed by electrophoresis on a 5% NuSieve GTG gel.

	Results

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assessment of buffer conditions compatible with rems-pcr
In REMS-PCR, the RE and the DNA polymerase must function in identical reaction conditions and retain activity during thermocycling. We developed assays to assess the activity of REs and DNA polymerases under various reaction conditions. BstNI remained fully active after 15 thermocycles and moderately active after 30 thermocycles in buffer 2, pH 8.3, with 4 mmol/L MgCl₂ and 1 mmol/L DTT, or buffer 2, pH 8.0–8.5, with 10 mmol/L MgCl₂ and 1 mmol/L DTT (Fig. 1 A and Table 1 ). BstNI was more thermostable in these buffer conditions than in the buffer recommended by the manufacturer (NEB2 with 0.1 g/L BSA; Fig. 1A ). The same reaction conditions were tested for Taq DNA polymerase. Buffer 2 (pH 8.3) with 4 mmol/L MgCl₂ and 1 mmol/L DTT permitted efficient PCR amplification while preserving BstNI activity.

View larger version (107K): [in this window] [in a new window]   Figure 1. Thermostability of BstNI under various conditions (A) and limit of detection of REMS-PCR using BstNI (B). (A), lanes 1 and 14, 100-bp marker; lanes 2, 6, and 10, NEB2 + 100 mg/L BSA (cycles 0, 15, and 30, respectively); lanes 3, 7, and 11, PCR buffer II (PE Biosystems) + 4 mmol/L MgCl2 (cycles 0, 15, 30, respectively); lanes 4, 8, and 12, buffer 2 (pH 8.3) + 4 mmol/L MgCl2 + 1 mmol/L DTT (cycles 0, 15, and 30, respectively); lanes 5, 9, and 13, buffer 2 (pH 8.3) + 10 mmol/L MgCl2 + 1 mmol/L DTT (cycles 0, 15, and 30, respectively). (B), lane 1, X-174 cut with HinfI, (Promega Corporation); lane 2, Calu 1 diluted 10-fold into K562; lane 3, Calu 1 diluted 100-fold into K562; lane 4, Calu 1 diluted 1000-fold into K562; lane 5, K562; lane 6, K562 with no BstNI.

Similar experiments identified conditions in which BslI could be used. BslI retained moderate activity after 30 thermocycles with DTT (1 mmol/L) in buffer systems that contained (a) PCR buffer II and 10 mmol/L MgCl₂; (b) Stoffel buffer and 10 mmol/L MgCl₂; (c) 50 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 8.5), and 10 mmol/L MgCl₂; (d) 100 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.3–8.5), and 10 mmol/L MgCl₂; or (e) 100 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.5), and 6 mmol/L MgCl_2. Conditions that preserved activity during thermocycling were determined for Tru9I, Tsp509I, TaiI, and BsiYI (not shown). We found no conditions that maintained activity of TthHB8I, MwoI, or TaqI. A single cycle of denaturation inactivated both MwoI and TaqI under all conditions tested.

analysis of K-ras CODON 12 USING REMS-PCR
A REMS-PCR protocol using BstNI was used to detect mutations in the first two bases within codon 12 of the K-ras gene (Fig. 1B ). These reactions contained three sets of primers: (a) diagnostic primers 5BKIT and 3K2, which amplify an 82-bp region of exon 1 of the K-ras gene (5BKIT induces a BstNI site spanning codon 12 in wild-type, but not mutant, amplicons); (b) RE control primers 5BK36 and 3K37, which amplify a 130-bp region in exon 3 of the K-ras gene (5BK36 induces a BstNI site in all RE control amplicons); and (c) PCR control primers 5BK38 and 3K39, which amplify a 215-bp region of exon 4b of the K-ras gene (this amplicon contains no BstNI sites).

