WO2020193368A1 - Method for improving the amplification efficiency of bead-based emulsion pcr (empcr) - Google Patents

Method for improving the amplification efficiency of bead-based emulsion pcr (empcr) Download PDF

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Publication number
WO2020193368A1
WO2020193368A1 PCT/EP2020/057619 EP2020057619W WO2020193368A1 WO 2020193368 A1 WO2020193368 A1 WO 2020193368A1 EP 2020057619 W EP2020057619 W EP 2020057619W WO 2020193368 A1 WO2020193368 A1 WO 2020193368A1
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Prior art keywords
beads
sulfate
empcr
bead
anionic surfactant
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PCT/EP2020/057619
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French (fr)
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Matthias Wahl
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Qiagen Gmbh
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Publication of WO2020193368A1 publication Critical patent/WO2020193368A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention is directed to an improvement of the amplification efficiency of bead-based emulsion PCR (emPCR).
  • the present invention relates to the field of molecular biology, more par ticularly to the amplification of nucleic acid molecules.
  • PCR polymerase chain reaction
  • PCR-based methods have been adapted to amplifying mole cules contained within water-in-oil emulsions.
  • a plurality of biological samples e.g. nucleic acid samples
  • PCR amplification is conducted on each of the plurality of encapsulated nucleic acid samples simultaneously.
  • Such reaction compartments or microcapsules are often referred to as "microreactors” because the amplification occurs within the reaction compartments.
  • This kind of PCR is often referred to as emulsion PCR (emPCR) or droplet PCR.
  • the small reaction compartments can include a template bead or micro sphere and the amplification process may be referred to as bead-based emPCR.
  • beads along with DNA templates are suspended in an aqueous reaction mixture and then encapsulated in an inverse (water-in-oil) emulsion.
  • the template DNA may be either bound to the bead prior to emulsification or may be included in solution in the amplification reaction mixture.
  • European patent application no. 3 170 903 discloses the use of an ani onic surfactant in the context the emPCR.
  • the anionic surfactant is proposed to be added at the so-called “breaking step” which is carried out after the amplification process is concluded.
  • this breaking step the oil phase is removed or the emulsion is "broken" by the anionic surfactant in order to isolate the amplified nucleic acids from the reaction compartments or aqueous droplets, respectively.
  • the anionic surfactant by adding the anionic surfactant the use of isopropanol or butanol as breaking agents can be avoided which have the disadvantages of light inflammability, acute toxicity, low flash point, causing corrosion, and unpleasant odor.
  • Bead-based emPCR is employed for target amplification for several next generation sequencing (NGS) platforms, such as Roche/545, ThermoFisher Scientific Ion TorrentTM, or the QIAGEN GeneReader. These systems employ bead-based emPCR for clonal amplification of the molecules to be sequenced prior to the actual sequencing reaction, i.e. single molecules are clonally amplified on the beads.
  • NGS next generation sequencing
  • the present invention satisfies these and other needs.
  • the present invention proposes the use of an anionic surfactant to im prove the amplification efficiency of bead-based emulsion PCR (emPCR).
  • the present invention further proposes a method for improving the am plification efficiency of bead-based emulsion PCR (emPCR), said method is comprising the following steps:
  • emPCR bead-based emulsion PCR
  • beads commonly used in bead- based emPCR vary in terms of their accumulation behavior. This variation results in a tendency of some populations of beads to accumulate in the aqueous phase or droplets while other populations tend to accumulate in the oil phase. This phenomenon can even be found among one and the same species of beads which, apparently, comprise sub populations having different hydrophobic or hydrophilic characteristics.
  • hydrophilic/ hydrophobic property of a bead As this has been realized by the inventor, whether a bead goes into the oil phase or contributes to a droplet or aqueous phase is dictated by the hydrophilic/ hydrophobic property of a bead. Importantly, hydrophobic beads which tend to accumulate in the oil phase will not contribute to the amplification in emPCR. To the contrary, hydro philic beads which tend to accumulate in the aqueous droplets do contribute to the amplification in emPCR.
  • the inventor has also realized that the low performance or amplification efficiency in bead-based emPCR may be additionally caused by a clumping of the beads.
  • the probable mechanism underlying the invention is that the anionic surfactant shields hydrophobic patches of low-performing beads, thereby increasing the hydrophilic properties of beads.
  • the current art detergents that are added to reduce clumping of beads include Tween 20 and Triton X 100. However, these detergents are not negatively charged and the inventor has realized that they do not seem to stay on the bead surface for long-term. Therefore, these detergents do not confer the positive effect of the inven tion.
  • an "anionic surfactant” refers to anionic com pounds that lower the surface tension or interfacial tension between two liquids or be tween a liquid and a solid.
  • Anionic surfactants contain anionic functional groups at their head, such as sulfate, sulfonate, phosphate, and carboxylates.
