Analysis of nucleic acids by tandem hybridization on oligonucleotide microarrays.
Explore the source record for details and available documents.
Nucleic Acids↗
Biomedical subjects
R Maldonado-Rodriguez
Publications and source records attributed to R Maldonado-Rodriguez.
Hybridization of glass-tethered oligonucleotide probes to target strands preannealed with labeled auxiliary oligonucleotides.
In this article we introduce a strategy of preannealing labeled auxiliary oligonucleotides to single-stranded target DNA, prior to hybridization of the DNA target to oligonucleotide arrays (genosensors) formed on glass slides for the purpose of mutation analysis. Human genomic DNA samples from normal individuals and cystic fibrosis (CF) patients (including homozygous delta F508 and heterozygous delta F508/wild type (wt) in the region examined) were used. A PCR fragment of length 138 bp (wt) or 135 bp (mutant) was produced from exon 10 in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, using a new pair of polymerase chain reaction (PCR) primers. This fragment contains four of the most frequent mutation sites causing the disease (Q493X, delta I507, delta F508, and V520F). Each of these mutations was tested using a pair of nonamer (9-mer) probes covalently attached to glass slides, representing the normal (wt) and the mutant alleles. Single-stranded target DNA was isolated from the PCR fragment using one PCR primer labeled with biotin and a streptavidin minicolumn to capture the biotin-labeled strand. Prior to hybridization to the 9-mer array on a glass slide, the unlabeled target strand was preannealed with one, three, or four auxiliary oligonucleotides, at least one being labeled with 32P. As observed previously in several laboratories, the discrimination between normal (wt) and mutant alleles at each site using oligonucleotide array hybridization ranged from very good to poor, depending on the number and location of mismatches between probe and target. Terminal mismatches along the probe were difficult to discriminate, internal mismatches were more easily discriminated, and multiple mismatches were very well discriminated. An exceptionally intense hybridization signal was obtained with a 9-mer probe that hybridized contiguously (in tandem) with one auxiliary oligonucleotide preannealed to the target DNA. The increased stability is apparently caused by strong base stacking interactions between the "capture probe" and the auxiliary oligonucleotide. The presence of the delta F508 mutation was detected with this system, including discrimination between homozygous and heterozygous conditions. Base mismatch discrimination using the arrayed 9-mer probes was improved by increasing the temperature of hybridization from 15 to 25 degrees C. Auxiliary oligonucleotides, preannealed to the single-stranded template, may serve several purposes to enable a more robust genosensor-based DNA sequence analysis: 1. A convenient means of introducing label into the target DNA molecule. 2. Disruption of interfering short-range secondary structure in the region of analysis. 3. Covering up of redundant binding sites in the target strand (i.e., where a given probe has more than one complement within the target). 4. Tandem hybridization with the capture probe (providing contiguous stacking) as a means for achieving efficient mismatch discrimination at the terminal position of the capture probe (adjacent to the auxiliary oligonucleotide). By use of multiple auxiliary oligonucleotides, all of the above benefits can be derived simultaneously.
Base Sequence↗
Mutation detection by stacking hybridization on genosensor arrays.
A new strategy for analysis of point mutations using oligonucleotide array (genosensor) hybridization was investigated. In the new approach, a single-stranded target strand is preannealed with a labeled "stacking oligonucleotide," and then the partially duplex labeled target molecule is hybridized to an array of glass-tethered oligonucleotide probes, targeted to the region on the target immediately adjacent to the stacking oligomer. In this configuration, the base-stacking interactions between the "capture probe" and the contiguously stacking oligomer stabilize the binding of the target molecule to its complementary probe on the genosensor array. The temperature of hybridization can be adjusted so that the target molecule will bind to the glass-tethered probe only in the presence of the stacking oligomer, and a single mismatch at or near the terminal position ol the capture probe disrupts the stacking interactions and thereby eliminates or greatly reduces the hybridization. This stacking hybridization approach was investigated using a collection of synthetic targets, probes, and stacking oligonucleotides, which permitted identification of conditions for optimal base mismatch discrimination. The oligonucleotide probes were tethered to the glass using a simple, improved attachment chemistry in which a 3'-aminopropanol function introduced into the probe during chemical synthesis binds covalently to silanol groups on clean, underivatized glass. "Operating parameters" examined in the stacking hybridization system included length of capture probe, position, type and number of mismatches between the probe and the target, temperature of hybridization and length of washing, and the presence of terminal phosphate group in the probe, at its junction with the stacking oligomer. The results suggest that in the stacking hybridization configuration: 1. Optimal mismatch discrimination with 9-mer probes occurs at 45 degrees C, after which little or no improvement in mispair rejection occurred on lengthy continued washing at 45 degrees C. 2. At 25 degrees C optimal mismatch discrimination occurred with 7- or 8-mer probes, or with 9-mer probes containing an additional internal mismatch. 3. The presence of a phosphate group on the 5'-end of the glass-tethered probe had no general effect on mismatch discrimination, but influenced the relative stability of different mismatches in the sequence context studied. These results provide a motivation for continued development of the stacking hybridization technique for nucleic acid sequence analysis. This approach offers several advantages over the traditional allele-specific oligonucleotide hybridization technique, and is distinct from the contiguous stacking hybridization sitrategy that the Mirzabekov laboratory has introduced (Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918; Parinov et al. (1996) Nucleic Acids Res. 24, 2998-3004).
