Methods for Amplifying a Complete Genome or Transcriptome

• US 20140274811A1
• Filed: 03/14/2014
• Published: 09/18/2014
• Est. Priority Date: 03/14/2013
• Status: Active Application

• Current Assignee: Aegea Biotechnologies, Inc.
• Original Assignee: Aegea Biotechnologies, Inc.
• Inventors: Arnold, Lyle J.
• Application Number:
  US14/214,634
• Publication Number:
  US 20140274811A1
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags

Abstract

The present invention provides methods for amplifying a complete genome or transcriptome. The genome or transcriptome is prepared as a set of target nucleic acids and mixed with a first primer. The first primer comprises a target-binding region having a first random sequence of about 6 to about 9 nucleotides and a tag sequence that contains one or more non-natural nucleotides. The first primer is annealed to the target nucleic acids and extended by polymerase to produce a first duplex nucleic acid. The target nucleic acid is removed from the first nucleic acid. A second primer is annealed to the first nucleic acid having a second random sequence of about 6 to about 9 nucleotides and a tag sequence that contains one or more non-natural nucleotides. The second primer is extended by polymerase to produce a second duplex nucleic acid. The second nucleic acid contains a tag sequence on one terminus and a complement of the tag sequence on the other. The first nucleic acid is removed from the second nucleic acid. A third primer is annealed to the second nucleic acid having the same sequence as the tag sequence or a portion thereof and at least one of the non-natural nucleotides of the tag sequence. The third primer is extended by polymerase to produce a third duplex nucleic acid. The second nucleic acid is removed from the third nucleic acid. The third primer is annealed to the second nucleic acid and the third nucleic acid. The third primer is extended by polymerase. Repeating these last three steps thereby results in amplification of the genome or transcriptome.

Claims

1. A method of amplifying a genome or transcriptome obtained from a sample, wherein said genome or transcriptome is provided as a set of target nucleic acids said method comprising the steps of:

A. annealing a first primer to a set of target nucleic acids in a mixture comprising said target nucleic acids and said first primer wherein said primer comprises a target binding region containing a first random sequence having about 6 to about 9 nucleotides and a tag sequence, wherein said tag sequence contains one or more non-natural nucleotides and extending said first primer to produce a first duplex nucleic acid containing a first nucleic acid and said target nucleic acid, and optionally removing said first nucleic acid from said target nucleic acid;

B. annealing a second primer to said first nucleic acid, wherein said second primer comprises a target binding region containing a second random sequence having about 6 to about 9 nucleotides and a tag sequence, wherein said tag sequence contains one or more non-natural nucleotides and extending said second primer by polymerase to produce a second nucleic acid duplex containing said first nucleic acid and a second nucleic acid containing a tag sequence on one end and a complement of said tag sequence on the other and, optionally removing said first nucleic acid from said second nucleic acid;

C. annealing a third primer having a substantially identical sequence as said tag sequence or a portion thereof and at least one of said non-natural nucleotides of said tag sequence to said second nucleic acid and extending said third primer by polymerase to produce a third nucleic acid duplex containing said second nucleic acid and a third nucleic acid and, optionally removing said second nucleic acid from said third nucleic acid;

D. annealing said third primer to said second nucleic acid and said third nucleic acid and extending said third primer bound to said second nucleic acid and said third nucleic acid by polymerase; and

E. repeating steps F and G thereby amplifying said genome or said transcriptome.

2. The method according to claim 1, wherein said first random sequence of said first primer and said second random sequence of said second primer are the same.

3. The method according to claim 1, wherein said first primer comprises a tag sequence and a target-binding region having a random sequence of about 1 to about 3 nucleotides and a poly-T sequence.

4. The method according to claim 1, wherein said first primer comprises a tag sequence and a target-binding region comprising a poly-T sequence.

5. The method according to claim 1, wherein said first primer further comprises an anchor sequence.