Compositions and methods for processing and amplification of DNA, including using multiple enzymes in a single reaction

Abstract

The present invention concerns preparation of DNA molecules, such as a library, using a stem-loop oligonucleotide. In particular embodiments, the invention employs a single reaction mixture and conditions. In particular, at least part of the inverted palindrome is removed during the preparation of the molecules to facilitate amplification of the molecules. Thus, in specific embodiments, the DNA molecules are suitable for amplification and are not hindered by the presence of the palindrome.

1 . A method of preparing a nucleic acid molecule, comprising: 
 providing a double stranded nucleic acid molecule; and    attaching one strand of a stem-loop oligonucleotide comprising an inverted repeat and a loop to the double stranded nucleic acid molecule to produce an oligonucleotide-attached nucleic acid molecule.    
     
     
         2 . The method of  claim 1 , wherein the double stranded nucleic acid molecule is a double stranded DNA molecule.  
     
     
         3 . The method of  claim 1 , wherein the attaching is further defined as attaching the oligonucleotide to the double stranded nucleic acid molecule under conditions to produce a non-covalent junction in the oligonucleotide-attached nucleic acid molecule.  
     
     
         4 . The method of  claim 3 , wherein the non-covalent junction comprises a nick, a gap, or a 5′ flap structure.  
     
     
         5 . The method of  claim 1 , wherein the attaching is further defined as ligating.  
     
     
         6 . The method of  claim 1 , further comprising displacing one strand of the oligonucleotide from the oligonucleotide-attached nucleic acid molecule by strand displacement or by nick translation polymerization.  
     
     
         7 . The method of  claim 1 , wherein at least part of the oligonucleotide-attached nucleic acid molecule is amplified.  
     
     
         8 . The method of  claim 7 , wherein the amplification comprises polymerase chain reaction.  
     
     
         9 . The method of  claim 7 , wherein the amplification comprises RNA transcription.  
     
     
         10 . The method of  claim 7 , wherein the amplification comprises strand displacement.  
     
     
         11 . The method of  claim 1 , further comprising amplifying the oligonucleotide-attached nucleic acid molecule, wherein at least part of the inverted repeat is excluded from the amplified oligonucleotide-attached nucleic acid molecule.  
     
     
         12 . The method of  claim 5 , wherein the ligating is further defined as comprising: 
 generating ligatable ends on the double stranded nucleic acid molecule;    generating a ligatable end on the stem-loop oligonucleotide; and    ligating one strand of the ligatable end of the stem-loop oligonucleotide to one strand of an end of the nucleic acid molecule, thereby generating a non-covalent junction in the oligonucleotide-attached nucleic acid molecule.    
     
     
         13 . The method of  claim 1 , further defined as comprising: 
 generating blunt ends on the nucleic acid molecule;    generating a blunt end on the stem-loop oligonucleotide; and    ligating one strand of the blunt end of the stem-loop oligonucleotide to one strand of a blunt end of the nucleic acid molecule, thereby generating a nick in the oligonucleotide-ligated nucleic acid molecule.    
     
     
         14 . The method of  claim 1 , wherein said stem-loop oligonucleotide comprises a known sequence.  
     
     
         15 . The method of  claim 1 , wherein said stem-loop oligonucleotide comprises a regulatory sequence.  
     
     
         16 . The method of  claim 2 , further comprising: 
 digesting the DNA molecule with one or more endonucleases to produce DNA fragments;    producing blunt ends on the DNA fragments;    producing a blunt end on the stem-loop oligonucleotide; and    ligating one strand of the blunt end of a stem-loop oligonucleotide to one strand of a blunt end of a DNA fragment, thereby generating a nick in a oligonucleotide-ligated DNA fragment.    
     
     
         17 . The method of  claim 16 , wherein the endonuclease is a restriction endonuclease, DNAse I, or an apoptotic endonuclease.  
     
     
         18 . The method of  claim 17 , wherein the restriction endonuclease is methylation-specific or methylation-sensitive.  
     
     
         19 . The method of  claim 1 , wherein the oligonucleotide-attached nucleic acid molecule comprises a nick having a 3′ hydroxy group, wherein there is polymerization from the 3′ hydroxy group of at least part of the oligonucleotide-attached nucleic acid molecule.  
     
     
         20 . The method of  claim 5 , wherein the strand displacement or nick translation polymerization is further defined as polymerization that ceases at a non-replicable base or region in the loop or in a region of the stem adjacent to the loop.  
     
     
         21 . The method of  claim 2 , further comprising the step of digesting the double stranded DNA molecule with an endonuclease to generate DNA fragments, wherein the oligonucleotide becomes ligated to one strand of the DNA fragment and wherein polymerization of an oligonucleotide-ligated DNA fragment excludes at least part of the inverted repeat by subjecting the oligonucleotide-ligated DNA fragment to strand displacement or nick translation polymerization that halts at a base or sequence in the loop or in a region of the stem adjacent to the loop.  
     
     
         22 . The method of  claim 1 , wherein the stem-loop oligonucleotide is further defined as comprising a non-replicable base or sequence.  
     
     
         23 . The method of  claim 22 , wherein at least part of the non-replicable base or sequence is present in the loop of the oligonucleotide or in a sequence of the stem adjacent to the loop.  
     
     
         24 . The method of  claim 22 , wherein the non-replicable base or sequence comprises an abasic site or sequence, hexaethylene glycol, or a bulky chemical moiety attached to the sugar-phosphate backbone or the base.  
     
