No abstract

The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean (3-thalassemia mutations (nine alleles).Differential hybridization with sequence-specific oligonucleotide probes has become a widely used technique for the detection of genetic mutations and polymorphisms (1)(2)(3)(4)(5). When hybridized under the appropriate conditions, these synthetic DNA probes (usually 15-20 bases in length) will anneal to their complementary target sequences in the sample DNA only if they are perfectly matched. In most cases, the destabilizing effect ofa single base-pair mismatch is sufficient to prevent the formation of a stable probe-target duplex (6). With an appropriate selection of oligonucleotide probes, the relevant genetic content of a DNA sample can be completely described.This very powerful method of DNA analysis has been greatly simplified by the in vitro DNA-amplification technique, the polymerase chain reaction (PCR) (7-9). The PCR can selectively increase the number of copies of a particular DNA segment in a sample by many orders of magnitude. As a result ofthis 106-to 108-fold amplification, more convenient assays and nonradioactive detection methods have become possible (10)(11)(12). These PCR-based assays are usually done by amplifying the target segment in the sample to be tested, fixing the amplified DNA onto a series of nylon membranes, and hybridizing each membrane with one of the labeled oligonucleotide probes under stringent hybridization conditions. However, each probe must still be individually hybridized to the amplified DNA and the process can easily become difficult in a system where many different mutations or polymorphisms occur.One approach t...

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.

We have targeted the d(G3A4G3).d(C3T4C3) duplex for triplex formation with d(G3T4G3) in the presence of MgCl2. The resulting triple helix, d(G3T4G3)*d(G3-A4G3).d(C3T4C3), is considerably weaker than the related triplex, d(G3A4G3)*d(G3A4G3).d(C3T4C3), and melts in a biphasic manner, with the third strand dissociating at temperatures about 20-30 degrees C below that of the remaining duplex. This is in distinct contrast to the d(G3A4G3)*d(G3A4G3).d(C3T4C3) triplex, which melts in essentially a single transition. Gel electrophoresis under non-denaturing conditions shows the presence of the d(G3T4G3)*d(G3A4G3).d(C3T4C3) triplex as a band of low mobility compared to the duplex or the single strand bands. Binding of the d(G3T4G3) third strand and the purine strand of the duplex can be monitored by imino proton NMR spectra. While these spectra are typically very broad for intermolecular triplexes, the line widths can be dramatically narrowed by the addition of two thymines to both ends of the pyrimidine strand. Thermodynamic analysis of UV melting curves shows that this triplex is considerably less stable than related triplexes formed with the same duplex. The orientation of the third strand was addressed by a combination of fluorescence energy transfer and UV melting experiments. Results from these experiments suggest that, in the unlabeled triplex, the preferred orientation of the third strand is parallel to the purine strand of the duplex.