  • Prominent and well-suited alkyl sulfates include ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate, another name for the compound) and the related alkyl-ether sulfates sodium laureth sulfate, also known as sodium lauryl ether sulfate (SLES), and sodium myreth sulfate.
  • ALS ammonium lauryl sulfate
  • SDS sodium lauryl sulfate
  • SLES sodium lauryl ether sulfate
  • sodium myreth sulfate sodium myreth sulfate
  • beads or “microsphere” which are used inter changeably, refer to a spherical microparticle commonly used in bead-based emPCR. Typical beads comprise diameters of between 0.1 and 100 pm and are commercially available in a wide variety of materials, including polymers, ceramics, glass, and metals. Polyethylene, polystyrene and expandable beads or microspheres are the most common types of polymer beads or microspheres.
  • Beads or microspheres used in bead-based emPCR have the capability to immobilize nucleic acid molecules on their surface, such as PCR primers.
  • the beads comprise magnetic properties.
  • the beads include DynabeadsTM My- OneTM Carboxylic Acid (Thermo Fisher Scientific, Cat. No. 65011). Such beads are uniform, monosized superparamagnetic and 1 pm in diameter, and are composed of highly cross-linked polystyrene with evenly distributed magnetic material. The beads are further coated with a hydrophilic layer of glycidyl ether, concealing the iron oxide inside them. Carboxylic acid groups are then introduced on the surface of the beads.
  • Amplification efficiency is a measure of the capacity of the polymerase chain reaction to synthesize nucleic acids. The procedural steps occur prior to the "breaking step".
  • An "improvement" of the amplification efficiency refers to an increase in amplification effectiveness in contrast to a reference bead-based emPCR which charac terized by the accumulation of at least a fraction of the beads in the oil phase.
  • a "buffer solution” as used herein refers to a liquid medium providing the environment for the incubation of beads and the anionic surfactant.
  • the buffer solution allows the carrying out of an emPCR and includes, without being limited thereto, aqueous TE buffer (Tris/EDTA buffer).
  • Incubating means the contacting of the surface of the beads and the anionic surfactant under conditions including time, temperature, and pressure, thereby ensuring the reacting of the beads' surface structures with the surfac tant.
  • Isolating refers to recovering of the beads from the buffer solution after incubation and may include washing steps to remove residues of non- reacted surfactant from the beads' surface.
  • said anionic surfactant is selected from the group consisting of: alkyl sulfates and alkyl carboxylates.
  • said alkyl sulfate is selected from the group consisting of: ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate), sodium laureth sulfate (SLES, sodium lauryl ether sulfate), and sodium myreth sulfate.
  • the bead-based emPCR involves beads comprising carboxylic acid groups and/or streptavidin on their surface.
  • the performance of such beads can be improved which are commonly used in emPCR.
  • carboxylic acid groups or streptavidin on the beads' surface allows a stable coupling of nucleic acid molecules such as primers for the PCR.
  • the interaction between the beads and nucleic acid molecules to capture the latter to the beads' surface may be mediated by covalent bonds, e.g. nucleic acid mole cules are covalently bound to the beads' surface, or by high non-covalent affinity interac tion, e.g. between biotin and streptavidin.
  • the beads comprise PCR primers on their surface, and it is preferred if the beads are pre-treated with said anionic surfactant before subjecting them to said bead-based emPCR. In another embodiment of the inven tion said pre-treatment is carried out by incubating said beads in a buffer solution contain ing said anionic surfactant.
  • said buffer solution contains said ani onic surfactant in a final concentration of between approx. 0.1 50 wt.-%, preferably approx. 0.5 40 wt.-%, more preferably approx. 1 0 - 30 wt.-%, more preferably approx. 1 5 - 20 wt.-%, more preferably approx. 2.0 - 15 wt.-%, most preferably approx. 10 wt.-%.
  • This embodiment has the advantage that said anionic surfactant is pro vided in the buffer solution in a concentration which ensures an effective increase of the beads' hydrophilic properties.
  • the skilled person can easily prepare a stock solution comprising the anionic surfactant in the concentration needed in order to end up with the final concentrations as prescribed in this embodiment.
  • the surfactant may be added to the buffer so lution in several steps where, possibly, different amounts or concentrations may be added. There may be a first step of adding the anionic surfactant in a considerably high concen tration and (a) following step(s) of adding the anionic surfactant in a lower concentration, e.g. 10 wt.-% followed by 5 wt.-%, possibly with a washing steps in between.
  • Another subject-matter of the invention relates to a kit for amplifying nu cleic acid molecules by bead-based emulsion PCR (emPCR), comprising
  • - beads preferably beads comprising carboxylic acid groups or streptavidin on their surface, further preferably beads comprising PCR primers on their surface; a buffer solution adapted for an incubation of said beads, and - an anionic surfactant, preferably selected from the group consisting of alkyl sulfates and alkyl carboxylates, further preferably said alkyl sulfate is se lected from the group consisting of: ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate), sodium laureth sulfate (SLES, sodium lauryl ether sulfate), and sodium myreth sulfate.
