Quantitation of C-Myc Gene Amplification by a Competitive PCR Assay System
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Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Quantitation of c-myc Gene Amplification by a Competitive PCR Assay System Seth P. Harlow and Carleton C. Stewart Departments of Surgical Oncology and Flow Cytometry, Roswell Park Cancer Institute, Buffalo, New York 14263 Gene amplification is a common Gene amplification, particularly am- being possible. A number of variables, event in the progression of human plification of the growth-promoting however, must be accounted for to de- cancers. The detection and quantita- proto-oncogenes, is a common event in termine the true gene amplification level tion of certain amplified oncogenes the progression of many human can- in a population of cells. These include has been shown to have prognostic cers. (~ Detection and quantitation of cell number, cell cycle phase (cells in G 2 importance in certain human malig- certain specific amplified genes may or mitosis will have twice the gene cop- nancies. A method is described that have the potential for predicting patient ies as cells in G O or G1), and chromo- utilizes the principles of competitive outcome or response to therapy for a some ploidy (genes on aneuploid chro- PCR for quantitation of the c-mu number of different human tumor mosomes generally are not considered gene copy number in relation to the types. (2'3) Recently, a number of meth- amplified). To account for all of these copy number of a reference gene (tis- ods have been described to accomplish variables, quantitation of an internal sue plasminogen activator It-PAl this goal by a variety of techniques (4-6~ control or reference gene has been used gene) located on the same chromo- differing from the standard Southern routinely in Southern techniques. The some (8) as the c-mu gene. This ratio blot technique. (7~ A significant advance reference gene ideally should be located gives the true level of amplification in the quantitation of specific nucleic on the same chromosome as the gene of of the c-myc gene, accounting for acid sequences was made with the intro- interest and should be located at a dis- variables such as cell number, cell cy- duction of quantitative PCR methods. tance from that gene such that it would cle phase, and chromosome 8 ploidy. The technique of competitive PCR, (s~ not likely be coamplified with it. In ad- The determination of gene amplifica- which uses an external DNA standard, dition, the reference gene should not be tion depends on the precise measure- has been well documented as a precise a gene that is known to be amplified or ment of the ratio of target and refer- and accurate method for quantitating deleted on its own as part of the known ence genes. An important feature of specific gene sequences. (9'1~ tumor progression. Gene amplification this assay is that the competitive ref- Competitive PCR methods rely on the can then be calculated simply as follows erence standards used for target addition of a known amount of external (number of gene copies/number of refer- gene c-mu and reference gene t-PA DNA standard to several PCR reaction ence gene copies) in a given sample. have been linked to form a hybrid. tubes containing equivalent amounts of In this paper we describe a novel This simple modification guarantees sample DNA. The external standard is a modification of the competitive PCR that both reference gene and target double-stranded DNA molecule identical methodology for quantitating gene am- gene assay tubes get identical to the genomic sequence of interest with plification of the c-myc proto-oncogene. amounts of the competitive template either a small deletion, insertion, or new This method utilizes a competitive refer- for each gene, thereby eliminating a restriction site, such that it can be easily ence standard (CRS) molecule, which is a significant source of error. This discriminated from the genomic se- competitive external DNA standard for method has the same desirable at- quence by size on gel electrophoresis. the c-myc gene (chromosome 8q24) (11~ tributes of standard PCR in that very Thus, the external standard has the same linked to a competitive external DNA small sample sizes are required and primer binding sites and has a similar standard for the tissue plasminogen that results can easily be obtained in amplification efficiency as the genomic activator (t-PA) gene (chromosome <24 hr. In addition, this technique sequence. Quantitation of the sample 8p12). 