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Representational difference analysis using minute quantities of DNA
References
Representational difference analysis using minute quantities of DNA
ABSTRACT
RDA has allowed the detection of distinct homozygously deleted or amplified regions in different cancer cell lines (4). When applied on xenografts of pancreatic carcinoma, RDA resulted in the detection of homozygous deleted regions located within the BRCA2 locus of heritable breast cancer susceptibility (5). RDA analysis of AIDS-associated Kaposi's sarcoma, Creutzfeldt-Jakob disease, multiple sclerosis and the GB hepatitis agent revealed novel herpes virus-like sequences, novel virus-like sequences, human herpes virus 6 sequences and flavivirus-like genomes, respectively (6-10). The drawback of the current RDA technique is the requirement of pure driver DNA and relatively pure tester DNA, thus excluding the analysis of tissue biopsies for the study of tumors (2). Indeed, tumor biopsies always contain normal stromal cells, endothelial cells and inflammatory cells. Thus, tumor samples cannot be used to prepare the driver amplicon. In this way, most deletions in the tumor genome cannot be detected. To circumvent this problem we modified the technique to study very small quantities of DNA, so that pure cell populations isolated by micromanipulation from tissue biopsy sections can be analyzed. In addition, this modified technique allows the study of genomic alterations in conditions where only a few cells are available for analysis such as e.g. epithelial dysplasias or the Reed-Sternberg cells of Hodgkin's disease.
The first major modification of the RDA technique compared to the technique originally described includes the omission of all DNA extraction procedures to prepare the genomic representations. This is necessary when analyzing very small DNA quantities in order not to lose any DNA. To inhibit the PCR amplification of dimers of the negative adaptor sequence (RBAM14), added at high concentration in the ligation reaction, with the positive adaptor and PCR primer sequence (RBAM24), two non-complementarynucleotides were added to the 3[prime] end of the RBAM14 negative adaptor sequence with respect to the one originally published (RBAM12, Table 1). A second major modification of the RDA technique is the introduction of a semi-nested PCR reamplification with an internal primer (RBAM24.int, Table 1) to obtain sufficient and almost unlimited quantities of specifically amplified genomic DNA. Indeed, a relatively large quantity of DNA is necessary for the differential hybridization steps. By our procedure, the first round PCR products provide an unlimited source of template for amplification to obtain sufficient DNA without having to use extra tissue or cells. The latter may indeed be very limited in conditions such as epithelial dysplasia or the Reed-Sternberg cells in Hodgkin's disease. The internal primer sequence used for the semi-nested PCR differs from that of the primer used in the first amplification round and includes the omission at the 5[prime] end and the addition at the 3[prime] end of three nucleotides. The latter nucleotide sequence is complementary to the restriction enzyme BamHI recognition sequence (RBAM24.int, Table 1). The subsequent differential hybridization and amplification steps were performed as previously published (1).
Table 1.
| Primer | Sequence |
| RBAM12 | 5[prime]-GATCCTCGGTGA-3[prime] |
| RBAM14 | 5[prime]-GATCCTCGGTGACT-3[prime] |
| RBAM24 | 5[prime]-AGCACTCTCCAGCCTCTCACCGAG-3[prime] |
| RBAM24.int | 5[prime]-ACTCTCCAGCCTCTCACCGAGGAT-3[prime] |
Figure 1. Agarose gel electrophoresis (1.5% NuSieve agarose, low melting point, FMC Bio Products, Rockland, ME) of the differential PCR products of the first round of hybridization/amplification of 10 ng (lane 1), 1 ng (lane 2) and 500 pg (lane 3) of Raji cell DNA admixed with pSV.Sport1 in a ratio equivalent to 10 molecules of pSV.Sport1/cell. Lane 4 represents the differential PCR products of a second round of hybridization/amplification using 500 pg of Raji cell DNA admixed with the plasmid in a ratio equivalent to one molecule pSV.Sport1/cell. M, DNA marker 50-1000 bp (FMC Bio Products). The modified RDA technique was tested using genomic Raji cell DNA as driver DNA and Raji cell DNA supplemented with the plasmid pSV.Sport1 (Life Technologies, Gaithersburg, MD) containing a unique BamHI fragment of 580 bp as tester DNA. Although pure DNA was used, every step of the modified RDA technique was performed as for the analysis of whole microdissected tissue or cells such as described above, including the proteinase K digestion of the samples. The sensitivity of the technique was tested by using different amounts of DNA, ranging from 10 ng to 100 pg, which were supplemented with 6 pg to 0.06 fg pSV.Sport1, respectively, for the tester sample. Successful isolation of the 580 bp fragment was repeatedly obtained after RDA analysis of as few as 500 pg of Raji cell DNA admixed with 0.3 fg pSV.Sport1 (Fig. To prepare the genomic representations or amplicons, tester and driver tissue or cell samples were first