US 2015O167065A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0167065 A1 Nelson et al. (43) Pub. Date: Jun. 18, 2015 (54) ISOTHERMAL AMPLIFICATION OF (22) Filed: Dec. 13, 2013 NUCLECACDS WITHNA POROUS MATRIX Publication Classification (71) Applicant: General Electric Company, (51) Int. Cl. Schenectady, NY (US) CI2O I/68 (2006.01) (52) U.S. Cl. (72) Inventors: John Richard Nelson, Clifton Park, NY CPC .................................... CI2O I/6844 (2013.01) (US); David Roger Moore, Rexford, NY (US); Bing Li, Clifton Park, NY (US); (57) ABSTRACT Robert Scott Duthie, Schenectady, NY Provided herein are methods for amplification a target (US); Patrick McCoy Spooner, dsDNA that is impregnated within a porous matrix using Slingerlands, NY (US) endonuclease-assisted DNA amplification. The amplicons may be subsequent detected within the porous matrix or may (73) Assignee: General Electric Company, be eluted out of the porous matrix. Methods for extracting a Schenectady, NY (US) genetic material from a biological sample using endonu clease-assisted DNA amplification within a porous matrix are (21) Appl. No.: 14/106,264 also provided. Patent Application Publication Jun. 18, 2015 Sheet 1 of 6 US 201S/O167065 A1 Inosine Inosine-containing f primer I Target DNA DNA Polymerase O Y Endonuclease& I I y Endonuclease nicking -uiem y y FIG. 1 Patent Application Publication Jun. 18, 2015 Sheet 2 of 6 US 201S/O167065 A1 Exonuclease-resistant, inosine-containing primer Exonuclease treatment Exonuclease inactivation Inosine I f Target DNA DNA Polymerase O Y Endonuclease& T y Endonuclease nicking FIG. 2 Patent Application Publication Jun. 18, 2015 Sheet 3 of 6 US 201S/O167065 A1 No Template 50 ng TB DNA — — — Thioation - -- 5% FiCOI - H | FIG. 3 Patent Application Publication Jun. 18, 2015 Sheet 4 of 6 US 201S/O167065 A1 SOUtion NC PEGNC 903 PEG 903 FUSiOn 5 CD o H d g 2 g 2 g 2 g Z Z Z Z FIG. 4 Patent Application Publication Jun. 18, 2015 Sheet 5 of 6 US 201S/O167065 A1 Seidoo Seldoo O. Seldoo OOL Seidoo O'OOL Seldoo 00'00 seidoo OOOOOL OLN Jexe/N JeyueW Seldoo seidoo Ol seidoo 00 seldoo O'OO seidoo OO'OOL Seidoo OOOOOL OLN Patent Application Publication Jun. 18, 2015 Sheet 6 of 6 US 201S/O167065 A1 OO-OfM. GuoSn GUOISn OO-OfM ON pe)009 ON pe)0OI9 OO-OfM ON ON OO-IO/W GuoSn GUOISn OO-OfM ON pe)009 ON pe)3O9 OO-O FM ON ON OO - O UAA IOIUOO em|ISO US 2015/O167065 A1 Jun. 18, 2015 ISOTHERMAL AMPLIFICATION OF 0005) DNA detection techniques employing simplified NUCLECACDS WITHNA POROUS DNA amplification methods that do not require target dsDNA MATRIX denaturation prior to its amplification would offer several potential advantages for processing and screening of a broad FEDERALLY SPONSORED RESEARCH & range of sample types. Further, along with simpler and robust DEVELOPMENT sample preparation and processing methods for trace and/or 0001. This invention was made with Government support dilute target nucleic acids, such techniques would greatly under contract number HR0011-11-2-0007, held by the Uni facilitate DNA-based assays in POC and field-deployed versity of Washington awarded by the Defense Advanced assayS. Research Projects Agency (DARPA). The Government has certain rights in the invention. BRIEF DESCRIPTION 0006. In some embodiments, a method for producing at FIELD OF INVENTION least one amplicon based on a target dsDNA within a porous matrix is provided. The method comprises the steps of pro 0002 The invention generally relates to isothermal ampli viding the porous matrix, impregnating the target dsDNA fication of a double stranded DNA (dsDNA) within a porous within the porous matrix and amplifying at least one portion matrix. It further relates to amplification of a dsDNA (e.g., a of the impregnated target dsDNA within the porous matrix to genomic DNA) that is impregnated within a porous matrix produce at least one amplicon within the porous matrix. The using an endonuclease-assisted nucleic acid amplification amplification is performed under isothermal conditions by and Subsequent detection of amplicons within the porous contacting the impregnated, target dsDNA with a DNA matrix. amplification reaction mixture comprising at least one BACKGROUND inosine-containing primer, at least one 5'-->3' exonuclease deficient DNA polymerase having strand displacement activ 0003. With the development of a variety of techniques for ity, at least one nuclease that is capable of nicking a DNA at isolation, amplification and detection of nucleic acids, a residue 3' to an inosine residue, and dNTP mixture. The nucleic acid-based assays have emerged over the years as DNA amplification may be performed under isothermal con powerful tools for various applications such as diagnostic and ditions without any prior denaturation of the dsDNA