Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries.

Abstract

An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A'T versus G C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.Short oligonucleotide probes [range, 14-20 base pairs (bp)] are commonly used to screen libraries of cloned DNA for genes of interest (1-3). Typically, these probes are pools representing all possible codon choices for a short amino acid sequence. Although this method has been successful, there is considerable uncertainty in the hybridization conditions because the binding of the oligonucleotides depends on two factors: (i) the length of the hybrid formed and (ii) the G-C content of the probe. Empirically determined formulas allow for estimation of the oligonucleotide dissociation temperature (Td) (4); however, these methods can be unsatisfactory when screening with pools of oligonucleotides. Although the length of the probes in the pool is constant, the individual probes differ considerably in G-C content, making suitably stringent and selective hybridization conditions difficult to find for all members of the pool. Thus, a large number of false positives can occur when screening highly complex libraries for genes of low abundance.We describe here the use of tetramethylammonium chloride (Me4NCl) in the hybridization of oligonucleotide probes to eliminate the dependence of Td on the G-C content of the probe, reducing the problem to a simple dependence on length of the hybrid. Tetraalkylammonium salts were found some years ago to bind to A+T-rich polymers of DNA (5) and have been used to abolish the preferential melting of A-T versus G-C base pairs for fragments of DNA (6). Me4NCl binds selectively to A-T base pairs, thus displacing the dissociation equilibrium and raising the melting temperature. At 3.0 M Me4NCl, this displacement is sufficient to shift the melting temperature of A-T base pairs to that of G-C base pairs (6). For the melting of long DNA, this shift results in a remarkable sharpening of the melting profile. Natural DNAs that melt over a range of 5 to 10°C in the presence of Na+ melt within 1°C in Me4NCl (6)(7)(8). The data presented here show the utility of hybridizations with Me4NCl when oligonucleotide probes are used. The Td values of probes from 11 to >1000 bp have been determined so that hybridization conditions for probes of various lengths can be chosen easily. This method is applicable to a variety of circumstances in which an exact match to a probe is desired. METHODS DNA Synthesis, Binding, and Labeling. In this procedure, nit...

Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides

“…Although mature miRNAs are only approximately 22 nt in length, they are much more abundant than mRNAs (Lim et al, 2003b), which suggested to us that an oligonucleotide probe with a single hapten could bind to a sufficient number of copies of a specific miRNA to compensate for the reduced number of haptens per probe. Highly stringent wash conditions have been developed for the detection of mismatches in DNA after hybridization with labeled DNA oligonucleotide probes, based on the use of tetramethyl ammonium chloride (TMAC) to make the melting temperature (Tm) of the oligonucleotide and its target a function of duplex length, independent of GC content (Wood et al, 1985;Jacobs et al, 1988;Litt et al, 1998). TMAC also reduces the melting temperature of an RNA:RNA duplex to that of an equivalent DNA: DNA duplex (Golas et al, 1980).…”

Southern molecular hybridization experiments with parallel complementary DNA probes

“…Encouraged by efficient hybridization of Southern blots containing cloned sequences with parallel complementary probes, we attempted to hybridize the probes with genomic Southern blots in 2x SSC solution, as well as in solutions containing 3 M tetramethylammonium chloride [16] or in 3 M tetramethylammonium bromide, but we observed a high background. Therefore we have used molecular hybridization in the agarose dried gels [10].…”

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

“…The design of the probes for screening mitochondrial LeuRS clones took into account the following considerations: (i) to minimize the number of oligonucleotide sequences represented, we omitted certain rare codons, found <5% of the time in most N. crassa genes (M. S. Sachs, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, 1986); (ii) to compensate for any losses of an exact match because of this omission, we relaxed the stringency of hybridization slightly; and (iii) to reduce the effects of differing G+C contents in the probes on hybridization, we used tetramethylammonium chloride, which stabilizes A T base pairing during the washes (64).…”