  • ALS ammonium lauryl sulfate
  • SDS sodium lauryl sulfate
  • SLES sodium laureth sulfate
  • sodium myreth sulfate sodium myreth sulfate.
  • kits are a combination of individual elements useful for carrying out the use and/or method of the invention, wherein the elements are optimized for use together in the methods.
  • the kits also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the invention.
  • kits unify all essential elements required to work the method according to the invention, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the invention.
  • the beads could be provided in surfac tant containing buffer, and the customer would just have to equilibrate (wash) beads with buffer required for application.
  • Fig. 1 illustrates clonal amplification of DNA molecules from a library by bead- based emPCR
  • Fig. 2 illustrates the surface of the beads and the potential mechanism of ani onic surfactants, exemplified by ALS;
  • Fig. 3 shows a microscopic image of emulsion after emulsion making. Exam ples of beads in oil phase are indicated by an arrow;
  • Fig. 4/5 illustrate the effect of anionic surfactant treatment as exemplified with
  • Fig. 6 shows the structure of ammonium lauryl sulfate (ALS);
  • Fig. 7 shows electron microscopic images taken from Thermo MyOne magnet ic beads coupled with emPCR primers
  • Fig. 8 shows the structure of ALS and Tween 20
  • Fig. 9 shows that the anionic surfactant, exemplified with ALS, remains on the beads for at least 4 weeks.
  • Emulsion PCR is a commonly used tool in molecular biology.
  • NGS next generation sequencing
  • emPCR is a commonly used tool in molecular biology.
  • Several next generation sequencing (NGS) systems employ emPCR on beads (bead- based emPCR) for clonal amplification of the molecules to be sequenced prior to the actual sequencing reaction (Fig. 1).
  • the manufacturing of beads is a multi-step process. As one of the steps, hydrophobic polystyrene beads become hydrophilic (charged) by polymerization. Electron microscopic images indicate that this polymerization is not uniformly (Fig. 7). Therefore spots with hydrophobic patches will still be present (Fig. 2).
  • NH2-modified oligonucleotides were coupled onto carboxylic acid beads using EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide).
  • Emulsion PCR was conducted using: Libraries (DNA fragments) gener ated using the QIAGEN GeneRead QIAact Lung DNA UMI Panel Kit (Cat No. /ID:
  • More primer loaded beads in the oil phase means that fewer beads will be used in emulsion PCR, leading to a lower number of live beads and, subsequently, a lower yield after enrichment of live beads.
  • Failing primer loaded beads can be rescued with anionic surfactant
  • Tween 20 was added to the beads. Tween 20 will bind to the hydrophobic patches via its nonpolar hydrocarbon chain. However, in contrast to anionic surfactants such as ALS its hydrophilic do main is not positively charged (see Fig. 8).
  • ALS also binds to the hydrophobic patches via its nonpolar hydrocarbon chain. In this way, the hydrophobic patches become negatively charged. This negative charge leads to an increased hydrophilic property compared to the non-charged Tween. Additionally, the negative charge is not binding to the phosphate backbone of the primers loaded onto the beads, i.e. phosphate background and negative charge of ALS lead to repulsion. Therefore, the PCR primer is accessible.
  • ALS may not be added to enzymatic reactions as it facilitates the dena- turation of proteins.
  • the inventor therefore tested the duration of the positive effect of ALS. For this, ALS treated beads were switched back to Tween-containing buffers and stored for several weeks.
  • Fig. 9 and Table 3 show that the positive effect of ALS lasts for at least 4 weeks. Table 3:

Abstract

The invention is directed to a novel use, method and kit to be employed to improve the amplification efficiency of bead-based emulsion PCR (emPCR).

Description

Method for improving the amplification efficiency of bead-based emulsion PCR (emPCR)
[0001] The present invention is directed to an improvement of the amplification efficiency of bead-based emulsion PCR (emPCR).
FIELD OF THE INVENTION
[0002] The present invention relates to the field of molecular biology, more par ticularly to the amplification of nucleic acid molecules.
BACKGROUND OF THE INVENTION
[0003] The polymerase chain reaction (PCR) is a technology in molecular biol ogy used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
[0004] Recently, PCR-based methods have been adapted to amplifying mole cules contained within water-in-oil emulsions. In such amplification methods a plurality of biological samples, e.g. nucleic acid samples, may be individually encapsulated in small aqueous micelles forming reaction compartments in the surrounding oil phase. PCR amplification is conducted on each of the plurality of encapsulated nucleic acid samples simultaneously. Such reaction compartments or microcapsules are often referred to as "microreactors" because the amplification occurs within the reaction compartments. This kind of PCR is often referred to as emulsion PCR (emPCR) or droplet PCR.
