Patent Application
20250002973
This application relates to methods and formulations for denaturing DNA.
Formamide and sodium hydroxide are currently used in a number of sequencing systems to both denature the DNA library before sequencing and de-hybridise sequencing strands during clustering/resynthesis between reads However, neither formamide nor sodium hydroxide are ideal denaturing agents. Formamide is a hazardous substance. It is a suspected carcinogen and may cause birth defects. It, therefore, necessitates specialized disposal routes, hinders efforts to reach Corporate Social Responsibility goals, and is a key complaint from customers. Also, manual denaturation by NaOH increases the hands-on time of sequencing, is a potential source of human error, and requires the customer to purchase and store additional chemicals and equipment. Operator error during denaturation is a frequent issue for customers using sodium hydroxide and is a significant cause of customer run failures. Additionally, sodium hydroxide solution is hazardous to users because of its corrosive properties and cannot be used in cartridges without additional measures such as alkaline-resistant foils.
Some examples herein provide a solution formulation for denaturing double-stranded DNA (dsDNA), the solution formulation including betaine; and at least one organic solvent.
In some examples, the at least one organic solvent includes diethylene glycol (DEG).
In some examples, a concentration of betaine in the solution formulation is between about 1M and about 5M, and a concentration of DEG in the solution formulation is between about 3% and about 40%.
In some examples, the concentration of DEG in the solution formulation is between about 20% and about 40%. In some examples, the concentration of DEG in the solution formulation is between about 20% and about 30%. In some examples, the concentration of DEG in the solution formulation is about 20%.
In some examples, the concentration of betaine in the solution formulation is between about 2M and about 5M. In some examples, the concentration of betaine in the solution formulation is between about 2M and about 4M. In some examples, the concentration of betaine in the solution formulation is about 2M. In some examples, the concentration of betaine in the solution formulation is about 3M.
In some examples, the at least one organic solvent includes diethylene glycol (DEG) and dimethyl sulfoxide (DMSO).
In some examples, a concentration of betaine in the solution formulation is between about 1M and about 5M, and a concentration of the DEG in the solution formulation is between about 3% and about 40%.
In some examples, a concentration of DMSO in the solution formulation is between about 5% and about 30%. In some examples, the concentration of the DMSO in the solution formulation is between about 7% and about 12%. In some examples, the concentration of DMSO in the solution formulation is about 10%. In some examples, the concentration of DMSO in the solution formulation is between about 17% and about 22%. In some examples, the concentration of DMSO in the solution formulation is about 20%.
In some examples, the concentration of the DEG in the solution formulation is between about 5% and about 25%. In some examples, the concentration of the DEG in the solution formulation is between about 13% and about 17%. In some examples, the concentration of the DEG in the solution formulation is about 15%.
In some examples, the concentration of betaine in the solution formulation is between about 2M and about 3M. In some examples, the concentration of betaine in the solution formulation is about 2M. In some examples, the concentration of betaine in the solution formulation is about 2.5M. In some examples, the concentration of betaine in the solution formulation is about 3M.
In some examples, the at least one organic solvent includes DMSO.
In some examples, a concentration of betaine in the solution formulation is between about 1M and about 5M. In some examples, the concentration of betaine in the solution formulation is between about 2M and about 3M. In some examples, the concentration of betaine in the solution formulation is about 2M. In some examples, the concentration of betaine in the solution formulation is about 2.5M. In some examples, the concentration of betaine in the solution formulation is about 3M.
In some examples, a concentration of DMSO in the solution formulation is between about 5% and about 30%. In some examples, the concentration of the DMSO in the solution formulation is between about 7% and about 12%. In some examples, the concentration of DMSO in the solution formulation is about 10%. In some examples, the concentration of DMSO in the solution formulation is between about 17% and about 22%. In some examples, the concentration of DMSO in the solution formulation is about 20%.
In some examples, any of the solution formulations further include a zwitterionic buffer. In some examples, the zwitterionic buffer produces a pH in the solution formulation at a range of between about 9.5 and about 11.5. In some examples, the zwitterionic buffer includes any one or more of 4. (cyclohexylamino)-1-butanesulfonic acid (CABS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (CHAPS).
