Patent Application
20180251826
The present disclosure relates to a method for amplifying DNA, in particular, a method for amplifying whole genome of a single cell.
Single-cell whole genome sequencing is a new technique for amplifying and sequencing whole-genome at single-cell level. Its principle is to amplify minute amount whole-genome DNA isolated from a single cell, and perform high-throughput sequencing after obtaining a high coverage of the complete genome. Two prerequisites are needed for establishment of this technique: 1. high-quality whole-genome amplification technique; and 2. a low-cost, high-throughput, sequencing technique.
Currently, there are four major types of whole-genome amplification techniques: Primer Extension Preamplification-Polymerase Chain Reaction (referred to as PEP-PCR, for detailed method see Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. 1992. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci USA. 89 (13):5847-51.), Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (referred to as DOP-PCR, for detailed method see Telenius H, Carter N P, Bebb C E, Nordenskjo M, Ponder B A, Tunnacliffe A. 1992. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13:718-25), Multiple Displacement Amplification (referred to as MDA, for detailed method see Dean F B, Nelson J R, Giesler T L, LaskenRS. 2001. Rapid amplification of plasmid and phageDNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11:1095-99), and Multiple Annealing and Looping Based Amplification Cycles (referred to as MALBAC, for detailed method see PCT patent application No. WO2012166425). However, some of the currently-available conventional amplification methods are easy to operate but ultimately lead to undesirable amplification results, while others have good amplification effect but the operation process of which are rather complicated. Taking MALBAC as an example, it mainly has the following defects: 1. Generally, amplification products can only be obtained after going through several steps like cell lysing, lysing termination (by increasing temperature/adding of a neutralizing reagent), pre-amplification, and amplification, etc. The entire process involves preparation of multiple reagents and addition of liquids with lid being opened, which increase the risk of introducing environmental contamination. 2. The entire experimental process lasts more than 4 hours, with a low efficiency concerning personnel and instrument, and thus cannot provide satisfactory results within a short period of time for samples that are clinically in urgent need of verification. 3. The entire experimental process requires a high degree of proficiency of the operators, and first-time users cannot obtain satisfactory amplification results in a short time.
Therefore, at present there is an urgent need for an improved amplification method that overcomes one, more, or all defects of the conventional amplification methods.
The present disclosure provides a method of amplifying genomic DNA of a cell and a kit for amplifying genomic DNA.
In one aspect of the present application, a method of amplifying genomic DNA of a cell is provided, said method comprises: (a) providing a reaction mixture, wherein the reaction mixture comprises the genomic DNA, a first type of primer, a second type of primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and wherein the second type of primer comprises the common sequence but not the variable sequence; (b) placing the reaction mixture in a first thermal cycle program such that the variable sequence of the first type of primer can pair with the genomic DNA and amplify the genomic DNA to obtain a genomic amplification product, wherein the genomic amplification product comprises the common sequence at its 5′ end and comprises complementary sequence of the common sequence at its 3′ end; (c) placing the reaction mixture obtained from step (b) in a second thermal cycle program, such that the common sequence of the second type of primer can pair with 3′ end of the genomic amplification product and amplify the genomic amplification product to obtain an expanded genomic amplification product, wherein the reaction mixture is provided prior to the step (b) and the step (c).
In some embodiments, the method further comprises analyzing the amplification product to identify disease- or phenotype-associated sequence features. In some embodiments, the disease- or phenotype-associated sequence features include chromosomal abnormalities, chromosomal translocation, aneuploidy, partial or complete chromosomal deletion or duplication, fetal HLA haplotypes and paternal mutations. In some embodiments, the disease or phenotype is selected from the group consisting of: beta-thalassemia, Down's syndrome, cystic fibrosis, sickle cell disease, Tay-Sachs disease, Fragile X syndrome, spinal muscular atrophy, hemoglobinopathy, Alpha-thalassemia, X-linked diseases (diseases dominated by genes on the X chromosome), spina bifida, anencephaly, congenital heart disease, obesity, diabetes, cancer, fetal sex, and fetal RHD.
In some embodiments, the genomic DNA is contained within a cell, and the reaction mixture further comprises a surfactant and/or a lyase capable of lysing the cell.
