Methods for simplifying microbial nucleic acids by chemical modification of cytosines
Inventors
Millar, Douglas Spencer • Miklos, George L. Gabor
Assignees
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Publication Number
US-7833942-B2
Publication Date
2010-11-16
Expiration Date
Abstract
A method for simplification of a microbial genome or microbial nucleic acid comprising treating microbial genome or nucleic acid with an agent that modifies cytosine to form derivative microbial nucleic acid and amplifying the derivative microbial nucleic acid to produce a simplified form of the microbial genome or nucleic acid.
Core Innovation
The invention simplifies microbial genomes and microbial nucleic acids by reducing base complexity through generating a derivative or simplified microbial genome or derivative or simplified microbial nucleic acid in which substantially all positions naturally occupied by cytosines are occupied by uracil or are replaced by thymine. This reduction creates a derivative or simplified nucleic acid whose nucleotide sequence is not found in the microbial genome or microbial nucleic acid in which the cytosines have not been occupied by uracil or thymine.
From the derivative or simplified microbial genome, the invention obtains a microbial specific nucleic acid that comprises at least a portion of the derivative or simplified microbial genome or derivative or simplified microbial nucleic acid. The invention also supports obtaining or identifying a microbial specific nucleic acid sequence by forming a simplified form with substantially bases selected from adenine, guanine and thymine such that the simplified form has a nucleotide sequence not found in the microbial genome or microbial nucleic acid without the cytosine conversion.
The invention further provides for producing and selecting microbial-specific nucleic acid molecules by treating microbial derived DNA or microbial nucleic acid with an agent that modifies cytosine to uracil, producing a simplified nucleic acid molecule by amplifying at least part of the derivative microbial nucleic acid, and selecting sequences from the simplified form that are not found in the unconverted microbial DNA. The documented approach includes reducing relative genomic complexity, including reduced probe/primer combinations after bisulfite conversion, and applying the resulting simplified sequences to detection strategies including primer/probe design and detection approaches such as polymerase chain reaction (PCR), isothermal/signal amplification, ligand-binding detection, electrophoresis, and microarrays.
Claims Coverage
The independent claims are clm-00001, clm-00014, clm-00019, clm-00029, clm-00047, and clm-00050. They focus on cytosine modification-driven sequence simplification, generating sequences not found in untreated microbial genomes, producing microbial-specific nucleic acids by amplification, selecting simplified-form microbial-specific sequences, and detecting microorganisms using amplification products that are not found in untreated microbial genomes.
Cytosine-to-uracil or cytosine-to-thymine sequence simplification for obtaining microbial specific nucleic acids
A method for obtaining a microbial specific nucleic acid by reducing the complexity of a microbial genome or microbial nucleic acid through generating a derivative or simplified microbial genome or derivative or simplified microbial nucleic acid in which substantially all positions naturally occupied by cytosines are occupied by uracil or replaced by thymine, so that the derivative or simplified nucleic acid has a nucleotide sequence not found in the untreated microbial genome or microbial nucleic acid; and obtaining a microbial specific nucleic acid from the derivative or simplified microbial genome, wherein the microbial-specific nucleic acid comprises at least a portion of the derivative or simplified microbial genome.
Changing substantially all cytosines to thymine for microbial-specific nucleic acid sequences
A method for obtaining or identifying a microbial specific nucleic acid sequence by obtaining a nucleic acid sequence; reducing base complexity by changing substantially all cytosines in the sequence to thymine to form a simplified nucleic acid sequence containing substantially only bases selected from adenine, guanine and thymine such that the simplified nucleic acid has a nucleotide sequence not found in the microbial genome or microbial nucleic acid where substantially all cytosines have not been changed to thymine; and obtaining or identifying a microbial specific nucleic acid sequence from the simplified nucleic acid sequence, wherein the microbial-specific nucleic acid comprises at least a portion of the simplified nucleic acid.
Cytosine-to-uracil modification and amplification of microbial nucleic acid
A method for simplification of a microbial nucleic acid by treating microbial nucleic acid with an agent that modifies cytosine to uracil to form derivative microbial nucleic acid such that the derivative microbial nucleic acid has a nucleotide sequence not found in the microbial nucleic acid which has not been treated with the agent; and producing a simplified form by amplifying the derivative microbial nucleic acid.
Producing microorganism-specific nucleic acids from cytosine-to-uracil treated microbial-derived DNA
A method for producing a microbial-specific nucleic acid molecule by treating a sample containing microbial derived DNA with an agent that modifies cytosine to uracil to form derivative microbial nucleic acid such that the derivative microbial nucleic acid has a nucleotide sequence not found in the untreated microbial derived DNA; and producing a simplified nucleic acid molecule by amplifying at least part of the derivative microbial nucleic acid, the simplified nucleic acid molecule having a reduced total number of cytosines compared with the corresponding untreated microbial nucleic acid and comprising a nucleic acid sequence specific for a microorganism or microorganism type.
Selecting microbial-specific sequences using simplified forms
A method for selecting a sequence of a microbial-specific nucleic acid molecule by obtaining a DNA sequence from a microorganism; forming a simplified form of the microbial DNA sequence by changing substantially all cytosines to thymine such that the simplified form comprises substantially bases adenine, guanine and thymine and has a nucleotide sequence not found in the untreated microbial DNA sequence, wherein the conversion mimics the action of an agent that converts cytosine to uracil; and selecting a microbial-specific nucleic acid sequence from the simplified form.
Detecting microorganisms by amplification of cytosine-to-uracil treated nucleic acid
A method for detecting the presence of a microorganism in a sample by treating a microbial nucleic acid with an agent that modifies cytosine to uracil to form derivative microbial nucleic acid; providing primers capable of allowing amplification of a desired microbial-specific nucleic acid molecule to the derivative microbial nucleic acid, wherein the desired microbial-specific nucleic acid comprises at least a portion of the derivative microbial nucleic acid; carrying out an amplification reaction on the derivative microbial nucleic acid to generate an amplification product having a nucleotide sequence not found in an untreated microbial genome; and assaying for the presence of the amplification product containing the desired microbial-specific nucleic acid molecule.
The core coverage is sequence simplification driven by cytosine modification to uracil or to thymine, creating sequences not found in untreated microbial genomes, followed by obtaining, producing, selection, or detection of microbial-specific nucleic acids by amplification or selection from simplified forms.
Stated Advantages
Reduces the complexity of the microbial genome or microbial nucleic acid by generating a derivative or simplified microbial genome or derivative or simplified microbial nucleic acid.
Produces a derivative or simplified nucleic acid whose nucleotide sequence is not found in a microbial genome or microbial nucleic acid that has not been subjected to the cytosine occupation or replacement.
Allows obtaining microbial specific nucleic acids comprising at least a portion of the derivative or simplified microbial genome or derivative or simplified microbial nucleic acid.
Allows detection of the presence of a microorganism by assaying for amplification products having nucleotide sequences not found in untreated microbial genomes, where detection is indicative of the presence of the microorganism.
Documented Applications
Primer/probe design and detection strategies for microbial-specific sequences, including PCR, isothermal/signal amplification, ligand-binding detection, electrophoresis, and microarrays.
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