Some examples herein provide a method of denaturing double-stranded DNA (dsDNA), the method including: contacting the dsDNA in a fluid-filled container with a denaturant including betaine.
In some examples, a ratio of denaturant to the dsDNA is between about 1:1 and about 3:1. In some examples, a ratio of denaturant to the dsDNA is about 1:1. In some examples, the ratio of the denaturant to the dsDNA is about 1.3:1. In some examples, the ratio of the denaturant to the dsDNA is about 1.5:1. In some examples, the ratio of the denaturant to the dsDNA is about 2:1. In some examples, the ratio of the denaturant to the dsDNA is about 2.5:1. In some examples, the ratio of the denaturant to the dsDNA is about 3:1.
In some examples, the denaturant further includes diethylene glycol (DEG).
In some examples, the denaturant further includes dimethyl sulfoxide (DMSO).
In some examples, the denaturant further includes a zwitterionic buffer. In some examples, the zwitterionic buffer produces a pH in the fluid-filled container between about 9.5 and about 11.5.
Some examples herein provide a kit for denaturing double-stranded DNA (dsDNA), the kit including: a denaturant including betaine and diethylene glycol (DEG) in a solution; and a buffer.
In some examples, a concentration of DEG in the solution is between about 20% and about 40%.
In some examples, the denaturant further includes dimethyl sulfoxide (DMSO).
In some examples, the concentration of DEG in the solution is between about 5% and about 20%.
In some examples, a concentration of betaine in the solution is between about 1M and 5M. In some examples, the concentration of betaine in the solution is between about 2M and about 3.5M. In some examples, the concentration of betaine in the solution is about 2M. In some examples, the concentration of betaine in the solution is about 2.5M. In some examples, the concentration of betaine in the solution is about 3.0M. In some examples, the concentration of betaine in the solution is about 3.5M.
It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein may be implemented together in any appropriate combination, and that any features/examples from any one or more of these aspects may be implemented together with any of the features of the other aspect(s) as described herein in any appropriate combination to achieve the benefits as described herein.
Current DNA denaturants include formamide and sodium hydroxide. Formamide is a serious health hazard and sodium hydroxide requires manual denaturation that can cause human error, in addition to being corrosive. Disclosed herein are alternative denaturing reagents (XDR formulations) that can be used to denature DNA. XDR formulations disclosed herein are not hazardous and can be used to effectively denature template DNA before sequencing, and/or to de-hybridize sequencing primers that are bound to surface DNA during sequencing.
In some examples, XDR formulations disclosed herein include betaine as well as other additive(s). In some examples, the XDR denaturants may include any one or more of a buffer, diethylene glycol (DEG), and dimethyl sulfoxide (DMSO). In some examples, the XDR denaturants include betaine and DEG. In some examples, the XDR denaturants include betaine, DEG, and a buffer. In some examples, the XDR formulations include betaine and DMSO. In some examples, the XDR formulations include betaine, DMSO, and a buffer. In some examples, the XDR formulations including betaine, DEG, and DMSO. In some examples, the XDR formulations include betaine, DEG, DMSO, and a buffer.
In some examples, any of the XDR formulations described herein can be used to denature libraries before sequencing. In some examples, any of the XDR formulations described herein can be used to de-hybridise strands in clustering and/or resynthesis, between reads. In some examples, any of the XDR formulations described herein can be used in bridge amplification. In some examples, any of the XDR formulations described herein can be used in exclusion amplification.
First, some terms used herein will be briefly explained. Then, some example compositions and example methods using the compositions will be described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components
The terms “substantially,” “approximately,” and “about” used throughout this specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they may refer to less than or equal to ±10% of the stated amount, such as less than or equal to ±5% of the stated amount, such as less than or equal to ±2% of the stated amount, such as less than or equal to ±1% of the stated amount, such as less than or equal to +0.5% of the stated amount, such as less than or equal to ±0.2% of the stated amount, such as less than or equal to ±0.1% of the stated amount, such as less than or equal to ±0.05% of the stated amount.
As used herein, the term “nucleotide” is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some examples also includes a nucleobase. A nucleotide that lacks a nucleobase may be referred to as “abasic.” Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).