In some embodiments, the method further comprises placing the reaction mixture in a lysing thermal cycle program prior to said steps (b) and (c), such that the cell is lysed and the genomic DNA is released.
In some embodiments, the common sequence is selected such that the common sequence does not substantially bind to genomic DNA to cause amplification. In some embodiments, the common sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
In some embodiments, the variable sequence comprises a random sequence. In some embodiments, the variable sequence has a length of 2-20 bases, 3-10 bases, 4-9 bases, or 5-8 bases. In some embodiments, three or more base positions in the variable sequence consist of one or more types of bases selected from G, A and T, or consist of one or more types of bases selected from C, A and T. In some embodiments, the three or more base positions are located at 3′ end or in the middle of the variable sequence. In some embodiments, the variable sequence is selected from the group consisting of (N)nGGG, (N)nTTT, (N)mTNTNG, (N)xGTGG(N)y, wherein N refers to any nucleotide that can pair with a naturally occurring nucleic acid, n is a positive integer selected from 3-17, m is a positive integer selected from 3-15, x and y are positive integers selected from 3-13, respectively. In some embodiments, the variable sequence is selected such that the variable sequence is homogeneously distributed in genome and with a high coverage.
In some embodiments, the first type of primer includes a sequence of SEQ ID NO: 11 [GTGAGTGATGGTTGAGGTAGTGTGGAG], SEQ ID NO: 12 [GTGAGTGATGGTTGAGGTAGTGTGGAG GGG], SEQ ID NO: 13 [GTGAGTGATGGTTGAGGTAGTGTGGAG TTT], SEQ ID NO: 14 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG] or SEQ ID NO: 15 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN], and the second type of primer, in a 5′ to 3′ orientation, has a sequence of SEQ ID NO: 1 [GTGAGTGATGGTTGAGGTAGTGTGGAG], wherein N is any nucleotide that can pair with a naturally occurring nucleic acid.
In some embodiments, the nucleic acid polymerase has thermostability and/or strand displacement activity. In some embodiments, the nucleic acid polymerase is selected from the group consisting of Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase (e.g., Thermococcus litoralis Vent polymerase, Deep Vent polymerase, Vent(-exo) polymerase, Deep Vent(-exo) polymerase), TOPOTaq DNA polymerase, 9° Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant (lacking 3′-5′ exonuclease activity), Phusion® High-Fidelity DNA polymerase, Taq polymerase, Bst DNApolymerase (full-length), E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase, and any combination thereof.
In some embodiments, the reaction mixture further comprises a pH regulator, such that the pH value of the reaction mixture is maintained between 7.0-9.0.
In some embodiments, the reaction mixture further comprises one or more components selected from the group consisting of: Mg2+, dTT, bovine serum albumin, DNase inhibitor, RNase, SO42−, Cl−, K+, Ca2+, Na+, and (NH4)+.
In some embodiments, the first thermal cycle program included: (b1) placing the reaction mixture in a thermal program capable of opening double strands of the genomic DNA; (b2) placing the reaction mixture in a thermal program that enables binding of the first type of primer to single-strand DNA template; (b3) placing the reaction mixture in a thermal program that enables extension of the length of the first type of primer that binds a single-strand DNA template under the action of the nucleic acid polymerase, to produce an amplification product; (b4) placing the reaction mixture in a thermal program capable of denaturing the amplification product into single strands; (b5) repeating steps (b2) to (b4) to a designated first cycle number. In some embodiments, the designated first cycle number is more than 2. In some embodiments, after proceeding to the second cycle, the amplification product comprises genomic amplification product comprising the common sequence at the 5′ end and a complementary sequence of the common sequence at the 3′ end. In some embodiments, the method further comprises a step (b4′) after step (b4) and prior to step (b5), wherein the reaction mixture is placed in a suitable thermal program enabling hybridization of the 3′ end and 5′ end of the genome amplification product to form a loop structure, or enabling binding of the 3′ end of the genomic amplification product to a primer. In some embodiments, the method directly proceeds to step (b5) after step (b4). In some embodiments, the first cycle number of the step (b5) is more than 3, more than 4, more than 5, or more than 6, and no more than 10.