02~ This CRS molecule acts as an does not require the use of radioac- gene dosage can then be made by a titra- external standard to control for the rate tivity or expensive DNA detection tion of external standard and extrapolat- and efficiency of the PCR reaction and as kits, and thus, may give it wider ap- ing where the genomic product molarity an internal standard to control for cell plicability for the study of human is equal to that of the external standard. number, cell cycle phase, and ploidy ab- ca n ce rs. Competitive PCR methods can there- normalities. This method shares the ad- fore give precise information on the vantages of standard PCR techniques in number of gene sequences in a given that it is relatively quick and can be used sample with sensitivities <1 attomole on samples of very small size (as few as 3:163-1689 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $5.00 PCR Methods and Applications 163 Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press 1000 cells). These benefits make this downstream primer and the comple- plifying from the small CRS templates. methodology attractive for analysis of ment to the t-PA upstream primer. The The primers used in this study are as fol- clinical samples where available tissue is resulting product was a combined c-myc! lows: c-myc (~1) primers--sense (5' end) often limited. t-PA CRS that could be used for the de- position 6721-6740, 5'-AAGGTCAGAG- termination of c-myc gene amplification TCTGGATCAC-3'; antisense (3' end) po- (Fig. 2). The templates were linked to re- sition 7059-7078, 5'-TAACTACCTTGG- MATERIALS AND METHODS duce the inaccuracies produced by hav- GGGCCTTT-3', PCR product gener- CRS ing to quantitate and dilute two inde- ated-- 358 bp. t-PA (~2) primers--sense (5' By using site-specific mutagenesis tech- pendent templates. Molar amounts of end) position 30,805-30,824, 5'-GC- niques, as described previously, (13) com- the CRS were determined by quantita- CACCTGCGGCCTGAGACA-3'; antisense petitive double-stranded DNA templates tion of OD at 260 nm after purification (3' end) position 30,959-30,978, 5'-AG- to the c-myc and t-PA genes were made. of the CRS on low-melting-point agarose AGAGAATCCAGCAGGAGC-3'; PCR prod- Each DNA template had a novel SmaI re- gels. uct generated = 174 bp. striction endonuclease recognition site (5'-CCCGGG-3') incorporated with mu- PCR Primers Cell Lines tagenesis primers (Fig. 1). Both tem- plates were found to digest completely Primers used for the competitive PCR re- Cell lines used in this study were ob- into two fragments by the SmaI restric- actions were selected to lie in a nested tained from ATCC (American Type Cul- tion enzyme before linking. The two fashion in relation to the ends of the ture Collection, Rockville, MD) and competitive templates were then linked CRS sequence. We have found that using grown according to the instructions sup- by PCR, using an oligonucleotide primer nested primers improves PCR amplifica- plied. The cell lines studied were HL60 that was a combination of the c-myc tion sensitivity and specificity when am- (human promyelocytic leukemia), SKCO1 (human colon adenocarcinoma), and Colo 320DM (human colon carcinoma). Cells were harvested without enzy- matic digestion or EDTA to avoid inter- A ference with the PCR. Cells were centri- fuged and resuspended in sterile Genomic Sequence 5' CCCAGG z' phosphate-buffered saline. They were Position (6940-6945) counted by hemocytometer and ali- quoted so that study solutions would Competitive Template contain the equivalent of -1000 cells/t~l Same Position 5' CCCGGG 3' of lysis solution (with the exception of the COLO320 DM cell line, where -500 cells/l~l of lysis solution were used). c-myc Competitive Template with 222 BP's 136 BP's sma-1 site ~ Cell Lysis Solution (lengths are those Prior to competitive PCR reactions, cells obtained with nested were lysed in the following solution, as primers) has been described previously: (14) 10 mM Tris-HC1 (pH 8.3), 2.5 mM MgC12, 50 mM KC1, 0.1 mg/ml gelatin, 0.45% NP-40 B (LKBProdukterAB, Bromma, Sweden), 0.45% Tween 20 (Sigma Chemical, St. Genomic Sequence Louis, MO), and 200 l~g/ml of proteinase Position (30922-30927) 5' CCCGGA , 3' K. Samples were incubated at 60~ for 60 min, then at 94~ for 10 min to inacti- vate proteinase K. Competitive Template CCCGGG Same Position Competitive PCR Reactions t-PA Competitive All competitive PCR reactions were per- Template with 120 BP's 54 BP's formed in 100 t~1 total volume and cy- sma-1 site ~ cled in a Perkin-Elmer Cetus (Norwalk, (lengths are those CT) DNA Thermal Cycler with an overlay obtained with of mineral oil. The reaction solution nested primers) constituents were as follows: 10 mM Tris- FIGURE 1 The sites of directed point mutations to create the competitive templates for the c-myc HC1 (pH 8.3), 1.5 mM MgC12, 50 mM KC1, gene (A) and the t-PA gene (B). The size of each portion of the template fragment after digestion 0.1 mg/ml of gelatin, 150 i~molefliter of with SmaI restriction endonuclease is also shown.