Unlock instant, AI-driven research and patent intelligence for your innovation.

Methods for assembling DNA molecules

a dna molecule and assembler technology, applied in biochemistry, organic chemistry, biochemical apparatus and processes, etc., can solve the problems of difficult assembling of oligos into higher-order dna assemblies, user encounters difficulty in assembly of multiple genes of interest, and difficult assembly of oligos

Pending Publication Date: 2021-11-04
TELESIS BIO INC
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach simplifies the assembly process, reduces error rates, and allows for the efficient construction of DNA molecules with minimal residual sequences, facilitating the assembly of complex DNA constructs such as plasmids and artificial chromosomes.

Problems solved by technology

While these techniques are often useful, the user encounters difficulty with the assembly of a construct containing multiple genes of interest.
These techniques are also often labor intensive, such as splicing by overlap extension or other methods of generating single-stranded overhangs.
These scar sequences can make it difficult to assemble the oligos into higher order DNA assemblies.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Methods for assembling DNA molecules
  • Methods for assembling DNA molecules
  • Methods for assembling DNA molecules

Examples

Experimental program
Comparison scheme
Effect test

example 1

Assembly of 500 bp Constructs

[0064]This example illustrates the method in the partitioning and assembly of 24 DNA constructs of about 500 bp each from an oligo pool. The starting oligonucleotides were synthesized on a microarray and harvested into the oligo pool. About 10 of the oligos, when arranged adjacent to each other and in order comprise one of the 500 bp DNA constructs to be assembled. The oligos were each about 78 bp in length having a 30 bp overlap with the adjacent oligonucleotide and contained 18 bp of poly-A 3′ and / or 5′ flanking primer binding sites. The harvested oligo pool was diluted to a 0.25-0.5 nM average for each oligo. 24 pairs of primers containing dU and a thermostable DNA polymerase that was uracil-literate was added to the mixture.

[0065]The mixture was subjected to a PCR procedure according to the protocol shown below with partitioning primers containing dUs and using VERASEQ® DNA polymers (VSU). The product was then diluted 2× and the flanking sequences re...

example 2

Assembly of A 1.2 kb Construct

[0110]This example shows the assembly of a 1.2 kb DNA construct from oligo pools with 16, 32, and 58 oligonucleotides according to the invention. The Example also shows the robustness of the method of the invention.

[0111]Fifty-eight oligonucleotides were designed to comprise the DNA molecule of desired sequence to be assembled. The oligonucleotides were each approximately 78 bp in length and had about a 30 bp overlap with the adjacent oligo, plus an 18 bp flanking sequence on the 3′ or 5′ end.

[0112]The oligos were divided into three pools as follows:[0113]a. Pool 1: The oligos were placed into four sub-pools with 16, 16, 16, and 10 oligos resulting in a concentration of 5 nM for each oligo.[0114]b. Pool 2: The oligos were placed into two sub-pools with 32 and 26 oligos resulting in a concentration of 2.5 nM for each oligo.[0115]c. Pool 3: All 58 oligos were provided as one pool at 1.25 nM concentration for each oligo.

[0116]A PCR procedure was set up usi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3′ or a 5′ primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3′ flanking sequence and a universal 5′ flanking sequence, respectively.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application of U.S. application Ser. No. 15 / 839,597 filed Dec. 12, 2017, now issued as U.S. patent Ser. No. 11 / 060,137; which claims the benefit under 35 USC § 119(e) to U.S. Application Ser. No. 62 / 434,300 filed Dec. 14, 2016, now expired. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.BACKGROUND OF THE INVENTIONField of the Invention[0002]The invention pertains to processes and compositions for the assembly of nucleic acid molecules.Background Invention[0003]The synthesis and assembly of DNA molecules remain critical technologies in the field of molecular biology. In particular, the ability to readily assemble multiple double-stranded DNA molecules in a correct order to yield a functional gene or nucleic acid construct (e.g., a vector) is presently an important objective in molecular biology. Existing techniq...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/686C12N15/10C07H19/173C07H19/073C12N9/22C12N15/09C12P19/34
CPCC12Q1/686C12N15/1031C07H19/173C07H19/073C12Y302/02027C12N9/22C12N15/09C12P19/34C12N15/10C12Q2521/101C12Q2533/101
Inventor GILL, JOHN E.GIBSON, DANIEL G.FU, LIXIA
Owner TELESIS BIO INC