In some embodiments, the step (c) comprises: (c1) placing the reaction mixture obtained from step (b) in a thermal program capable of opening DNA double strands; (c2) placing the reaction mixture in a thermal program that enables binding of the second type of primer to single strands of the genomic amplification product obtained from step (b); (c3) placing the reaction mixture in a thermal program that enables extension of the length of the second type of primer that binds to single strands of the amplification products, under the action of the nucleic acid polymerase; (c4) repeating steps (c1) to (c3) to a designated second cycle number. In some embodiments, the second cycle number in the step (c4) is greater than the first cycle number in the step (b5). In some embodiments, the thermal program in the step (b1) comprises allowing reacting for 1-10 minutes at a temperature between 90-95° C. In some embodiments, the step (b2) comprises placing the reaction mixture in more than one thermal program to promote sufficient and efficient binding of the first type of primer to the DNA template; in some embodiments, the more than one thermal program comprises: a first temperature between 5-10° C., a second temperature between 25-30° C., and a third temperature between 45-50° C.
In some embodiments, the step (b2) comprise allowing reacting at a first temperature for 3-50 s, allowing reacting at a second temperature for 3-50 s, and allowing reacting at a third temperature for 3-50 s. In some embodiments, the thermal program in the step (b3) comprises allowing reacting at a temperature of 60-90° C. for 1-15 minutes. In some embodiments, the thermal program in the step (b4) comprises allowing reacting at a temperature of 90-95° C. for 10-50 s. In some embodiments, the thermal program in the step (c1) comprises allowing reacting at a temperature of 90-95° C. for 10-30 s. In some embodiments, the thermal program in the step (c2) comprises allowing reacting at a temperature of 45-65° C. for 10-30 s. In some embodiments, the thermal program in the step (c3) comprises allowing reacting at a temperature of 60-80° C. for 1-15 minutes. In some embodiments, the genomic DNA in the step (a) is released from a lysed cell, the lysing includes thermal lysing, base lysing, enzymatic lysing or mechanical lysing.
In some embodiments, the thermal lysing comprises lysing at a temperature between 20-100° C. for 10-100 minutes. In some embodiments, the thermal lysing is carried out in presence of a lysing reagent. In some embodiments, the lysing reagent includes one or more surfactants selected from the group consisting of: NP-40, Tween, SDS, Triton X-100, EDTA, and guanidinium isothiocyanate, and/or lyase.
In another aspect of the present application, a method of amplifying genome of a cell is provided, said method comprises: (a) providing a reaction mixture, wherein the reaction mixture comprises DNA of said genome, a first type of primer, a second type of primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and wherein the second type of primer comprises the common sequence but not the variable sequence; (b) placing the reaction mixture in a first thermal cycle program such that the variable sequence of the first type of primer can pair with the DNA of the genome and amplify the DNA of the genome to obtain a genomic amplification product, wherein the genomic amplification product comprises the common sequence at its 5′ end and comprises complementary sequence of the common sequence at its 3′ end; wherein the first thermal cycle program comprises: (b1) allowing reacting at a first denaturing temperature between 90-95° C. for 1-10 minutes; (b2) allowing reacting at a first annealing temperature between 5-10° C. for 3-50 s, at a second annealing temperature between 25-30° C. for 3-50 s, and at a third annealing temperature between 45-50° C. for 3-50 s; (b3) allowing reacting at a first elongation temperature between 60-90° C. for 1-15 minutes; (b4) allowing reacting at a first melting temperature between 90-95° C. for 10-50 s; (b5) repeating steps (b2) to (b4) for 6-9 cycles; (c) placing the reaction mixture obtained from step (b) in a second thermal cycle program, such that the common sequence of the second type of primer can pair with 3′ end of the genomic amplification product and amplify the genomic amplification product to obtain an expanded genomic amplification product; wherein the second thermal cycle program comprises: (c1) allowing reacting at a second denaturation temperature between 90-95° C. for 1-10 minutes; (c2) allowing reacting at a second melting temperature between 90-95° C. for 10-30 s; (c3) allowing reacting at a fourth annealing temperature between 45-65° C. for 10-30 s; (c4) allowing reacting at a second elongation temperature between 60-80° C. for 1-15 minutes; (c5) repeating steps (c2) to (c4) for 5-30 cycles; (d) obtaining amplification product from the step (c); wherein the reaction mixture is provided prior to the step (b) and the step (c).
