Methods for fragmenting nucleic acid
AssigneeAffymetrix, Inc.
InventorsKai Wu, Charles Miyada, Thong Nguyen
Life of this patent
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Abstract
Methods for using an apurinic/apyrimidinic endonuclease, capable of cleaving both single- and double-stranded cDNA, for fragmentation and labeling of single stranded or double stranded DNA molecules are provided. Amplification methods that generate single-stranded amplified cDNA are also disclosed. In the subject methods AP sites in a population of nucleic acids are cleaved by an AP endonuclease that is active on both double and single stranded DNA. Fragments may be end labeled. In preferred embodiments APE 1 is used. The methods may be used in a variety of applications where end-labeling single or double stranded DNA is desired.
Claims41
A method for obtaining a nucleic acid amplification product comprising labeled cDNA fragments from a nucleic acid sample containing RNA, the method comprising:
- a.) providing a first nucleic acid sample comprising RNA;
- b.) amplifying the first nucleic acid sample to obtain a second nucleic acid sample comprising single stranded cDNA, wherein said single stranded cDNA contains uracil;
- c.) cleaving the single stranded cDNA by a method comprising incubating the single stranded cDNA in a reaction with UDG and an AP endonuclease, wherein said AP endonuclease is active on single stranded cDNA, to generate single-stranded cDNA fragments;
- and
- d.) labeling said single stranded cDNA fragments in a reaction comprising TdT and at least one labeled nucleotide to obtain labeled cDNA fragments.
A method for analyzing the expression of a plurality of genes in a sample, said method comprising:
- a.) obtaining a first nucleic acid sample comprising mRNA from said sample;
- b.) generating a second nucleic acid sample comprising cDNA by a method comprising mixing said first nucleic acid sample in a reaction comprising a primer including a 3′ portion comprising random sequence and a 5′ portion including an RNA polymerase promoter sequence and a reverse transcriptase;
- c.) generating a third nucleic acid sample comprising second strand cDNA by a method comprising mixing said second nucleic acid sample in a reaction comprising RNase H and a DNA polymerase:
- d.) generating a fourth nucleic acid sample comprising cRNA by a method comprising mixing said third nucleic acid sample with an RNA polymerase;
- e.) generating a fifth nucleic acid sample comprising first strand cDNA by a method comprising mixing said fourth nucleic acid sample with random primers, a reverse transcriptase, dTTP, dGTP, dCTP, dATP and dUTP;
- f.) generating a sixth nucleic acid sample comprising sense orientation single stranded cDNA by a method comprising mixing said fifth nucleic acid sample in a reaction comprising RNase H;
- g.) fragmenting said sixth nucleic acid sample in a reaction comprising UDG and APE 1 to obtain single stranded cDNA fragments;
- h.) labeling said single stranded cDNA fragments in a reaction comprising terminal transferase and a labeled nucleotide to obtain labeled fragments;
- i.) hybridizing said labeled fragments to an array comprising more than 100,000 probes to generate a hybridization pattern;
- and
- j.) analyzing said hybridization pattern.
A method to determine the efficiency of fragmentation of a complex nucleic acid sample by a UDG and APE 1 mediated fragmentation process comprising:
- a.) obtaining a control oligonucleotide wherein said control oligonucleotide comprises a 5′ first region and a 3′ second region separated by at least one uracil or at least one abasic position, wherein the 3′ end of the control oligonucleotide is not a substrate for terminal labeling by TdT;
- b.) adding an aliquot of said control oligonucleotide to said complex nucleic acid sample to generate a mixture;
- c.) treating said mixture with a UDG activity and an APE 1 activity to obtain fragments, wherein said control oligonucleotide is cleaved into a first fragment comprising said first region and a second fragment comprising said second region;
- d.) labeling at least some of the products of step c.) in a reaction comprising TdT;
- e.) hybridizing at least some of the products of step d.) to a microarray, wherein the microarray comprises probes for the first region of the control oligonucleotide;
- and
- f.) analyzing the hybridization pattern to determine the efficiency of fragmentation of the control oligonucleotide.
A control oligonucleotide comprising from the 5′ end, a first region, a cleavage position, a second region and a 3′ terminal modification blocking 3′ extension or labeling of the control oligonucleotide at its 3′ end.
A method for obtaining a nucleic acid amplification product comprising labeled cDNA fragments from a nucleic acid sample containing RNA, the method comprising:
- a.) providing a first nucleic acid sample comprising RNA;
- b.) synthesizing single-stranded cDNA containing uracil from said RNA in a reaction comprising a reverse transcriptase, random primers, dUTP, dGTP, dCTP, dATP and dTTP;
- c.) cleaving the single stranded cDNA by a method comprising incubating the single stranded cDNA in a reaction with UDG and an AP endonuclease, wherein said AP endonuclease is active on single stranded cDNA, to generate single-stranded cDNA fragments;
- and
- d.) labeling said single stranded cDNA fragments in a reaction comprising TdT and at least one labeled nucleotide to obtain labeled cDNA fragments.
A kit comprising a solution of T7-N6 primers, buffer, DTT, dGTP, dCTP, dATP, a solution of dTTP and dUTP, an RNase inhibitor, a reverse transcriptase, a DNA polymerase, APE 1, and random primers, wherein the ratio of dTTP to dUTP in the solution of dTTP and dUTP is about 4 to 1.
The kit of claim 39 further comprising a solution of random primers.
Dependencies are inferred from the claim text.