In the reactions containing BstNI, the presence of the 82-bp fragment was diagnostic for the presence of K-ras codon 12 mutations. This fragment was visible in reactions containing Calu 1:K562 DNA at ratios of 1:10, 1:10², and 1:10³, but was not visible in reactions containing K562 DNA alone. The RE control fragment was not visible in any reactions containing BstNI, indicating that the RE activity was sufficient throughout the PCR to inhibit amplification of all fragments containing BstNI sites. The 215-bp PCR control fragment was visible in all reactions, including the reaction containing K562 DNA, confirming that the reactions conditions were adequate for amplification. In the control reaction containing K562 DNA in the absence of BstNI, all three fragments were clearly visible, indicating efficient amplification with all three primer sets.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In REMS-PCR, both the RE and the DNA polymerase must function under the same reaction conditions and be sufficiently thermostable in these conditions to retain activity during thermocycling. The RE must actively cleave at temperatures compatible with the stringent conditions required for annealing of primers during the PCR, typically 50–65 °C. Although many REs fulfil this criterion, not all are suitable for REMS-PCR. Manufacturers’ data on the thermal stability of REs in buffers supplied with the enzymes do not predict whether conditions can be found that preserve RE activity during thermocycling. For example, MwoI is listed as not being heat inactivated by incubation for 20 min at 80 °C (New England Biolabs, 1998–1999 catalog), but in the RE thermostability assay, this enzyme was fully inactivated by denaturation at 92 °C for 2 min in all conditions analyzed. BslI is listed as being heat inactivated after incubation at 80 °C for 20 min (New England Biolabs, 1998–1999 catalog), but the thermostability assay identified conditions under which BslI still cleaved after 30 cycles of PCR.

The pH and ionic strength of the buffer, the choice and concentration of monovalent cation, the concentration of free Mg²⁺, and the presence of other additives, particularly DTT, can all influence the thermostability of different REs to different extents. The influence of each of these components depends on the other components in the buffer. BstNI, BslI, Tru9I, Tsp509I, and TaiI were sufficiently thermostable to maintain activity while being thermocycled during the PCR.

Multiplex REMS-PCR systems, which incorporate primer sets for amplification of both diagnostic and control fragments, allow unequivocal analysis of codon status. REMS-PCR systems contain two controls, which test for the function of the DNA polymerase (PCR control fragment) and the RE (RE control fragment). The PCR control primers amplify a region devoid of RE sites. The presence of this fragment indicates efficient PCR, and the absence of these amplicons indicates the potential for false-negative results. The PCR control fragments should be the largest of the three fragments because longer fragments will be more sensitive to the integrity of the DNA and hence will serve as a control for all PCR conditions. The RE control amplicons all harbor RE sites. The presence of these amplicons indicates the potential for false-positive results because RE control primers amplify only when the RE activity is inadequate for REMS-PCR. The efficiency of amplification with the RE control primers should be slightly greater than that of the diagnostic primers. This ensures that RE control amplicons will become visible at an earlier cycle number than the diagnostic amplicons in the event of failure of the RE to inhibit PCR.

Our REMS-PCR system for analysis of mutations at K-ras codon 12 detected 1 mutant allele in a background of 1000 wild-type alleles. Specific applications proposed for assays for codon 12 include (a) diagnostics tests (pancreatic cancer); (b) prognostic tests that detect micrometastatic disease in lymph nodes and other specimens (colorectal and pancreatic cancer); (c) screening tests that detect exfoliated tumor cells or soluble tumor DNA in body fluids such as blood, stools (colorectal and pancreatic cancer), or sputum (lung cancer); and (d) tests that may assist clinicians in tailoring therapy (10)(11)(12). Some of these applications require detection of small numbers of tumor cells present in a 1000-fold excess of nondiseased cells. The sensitivity afforded by REMS-PCR satisfies this criterion. The clinical utility of the REMS-PCR K-ras codon 12 system has been published previously (7).