[0005] The small reaction compartments can include a template bead or micro sphere and the amplification process may be referred to as bead-based emPCR. In such a technique, beads along with DNA templates are suspended in an aqueous reaction mixture and then encapsulated in an inverse (water-in-oil) emulsion. The template DNA may be either bound to the bead prior to emulsification or may be included in solution in the amplification reaction mixture.
[0006] Techniques for bead-based emPCR are e.g. disclosed in PCT publica tion WO 2005/073410 A2 which is incorporated herein by reference in its entirety.
[0007] European patent application no. 3 170 903 discloses the use of an ani onic surfactant in the context the emPCR. There, the anionic surfactant is proposed to be added at the so-called "breaking step" which is carried out after the amplification process is concluded. In this breaking step the oil phase is removed or the emulsion is "broken" by the anionic surfactant in order to isolate the amplified nucleic acids from the reaction compartments or aqueous droplets, respectively. According to the authors, by adding the anionic surfactant the use of isopropanol or butanol as breaking agents can be avoided which have the disadvantages of light inflammability, acute toxicity, low flash point, causing corrosion, and unpleasant odor.
[0008] Bead-based emPCR is employed for target amplification for several next generation sequencing (NGS) platforms, such as Roche/545, ThermoFisher Scientific Ion TorrentTM, or the QIAGEN GeneReader. These systems employ bead-based emPCR for clonal amplification of the molecules to be sequenced prior to the actual sequencing reaction, i.e. single molecules are clonally amplified on the beads. [0009] In order to facilitate clonal amplification of DNA on beads, it is important that one single DNA molecule and one single bead are present in a reaction compartment or droplet in emPCR, respectively. This is achieved by optimizing or "fine-tuning" DNA molecule and bead concentrations. The number of beads in a droplet can be mediated e.g. by the number of beads and/or the number and size of the droplets,
[0010] However, despite theses measures occasionally in bead-based emPCR certain batches or subpopulations of beads do not perform well. This poor performance of beads is characterized in a decrease of the yield of amplified nucleic acid molecules in the emPCR. The reasons for this low performance are so far not perfectly clear.
[0011] Against this background, it is an object underlying the present invention to improve the low performance observable in bead-based emPCR.
[0012] The present invention satisfies these and other needs.
SUMMARY OF THE INVENTION
[0013] The present invention proposes the use of an anionic surfactant to im prove the amplification efficiency of bead-based emulsion PCR (emPCR).
[0014] The present invention further proposes a method for improving the am plification efficiency of bead-based emulsion PCR (emPCR), said method is comprising the following steps:
1) providing a buffer solution containing an anionic surfactant,
2) adding beads to said buffer solution,
3) incubating said beads-containing buffer solution,
4) isolating said beads from said buffer solution, and
5) subjecting said isolated beads to a bead-based emulsion PCR (emPCR). [0015] The inventor has realized that the amplification efficiency of a bead- based emPCR is determined by the fraction of beads that are distributed into the aqueous droplets. Beads going into the oil phase will not be amplified during emPCR.
[0016] The inventor has further found that beads commonly used in bead- based emPCR vary in terms of their accumulation behavior. This variation results in a tendency of some populations of beads to accumulate in the aqueous phase or droplets while other populations tend to accumulate in the oil phase. This phenomenon can even be found among one and the same species of beads which, apparently, comprise sub populations having different hydrophobic or hydrophilic characteristics.
[0017] As this has been realized by the inventor, whether a bead goes into the oil phase or contributes to a droplet or aqueous phase is dictated by the hydrophilic/ hydrophobic property of a bead. Importantly, hydrophobic beads which tend to accumulate in the oil phase will not contribute to the amplification in emPCR. To the contrary, hydro philic beads which tend to accumulate in the aqueous droplets do contribute to the amplification in emPCR.
[0018] The inventor found that the low performance observable in bead-based emPCR is caused by the hydrophobic properties of some, poor performing bead lots. The inventor has further observed that this phenomenon is batch-dependent, i.e. it can be found in some batches of beads while it is missing in other batches. It can also be varying in strength, i.e. some batches only perform slightly poorer than the reference standard beads while others are heavily poorer in performance.
[0019] The inventor has also realized that the low performance or amplification efficiency in bead-based emPCR may be additionally caused by a clumping of the beads.
[0020] The addition of an anionic surfactant to the beads according to the in vention rescues this low performance or low amplification efficiency, respectively.
[0021] This finding was surprising and could not be expected. [0022] In the art, e.g. as disclosed in EP 3 170 903 (loc. cit.), the use of an ani onic surfactant is proposed as a "breaking" agent to be added to the emulsion after the actual PCR reaction is already completed. In the art the addition of the anionic surfactant is proposed to isolate the already amplified nucleic acids. The anionic surfactant is, however, not used to increase the amplification efficiency or performance of the bead- based emPCR, respectively.