As used herein, the term “nucleotide” also is intended to encompass any nucleotide analogue which is a type of nucleotide that includes a modified nucleobase, sugar and/or phosphate moiety compared to naturally occurring nucleotides. Example modified nucleobases include inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5′-phosphosulfate. Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates.
As used herein, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are bonded to one another. A polynucleotide is one nonlimiting example of a polymer. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues thereof. A polynucleotide may be a single stranded sequence of nucleotides, such as RNA or single stranded DNA, a double stranded sequence of nucleotides, such as double stranded DNA, or may include a mixture of a single stranded and double stranded sequences of nucleotides. Double stranded DNA (dsDNA) includes genomic DNA, and PCR and amplification products. Single stranded DNA (ssDNA) can be converted to dsDNA and vice-versa. Polynucleotides may include non-naturally occurring DNA, such as enantiomeric DNA. The precise sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the foregoing.
The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description the terms may be used to distinguish one species of polynucleotide from another when describing a particular method or composition that includes several polynucleotide species.
As used herein, the term “XDR”, the phrase “XDR solution formulation”, or the phrase “XDR formulation”, each refer to a DNA denaturant formulation in which betaine is used as the primary additive to denature the DNA. In some examples, an XDR formulation includes additional component(s) such as any one or more of a buffer, di-ethylene glycol (DEG), and dimethyl sulfoxide (DMSO). Various “XDR formulations” are disclosed herein; these various “XDR formulations” are labeled, for example, as XDR1, XDR2, XDR3, etc. . . .
As used herein, the term “betaine” is intended to refer to glycine betaine, also known as trimethylglycine (TMG), and has the following structure:
When a composition or solution herein is referred to as including a range % or a particular % of a compound, it is meant that such compound is present at that range % or particular % by volume.
As used herein, the term “LDR” refers to a denaturant that includes 99.9% formamide.
Some examples herein provide a solution formulation for denaturing double-stranded DNA (dsDNA) that includes betaine. In some examples, the solution formulation de-hybridizes a primer bound to surface DNA.
In some examples, the solution formulation includes betaine. In some examples, the solution formulation includes betaine and diethylene glycol (DEG). In some examples, the solution formulation is an XDR formulation.
In some examples, a concentration of betaine in the solution formulation is between about 1M and about 5M, for example about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2M, about 2.1M, about 2.2M, about 2.3M, about 2.4M, about 2.5M, about 2.6M, about 2.7M, about 2.9M, about 3.0M, about 3.1M, about 3.2M, about 3.3M, about 3.4M, about 3.5M, about 3.6M, about 3.7M, about 3.8M, about 3.9M, about 4.0M, about 4.1M, about 4.2M, about 4.3M, about 4.4M, about 4.5M, about 4.6M, about 4.7M, about 4.8M, about 4.9M, or about 5M. In some examples, the concentration of betaine in the solution formulation is between about 2M and about 5M. In some examples, the concentration of betaine in the solution formulation is between about 2M and about 4M. In some examples, the concentration of betaine in the solution formulation is between about 2M and about 3.5M. In some examples, the concentration of betaine in the solution formulation is less than 1M. In some examples, the concentration of betaine in the solution formulation is greater than 5M.
In some examples, a concentration of DEG in the solution formulation is between about 3% and about 40%. In some examples, the concentration of DEG in the solution formulation is between about 20% and about 40%, for example about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about, 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%. In some examples, the concentration of DEG in the solution formulation is greater than 40%. In some examples, the concentration of DEG in the solution formulation is between about 20% and about 30%.
In some examples, the solution formulation further includes dimethyl sulfoxide (DMSO). In some examples, a concentration of DMSO in the solution formulation is between about 5% and about 30%, for example, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%. In some examples, the concentration of the DMSO in the solution formulation is between about 7% and about 12%. In some examples, the concentration of DMSO in the solution formulation is about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%. In some examples, the concentration of DMSO in the solution formulation is between about 17% and about 22%. In some examples, the concentration of DMSO in the solution formulation is about 17%, about 18%, about 19%, about 20%, about 21%, or about 22%.