In some embodiments, the common sequence comprises or consists of SEQ ID NO: 1; the variable sequence comprises or consists of NNNNNTTT or NNNNNGGG and N is any nucleotide that can pair with a naturally-occurring nucleic acid.
In some embodiments, a kit for amplifying genomic DNA is provided, said kit comprises a mixture containing a first type of primer and a second type of primer, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and wherein the second type of primer comprises the common sequence but not the variable sequence.
In some embodiments, the mixture further comprises a mixture of nucleotide monomers and Mg2+.
In some embodiments, the mixture further comprises one or more components of the following: dTT, bovine serum albumin (BSA), pH regulator (e.g. Tris HCl), DNase inhibitor, RNase, SO42−, Cl−, K+, Ca2+, Na+, and/or (NH4)+.
In some embodiments, the mixture further comprises a nucleic acid polymerase.
In some embodiments, the kit further comprises a surfactant and/or a lyase capable of lysing a cell. In some embodiments, the surfactant is selected from one or more of NP-40, Tween, SDS, TritonX-100, EDTA, and guanidine isothiocyanate. In some embodiments, the lyase is selected from one or more of protease K, pepsin, and papain.
In some embodiments, the mixture further comprises a surfactant and/or a lyase capable of lysing a cell.
In another aspect of the present application, a kit for amplifying genomic DNA is provided, said kit comprises a first type of primer and a second type of primer, and further comprises an instruction for users, said instruction records the following steps: mixing the first type of primer and the second type of primer in the same container before said amplifying, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and wherein the second type of primer comprises the common sequence but not the variable sequence.
The above and other features of the present disclosure will be more comprehensively described through the following specification and claims appended, in combination with the drawings. It is understood that these drawings only depict several embodiments of the present disclosure and therefore should not be considered as limiting the scope of the disclosure. By applying the drawings, the present disclosure will be described more clearly and in more details.
The present disclosure provides a method of amplifying genomic DNA, in particular a method of amplifying whole genome of a single cell.
The present disclosure is based, at least in part, on the discovery that prior to amplification reaction of genomic DNA, all reagents required for amplification can be added into a single reaction mixture, and then this reaction mixture is placed under a condition for amplification reaction, until completion of amplification. This method eliminates the need of adding reagents to a reaction mixture after initiation of amplification reaction, and thereby greatly reduces additional operations caused by, and contamination which might be resulted from addition of reagents, and greatly shortens the reaction time required.
Before the present disclosure, when two or more primers are used for genomic DNA, the amplification reaction needs to be separated into at least two steps, where different primers are used in each step, and thereby different amplification purposes are achieved. It was previously believed that, in order to avoid interferences between primers which in turns impact the amplification effect, primers required for the second step can only be added to the reaction mixture after the first step is completed, or the first step may only employ a very low cycle number (e.g., 1 cycle). Therefore, in the methods prior to the present disclosure, one can either add only the primers needed for the first step before an amplification reaction and after the first-step amplification reaction ends, add primers required for the second step to the reaction system, and perform the second-step amplification reaction (see, e.g., WO2012/166425); or only employ a very low cycle number in the first step, by which a desirable amplification efficiency can hardly be achieved. Surprisingly, the inventors of the present application found that when placing all primers originally believed to interfere with each other at once into a single reaction mixture and performing amplification under the reaction conditions of the present disclosure, an amplification effect which is comparable to that where primers are separately added can be unexpectedly obtained. Therefore, the present disclosure greatly improves reaction efficiency, shortens reaction time, and reduces the risk of sample contamination, and improves reliability of results.
In one aspect, the present application provides a method of amplifying genomic DNA of a cell, comprising: (a) providing a reaction mixture, wherein the reaction mixture comprises the genomic DNA, a first type of primer, a second type of primer, a mixture of nucleotide monomers, and nucleic acid polymerase, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and wherein the second type of primer comprises the common sequence but not the variable sequence; (b) placing the reaction mixture in a first thermal cycle program such that the variable sequence of the first type of primer can pair with the genomic DNA and amplify the genomic DNA to obtain a genomic amplification product, wherein the genomic amplification product comprises the common sequence at its 5′ end and comprises complementary sequence of the common sequence at its 3′ end; (c) placing the reaction mixture obtained from step (b) in a second thermal cycle program, such that the common sequence of the second type of primer can pair with 3′ end of the genomic amplification product and amplify the genomic amplification product to obtain an expanded genomic amplification product. See
Step (a): Providing a Reaction Mixture
The method of the present application is broadly applicable for amplification of genomic DNA, particularly for rapid and accurate amplification of minute amount genomic DNA.