[0023] Without being bound to this theory, the probable mechanism underlying the invention is that the anionic surfactant shields hydrophobic patches of low-performing beads, thereby increasing the hydrophilic properties of beads.
[0024] The current art detergents that are added to reduce clumping of beads include Tween 20 and Triton X 100. However, these detergents are not negatively charged and the inventor has realized that they do not seem to stay on the bead surface for long-term. Therefore, these detergents do not confer the positive effect of the inven tion.
[0025] According to the invention an "anionic surfactant" refers to anionic com pounds that lower the surface tension or interfacial tension between two liquids or be tween a liquid and a solid. Anionic surfactants contain anionic functional groups at their head, such as sulfate, sulfonate, phosphate, and carboxylates. Prominent and well-suited alkyl sulfates include ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate, another name for the compound) and the related alkyl-ether sulfates sodium laureth sulfate, also known as sodium lauryl ether sulfate (SLES), and sodium myreth sulfate.
[0026] As used herein, the terms "bead" or "microsphere" which are used inter changeably, refer to a spherical microparticle commonly used in bead-based emPCR. Typical beads comprise diameters of between 0.1 and 100 pm and are commercially available in a wide variety of materials, including polymers, ceramics, glass, and metals. Polyethylene, polystyrene and expandable beads or microspheres are the most common types of polymer beads or microspheres. [0027] Beads or microspheres used in bead-based emPCR have the capability to immobilize nucleic acid molecules on their surface, such as PCR primers.
[0028] Preferably, the beads comprise magnetic properties.
[0029] In an embodiment of the invention the beads include Dynabeads™ My- One™ Carboxylic Acid (Thermo Fisher Scientific, Cat. No. 65011). Such beads are uniform, monosized superparamagnetic and 1 pm in diameter, and are composed of highly cross-linked polystyrene with evenly distributed magnetic material. The beads are further coated with a hydrophilic layer of glycidyl ether, concealing the iron oxide inside them. Carboxylic acid groups are then introduced on the surface of the beads.
[0030] "Amplification efficiency" as used herein is a measure of the capacity of the polymerase chain reaction to synthesize nucleic acids. The procedural steps occur prior to the "breaking step".
[0031] An "improvement" of the amplification efficiency refers to an increase in amplification effectiveness in contrast to a reference bead-based emPCR which charac terized by the accumulation of at least a fraction of the beads in the oil phase.
[0032] A "buffer solution" as used herein refers to a liquid medium providing the environment for the incubation of beads and the anionic surfactant. In an embodiment of the invention the buffer solution allows the carrying out of an emPCR and includes, without being limited thereto, aqueous TE buffer (Tris/EDTA buffer).
[0033] "Incubating" as referred to herein means the contacting of the surface of the beads and the anionic surfactant under conditions including time, temperature, and pressure, thereby ensuring the reacting of the beads' surface structures with the surfac tant. [0034] "Isolating" as used herein refers to recovering of the beads from the buffer solution after incubation and may include washing steps to remove residues of non- reacted surfactant from the beads' surface.
[0035] The object underlying the invention is herewith completely met.
[0036] In an embodiment of the invention said anionic surfactant is selected from the group consisting of: alkyl sulfates and alkyl carboxylates. Preferably, said alkyl sulfate is selected from the group consisting of: ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate), sodium laureth sulfate (SLES, sodium lauryl ether sulfate), and sodium myreth sulfate.
[0037] This measure has the advantage that such kind of anionic surfactant is employed which, according to the findings of the inventor, yields especially good results.
[0038] In an embodiment of the invention the bead-based emPCR involves beads comprising carboxylic acid groups and/or streptavidin on their surface.
[0039] By this measure the performance of such beads can be improved which are commonly used in emPCR. The provision of carboxylic acid groups or streptavidin on the beads' surface allows a stable coupling of nucleic acid molecules such as primers for the PCR. The interaction between the beads and nucleic acid molecules to capture the latter to the beads' surface may be mediated by covalent bonds, e.g. nucleic acid mole cules are covalently bound to the beads' surface, or by high non-covalent affinity interac tion, e.g. between biotin and streptavidin.
[0040] In an embodiment of the invention the beads comprise PCR primers on their surface, and it is preferred if the beads are pre-treated with said anionic surfactant before subjecting them to said bead-based emPCR. In another embodiment of the inven tion said pre-treatment is carried out by incubating said beads in a buffer solution contain ing said anionic surfactant. [0041] These measures result in the advantage that a non-desired denaturation of proteins in the actual PCR is avoided. The interaction between the hydrophobic patches of the beads and e.g. a long nonpolar hydrocarbon chain of an anionic surfactant, such as ALS, is strong. Therefore, the surfactant will remain on the beads for several hours and the desired effect will be maintained for subsequent PCR. No additional surfactant has to be added to the actual PCR buffer or reaction itself, respectively.