In some examples, a concentration of the DEG is between about 5% and about 25% in any of the solution formulations containing DMSO described herein. In some examples, the concentration of DEG is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25%. In some examples, the concentration of the DEG in the solution formulation is between about 13% and about 17%. In some examples, the concentration of the DEG in the solution formulation is about 13%, about 14%, about 15%, about 16%, or about 17%.
In some examples, a concentration of betaine is between about 2M and 3M in any of the solution formulations containing DMSO described herein. In some examples, the concentration of betaine in the solution formulation is about 2M, about 2.1M, about 2.2M, about 2.3M, about 2.4M, about 2.5M, about 2.6M, about 2.7M, about 2.8M, about 2.9M, or about 3M.
In some examples, any of the solution formulations described herein further includes a buffer. In some examples, the buffer includes a zwitterionic buffer. In some examples, the zwitterionic buffer produces a pH in the solution formulation at a range of between about 9.5 and about 11.5. In some examples, the zwitterionic buffer produces a pH in the solution formulation at a range of between about 8.5 and about 12.5. In some examples, the zwitterionic buffer produces a pH in the solution formulation of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11, about 11.1, about 11.2, about 11.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12.0, about 12.1, about 12.2, about 12.3, about 12.4, or about 12.5.
In some examples, the zwitterionic buffer includes any one or more of 4-(cyclohexylamino)-1-butanesulfonic acid (CABS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (CHAPS). In some examples, the zwitterionic buffer includes any zwitterionic buffer known in the art.
In some examples, the solution formulations described herein are any of XDR1, XDR2, XDR3, XDR4, XDR5, XDR6, XDR7, XDR8, XDR9, XDR10, XDR11, XDR12, XDR13, XDR14, XDR15, XDR16, XDR17, XDR18, XDR19, XDR20, XDR21, XDR22, XDR23, XDR24, XDR25, XDR26, XDR27, and XDR30. The components that make up each of these XDR formulations, which are aqueous solutions, are as follows:
Some examples herein provide a method of denaturing double-stranded DNA (dsDNA), including contacting the dsDNA in a fluid-filled container with a denaturant that includes betaine. In some examples, the denaturant that includes betaine is any of the XDR formulations described herein.
In some examples, a ratio of the denaturant to the dsDNA is between about 1:1 and about 3:1. Illustratively, in some examples, a ratio of the denaturant to the dsDNA is about 1:1. In some examples, the ratio of denaturant to the dsDNA is about 1.2:1. In some examples, the ratio of the denaturant to the dsDNA is about 1.3:1. In some examples, the ratio of the denaturant to the dsDNA is about 1.4:1. In some examples, the ratio of the denaturant to the dsDNA is about 1.5:1. In some examples, the ratio of the denaturant to the dsDNA is about 1.6:1. In some examples, the ratio of the denaturant to the dsDNA is about 2:1. In some examples, the ratio of the denaturant to the dsDNA is about 2.5:1. In some examples, the ratio of the denaturant to the dsDNA is about 3:1.
In some examples, the denaturant further includes diethylene glycol (DEG). In some examples, the concentration of DEG is between about 3% and about 40%, for example, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%. In some examples, the concentration of DEG in the solution is less than 3%. In some examples, the concentration of DEG in the solution is greater than 40%.
In some examples, the denaturant further includes dimethyl sulfoxide (DMSO). In some examples, a concentration of DMSO in the solution formulation is between about 5% and about 30%, for example, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.
In some examples, the denaturant further includes a zwitterionic buffer. In some examples, the zwitterionic buffer produces a pH in the fluid-filled container between about 9.5 and about 11.5. In some examples, the zwitterionic buffer produces a pH in the solution formulation at a range of between about 8.5 and about 12.5. In some examples, the zwitterionic buffer produces a pH in the solution formulation of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11, about 11.1, about 11.2, about 11.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12.0, about 12.1, about 12.2, about 12.3, about 12.4, or about 12.5.
In some examples, the zwitterionic buffer includes any one or more of 4-(cyclohexylamino). 1-butanesulfonic acid (CABS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (CHAPS). In some examples, the zwitterionic buffer includes any zwitterionic buffer known in the art.