Base Pair Mismatch↗
Mutagenic consequences of the incorporation of 6-thioguanine into DNA.
6-Thioguanine (S6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells. The cytotoxicity of S6G is thought to derive from its incorporation into DNA in place of guanine. The deoxyribonucleoside triphosphate of S6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508-514, 1991). Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine. To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M13lacISaXb in reactions that omitted dGTP (-G) or dATP (-A). LacI mutation frequency was determined by beta-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducer). When a high concentration of SdGTP (24 microM) was used in the DNA polymerase reaction, phage infectivity was inhibited. When a relatively low concentration (2.4 nM) was added to the -G and -A reactions, mutagenic effects were observed. DNA sequencing of mutant progeny arising from the -G + S6G reaction revealed C-to-T base transitions and some C-to-A transversions. Similarly, the presence of SdGTP in the -A reactions led to mutants with T-to-C transitions. No insertions or deletions were observed. These data indicate that mispairing of S6G with thymine leads to mutagenic effects in this assay.
Bacterial Proteins↗
In vitro mutagenesis in the lacI gene of Escherichia coli: fate of 3'-terminal mispairs versus internal base mispairs in a transfection assay.
The fate of G.T mismatches and frameshifts, present at the 3'-terminus of primer-template or internally, has been studied with a combined transfection and electrophoretic assay following in vitro polymerization by DNA polymerase I (Klenow enzyme) of Escherichia coli. Several synthetic oligodeoxynucleotide primers were synthesized and annealed to uracil-containing single-stranded DNA of M13 phage bearing the lacI gene, to produce 1-3 consecutive G.T mismatches in the middle of the duplex region or at the 3'-OH end of the primer. Additional mismatched primer-templates were prepared, in which the primer had a deleted nucleotide, an extra nucleotide or both G.T mismatch and an extra nucleotide. The extension or degradation of these primers during in vitro DNA synthesis in the presence of all 4 dNTPs ('complete' reaction) or in the absence of dATP ('-A' reaction) was monitored by gel electrophoresis. Duplex DNA products were used in a transfection assay and the nucleotide changes in i-mutant progeny were determined by sequence analysis. The results suggest that whereas a single 3'-terminal G.T mismatch is relatively stable in chain elongation by Klenow enzyme, multiple terminal G.T mismatches are degraded by the 3'-exonuclease activity of this polymerase prior to primer extension. This editing activity is increased with the number of 3'-terminal mispairs. Single, double and triple T----C base substitutions were efficiently recovered when the mismatches occurred internally. Also, single-base eliminations or additions were readily recovered when the mutagenic primers contained an internal base deletion or addition, respectively. When products of the '-A' misincorporation reaction (catalyzed by Klenow enzyme) were assayed by transfection, base substitutions (exclusively T----C), but no frameshifts, were recovered. The results indicate that the absence of multiple tandem base substitutions among i- mutants recovered following primer elongation under mutagenic 'minus' conditions was due to the efficient action of the 3'-exonuclease activity of the Klenow enzyme on multiple terminal mismatches during in vitro polymerization, rather than to in vivo events (lack of expression or occurrence of mismatch repair) in the M13-lacI transfection assay.
Bacterial Proteins↗
Genetic assay of misincorporation.
