MATERIALS AND METHODS FOR NUCLEIC ACID FRACTIONATION BY SOLID PHASE ENTRAPMENT AND ENZYME-MEDIATED DETACHMENT

• US 20110269194A1
• Filed: 04/20/2011
• Published: 11/03/2011
• Est. Priority Date: 04/20/2010
• Status: Abandoned Application

• Current Assignee: Swift Biosciences, Inc. (Integrated DNA Technologies, Inc.)
• Original Assignee: Swift Biosciences, Inc. (Integrated DNA Technologies, Inc.)
• Inventors: MAKAROV, VLADIMIR
• Application Number:
  US13/090,966
• Publication Number:
  US 20110269194A1
• Time in Patent Office: Days
• US Class Current: 435/91.53
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Materials and methods are provided for the gel-free fractionation of polynucleotide molecules. According to the present invention, fractionation is size-based or sequence-based.

Claims

1. A method of preparing a desired polynucleotide comprising contacting a labeled polynucleotide having a double-stranded region comprising a first strand and a second strand wherein either the first strand or second strand is labeled and immobilized with an enzyme or enzyme mixture under conditions wherein the interaction of the first strand with the second strand is reduced, thereby resulting in dissociation of the second strand from the first strand, wherein either an unlabeled first strand or unlabeled second strand is the desired polynucleotide.

2-92. -92. (canceled)

93. A composition comprising a labeled polynucleotide having a double-stranded region, the labeled polynucleotide comprising a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, the labeled polynucleotide further comprising a double-stranded target sequence between a double-stranded 5′

terminal sequence and a double-stranded 3′

terminal sequence, the 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent, the 5′

terminal sequence comprising one or more nuclease-resistant bases or one or more RNA residues.

94. A composition comprising a labeled polynucleotide having a double-stranded region, the labeled polynucleotide comprising a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, the labeled polynucleotide further comprising a double-stranded target sequence between a double-stranded 5′

terminal sequence and a single stranded 3′

terminal sequence, the 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent, the 5′

terminal sequence comprising one or more nuclease-resistant bases or one or more RNA residues.

95. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) optionally introducing a nick or gap at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner in the case of 5′

end attachment in step b);

d) introducing a nick or gap at the 5′

end of the double-stranded 3′

terminal sequence on the second strand distal to the solid phase binding partner;

thereby creating an extendable break in the 5′

end of the double-stranded 3′

terminal sequence on the second strand distal to the solid phase binding partner; and

f) contacting the labeled polynucleotide with an enzyme with 5′

-3′

exonuclease and polymerase activity under conditions wherein the second strand of the labeled polynucleotide is degraded up to a non-degradable location and a new second strand is synthesized up to a non-extendable location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

96. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing a nick or gap at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner;

d) introducing a nick or gap at the 5′

end of the double-stranded 3′

terminal sequence on the second strand distal to the solid phase binding partner; and

e) contacting the labeled polynucleotide with an enzyme with 5′

-3′

exonuclease and polymerase activity under conditions wherein the first strand and the second strand of the labeled polynucleotide are degraded, and a new first strand and a new second strand are synthesized, up to an enzyme collision location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

97. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) optionally introducing a nick or gap at the 5′

end of the double-stranded 5′

terminal sequence on the first′

strand proximal to the solid phase binding partner in the case of 5′

end attachment in step b);

d) introducing nick or gap at the 5′

end of the double-stranded 3′

terminal sequence on the second′

strand distal to the solid phase binding partner; and

e) contacting the labeled polynucleotide with an enzyme with polymerase activity under conditions wherein the second strand of the labeled polynucleotide is displaced, and a new second strand is synthesized up to a non-extendable location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

98. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing nick or gap at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner;

d) introducing nick or gap at the 5′

end of the double-stranded 3′

terminal sequence on the second strand distal to the solid phase binding partner; and

e) contacting the labeled polynucleotide with an enzyme with polymerase activity under conditions wherein the first strand and second strand of the labeled polynucleotide are displaced, and a new first strand and a new second strand are synthesized up to an enzyme collision location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

99. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing a nick or gap at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner;

d) introducing nuclease-resistant bases at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner, thereby creating a non-degradable break in the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner; and

e) contacting the labeled polynucleotide with an enzyme with 5′

-3′

exonuclease activity under conditions wherein the second strand of the labeled polynucleotide is degraded up to a non-degradable location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

100. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing nick or gap at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner; and

d) contacting the dsDNA fragment with an enzyme with 5′

-3′

exonuclease activity under conditions wherein the first strand and the second strand of the labeled polynucleotide are degraded up to an enzyme collision location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

101. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing nick or gap at the 3′

end of the double-stranded 5′

terminal sequence on the second strand proximal to the solid phase binding partner;

d) introducing nuclease-resistant bases at the 3′

end of the double-stranded 5′

terminal sequence on the second strand proximal to the solid phase binding partner, thereby creating a non-degradable break in the 3′

end of the double-stranded 5′

terminal sequence on the second strand proximal to the solid phase binding partner; and

e) contacting the dsDNA fragment with an enzyme with 3′

-5′

exonuclease activity under conditions wherein the first strand of the labeled polynucleotide is degraded up to a non-degradable location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

102. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing nick or gap at the 3′

end of the double-stranded 5′

terminal sequence on the second strand proximal to the solid phase binding partner; and

d) contacting the dsDNA fragment with an enzyme with 3′

-5′

exonuclease activity under conditions wherein the first strand and the second strand of the labeled polynucleotide are degraded up to an enzyme collision location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

103. (canceled)

104. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing a 5′

single-stranded overhang at the second strand of the double-stranded 3′

terminal sequence distal to the label; and

d) contacting the single-stranded overhang with an enzyme with 5′

-3′

helicase activity under conditions wherein the first strand of the labeled polynucleotide is separated from the second strand up to the end proximal to the solid phase binding partner, at which point the desired polynucleotide is separated from the labeled polynucleotide.

105. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus and a second strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises a double-stranded target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a double-stranded 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent and the 3′

terminus of the second strand and a sequence adjacent, the double-stranded 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent and the 5′

terminus of the second strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner or, in the alternative, attaching the 3′

end of the double-stranded 5′

terminal sequence on the second strand to a solid phase binding partner;

c) introducing a 3′

single-stranded overhang at the first strand of the double-stranded 3′

terminal sequence distal to the label; and

d) contacting the single-stranded overhang with an enzyme with 3′

-5′

helicase activity under conditions wherein the first strand of the labeled polynucleotide is separated from the second strand up to the end proximal to the solid phase binding partner, at which point the desired polynucleotide is separated from the labeled polynucleotide.

106. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand and a sequence adjacent which includes one or more RNA bases, the 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent;

b) immobilizing the labeled polynucleotide, said immobilizing comprising attaching the 5′

end of the double-stranded 5′

terminal sequence on the first strand to a solid phase binding partner;

c) hybridizing a first target-specific primer to the target sequence;

d) contacting the labeled polynucleotide and hybridized primer with an enzyme with polymerase activity under conditions wherein a second strand is synthesized, thereby creating a partially double-stranded fragment;

e) introducing a nick or gap in the partially double-stranded fragment at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner at the site of the one or more RNA bases;

f) introducing one dideoxynucleotide in the nick or gap of step e), thereby creating a non-extendable break in the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner;

g) removing any enzymes and unincorporated dideoxynucleotides from step f);

h) hybridizing a second primer to a sequence upstream of the first target-specific primer, said second primer selected fro the group consisting of;

(i) a second target-specific primer that hybridizes to a sequence on the target sequence; and

(ii) a universal primer that hybridizes to a sequence on the 3′

terminal sequence; and

i) contacting the labeled polynucleotide and hybridized second primer with an enzyme with exonuclease and polymerase activity or, in the alternative, with an enzyme with polymerase-strand-displacement activity, under conditions wherein a new second strand is synthesized up to a non-extendable location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

107. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a labeled polynucleotide, wherein the labeled polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus, and wherein the labeled polynucleotide comprises target sequence between a double-stranded 5′

terminal sequence located proximal to the label, and a 3′

terminal sequence located distal to the label, the double-stranded 5′

terminal sequence including the 5′

terminus of the first strand that contains 5 or more RNA bases, the 3′

terminal sequence including the 3′

terminus of the first strand and a sequence adjacent;

b) hybridizing a first target-specific primer to the target sequence;

c) contacting the labeled polynucleotide and hybridized primer with an enzyme with polymerase activity under conditions wherein a second strand is synthesized, thereby creating a partially double-stranded fragment;

d) removing any enzymes and unincorporated deoxynucleotides from step c);

e) introducing a break at the 5′

end of the double-stranded 5′

terminal sequence on the first strand proximal to the solid phase binding partner by removing the 5 or more RNA bases;

f) hybridizing labeled probe in the break introduced in step e), wherein said probe is blocked from extension on the 3′

end and is attached to a solid support, thereby immobilizing the labeled polynucleotide;

g) hybridizing a second primer to a sequence upstream of the first target-specific primer, said second primer selected fro the group consisting of;

(i) a second target-specific primer that hybridizes to a sequence on the target sequence; and

(ii) a universal primer that hybridizes to a sequence on the 3′

terminal sequence; and

h) contacting the labeled polynucleotide and hybridized second primer with an enzyme with exonuclease and polymerase activity under conditions wherein a new second strand is synthesized up to a non-extendable location, at which point the desired polynucleotide is separated from the labeled polynucleotide.

108. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a polynucleotide, wherein the polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus;

b) hybridizing a labeled probe to the 5′

end of the first strand, wherein said probe is attached to a solid support, thereby immobilizing the labeled polynucleotide;

c) hybridizing a target-specific primer to a sequence on the first strand that is 3′

to the hybridized probe; and

d) contacting the labeled polynucleotide and hybridized primer with an enzyme with polymerase activity under conditions wherein a second strand is synthesized, thereby creating a partially double-stranded fragment that is separated from the probe.

109. A method of preparing a desired polynucleotide comprising the steps of:

a) preparing a polynucleotide, wherein the polynucleotide comprises a first strand having a 5′

terminus and a 3′

terminus;

b) hybridizing a target-specific, labeled probe to a sequence on the first strand, wherein said probe is attached to a solid support, thereby immobilizing the labeled polynucleotide;

c) hybridizing a target-specific primer to a sequence on the first strand that is 3′

to the hybridized probe; and

d) contacting the labeled polynucleotide and hybridized primer with an enzyme with polymerase activity under conditions wherein a second strand is synthesized, thereby creating a partially double-stranded fragment that is separated from the probe.