CELL FREE CLONING OF NUCLEIC ACIDS

• US 20160251651A1
• Filed: 05/16/2016
• Published: 09/01/2016
• Est. Priority Date: 08/05/2014
• Status: Active Application

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: BANYAI, William, PECK, Bill James, FERNANDEZ, Andres, CHEN, Siyuan, INDERMUHLE, Pierre
• Application Number:
  US15/156,134
• Publication Number:
  US 20160251651A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/626 being a flow cytometer

Abstract

Methods and devices for cell-free sorting and cloning of nucleic acid libraries are provided herein.

Claims

1-93. -93. (canceled)

94. A method for nucleic acid sorting comprising:

(a) providing a plurality of circular double-stranded nucleic acids, each of the plurality of circular double-stranded nucleic acids comprising a first strand that is a continuous circle and a second strand that comprises a gap, wherein the gap has a length of at least one base;

(b) diluting the plurality of circular double-stranded nucleic acids to a concentration of less than 100 nM;

(c) extending the second strand a first amplification reaction, wherein the first strand is a template strand, thereby forming a plurality of amplicon nucleic acids comprising a plurality of copies of the first strand; and

(d) partitioning such that on average there are 0.1 to 10 amplicon nucleic acids per fraction.

95. The method of claim 94, wherein the second strand is a primer in the first amplification reaction, and wherein the first amplification reaction is performed without an additional primer sequence.

96. The method of claim 94, wherein the second strand is at least about 500 bases in length.

97. The method of claim 94, wherein the plurality of circular double-stranded nucleic acids comprises at least about 100 nucleic acids comprising at least 500 bases in length.

98. The method of claim 97, wherein the second strand comprises a nucleic acid sequence that differs in at least 7 bases from another second strand in the plurality of circular double-stranded nucleic acids.

99. The method of claim 94, wherein the gap is 1 to 5 bases in length.

100. The method of claim 94, wherein the plurality of circular double-stranded nucleic acids is formed by ligating a double-stranded vector to a double-stranded non-circularized nucleic acid, and wherein the vector anneals to a 5′

end and a 3′

end of the double-stranded non-circularized nucleic acid.

101. The method of claim 100, wherein the non-circularized double-stranded nucleic acid or the double-stranded vector comprises a strand having 1 to 10 fewer bases than a complementary strand, and wherein the 1 to 10 fewer bases corresponds to the length of the gap in the circular double-stranded nucleic acid.

102. The method of claim 101, wherein the gap is formed at a juncture between the double-stranded vector and each of the plurality of non-circularized double-stranded nucleic acids.

103. The method of claim 100, wherein each of the plurality of non-circularized double-stranded nucleic acids comprises an overhang formed by excision of a non-canonical base positioned at an end of a single strand of a precursor non-circularized double-stranded nucleic acid.

104. The method of claim 103, wherein the non-canonical base is positioned 4 to 10 bases from the end of the single strand of the precursor non-circularized double-stranded nucleic acid.

105. The method of claim 103, wherein the non-canonical base is uracil.

106. The method of claim 103, wherein one of the strands of the non-circularized double-stranded nucleic acid or double-stranded vector lacks a 5′

phosphate.

107. The method of claim 94, wherein the plurality of circular double-stranded nucleic acids is diluted to a concentration of less than about 100 pM prior to extending the second strand of each of the circular double-stranded nucleic acids.

108. The method of claim 94, wherein partitioning comprises diluting the plurality of amplicon nucleic acids to about 0.3 to 1.5 amplicon nucleic acids per fraction.

109. The method of claim 94, comprising a second amplification reaction, wherein the second amplification reaction is performed after partitioning.

110. The method of claim 94, wherein the circular double-stranded nucleic acids are heat denatured prior to amplification.

111. A method for nucleic acid sorting comprising:

(a) providing a plurality of circular double-stranded nucleic acids, each of the plurality of circular double-stranded nucleic acids comprising a first strand that is a continuous circle and a second strand comprising a gap, wherein the gap has a length of at least one base;

(b) partitioning such that on average there are about 0.1 to 10 circular double-stranded nucleic acids from the plurality of circular double-stranded nucleic acids per fraction; and

(c) amplifying the partitioned circular double-stranded nucleic acids in the presence of a random primer to generate a plurality of amplicon nucleic acids, wherein the random primer comprises 4 to 8 bases in length.

112. The method of claim 111, comprising forming each circular double-stranded nucleic acid by ligating a double-stranded vector to a double-stranded non-circularized nucleic acid, wherein the vector anneals to a 5′

end and a 3′

end of the double-stranded non-circularized nucleic acid.

113. The method of claim 112, wherein the double-stranded non-circularized nucleic acid or the double-stranded vector comprises a strand lacking a 5′

phosphate.

114. The method of claim 112, wherein the double-stranded non-circularized nucleic acid or the double-stranded vector comprises a strand having 1 to 10 fewer bases than a complementary strand, wherein the 1 to 10 fewer bases corresponds to the length of the gap in the circular double-stranded nucleic acids.

115. The method of claim 111, wherein the gap 1 to 5 bases in length.

116. The method of claim 111, wherein partitioning comprises diluting such that on average there are about 0.5 to 2 of the circular double-stranded nucleic acids per fraction.

117. The method of claim 111, wherein partitioning comprises diluting such that on average there is about 1 of the circular double-stranded nucleic acids per fraction.

118. The method of claim 111, wherein partitioning comprises diluting to a concentration of about 1.5 to 17 of the circular double-stranded nucleic acids per 1 μ

l of solution.

119. The method of claim 111, wherein the plurality of circular double-stranded nucleic acids comprises at least 100 circular double-stranded nucleic acids at least 500 bases in length.

120. The method of claim 111, wherein the plurality of circular double-stranded nucleic acids comprises nucleic acids that differ in at least 7 bases.

121. A method for nucleic acid sorting comprising:

(a) forming a plurality of circular single-stranded nucleic acids by joining a double-stranded non-circularized nucleic acid and two adaptor sequences, wherein each of the two adaptor sequences encodes for a hairpin secondary structure;

(b) diluting the plurality of circular single-stranded nucleic acids to a concentration of at most 1 nM;

(c) amplifying the plurality of circular single-stranded nucleic acids in the presence of a primer having sequence complementary to one of the two adaptor sequences; and

(d) partitioning the amplification reaction such that on average there are 0.1 to 10 amplicon nucleic acids per fraction.