Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols.

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4'-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5' hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended.

Improving random mutagenesis by purification of the oligonucleotide variants.

An oligonucleotide-based mutagenesis method is presented where, contrary to most classical mutagenic approaches, preselection of the variants is performed at the oligonucleotide level to avoid cloning of non-desired sequences. The method relies on the generation of differentially phosphate-protected oligonucleotides. Protection of the phosphates is accomplished by substoichiometric incorporation of an Fmoc-protected and n-propyl-protected trinucleotide phosphoramidite during ordinary oligonucleotide assembly. Instead of the alkali-labile beta-cyanoethyl group introduced in ordinary assembly, the trinucleotide introduces the alkali-stable n-propyl group. As a result, single mutants carry three ionic phosphates less than the wild-type sequence, double mutants carry six ionic phosphates less and so on. This difference in ionic ratio enables separation of the variants by conventional polyacrilamide gel electrophoresis. In the exemplified library described herein, two sub-populations containing mainly triple and quadruple mutants were selected out of five possible sub-populations.

Combination of DMT-mononucleotide and Fmoc-trinucleotide phosphoramidites in oligonucleotide synthesis affords an automatable codon-level mutagenesis method.

BACKGROUND: Synthetic DNA has been used to introduce variability into protein-coding regions. In protocols that produce a few mutations per gene, the sampling of amino-acid sequence space is limited by the bias imposed by the genetic code. It has long been apparent that the incorporation of trinucleotides in the synthetic regime would circumvent this problem and significantly enhance the usefulness of the technique. RESULTS: A new method is described for the creation of codon-level degenerate oligodeoxyribonucleotides that combines conventional dimethoxytrityl (DMT) mononucleoside phosphoramidite chemistry with 9-fluorenylmethoxycarbonyl (Fmoc) trinucleotide phosphoramidites (whose synthesis is reported in the paper). The substoichiometric use of these Fmoc-trinucleotides in an automatable, solid-phase synthesis procedure afforded DNA fragments comprising the wild-type sequence and a controllable distribution of mutants within two- and three-codon stretches of DNA, within the multiple cloning site of the conventional cloning vector pUC19. CONCLUSIONS: DMT and Fmoc are compatible protecting groups in conventional oligonucleotide synthesis methods, resulting in controllable levels of codon-based mutagenesis.

Sequence-specific detection of PCR-amplified DNA by restriction enzyme release of hybrids.

We have developed a method to detect PCR amplicons that contain a restriction enzyme site. The method is called Polymerase Chain Reaction-Hybridization and Enzymatic Release Assay (PCR-HERA). The PCR product is hybridized to a synthetic oligonucleotide that is immobilized on a microtitre plate. Specific cleavage of the resulting hybrid by a restriction endonuclease liberates a fluorescein labeled arm of the immobilized DNA. We demonstrate the use of PCR-HERA to detect species-specific sequences of extra-chromosomal, ribosomal DNA, of Entamoeba histolytica. This highly specific assay does not require any washing steps and can be easily automated.

Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia.

Multicolor spectral karyotyping allows simultaneous visualization of all human chromosomes and screening for chromosomal rearrangements without a priori knowledge of any abnormalities involved. Based on this potentially increased sensitivity, we investigated, in a preliminary manner, whether spectral karyotyping could detect cytogenetic aberrations in karyotypically normal leukemia. The test population was comprised of 28 cryopreserved, cytogenetically normal acute myeloid leukemia (AML) samples from patients registered to a randomized trial for previously untreated AML (SWOG 9031). Two normal and 12 samples with known cytogenetic aberrations were used to validate and establish the diagnostic accuracy of the spectral karyotyping assay and instrumentation in a clinical setting. Enumeration and region-specific DNA fluorescence in situ hybridization (FISH) probes verified discrepant results. In the validation data set, spectral karyotyping refined complex karyotypic rearrangements in six cases and defined the chromosomal origin of a "jumping" homogeneously staining region; however, the technology was less sensitive in the detection of subtelomeric rearrangements and double minute chromosomes. In the test population, spectral karyotyping identified previously undetected cytogenetic aberrations in two cases (7%) of karyotypically normal AML: a cryptic 11q23 translocation in 20/20 cells and a minor monosomy 7 clone in 3/21 cells (FISH, 10.5%). Both of these abnormalities are considered to confer a poor prognosis when based on classical cytogenetic prognostic criteria. As an adjunct to classical cytogenetics and standard FISH analyses, the additive resolution of spectral karyotyping, in particular, with chromosome paints spiked with subtelomeric and/or locus-specific probes, may allow significant gains to be made in diagnostic accuracy and recognition of genotype/phenotype prognostic relationships, and in defining underlying biologic mechanisms in cancer. Genes Chromosomes Cancer 28:318-328, 2000.