digested with proteinase K (200 µg/ml in a volume of 5 µl buffer: 10 mM Tris-HCl pH 8, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, 0.01% gelatin and 0.45% Tween-20) overnight at 37°C. After inactivation of the proteinase K by heating for 15 min at 80°C, all the reactions for the genomic representations including the restriction digestion, the ligation to the adaptors and the PCR amplification were subsequently performed in the same tube by adding the respective reagents. The restriction digestion was with 10 U of BamHI (Life Technologies) for 2 h at 37°C in a 10 µl volume. The final buffer concentration is identical to that of the proteinase K digestion. After inactivation of the restriction enzyme (10 min, 80°C), the DNA was ligated to non-phosphorylated RBAM14 and RBAM24 oligonucleotides in a 20 µl volume containing 20 µM of each oligonucleotide. The ligation is achieved by successively heating the reaction mix for 3 min at 72°C, cooling it from 50 to 10°C during 1 h and by the addition of 12 U of T4 DNA ligase (Stratagene, La Jolla, CA) in a buffer (100 mM Tris-HCl pH 7.5, 9 mM MgCl2, 1 mM DTT, 0.01% gelatin and 0.45% Tween-20) containing 1 mM of rATP. The mixture was incubated at 4°C overnight. After inactivation of T4 DNA ligase (10 min, 80°C) the DNA samples were PCR amplified in a 400 µl volume. The final buffer concentration was 68 mM Tris-HCl pH 8.8, 0.01% gelatin, 0.45% Tween-20, 4 mM MgCl2 and 300 µM of each dNTP. The tubes were incubated for 10 min at 72°C in a thermal cycler (480 thermal cycler, Perkin Elmer, Branchburg, NJ), while adding 15 U of Taq DNA polymerase (Promega, Madison, WI) to fill in the 5[prime] protruding ends of the ligated adaptors. The samples were then incubated for 5 min at 94°C and the RBAM24 primer was added to a final concentration of 1 µM. The samples were amplified for 30 cycles (1 min incubation at 95°C and 3 min at 72°C) followed by a final extension step at 72°C for 10 min. Sixteen µl of the 1/200 diluted PCR products were reamplified in a 400 µl volume using 1 µM of the modified RBAM24.int primer in the following buffer: 67 mM Tris-HCl pH 8.8, 16 mM (NH4)2SO4, 10 mM [beta]-mercaptoethanol, 100 µg/ml BSA, 4 mM MgCl2 and 300 µM of each dNTP. After incubation of the tubes for 10 min at 95°C and adding 15 U of Taq DNA polymerase, the samples were amplified for 20 cycles. To remove the adaptors of the PCR product, purified tester and driver amplicons were digested with 30 U of BamHI at 37°C overnight. Before the differential hybridization step, the tester amplicon was ligated to the JBAM12 and JBAM24 oligonucleotides in the same way as described for the amplicon preparation. Ligated tester (0.5 µg) and driver (40 µg) amplicons were mixed and dissolved in 4 µl of 3× EE buffer [30 mM EPPS (N-(2-hydroxyethyl)piperazine-N[prime]-3-propanesulfonic acid) pH 8.25, 3 mM EDTA]. This sample was denaturated for 5 min at 95°C. One µl of a 5 M NaCl solution was added and the sample was hybridized for 20 h at 67°C. The subsequent amplification and differential hybridization steps were performed exactly as described by Lisitsyn et al. (1). We have used this adapted RDA technique to analyze Reed-Sternberg cells, the malignant cells of Hodgkin's disease. These cells are relatively scarce and highly admixed with inflammatory cells in the tissues. Ten Reed-Sternberg cells and 100 lymphocytes were isolated from tissue slides and used as tester and driver, respectively. Reed-Sternberg cells are usually polyploid and therefore only 10 cells were considered to yield enough DNA for our test. Both Reed-Sternberg cells and lymphocytes were isolated from frozen tissue sections using a micromanipulator (MMW-202D micromanimulator, Narishige, Tokyo, Japan) mounted on an inverted microscope (Leica DMIRB, Deerfield, IL). The tissue sections were previously immunostained for CD15, a Reed-Sternberg cell marker, to unequivocally identify tester and driver cells. Differential sequences were obtained as illustrated in Figure Figure 2. Agarose gel electrophoresis (1.5% NuSieve, FMC BioProducts) of the genomic representations of lymphocytes (~100 cells, lane 1) and Reed-Sternberg cells (~10 cells, lane 2), as well as the differential PCR products (arrows) of the first, second, third and fourth round of hybridization/amplification (lanes 3-6 respectively). M, DNA marker 50-1000 bp (FMC Bio Products). Figure 3. Southern blotting of the amplicons derived from lymphocytes (lane 1) and Reed-Sternberg cells (lane 2), hybridized with a probe representing the 880 bp differential sequence (see Fig. 2). In conclusion, the modification of the RDA technique for the analysis of minute quantities of DNA allows the analysis of tissue biopsies, including those where only few cells of interest are present for the analysis. Therefore, this technique may also be very useful for the genetic analysis of the early stages of carcinogenesis in man, of which usually only small amounts of tissue are available.
REFERENCES
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Last modification: 20 Jul 1998
Copyright©Oxford University Press, 1998.
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