Analysis of other codons and/or other ras genes could extend the utility of the REMS-PCR system. BslI recognizes and cleaves the sequence CCN₇GG, where N is any nucleotide. This RE could theoretically be used to target any codon that codes for either glycine (GGN) or proline (CCN). BslI could be used in systems that analyze mutations that occur at codons 12 or 13 of any of the three ras oncogenes, K-ras, H-ras, and N-ras, because these loci all encode glycine. This RE has been used successfully in a second REMS-PCR system targeting codon 12 of K-ras, again demonstrating a sensitivity that allowed detection of 1 mutant in a 1000-fold excess of wild-type alleles (data not shown). REMS-PCR systems using BslI could potentially be used to screen for the vast majority of ras mutations.

REMS-PCR provides a sensitive, rapid, and reliable method that is suitable for analysis of genetic variations that are associated with disease. Because REMS-PCR mixtures contain all reagents at the initiation of reactions performed in closed vessels, the opportunity for contamination during PCR is eliminated, and the protocol is amenable to automation. Although the reactions do not require further manipulation before detection, the REMS-PCR method does not preclude subsequent analysis of diagnostic amplicons for identification of the exact nucleotide substitution (9). Multiple controls can be added to multiplexed REMS-PCR protocols to provide unequivocal analysis of mutant status of the bases screened. We conclude that REMS-PCR allows rapid, sensitive, and reliable screening of clinical samples for genetic mutations.

	Acknowledgments

 
The PCR process is covered by patents held by F. Hoffmann-La Roche.

	Footnotes

 
¹ Nonstandard abbreviations: RE, restriction endonuclease; REMS-PCR, restriction endonuclease-mediated selective-PCR; DTT, dithiothreitol; and BSA, bovine serum albumin.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Perucho M. PCR and cancer diagnostics: detection and characterization of single point mutations in oncogenes and antioncogenes. Mullis KB Ferre T Gibbs RA eds. The polymerase chain reaction 1994:269-394 Birkhauser Boston. .
2. Todd A, Iland HJ. Rapid screening of mutant N-ras alleles by analysis of PCR-induced restriction sites: allele specific restriction analysis (ASRA). Leuk Lymphoma 1991;3:293-330.
3. Todd A, Ireland CM, Radloff TJ, Kroenberg H, Iland HJ. Analysis of N-ras gene mutations in acute myeloid leukemia by allele specific restriction analysis. Am J Hematol 1991;38:207-213. [Web of Science][Medline] [Order article via Infotrieve]
4. Cohen JB, Levinson AD. A point mutation in the last intron responsible for increased expression and transforming activity of the c-Ha-ras oncogene. Nature 1988;334:119-124. [Medline] [Order article via Infotrieve]
5. Todd A. Allele-specific enrichment: a method for the detection of low level N-ras gene mutations in acute myeloid leukemia. Leukemia 1991;5:160-161. [Web of Science][Medline] [Order article via Infotrieve]
6. Levi S, Urbano-Ispizua A, Gill R, Thomas DM, Gilbertson J, Foster C, Marshall CJ. Multiple K-ras codon 12 mutations in cholangiocarcinomas with a sensitive polymerase chain reaction technique. Cancer Res 1991;51:3497-3502. [Abstract/Free Full Text]
7. Ward R, Hawkins N, O’Grady R, Sheehan C, O’Connor T, Impey H, et al. Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples. Am J Pathol 1998;53:373-379.
8. Roberts NJ, Impey HI, Applegate TL, Fuery CJ, Ward RL, Todd AV. Rapid, sensitive detection of mutant alleles in codon 12 of K-ras by REMS-PCR. Biotechniques 1999;27:418-420. [Web of Science][Medline] [Order article via Infotrieve]
9. Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 1990;18:999-1005. [Abstract/Free Full Text]
10. Sidransky D, Tokino Y, Hamilton SR, Kinzler KW, Levin B, Frost P, Vogelstein B. Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 1992;256:102-105. [Abstract/Free Full Text]
11. Mao L, Hruban RH, Boyle JO, Tockman M, Sidransky D. Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 1994;54:1634-1637. [Abstract/Free Full Text]
12. Duffy MJ. Can molecular markers now be used for early diagnosis of malignancy?. Clin Chem 1995;41:1410-1413. [Abstract/Free Full Text]

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