The invention describes a new method to sequence DNA. The improvements over the existing DNA sequencing technologies are high speed, high throughput, no electrophoresis and gel reading artifacts due to the complete absence of an electrophoretic step, and no costly reagents involving various substitutions with stable isotopes. The invention utilizes the Sanger sequencing strategy and assembles the sequence information by analysis of the nested fragments obtained by base-specific chain termination via their different molecular masses using mass spectrometry, as for example, MALDI or ES mass spectrometry. A further increase in throughput can be obtained by introducing mass-modifications in the oligonucleotide primer, chain-terminating nucleoside triphosphates and/or in the chain-elongating nucleoside triphosphates, as well as using integrated tag sequences which allow multiplexing by hybridization of tag specific probes with mass differentiated molecular weights.

Dna sequencing by mass spectrometry

The invention provides fast and highly accurate mass spectrometer based processes for detecting a particular nucleic acid sequence in a biological sample. Depending on the sequence to be detected, the processes can be used, for example, to diagnose a genetic disease or chromosomal abnormality; a predisposition to a disease or condition, infection by a pathogenic organism, or for determining identity or heredity.

DNA diagnostics based on mass spectrometry

Methods for determining the sequence of nucleic acids by cleaving the nucleic acid unilaterally from a first end with an exonuclease activity to sequentially release individual nucleotides, identifying each of the sequentially release nucleotides by mass spectrometry, and determining the sequence of the nucleic acid from the identified nucleotides are disclosed. The method is amenable to multiplexing for simulataneously determining more than one nuleic acid sequence.

Dna sequencing by mass spectrometry via exonuclease degradation

β-Cyanoethyl N,N-dialkylamino/N-morpholinomonochloro phosphoamidites, new phosphitylating agents facilitating ease of deprotection and work-up of synthesized oligonucleotides

High-resolution gel electrophoresis has been used to detect and quantitate promoter-specific oligonucleotides produced during initiation of transcription in vitro at the lactose operon (lac) UV5 promoter The resolved products are RNA species of various lengths which correspond to the initial lac mRNA sequence Quantitation shows that many oligonucleotides can be formed per preinitiation complex, including species as long as hexanucleotide Synthesis occurs without dissociation of the enzyme, as evidenced by levels of synthesis in the presence of heparin, a selective inhibitor of free RNA polymerase Thus, RNA polymerase cycles at this promoter in vitro producing oligonucleotides reiteratively In general, the yield of oligonucleotides decreases when the total concentration of all four substrates is increased or when a missing nucleoside triphosphate substrate is added Nevertheless, oligonucleotide synthesis persists under all conditions tested Strikingly, the dinucleotide always represents 50% of the total of all oligonucleotides, even when conditions are manipulated to cause a 100-fold variation in this total This shows that, after formation of the first phosphodiester bond at the lac UV5 promoter, dissociation of the dinucleotide is as likely as formation of the second phosphodiester bond As discussed above, after release of a small RNA, RNA polymerase may then begin another RNA chain, which is again subject to premature release These considerations lead to a model in which RNA polymerase cycles to produce oligonucleotides during initiation of transcription at the lac UV5 promoter in vitro Production of a long RNA transcript is then essentially an escape from this cycling reaction The drug rifampicin, which drastically inhibits escape to produce RNA, limits, but dose not prevent, the cycling reaction

Cycling of ribonucleic acid polymerase to produce oligonucleotides during initiation in vitro at the lac UV5 promoter.

The specific influence of the four nucleobases on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels under denaturing and nondenaturing conditions has been investigated using homooligomers from the four deoxyribonucleotides as chain length standards. Homooligomers of same chain lengths exhibit remarkable differences in mobility. Specific retardation of any other oligonucleotide investigated was found to be mainly dependent on base composition but not on sequence. A simple procedure is presented for calculating mobilities relative to the standards on denaturing gels. This allows a reliable identification of oligonucleotides on acrylamide-gels by exact chain length determination with respect to base composition and furthermore a detailed interpretation of complex reaction mixtures. The homooligomers also show the same differences in mobility on nondenaturing gels. The significance of this effect for strand separation is discussed.

/pdf/dna-chain-length-markers-and-the-influence-of-base-3xfia5vdwy.pdf

R. Frank

Papers

DNA chain length markers and the influence of base composition on electrophoretic mobility of oligodeoxyribonucleotides in plyacrylamide-gels