Background

The established methods for chemical synthesis of DNA work well for shorter oligonucleotides, but synthesizing DNA strands longer than one kilobase is expensive, low yield, and is prone to error. A hybrid approach using synthetic oligos and conventional PCR can be used for the synthesis of loner DNA.

Description

LLNL scientists have developed a method to synthesize long DNA sequences of varying length starting from short oligos. Synthetic oligos are generated using bioinformatics tools by overlapping multiple small segments, such as 4-mers or 6-mers, derived from both strands of the source DNA strand. DNA polymerases fill the gaps between these short n-mers to create the new, longer DNA strand. This process can be repeated multiple times using same or different length n-mers until the DNA strand of user specific length is synthesized within the microwell where this reaction takes place. An alternative version of this method, which separates the groups of different length n-mers spatially into distinct wells prior to the polymerase reaction occurring, is to separate them temporally. By sequentially adding the n-mers to the reaction mix, this alternative method may increase the probability the n-mers will bind in the correct order to create the desired long DNA strand.

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Advantages
  • Synthesis of long DNA strands with lower cost and higher yield
  • Spatial or temporal separation of added oligos as well as the ability to start with oligos of odd or even-length allows for flexibility in performing the method
Potential Applications
  • Synthesis of longer DNA strands
  • Manual introduction of specific n-mer component sequences into desired long DNA strand
Development Status

LLNL currently holds patents 8,470,537 and 7,871,799 for "Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions" (LLNL internal case # IL-11191).

Reference Number
43419