# US 10,501,782 B1 · Cell-free synthesis of DNA by strand displacement > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,501,782 B1 - Document ID: US10501782B1 - Status: Granted - Kind: B1 - Application number: 15/508,766 - Filed: 2015-08-21 - Granted: 2019-12-10 - Earliest claimed: 2014-09-05 - Assignees: Touchlight IP Limited - Applicants: Touchlight IP Limited - Inventors: Neil Porter; Paul Rothwell; Jonathan Extance - Web page: https://patentgrep.com/patent/US10501782B1 ## CPC - C12Q 1/6844: Nucleic acid amplification reactions - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention relates to an improved process for synthesis of DNA, RNA, proteins and like molecules, in particular cell-free enzymatic synthesis of DNA, preferably in large scale. The present invention relates to the synthesis of DNA using strand-displacement replication and the addition of nucleotides to the reaction mixture is controlled, thus controlling yield. The reaction mixture contains a starting amount of nucleotides, polymerase and DNA template, to which further nucleotides are supplied in a controlled manner. ## Claims ### Claim 1 A cell-free process for synthesising DNA comprising contacting a DNA template with at least one polymerase in the presence of nucleotides to form a reaction mixture, wherein the DNA template is amplified by strand displacement replication, and wherein further nucleotides are supplied to the reaction mixture continuously or at intervals during the process. ### Claim 2 (depends on 1) A process according to claim 1 wherein said reaction mixture further comprises one or more primers. ### Claim 3 A cell-free process for synthesising DNA comprising contacting a DNA template with at least one polymerase in the presence of nucleotides and at least one primer to form a reaction mixture, wherein the DNA template is amplified by strand displacement replication, and wherein further nucleotides are supplied to the reaction mixture continuously or at intervals during the process. ### Claim 4 (depends on 1) A cell-free process for synthesising DNA according to claim 1, wherein the reaction mixture further comprises one or more metal cations and further metal cations are supplied to the reaction mixture continuously or at intervals during the process. ### Claim 5 (depends on 4) The process according to claim 4 wherein said further nucleotides and further metal cations are supplied independently to the reaction mixture. ### Claim 6 (depends on 1) The process according to claim 1 wherein said DNA template is circular, and amplification of said DNA template is by rolling circle amplification. ### Claim 7 (depends on 1) The process according claim 1, wherein said DNA template is a closed linear DNA. ### Claim 8 (depends on 1) The process according to claim 1 wherein said DNA template comprises at least one processing enzyme target sequence. ### Claim 9 (depends on 8) The process according to claim 8 wherein a processing enzyme is supplied at regular or irregular intervals during the process to the reaction mixture. ### Claim 10 (depends on 1) The process according to claim 1 wherein said further nucleotides are supplied in a plurality of aliquots to the reaction mixture. ### Claim 11 (depends on 1) The process according to claim 1 wherein said further nucleotides are supplied as aliquots at regular intervals throughout the duration of the process, optionally at least every 30 minutes. ### Claim 12 (depends on 4) The process according to claim 4 wherein said metal cations are supplied as aliquots at regular intervals throughout the duration of the process, optionally at least every 30 minutes. ### Claim 13 (depends on 11) The process according to claim 11 wherein at least 3, 4, 5, 6, 7, 8, 9 or 10 aliquots are supplied to the reaction mixture. ### Claim 14 (depends on 10) The process according to claim 10 wherein said aliquots are fed from an external source using a pump or are supplied to the reaction mixture by use of an osmotic pump. ### Claim 15 (depends on 1) The process according to claim 1 wherein said nucleotides or further nucleotides comprise biologically inactive nucleotides. ### Claim 16 (depends on 1) The process according to claim 1 wherein said further nucleotides are supplied in response to a signal related to the concentration of DNA in the reaction mixture. ### Claim 17 (depends on 16) The process according to claim 16 wherein the signal indicating the concentration of DNA is generated by measuring a pressure difference created by a reciprocating pump that removes and returns a portion of the reaction mixture, optionally over a pressure sensor. ### Claim 18 (depends on 1) The process according claim 1 wherein said process is performed in a synthesis apparatus, said apparatus comprising: a reaction vessel having at least a first port and a second port; a temperature control device for controlling the temperature of the reaction vessel; a plurality of reservoirs for holding reaction components; a plurality of conduits, each of the plurality of conduits connecting the first port of the reaction vessel with a respective one of the plurality of reservoirs; at least one pressure sensor located proximate the first port or second port of the reaction vessel; supply means for selectively supplying controlled amounts of reaction components held in the reservoirs to the reaction vessel; agitation means for selectively withdrawing an amount of content from the reaction vessel through the second port thereof and selectively returning the contents of the reaction vessel to the reaction vessel through the second port thereof; and control means for controlling the supply means and the agitation means, wherein, the pressure sensor detects a change in pressure externally of the reaction vessel and sends a signal to the control means and the control means controls the supply means and/or agitation means in response to a change in observed pressure. ### Claim 19 (depends on 18) The process according to claim 18 wherein said reaction components comprise said further nucleotides and/or metal cations. ### Claim 20 (depends on 4) The process according to claim 4 wherein said metal cations comprise one or more metals selected from the list consisting of: magnesium (Mg2+), manganese (Mn2+), calcium (Ca2+), beryllium (Be2+), zinc (Zn2+) and strontium (Sr2+), lithium (Li+), sodium (Na+) and potassium (K+). ### Claim 21 (depends on 4) The process according to claim 4, wherein said metal cation is a divalent cation and the ratio between said divalent metal cations and the nucleotides is maintained at about 3:1 in the reaction mixture. ### Claim 22 (depends on 1) The process according to claim 1 wherein the concentration of nucleotides in the reaction mixture is maintained between 0.001 mM and 6 mM, optionally at about 3 mM. ### Claim 23 (depends on 1) The process according to claim 1 wherein said nucleotides and/or further nucleotides are deoxyribonucleoside triphosphates (dNTPs), or a derivative or modified version thereof. ### Claim 24 (depends on 1) The process according to claim 1 wherein said nucleotides and/or further nucleotides are one or more of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP) and derivatives thereof. ### Claim 25 (depends on 1) The process according to claim 1 wherein said nucleotides are provided as one or more of free acids, their salts or chelates thereof, optionally wherein said salts or chelates includes one or more of the following metal ions: Mg2+, Be2+, Ca2+, Sr2+, Li+, Na+, K+, Mn2+ or Zn2+. ### Claim 26 (depends on 8) The process according to claim 8 wherein said processing enzyme is supplied in response to a signal related to the concentration of the DNA in the reaction mixture. ### Claim 27 (depends on 20) The process according to claim 20, wherein said metal cations comprise Mg2+. ### Claim 28 (depends on 7) The process of claim 7, wherein said DNA template is incubated under denaturing conditions to form a closed circular single stranded DNA. ### Claim 29 (depends on 8) The process according to claim 8, wherein the at least one processing enzyme target sequence comprises a recombinase targeting sequence or a protelomerase target sequence. ### Claim 30 (depends on 15) The method of claim 15, wherein said nucleotides or further nucleotides are supplied to the reaction mixture via activation. ### Claim 31 (depends on 16) The method of claim 16, wherein the concentration of the DNA in the reaction mixture is monitored by measuring the difference in pressure of the reaction mixture, or a portion thereof, which can optionally generate a signal.