[0042] In an embodiment of the invention said buffer solution contains said ani onic surfactant in a final concentration of between approx. 0.1 50 wt.-%, preferably approx. 0.5 40 wt.-%, more preferably approx. 1 0 - 30 wt.-%, more preferably approx. 1 5 - 20 wt.-%, more preferably approx. 2.0 - 15 wt.-%, most preferably approx. 10 wt.-%.
[0043] This embodiment has the advantage that said anionic surfactant is pro vided in the buffer solution in a concentration which ensures an effective increase of the beads' hydrophilic properties. The skilled person can easily prepare a stock solution comprising the anionic surfactant in the concentration needed in order to end up with the final concentrations as prescribed in this embodiment.
[0044] It is to be understood that the surfactant may be added to the buffer so lution in several steps where, possibly, different amounts or concentrations may be added. There may be a first step of adding the anionic surfactant in a considerably high concen tration and (a) following step(s) of adding the anionic surfactant in a lower concentration, e.g. 10 wt.-% followed by 5 wt.-%, possibly with a washing steps in between.
[0045] Another subject-matter of the invention relates to a kit for amplifying nu cleic acid molecules by bead-based emulsion PCR (emPCR), comprising
- beads, preferably beads comprising carboxylic acid groups or streptavidin on their surface, further preferably beads comprising PCR primers on their surface; a buffer solution adapted for an incubation of said beads, and - an anionic surfactant, preferably selected from the group consisting of alkyl sulfates and alkyl carboxylates, further preferably said alkyl sulfate is se lected from the group consisting of: ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate), sodium laureth sulfate (SLES, sodium lauryl ether sulfate), and sodium myreth sulfate.
[0046] A kit is a combination of individual elements useful for carrying out the use and/or method of the invention, wherein the elements are optimized for use together in the methods. The kits also contain additional reagents, chemicals, buffers, reaction vials etc. which may be useful for carrying out the method according to the invention.
Such kits unify all essential elements required to work the method according to the invention, thus minimizing the risk of errors. Therefore, such kits also allow semi-skilled laboratory staff to perform the method according to the invention.
[0047] It is of particular advantage that the beads could be provided in surfac tant containing buffer, and the customer would just have to equilibrate (wash) beads with buffer required for application.
[0048] The features, characteristics, advantages and embodiments specified for the use and method according to the invention apply likewise to the kit according to the invention and vice versa.
[0049] It is to be understood that the before-mentioned features and those to be mentioned in the following cannot only be used in the combination indicated in the respec tive case, but also in other combinations or in an isolated manner without departing from the scope of the invention.
[0050] The invention is now further explained by means of embodiments result ing in additional features, characteristics and advantages of the invention. The embodi ments are of pure illustrative nature and do not limit the scope or range of the invention. The features mentioned in the specific embodiments are general features of the invention which are not only applicable in the specific embodiment but also in an isolated manner in the context of any embodiment of the invention.
[0051] The invention is now described and explained in further detail by refer ring to the following non-limiting examples and drawings.
Fig. 1 : illustrates clonal amplification of DNA molecules from a library by bead- based emPCR;
Fig. 2: illustrates the surface of the beads and the potential mechanism of ani onic surfactants, exemplified by ALS;
Fig. 3: shows a microscopic image of emulsion after emulsion making. Exam ples of beads in oil phase are indicated by an arrow;
Fig. 4/5: illustrate the effect of anionic surfactant treatment as exemplified with
ALS on the performance in GeneReader workflow.
Fig. 6: shows the structure of ammonium lauryl sulfate (ALS);
Fig. 7: shows electron microscopic images taken from Thermo MyOne magnet ic beads coupled with emPCR primers;
Fig. 8: shows the structure of ALS and Tween 20;
Fig. 9: shows that the anionic surfactant, exemplified with ALS, remains on the beads for at least 4 weeks. Examples
1. Introduction
[0052] Emulsion PCR (emPCR) is a commonly used tool in molecular biology. Several next generation sequencing (NGS) systems employ emPCR on beads (bead- based emPCR) for clonal amplification of the molecules to be sequenced prior to the actual sequencing reaction (Fig. 1).
[0053] The manufacturing of beads is a multi-step process. As one of the steps, hydrophobic polystyrene beads become hydrophilic (charged) by polymerization. Electron microscopic images indicate that this polymerization is not uniformly (Fig. 7). Therefore spots with hydrophobic patches will still be present (Fig. 2).
[0054] For the clonal amplification of the GeneReader system (Cat
No./ID: 185001 ; GeneRead Clonal Amp Q Kit), magnetic beads procured from Thermo Fisher Scientific (Dynabeads™ MyOne™ Carboxylic Acid, Cat. No 65011) are used.