to single forensic analysis. However, even today, immunoassays have stranded DNA (ssDNA). more widespread acceptance then nucleic acid-based assays 0007. In some embodiments, a method for extracting a due to their easy formats and lower operational costs Immu genetic material from a biological sample using a nuclease noassays are less complex than nucleic acid-based assays assisted DNA amplification assay is provided. The biological since they are simple detection assays and do not involve any sample is first contacted with a porous matrix comprising target amplification step. In contrast, amplification of a chemicals that lyse the biological sample and preserve the nucleic acid target (e.g., DNA amplification) is a critical step genomic DNA within the porous matrix. At least one portion in many of the nucleic acid-based assays. DNA amplification of the preserved genomic DNA is then amplified within the is a process of replicating a target DNA to generate multiple porous matrix to produce the at least one amplicon within the copies of it. Since individual strands of a dsDNA are antipar porous matrix. The at least one amplicon is interrogated or allel and complementary, each Strand may serve as a template eluted out of the porous matrix for interrogation. The ampli strand for the production of its complementary strand. The fication of the preserved genomic DNA is performed within template strand is preserved as a whole or as a truncated the porous matrix by contacting the preserved genomic DNA portion and the complementary Strand is assembled from within the porous matrix with a DNA amplification reaction deoxynucleoside triphosphates (dNTPs) by a DNA poly mixture comprising at least one inosine-containing primer, at merase. The complementary strand synthesis proceeds in least one 5'-->3' exonuclease-deficient DNA polymerase hav 5'-->3' direction starting from the 3' terminal end of a primer ing strand displacement activity, at least one nuclease that is sequence that is hybridized to the template strand. capable of nicking a DNA at a residue 3' to an inosine residue, 0004 For most of the currently known DNA amplification and dNTP mixture. The DNA amplification may be per techniques, expensive and/or complex equipment and higher formed under isothermal conditions without any prior dena levels of skilled labor are required. For nucleic acid-based turation of the dsDNA to single stranded DNA (ssDNA). analysis to become widely used for clinical or industrial applications, complexity of assays/instrumentation and their DRAWINGS cost need to be reduced. Specifically, if nucleic-acid based tests were to be performed at the point of care (POC) level 0008. These and other features, aspects and advantages of (e.g., near patient or near process), simple, easy to use, cost the present invention will become better understood when the competitive systems are essential. DNA-based assays involv following detailed description is read with reference to the ing thermal cycling amplification have been performed in a accompanying figures. lateral flow stick employing an associated thermally-regulat 0009 FIG. 1 illustrates a schematic representation of able apparatus. However, such thermal cycling amplification endonuclease-assisted DNA amplification usinginosine-con methods are less than ideal, for example, for POC applica taining primers. tions. Even though isothermal nucleic acid amplification 0010 FIG. 2 illustrates a schematic representation of (e.g., SMART reaction) of a target DNA putatively immobi endonuclease-assisted DNA amplification using exonu lized in a porous matrix (e.g., FTA paper) has been attempted, clease-resistant, inosine-containing primers. such methods involved prior heating of the immobilized, 0011 FIG. 3 illustrates enhanced efficiency of endonu target dsDNA to thermally denature the target dsDNA to its clease-assisted DNA amplification using exonuclease-resis single stranded counterparts. tant, inosine-containing primers. US 2015/O167065 A1 Jun. 18, 2015 0012 FIG. 4 illustrates endonuclease assisted isothermal agents like albumin (e.g., BSA) or pyrolidine) that may be amplification of a target dsDNA in different porous matrices required for a DNA amplification reaction. and in Solution. 0020. As used herein, the term “primer' refers to a short 0013 FIG. 5A illustrates detection limits when endonu linear oligonucleotide that hybridizes to a target DNA to clease assisted amplification is performed in Solution. prime a DNA synthesis reaction. The primer may be an RNA 0014 FIG. 5B illustrates detection limits when endonu oligonucleotide, a DNA oligonucleotide, or a chimeric clease assisted amplification is performed in a PEG-modified sequence. The primer may contain natural, synthetic, or nitrocellulose