A system to characterize mutations arising from in vitro nucleotide misincorporation, which avoids the effects of in vivo mismatch repair on recovery of mutants, was constructed and evaluated. The lacI gene of Escherichia coli was inserted into phage M13 and the M13-lacI recombinant was introduced into a strain of E. coli lacking a resident lacI gene. In this system the function of the M13-bearing lacI gene can be detected by plaque color. Mutants in the 5'-region of the lacI gene (encoding operator-binding domain) are seen as blue plaques when the host strain is grown in the presence of chromogenic substrate, X-gal, in the absence of inducer. The use of uracil-containing single stranded DNA from M13-lacI as template for DNA synthesis avoids the contribution of mismatch repair (in transfection recipients) on the recovery of mutants. To demonstrate the usefulness of the M13-lacI system we produced nucleotide misincorporations by in vitro DNA synthesis in the N-terminal region of the lacI template in the presence of only 3 deoxynucleoside triphosphates (dNTPs). Such mutagenic reactions were conducted in the absence of dATP with 4 different primers and in the absence of dGTP with 2 primers. The type of mutants produced by these reactions were identified through sequencing of DNA from progeny phage after screening for i- (blue plaque) phenotype. Mutations recovered in this system consisted of single and multiple base substitutions in the region of the template near the 3'-terminus of the primer. Nearly all of the mutants induced by '-A' conditions were T----C base substitutions, and those induced by '-G' conditions were C----T transitions. In general, the results were consistent with the spectrum of spontaneous mutants produced in strains deficient in mismatch repair, although some differences were noted. Several new base substitutions within the lacI gene (producing i- phenotype and unobserved by others) were isolated by the procedures described in this paper.
Bacteriophages↗
Influence of neighboring base sequence on mutagenesis induced by in vitro misincorporation in the lacI gene of Escherichia coli.
Genetic and electrophoretic assays of misincorporation were used to assess the effect of DNA sequence on mutagenesis arising from in vitro DNA synthesis within the lacI gene of Escherichia coli. The viral strand of a derivative of phage M13 containing the entire lacI gene was annealed with a series of synthetic oligonucleotides complementary to the N-terminal region of the lacI gene. Each primer-template was incubated with E. coli DNA polymerase I (Klenow fragment) under conditions favoring misincorporation, wherein one of the 4 dNTPs was lacking ('minus' reaction) or present at very low concentration ('micro' reaction). The extent of elongation of each primer was assessed by gel electrophoresis, and lacI mutants arising during the misincorporation reactions were detected by a transfection assay in which i- base substitutions within the in vitro synthesized strand were selectively recovered by the use of uracil-containing templates. Direct dideoxy sequencing of the '-A' reaction products and sequence analysis of i- mutant progeny revealed a vast predominance of single and non-tandem multiple base transitions. The addition of small quantities of dATP to a '-A' reaction increased the mutation yield and broadened the distribution of base substitutions along the template. We detected a general bias towards increased base substitution at template positions flanked by G.C base pairs or 5'-pyrimidine, 3'-purine nearest neighbors, although considerable site-to-site variation in the occurrence of base substitutions was seen, even within identical nearest neighbor contexts.
Base Sequence↗
Gene synthesis technology: recent developments and future prospects.
Gene synthesis is a potentially powerful tool in molecular biology that has not yet reached widespread use because of the relatively high cost and labor-intensive nature of the process. This paper reviews some recent technological developments and current research activities of this laboratory which promise to greatly reduce the cost of gene synthesis and to increase the speed and efficiency of the process. We recently developed an improved device for "segmented" synthesis of oligonucleotides, which utilizes porous Teflon wafers containing derivatized controlled pore glass supports to simultaneously synthesize up to 100 different DNA sequences. The stepwise coupling efficiency with the "wafer synthesis device" is as high as that attained with current automated "gene machines" producing 1-4 oligonucleotides at a time, whereas the reagent usage is only 20-50% that of the current DNA synthesizers. At present, we are optimizing the conditions for rapid, efficient assembly of genes on a solid-phase support, wherein ordered, stepwise annealing/washing is performed to segmentally elongate a "starting" oligonucleotide attached to a solid-phase support. We expect that the wafer synthesis device (operated at reduced scale of synthesis), together with solid-phase gene assembly, will permit the synthesis and assembly of an average size gene (1 kb) in one week at a cost of less than $1000. These developments should make gene synthesis a routine and powerful tool in molecular biology.
Biotechnology↗