These beads are then loaded with library specific primers using EDC chemistry.
[0055] During the life of the product, the inventor has observed batch- dependent performance differences correlating with batches procured from Thermo Fisher Scientific. The poorer performance of some lots coincides with the segregation of beads into the oil phase (Fig. 3; beads in oil phase are indicated by an arrow). Beads in the oil phase cannot be amplified during emulsion PCR as the reagents for clonal amplification are only present in the aqueous phase.
[0056] The inventor's data indicates that this failure mode is caused by the hy drophobic properties of these poor performing bead lots. The addition of an anionic surfactant, such as ammonium lauryl sulfate (ALS), rescues this failure mode. 2. Experiments
Some lots of carboxylated primer loaded beads are not compatible with emulsion PCR
[0057] NH2-modified oligonucleotides were coupled onto carboxylic acid beads using EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide).
[0058] Emulsion PCR was conducted using: Libraries (DNA fragments) gener ated using the QIAGEN GeneRead QIAact Lung DNA UMI Panel Kit (Cat No. /ID:
181931). Reagents for emulsion PCR and enrichment: GeneRead Clonal Amp Q Kit (Cat No./ID: 185001).
[0059] Starting with a new lot of Dynabeads™ MyOne™ Carboxylic Acid, the inventor was no longer able to synthesize primer loaded beads that perform in emulsion PCR: All lots synthesized with this lot and subsequent lots showed very low yield after enrichment of live beads during clonal amplification (see Figs. 4 and 5, and Table 1 ; note: minimum amount of live beads after enrichment is 140 Million). It has been found by the inventor that this was caused by the fact that a significant amount of primer loaded beads segregated into the oil phase (Fig. 3).
Table 1 :
Figure imgf000013_0001
[0060] More primer loaded beads in the oil phase means that fewer beads will be used in emulsion PCR, leading to a lower number of live beads and, subsequently, a lower yield after enrichment of live beads.
Failing primer loaded beads can be rescued with anionic surfactant
[0061] Extensive troubleshooting showed that this failure mode could be res cued with anionic surfactant, exemplified by amounium lauryl sulfate (ALS, for structure, see Fig. 6) as it is shown in Table 2:
Table 2:
Figure imgf000014_0001
* Measured using light scattering assay
No treatment group: beads stored in Tween 20 containing buffer;
• ALS treatment: beads washed with 10% ALS solution.
[0062] The ALS treatment not only rescued these beads, it strongly increased the yield after enrichment, therefore increasing the robustness of the complete Gene- Reader worklow. E.g. for a potential failure mode (operator error) where library input before clonal amplification is not correctly measured or pipetted (too low amount of library), there will still be enough beads for the sequencing reaction. [0063] The hypothesis for this observation is:
• Dynabeads™ MyOne™ Carboxylic Acid have hydrophobic patches. Elec tron microscopic images show that the coatings mediating the hydrophilic properties are not homogeneous (see Fig. 7). Most likely, some spots still have hydrophilic properties of the polystyrene.
• With the previous process, Tween 20 was added to the beads. Tween 20 will bind to the hydrophobic patches via its nonpolar hydrocarbon chain. However, in contrast to anionic surfactants such as ALS its hydrophilic do main is not positively charged (see Fig. 8).
• ALS also binds to the hydrophobic patches via its nonpolar hydrocarbon chain. In this way, the hydrophobic patches become negatively charged. This negative charge leads to an increased hydrophilic property compared to the non-charged Tween. Additionally, the negative charge is not binding to the phosphate backbone of the primers loaded onto the beads, i.e. phosphate background and negative charge of ALS lead to repulsion. Therefore, the PCR primer is accessible.
The positive effect of ammonium lauryl sulfate is stable over several weeks
[0064] ALS may not be added to enzymatic reactions as it facilitates the dena- turation of proteins. The inventor therefore tested the duration of the positive effect of ALS. For this, ALS treated beads were switched back to Tween-containing buffers and stored for several weeks.
[0065] Fig. 9 and Table 3 show that the positive effect of ALS lasts for at least 4 weeks. Table 3:
Figure imgf000016_0001
* Measured using Color Chart

Claims

Claims
1. Use of an anionic surfactant to improve the amplification efficiency of bead-based emulsion PCR (emPCR).
2. The use of claim 1 , characterized in that said anionic surfactant is selected from the group consisting of: alkyl sulfates and alkyl carboxylates.
3. The use of claim 2, characterized in that said alkyl sulfate is selected from the group consisting of: ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate), sodium laureth sulfate (SLES, sodium lauryl ether sul fate), and sodium myreth sulfate.
4. The use of any of the preceding claims, characterized in that the bead-based emPCR involves beads comprising carboxylic acid groups and/or streptavidin on their surface.
