Other

Yang, Zunyi

Benner, Steven a.

Zunyi Yang

Steven Albert Benner

Nicole Aurora Leal

Roberto Laos

Myong Jung Kim

Hyo-Joong Kim

Nilesh Karalkar

Steven A Benner

This invention covers processes that amplify multiple DNA and RNA targets, solving the 'multiplexed PCR problem', as well as supporting isothermal amplification and delivering other capabilities in multiplexed molecular analysis. It uses inventive DNA analogs.

Nucleotide analogs for efficient DNA and RNA amplification

The disclosed invention teaches processes to create a Watson-Crick complementary copy of a preselected oligonucleotide that contain non-standard nucleotides, which form nucleobase pairs fitting the standard Watson-Crick geometry, but here said pairs are joined by hydrogen bonding patterns different from those that join standard A:T and G:C pairs. The invention further relates to polymerases that incorporate those non-standard nucleotide analogs into oligonucleotide products using the corresponding triphosphate derivatives.

Polymerase incorporation of non-standard nucleotides

This invention relates to processes that amplify oligonucleotide analogs that incorporate non-standard nucleobase analogs from an artificially expanded genetic information system. These pair in DNA duplexes via patterns of hydrogen bonds that differ from patterns that join the thymine-adenine and guanine-cytosine nucleobase pairs.

Amplification of oligonucleotides containing non-standard nucleotides

This invention relates to the use of analogs of purines that have two tautomeric forms, one formally complementary to thymidine, the other formally complementary to cytidine, and analogs of pyrimidines that have two tautomeric forms, one formally complementary to guanosine, the other formally complementary to adenosine. These uses include the polymerase chain reaction amplification of targets having polymorphisms in primer binding sites and ligation.

Applications for biversal nucleoside analogs

Disclosed are processes that use DNA polymerases to extend primers annealed to templates, wherein a standard nucleotide in the template guiding the extension directs the incorporation of a nonstandard nucleotide analog into the product duplex opposite that standard template nucleotide, and processes wherein a nonstandard nucleotide in the template guiding the extension directs the incorporation of a standard nucleotide analog opposite the nonstandard template nucleotide. Conditions, including the pH of the mixture where the primer extension is done and the composition of triphosphate mixtures where the primary extensions done, are disclosed.

In vivo conversion of nucleosides in plasmid DNA

This invention provides for products and processes for binding to a preselected target, where the process involves contacting this target to an oligonucleotide molecule that contains one or more 'non-standard' nucleotides, which are nucleotide analogs that, when incorporated into oligonucleotides (DNA or RNA, collectively xNA), present to a complementary strand in a Watson-Crick pairing geometry a pattern of hydrogen bonds that is different from the pattern presented by adenine, guanine, cytosine, and uracil. This disclosure provides an example where such an oligonucleotide molecule contains a single 2-amino-8-(1'-&#x3b2;-D-2&#x2032;-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)one and a single 6-amino-5-nitro-3-(1&#x2032;-&#x3b2;-D-2&#x2032;-deoxyribofuranosyl)-2(1H)-pyridone, and where the target is a cell, and is obtained by a process of in vitro selection.

In vitro selection with expanded genetic alphabets

This invention relates to processes that amplify, in a polymerase chain reaction architecture, oligonucleotide analogs that incorporate non-standard nucleobase analogs from an artificially expanded genetic information system. These pair in DNA duplexes via patterns of hydrogen bonds that are different from patterns that join the thymine-adenine and guanine-cytosine nucleobase pairs.

Amplification of oligonucleotides containing non-standard nucleotides by polymerase chain reactions

This invention covers processes for the isothermal amplification of DNA molecules having a preselected sequence. It is based on the unexpected discovery that primers having, at some positions, adenine substituted by 2-aminopurine or diaminopurine, guanine by inosine, thymine by 2-thiothymine, and cytosine by N4-ethylcytosine ('SAMRS nucleotides') were accepted by enzymes used in the standard helicase-dependent amplification (HDA). Further unexpected was the discovery that target nucleotides are efficiently amplified in an HDA-like process (hereinafter abbreviated as simply HDA) using substituted primers. Also discovered was the diminution of spurious products through the use of SAMRS-substituted primers.

Helicase dependent amplification of DNA molecules using nucleotide analogs

The disclosed invention teaches processes to amplify oligonucleotides by contacting templates and primers with DNA polymerases and triphosphates of non-standard nucleotides, which form nucleobase pairs fitting the standard Watson-Crick geometry, but joined by hydrogen bonding patterns different from those that join standard A:T and G:C pairs. Thus, this invention relates to nucleotide analogs and their derivatives that, when incorporated into DNA and RNA, expand the number of replicatable nucleotides beyond the four found in standard DNA and RNA. The invention further relates to polymerases that incorporate those non-standard nucleotide analogs into oligonucleotide products using the corresponding triphosphate derivatives, and more specifically, polymerases and non-standard nucleoside triphosphates that support the polymerase chain reaction (PCR), including PCR where the products contain more than one non-standard nucleotide unit.

This invention enables processes for extracting from a mixture of oligonucleotide molecules individuals that bind to a preselected target (aptamers) or catalyze a preselected reaction (xNAzymes) that contain one or more non-standard nucleotides. These pair with their complements in a Watson-Crick geometry with a pattern of hydrogen bonds different from that pairing adenine and thymine, and guanine and cytosine. The processes comprise (a) obtaining this mixture containing non-standard nucleotides within preselected regions, (b) contacting the mixture with a preselected target or one or more reactants for the reaction whose catalysis is desired, (c) separating oligonucleotides having a greater affinity to the target or catalytic effectiveness from the remainder that have less affinity or catalytic effectiveness, and (d) amplifying the separated oligonucleotides. The process exploits PCR conditions that amplify oligonucleotides with less than 5% loss of the non-standard nucleotide per cycle, and processes to determine the sequences of the amplified oligonucleotides.

Growing community of inventors

Gainesville, FL, United States of America

Zunyi Yang