     
         25 . The method of  claim 24 , wherein the abasic site or sequence is introduced by one or more enzymes in the single solution.  
     
     
         26 . The method of  claim 1 , wherein the loop of the stem-loop oligonucleotide comprises at least one deoxy-uridine.  
     
     
         27 . The method of  claim 6 , wherein the strand displacement or nick translation polymerization of the oligonucleotide-attached nucleic acid molecule generates an endonuclease site.  
     
     
         28 . The method of  claim 2 , further comprising the step of digesting the double stranded DNA molecule with an endonuclease to generate DNA fragments, wherein the oligonucleotide becomes ligated to one strand of the DNA fragment to produce an oligonucleotide-ligated DNA fragment, and wherein strand displacement or nick translation polymerization of the oligonucleotide-ligated DNA fragment generates an endonuclease site.  
     
     
         29 . The method of  claim 27 , wherein the endonuclease site is a site-specific restriction endonuclease site and wherein at least part of the inverted repeat is removed by cleavage with said restriction endonuclease.  
     
     
         30 . The method of  claim 27 , wherein the endonuclease site resides within the stem and loop regions.  
     
     
         31 . The method of  claim 27 , wherein the endonuclease site resides within the loop region.  
     
     
         32 . The method of  claim 27 , wherein the endonuclease site resides within the stem region.  
     
     
         33 . The method of  claim 27 , wherein the endonuclease site is a homing endonuclease site.  
     
     
         34 . The method of  claim 15 , wherein RNA transcription is initiated from the regulatory sequence, thereby producing at least one transcribed polynucleotide.  
     
     
         35 . The method of  claim 34 , wherein the transcribed polynucleotide is replicated by a reverse transcriptase.  
     
     
         36 . The method of  claim 35 , wherein reverse transcription is initiated by hybridization of the oligonucleotide complementary to the 3′ end of the transcribed polynucleotide.  
     
     
         37 . The method of  claim 1 , wherein the stem-loop oligonucleotide further comprises a recognition sequence for a nicking endonuclease and wherein the oligonucleotide-attached nucleic acid molecule is amplified by strand displacement synthesis and second strand synthesis.  
     
     
         38 . The method of  claim 37 , wherein the recognition sequence for a nicking endonuclease resides at least in part within the stem of the stem-loop oligonucleotide.  
     
     
         39 . The method of  claim 37 , wherein the recognition sequence for a nicking endonuclease resides at least in part within the loop of the stem-loop oligonucleotide.  
     
     
         40 . The method of  claim 1 , wherein a 5′ end of the stem-loop oligonucleotide lacks a phosphate.  
     
     
         41 . The method of  claim 1 , wherein the oligonucleotide-attached nucleic acid molecule is further modified.  
     
     
         42 . The method of  claim 41 , wherein the further modification comprises cloning.  
     
     
         43 . The method of  claim 42 , further defined as comprising incorporation of the modified molecule into a vector, said incorporation occurring at ends in the modified molecule generated by endonuclease cleavage within the inverted repeat.  
     
     
         44 . The method of  claim 1 , further defined as occurring in a single suitable solution, wherein the process occurs in the absence of exogenous manipulation.  
     
     
         45 . The method of  claim 44 , further defined as occurring at one temperature.  
     
     
         46 . The method of  claim 44 , wherein the solution comprises one or more of the following: 
 ligase;    DNA polymerase;    one or more endonucleases;    RNA polymerase;    reverse transcriptase;    RNase H;    deoxy-uridine glycosylase;    nickase;    thermophilic DNA polymerase;    ATP;    rNTPs; and    dNTPs.    
     
     
         47 . The method of  claim 7 , further defined as occurring in a single suitable solution, wherein the process of preparing and amplifying of a oligonucleotide-attached nucleic acid molecule occurs in the absence of exogenous manipulation.  
     
     
         48 . The method of  claim 1 , wherein the oligonucleotide-attached nucleic acid molecule is immobilized to a solid support.  
     
     
         49 . The method of  claim 48 , wherein the molecule is immobilized non-covalently.  
     
     
         50 . The method of  claim 48 , wherein the molecule is immobilized covalently.  
     
     
         51 . The method of  claim 2 , further comprising the step of digesting the DNA molecule with an endonuclease to generate DNA fragments, wherein the oligonucleotide becomes attached to one strand of the DNA fragment, wherein strand displacement polymerization of the oligonucleotide-ligated DNA fragment and its arrest at a base or sequence in the loop or in a region of the stem adjacent to the loop generates a 5′ overhang, and wherein the single stranded 5′ overhang hybridizes to a complementary oligonucleotide covalently immobilized to a solid support.  
     
     
         52 . A method of preparing a DNA molecule, comprising: 
 providing an oligonucleotide; and    mixing the oligonucleotide with a double stranded DNA molecule, such that upon attachment of the oligonucleotide to the double stranded DNA molecule, the oligonucleotide attached to the DNA molecule is capable of demonstrating a function that it was incapable of before the attachment to the double stranded DNA molecule, thereby producing an oligonucleotide-attached DNA molecule suitable for modification.    
     
     
         53 . The method of  claim 52 , further defined as occurring in a single suitable solution, wherein the process occurs in the absence of exogenous manipulation.  
     
     
         54 . The method of  claim 52 , wherein the modification comprises site-specific nicking, site-specific double-strand cleavage, transcription, recombination, amplification, polymerization, nick translation, strand displacement, immobilization or a combination thereof.