Some examples herein provide a kit for denaturing double-stranded DNA (dsDNA), including a denaturant that includes betaine and diethylene glycol (DEG) in a solution; and a buffer.
In some examples, a concentration of DEG in the solution is between about 20% and about 40%. In some examples, the concentration of DEG in the solution is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%.
In some examples, the denaturant further includes dimethyl sulfoxide (DMSO). In some examples, the concentration of DEG in the solution is between about 5% and about 20%. In some examples, the concentration of DEG in the solution is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%.
In some examples, any of the kits described herein include a concentration of betaine in the solution that is between about 1 M and 5 M. In some examples, the concentration of betaine in the solution is about 2 M, about 2.5 M, about 3.0 M, about 3.5 M, about 4.5 M, or about 5.0 M.
In some examples, the buffer includes a zwitterionic buffer. In some examples, the zwitterionic buffer produces a pH in the solution formulation at a range of between about 9.5 and about 11.5. In some examples, the zwitterionic buffer produces a pH in the solution formulation at a range of between about 8.5 and about 12.5. In some examples, the zwitterionic buffer produces a pH in the solution formulation of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9, about 11, about 11.1, about 11.2, about 11.3, about 11.4, about 11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12.0, about 12.1, about 12.2, about 12.3, about 12.4, or about 12.5.
In some examples, the zwitterionic buffer includes any one or more of 4-(cyclohexylamino)-1-butanesulfonic acid (CABS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate) (CHAPS). In some examples, the zwitterionic buffer includes any zwitterionic buffer known in the art.
The XDR solution formulations, the solution formulations, and the methods of denaturing double-stranded DNA described herein can be used in the context of amplification methods, such as bridge amplification and exclusion amplification. Bridge amplification and exclusion amplification can be carried out in a flow cell.
Bridge Amplification: Bridge amplification can be performed on a flow cell. Single stranded template DNA is hybridised to lawn primers in a flow cell, and a polymerase is used to extend the primer to form double-stranded DNA. The double-stranded DNA is denatured, and the original template strand of the DNA molecule is washed away. This results in a single-stranded DNA molecule being bound to the lawn primers of the flow cell. The single-stranded DNA molecule turns over and forms a “bridge” by hybridising to a nearby lawn primer that is complementary to a sequence of the single-stranded DNA molecule. Polymerase extends the hybridised primer resulting in bridge amplification of the DNA molecule and the creation of a double-stranded DNA molecule. The double-stranded DNA molecule is then denatured.
The double-stranded DNA molecule can be denatured using (i) any of the XDR solution formulations described herein, (ii) any of the solution formulations described herein, or (iii) any of the methods of denaturing double-stranded DNA described herein. Denaturing results in two copies of single-stranded templates, one of which is immobilised to the support and the other of which may be washed away.
The one strand that is immobilised on the support may be used in further bridge amplification operations so as to generate a cluster that subsequently may be sequenced. Cluster generation produces clusters of double-stranded DNA. These clusters can be denatured using (i) any of the XDR solution formulations described herein, (ii) any of the solution formulations described herein, or (iii) any of the methods of denaturing double-stranded DNA described herein.
Exclusion Amplification: Exclusion amplification methods may allow for the amplification of a single target polynucleotide per substrate region and the production of a substantially monoclonal population of amplicons in a substrate region. For example, the rate of amplification of the first captured target polynucleotide within a substrate region may be more rapid relative to much slower rates of transport and capture of target polynucleotides at the substrate region. As such, the first target polynucleotide captured in a substrate region may be amplified rapidly and fill the entire substrate region, thus inhibiting the capture of additional target polynucleotide in the same substrate region. Alternatively, if a second target polynucleotide attaches to same substrate region after the first polynucleotide, the relatively rapid amplification of the first polynucleotide may fill enough of the substrate region to result in a signal that is sufficiently strong to perform sequencing by synthesis (e.g., the substrate region may be at least functionally monoclonal). The use of exclusion amplification may also result in super-Poisson distributions of monoclonal substrate regions; that is, the fraction of substrate regions in an array that are functionally monoclonal may exceed the fraction predicted by the Poisson distribution.
