System and method for synthesis of DNA particles and use thereof
Disclosed is a system and method for production of DNA particles and use thereof. The DNA particles can be produced by amplification of nucleic acid molecule(s). Alternatively, DNA particles can be prepared by condensing multiple DNA molecules. The DNA condensation into a particle is mainly triggered by pyrophosphate and positively charged cations (e.g. magnesium). DNA particles can be applied for numerous biological applications but not limited to directed evolution, proteomics, drug delivery and imaging. DNA particles can be used to synthesize proteins using in vitro transcription/translation reaction.
1. A method of generating individual particles each using a single nucleic acid molecule template by means of enzymatic amplification in aqueous micro-droplets, wherein each particle comprises nucleic acid(s), pyrophosphate(s), and metal ions, wherein said method of producing the individual particles comprises:
(i) providing one or more nucleic acid molecule templates to be amplified;
(ii) providing nucleic acid amplification reaction ingredients including polymerase, primers, dNTP, salts, non-ionic detergent(s);
(iii) partitioning nucleic acid molecules and amplification reaction ingredients in the aqueous micro-droplets using a microfluidics system such that one aqueous micro-droplet contains, on average, no more than one nucleic acid molecule template;
(iv) incubating for a given period of time to allow nucleic acid amplification and formation of the particles to occur; and
(v) releasing particles from the aqueous micro-droplets.
2. The method of claim 1 , wherein the nucleic acid molecule template is DNA, RNA, or a modified nucleic acid.
3. The method of claim 1 , wherein at least one nucleic acid molecule in each particle has a nucleotide sequence of one of the nucleic acid molecule templates.
4. The method of claim 1 , wherein the step of forming a particle further comprises amplifying the nucleic acid molecule template using DNA polymerase or RNA polymerase.
5. The method according to claim 4 , wherein a plurality of micro-channels of a fluid merge into a single micro-channel, and wherein a depth of the micro-channels is in a range from 1 μm to 1000 μm.
6. The method of claim 1 , wherein the nucleic acid molecule(s) within a particle comprise single-stranded, double-stranded, triple-stranded nucleic acids or a combination thereof.
7. The method of claim 1 , wherein the size of a single particle is greater than 10 nm, 100 nm, 1000 nm or 10 μm.
8. The method of claim 1 , wherein said particles are purified from the reaction mixture.
9. The method according to claim 1 , wherein the microfluidics system comprises:
(i) at least two inlets;
(ii) at least two microfluidic channels;
(iii) a flow focusing junction;
(iv) droplet collection outlet; and
(v) at least one of the at least two microfluidics channels connect the flow focusing junction with the outlet.
10. The method of claim 1 , wherein the formation of the particle(s) occurs in aqueous droplets no larger than 1 microliter volume and no smaller than 1 femtoliter volume.