5. The use of any of the preceding claims, characterized in that the beads comprise PCR primers on their surface.
6. The use of any of the preceding claims, characterized in that the beads are pre treated with said anionic surfactant before subjecting them to said bead-based emPCR.
7. The use of claim 6, characterized in that said pre-treatment is carried out by incubating said beads in a buffer solution containing said anionic surfactant.
8. The use of claim 7, characterized in that said buffer solution contains said anionic surfactant in a final concentration of between approx. 0.1 - 50 wt.-%, preferably approx. 0.5 - 40 wt.-%, more preferably approx. 1.0 - 30 wt.-%, more preferably approx. 1.5 - 20 wt.-%, more preferably approx. 2.0 - 15 wt.-%, most preferably approx. 10 wt.-%.
9. A method for improving the amplification efficiency of bead-based emulsion PCR (emPCR), comprising the following steps:
1) providing a buffer solution containing an anionic surfactant,
2) adding beads to said buffer solution,
3) incubating said beads-containing buffer solution,
4) isolating said beads from said buffer solution, and
5) subjecting said isolated beads to a bead-based emulsion PCR (emPCR).
10. The method of claim 9, characterized in that said anionic surfactant is selected from the group consisting of: alkyl sulfates and alkyl carboxylates.
11. The method of claim 10, characterized in that said alkyl sulfate is selected from the group consisting of: ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate), sodium laureth sulfate (SLES, sodium lauryl ether sul fate), and sodium myreth sulfate.
12. The method of any of claims 9 to 11 , characterized in that the beads comprise carboxylic acid groups and/or streptavidin on their surface.
13. The method of any of claims 9 to 11 , characterized in that said buffer solution
contains said anionic surfactant in a final concentration of between approx. 0.1 - 50 wt.-%, preferably approx. 0.5 - 40 wt.-%, more preferably approx. 1.0 - 30 wt.-%, more preferably approx. 1.5 - 20 wt.-%, more preferably approx. 2.0 - 15 wt.-%, most preferably approx. 10 wt.-%.
14. A kit for amplifying nucleic acid molecules by bead-based emulsion PCR (emPCR), comprising - beads, preferably beads comprising carboxylic acid groups on their surface, further preferably beads comprising PCR primers on their surface;
- a buffer solution adapted for an incubation of said beads, and
- an anionic surfactant, preferably selected from the group consisting of alkyl sulfates and alkyl carboxylates, further preferably said alkyl sulfate is se lected from the group consisting of: ammonium lauryl sulfate (ALS), sodium lauryl sulfate (SDS, sodium dodecyl sulfate), sodium laureth sulfate (SLES, sodium lauryl ether sulfate), and sodium myreth sulfate.
15. The kit of claim 14, further comprising a manual having instructions for carrying out the method of any of claims 9 to 13.
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Citations (7)

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WO2005073410A2 (en) 2004-01-28 2005-08-11 454 Corporation Nucleic acid amplification with continuous flow emulsion
US20130210991A1 (en) * 2012-02-09 2013-08-15 Life Technologies Corporation Hydrophilic Polymeric Particles and Methods for Making and Using Same
US20130260447A1 (en) * 2006-05-11 2013-10-03 Darren R. Link Systems and methods for handling microfluidic droplets
US20150018236A1 (en) * 2012-02-02 2015-01-15 Invenra Inc. High throughput screen for biologically active polypeptides
US20170044523A1 (en) * 2014-02-14 2017-02-16 Memo Therapeutics Ag Method for recovering two or more genes, or gene products, encoding an immunoreceptor
EP3170903A1 (en) 2015-11-20 2017-05-24 Qiagen GmbH Method for processing a water-in-oil emulsion
US20180044726A1 (en) * 2012-06-15 2018-02-15 Board Of Regents, The University Of Texas System High throughput sequencing of multiple transcripts

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Publication number Priority date Publication date Assignee Title
WO2005073410A2 (en) 2004-01-28 2005-08-11 454 Corporation Nucleic acid amplification with continuous flow emulsion
US20130260447A1 (en) * 2006-05-11 2013-10-03 Darren R. Link Systems and methods for handling microfluidic droplets
US20150018236A1 (en) * 2012-02-02 2015-01-15 Invenra Inc. High throughput screen for biologically active polypeptides
US20130210991A1 (en) * 2012-02-09 2013-08-15 Life Technologies Corporation Hydrophilic Polymeric Particles and Methods for Making and Using Same
US20180044726A1 (en) * 2012-06-15 2018-02-15 Board Of Regents, The University Of Texas System High throughput sequencing of multiple transcripts
US20170044523A1 (en) * 2014-02-14 2017-02-16 Memo Therapeutics Ag Method for recovering two or more genes, or gene products, encoding an immunoreceptor
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