Exclusion amplification can generate clusters of double-stranded DNA. The double-strand DNA can be denatured using (i) any of the XDR solution formulations described herein, (ii) any of the solution formulations described herein, or (iii) any of the methods of denaturing double-stranded DNA described herein.
In some examples, sequencing occurs after cluster generation of DNA. In some examples, the DNA clusters are produced from bridge amplification or from exclusion amplification. In some examples, the DNA clusters are attached to the surface of a flow cell. To facilitate sequencing, it is preferable if one of the strands of DNA is removed from the surface to allow efficient hybridization of a sequencing primer to the remaining immobilised strand. Any of the solution formulations, XDR solution formulations, or any of the methods of denaturing double-stranded DNA described herein can be used to de-hybridized DNA, thereby allowing removal of a strand of DNA from the surface.
Sequence data can be obtained from both ends of a template duplex by obtaining a sequence read from one strand of the template from a primer in solution, copying the strand using immobilised primers, releasing the first strand and sequencing the second, copied strand. For example, sequence data can be obtained from both ends of the immobilised duplex by a method wherein the duplex is treated to free a 3′-hydroxyl moiety that can be used an extension primer. The extension primer can then be used to read the first sequence from one strand of the template. After the first read, the strand can be extended to fully copy all the bases up to the end of the first strand. This second copy remains attached to the surface at the 5′-end. If the first strand is removed from the surface, the sequence of the second strand can be read. This gives a sequence read from both ends of the original fragment. The process whereby the strand is regenerated after the first read is known as “Paired-end resynthesis” or “PE resynthesis”. The typical steps of pairwise sequencing are known and have been described in WO 2008/041002, the entire contents of which are incorporated by reference herein.
Sequencing can be carried out using any suitable “sequencing-by-synthesis” technique, wherein nucleotides are added successively to the free 3′ hydroxyl group, resulting in synthesis of a polynucleotide chain in the 5′ to 3′ direction. The nature of the nucleotide added is preferably determined after each addition. One particular sequencing method relies on the use of modified nucleotides that can act as reversible chain terminators. Such reversible chain terminators include removable 3′ blocking groups. Once such a modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced there is no free 3′-OH group available to direct further sequence extension and therefore the polymerase cannot add further nucleotides. Once the nature of the base incorporated into the growing chain has been determined, the 3′ block may be removed to allow addition of the next successive nucleotide. By ordering the products derived using these modified nucleotides it is possible to deduce the DNA sequence of the DNA template. Such reactions can be done in a single experiment if each of the modified nucleotides has attached thereto a different label, known to correspond to the particular base, to facilitate discrimination between the bases added at each incorporation step. Suitable labels are described in PCT application PCT/GB2007/001770, the entire contents of which are incorporated by reference herein. Alternatively, a separate reaction may be carried out containing each of the modified nucleotides added individually.
Any of the solution formulations, XDR solution formulations, or any of the methods of denaturing double-stranded DNA described herein can be used to remove sequencing primers from strands of DNA.
The examples provided below are include for illustrative purposes and are not meant to be limiting of any of the disclosures described herein.
New denaturant solutions with various XDR formulations described in Examples 2-6 below were tested during sequencing reactions. The libraries were sequenced at 2×151 cycles on a modified HiSeq X instrument and then tested on a NextSeq 2000. The sequencing was performed with standard workflows and reagents, except with new denaturant solutions (XDR formulations) replacing LDR in the cartridge, reagent plate, or reagent tubes.
As shown in the data described below in Examples 2-6 and
Human 450 bp libraries were sequenced as described in Example 1. DNA was denatured using different denaturant formulations containing betaine and using denaturants that utilized formamide, which functioned as a control. Sequencing metrics were performed on both Read 1 and Read 2 to determine the quality of the sequencing.
The results in
The label “LDR” in each of
Formulations with 20% DEG with DMSO and without DMSO (Label “20”)
Formulation with DMSO and 20% DEG:
Denaturing formulations containing betaine were tested in bridge amplification. Human 450 bp libraries were sequenced as described in Example 1.
