Combined extension and ligation for nucleic acid assembly
Certain aspects of the present invention provide methods for assembling nucleic acid molecules. Some embodiments involve analyzing nucleic acid sequences and determining appropriate assembly strategies based on the presence or absence of sequence features that are known or predicted to interfere with extension-based and/or ligation-based assembly techniques. Aspects of the invention also provide kits, compositions, devices, and systems for assembling synthetic nucleic acids using polymerase-based techniques, ligase-based techniques, or combinations thereof.
1 . A method of synthesizing a target nucleic acid, the method comprising:
analyzing a sequence of a target nucleic acid for the presence of one or more predetermined interfering sequence features that are predicted to disrupt a multiplex oligonucleotide assembly reaction,
assembling one or more first fragments of the target nucleic acid using a polymerase-mediated oligonucleotide assembly reaction, wherein no predetermined interfering sequence features are present in any of the one or more first fragments,
if one or more predetermined interfering sequence features are present in the target nucleic acid, assembling one or more second fragments of the target nucleic acid using a ligase-mediated oligonucleotide assembly reaction, wherein the one or more second fragments comprise the one or more predetermined interfering sequence features, and
assembling the target nucleic acid from the one or more first fragments and the one or more second fragments if the target nucleic acid contained one or more predetermined interfering sequence features.
2 . A method of designing a target nucleic acid assembly, the method comprising:
analyzing a sequence of a target nucleic acid for the presence of one or more predetermined interfering sequence features that are predicted to disrupt a multiplex oligonucleotide assembly reaction,
selecting one or more first fragments of the target nucleic acid for assembly in a polymerase-mediated oligonucleotide assembly reaction, wherein no predetermined interfering sequence features are present in any of the one or more first fragments, and
if one or more predetermined interfering sequence features are present in the target nucleic acid, selecting one or more second fragments of the target nucleic acid for assembly in a ligase-mediated oligonucleotide assembly reaction, wherein the one or more second fragments comprise the one or more predetermined interfering sequence features,
wherein the target nucleic acid can be assembled from the one or more first fragments and the one or more second fragments if they are selected.
3 . The method of claim 2 , further comprising the act of assembling the one or more first fragments.
4 . The method of claim 3 , further comprising the act of assembling the one or more second fragments.
5 . The method of claim 2 , further comprising the act of assembling the target nucleic acid.
6 . The method of claim 1 , wherein each of the one or more predetermined interfering sequence features is, independently, a region of high GC content, a region of low GC content, a region containing a repeated sequence, a region containing a partially repeated sequence, a region containing a polymerase stall sequence, a region containing a sequence likely to be replicated erroneously by a polymerase, or any other sequence feature predicted to disrupt a multiplex oligonucleotide assembly reaction.
7 . The method of claim 6 , wherein the region of high GC content contains greater than 70% GC over a length of at least 10 to 20 nucleotides.
8 . The method of claim 6 , wherein the region of low GC content contains less than 30% GC over a length of at least 10-20 nucleotides.
9 . The method of claim 6 , wherein the repeated sequence is at least 7 bases long.
10 . The method of claim 6 , wherein the repeated sequence is a direct repeat.
11 . The method of claim 6 , wherein the repeated sequence is an inverted repeat.
12 . The method of claim 1 , wherein the act of analyzing the sequence of the target nucleic acid comprises calculating hybridization binding energies between pairs of test sequences selected from different regions of the target nucleic acid.
13 . The method of claim 12 , wherein each test sequence is a randomly selected sequence of between 20 and 60 consecutive bases of the target nucleic acid.
14 . The method of claim 12 , wherein each test sequence is selected according to a computer-implemented algorithm.
15 . The method of claim 12 , wherein an interfering sequence feature is the presence of a pair of test sequences having a predicted hybridization binding energy greater than a threshold level.
16 . The method of claim 1 , wherein the act of analyzing the sequence of the target nucleic acid comprises searching for potential stem-loop structures between pairs of test sequences selected from different regions of the target nucleic acid.
17 . The method of claim 16 , wherein each test sequence is a randomly selected sequence of between 20 and 60 consecutive bases of the target nucleic acid.
18 . The method of claim 16 , wherein each test sequence is selected according to a computer-implemented algorithm.
19 . The method of claim 14 , wherein an interfering sequence feature is the presence of a pair of test sequences that are predicted to form a stem-loop structure wherein at most two bases are unpaired at the 3′ end of one of the sequences.
20 - 55 . (canceled)
56 . A system for assembling a target nucleic acid, the system comprising:
a means for analyzing a sequence of a target nucleic acid for the presence of one or more predetermined interfering sequence features that are predicted to disrupt a multiplex oligonucleotide assembly reaction,
a means for assembling one or more first fragments of the target nucleic acid using a polymerase-mediated oligonucleotide assembly reaction, wherein no predetermined interfering sequence features are present in any of the one or more first fragments,
a means for assembling one or more second fragments of the target nucleic acid using a ligase-mediated oligonucleotide assembly reaction if one or more predetermined interfering sequence features are present in the target nucleic acid, wherein the one or more second fragments comprise the one or more predetermined interfering sequence features, and
a means for assembling the target nucleic acid from the one or more first and second fragments.
57 - 61 . (canceled)