The label “LDR” in
As shown in the sequencing data in
Sequencing metrics were evaluated between denaturants containing betaine and denaturants that contain formamide. The ratios of library:denaturant:neutralizing buffer were adjusted and tested. Different formulations were tested as shown below:
Each of the labels “LDR”, “DEG BET DMSO”, “DEG30 BET”, and “DEG BET 3.5” correspond to the labels on the x-axis on
In
As shown in
Human 450 bp libraries were sequenced as described in Example 1. DMSO-free versions of the denaturants were tested. Different formulations were tested as shown below:
The “LDR”, “DEG BET DMSO”, “DEG30 BET”, and “DEG BET3.5” labels correspond to the labels on the x-axis of
The sequencing data in
Human 450 bp libraries were sequenced as described in Example 1. Sequencing metrics were tested for the reagents described below over a 12-month accelerated stability study at 60° C.
As shown in
In this example, the following denaturants were tested:
The labels “Formamide”, “Betaine,” “Betaine+Buffer”, and “Betaine+Buffer+DEG” correspond to the labels on the x-axes on both
Denaturing after clustering: HiSeq X sequencing of human 450 bp libraries was performed using betaine as the denaturant along with other additives. 3.5 M betaine was used was used as a denaturant in place of formamide. The denaturant's ability to denature was measured with the ‘intensity’ metric (see
The data shown in
Denaturing libraries: HiSeq X sequencing of human 450 bp libraries was performed using betaine as the denaturant along with other additives. 3.5 M betaine was used as a denaturant in place of formamide. The ability of the denaturant to denature libraries was measured using percent GC (see
As shown in
In this example, the following denaturants were tested.
Denaturants tested in
Denaturants without DMSQ
HiSeq X sequencing of human 450 bp libraries was performed using various XDR formulations as the denaturant. The DNA was denatured after clustering. Percent GC coverage was used as the metric, as this metric is sensitive to incomplete denaturation.
In
In
In this example, the following denaturants were tested
Denaturants with 25 mM Buffer Molarity
HiSeq X sequencing of human 450 bp libraries was performed using XDR formulations as the denaturant. The DNA was denatured after clustering. Percent GC coverage was used as one of the sequencing metrics, as this metric is the most sensitive to incomplete denaturing. In addition, sequence intensity was tested; when there is no denaturing the sequence intensity will be zero. The data in
In this example, the following denaturants were tested.
HiSeq X sequencing of human 450 bp libraries was performed using various XDR formulations as the denaturant. The DNA was denatured after clustering. Percent GC coverage was used as the metric, as this metric is sensitive to incomplete denaturing. The data in
HybE is a surface-based assay that quantifies how much of a DNA library hybridized to the surface relative to the input concentration via a qPCR readout. In each of the tested formulations, a pH of 10.4 was used. As shown in
A mini high-powered DOE was generated using JMP software. The mini high-powered DOE was used to explore the effects of DMSO on library denaturation. The purpose of the assay was to determine whether formulations containing betaine that have lower concentrations of DMSO were still effective in denaturing DNA. Percent GC coverage was used as the metric, as this metric is sensitive to incomplete denaturing.
The data in
A 3-plex TruSeq DNA Nano human 450 bp DNA library was run on a NextSeq 2000 P3 kit 2×151+8+8. Analysis was performed using DRAGEN 3.9.5 Fluente orchestrated (N+3 shown are data from each index, only 1 run per condition).
As shown in
Various denaturing formulations containing betaine were tested using HiSeq X.
XDR formulations that contained no DEG were also tested using the HiSeq X. The purpose of testing XDR formulations without DEG was to mitigate high viscosities in certain XDR formulations.
XDR formulations that contained different buffers were also tested using the HiSeq X.
Using a viscometer, XDR formulations were tested to determine their viscosity.
While various illustrative examples are described above, it will be apparent to one skilled in the art that various changes and modifications may be made therein without departing from the disclosure. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the examples provided herein.
It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein may be implemented together in any appropriate combination, and that any features/examples from any one or more of these aspects may be implemented together with any of the features of the other aspect(s) as described herein in any appropriate combination to achieve the benefits as described herein.
This application claims the benefit of U.S. Provisional Patent Application No. 63/511,076, filed Jun. 29, 2023 and entitled “Methods and Formulations for Denaturing DNA,” the entire contents of which are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63511076 | Jun 2023 | US |