i. Genomic DNA
The method of the present application is preferably useful for genomic DNA. In certain embodiments, the initial amount of genomic DNA contained in a reaction mixture is no more than 10 ng, no more than 5 ng, no more than 1 ng, no more than 500 pg, no more than 200 pg, no more than 100 pg, no more than 50 pg, no more than 20 pg, or no more than 10 pg.
A genomic DNA may be from a biological sample, e.g., biological tissue, or body fluid that contains cells or free DNA. Samples containing genomic DNA can be obtained through known methods, e.g. obtained through oral mucosal samples, nasal samples, hair, mouthwash, cord blood, plasma, amniotic fluid, embryonic tissue, endothelial cells, nail samples, hoof samples, etc. A biological sample can be provided in any suitable form, for example, in paraffin embedded form, in freshly isolated form, etc. Genomic DNA may be from any species or biological species, including, but not limited to, humans, mammals, cattle, pigs, sheep, horses, rodents, birds, fish, zebrafish, shrimp, plants, yeasts, viruses or bacteria.
In certain embodiments, genomic DNA is that from a single cell, or that from two or more cells of the same type. Single cells or cells of the same type may be from, e.g., pre-implantation embryos, embryonic cells in peripheral blood of pregnant women, single sperms, egg cells, fertilized eggs, cancer cells, bacterial cells, tumor circulating cells, tumor tissue cells, or single cells or multiple cells of the same type obtained from any tissue. The method of the present application can be used to amplify DNA in some valuable samples or samples with low initial amount, e.g., human egg cells, germ cells, tumor circulating cells, tumor tissue cells, etc.
Methods for obtaining single cells are also known in the art, e.g., by the method of flow cytometry sorting (Herzenberg et al., Proc Natl Acad Sci USA 76:1453-55, 1979; Iverson et al., Prenatal Diagnosis 1:61-73, 1981; Bianchi et al., Prenatal Diagnosis 11:523-28, 1991), fluorescence-activated cell sorting, the method of separation using magnetic beads (MACS, Ganshirt-Ahlert et al., Am J Obstet Gynecol 166:1350, 1992), by using a semi-automatic cell picker (e.g., the Quixell™ cell transfer system by Stoelting Co.) or a combination thereof. In some embodiments, gradient centrifugation and flow cytometry techniques can be used to increase the efficiency of separation and sorting. In some embodiments, cells of particular types, such as cells expressing particular biomarkers, can be selected according to different properties of single cells.
Methods for obtaining genomic DNA are also well known in the art. In certain embodiments, genomic DNA can be released and obtained by lysing cells from biological samples or single cells. Lysing may be performed using any suitable method known in the art, for example, lysing can be performed by means of thermal lysing, base lysing, enzymatic lysing, mechanical lysing, or any combination thereof (see, specifically, e.g., U.S. Pat. No. 7,521,246, Thermo Scientific Pierce Cell Lysis Technical Handbook v2 and Current Protocols in Molecular Biology (1995). John Wiley and Sons, Inc. (supplement 29) pp. 9.7.1-9.7.2.).
Mechanical lysing includes methods that break cells using mechanical forces such as using ultrasonication, high speed stirring, homogenization, pressurization (e.g., French press), decompression and grinding. The most commonly used mechanical lysing method is the liquid homogenization method, which compels cell suspension to pass through a very narrow space, and thus shear force is applied on cell membrane (WO2013153176 A1).
In certain embodiments, mild lysing methods may be used. For example, cells can be lysed by being heated in a Tween-20-containing solution at 72° C. for 2 min, heated in water at 65° C. for 10 min (Esumi et al., Neurosci Res 60(4):439-51 (2008)), heated in PCR buffer II (Applied Biosystems) containing 0.5% NP-40 at 70° C. for 90 s (Kurimoto et al., Nucleic Acids Res 34(5):e42 (2006)), or using Protease (e.g. Protease K) or a chaotropic salt solution (e.g. guanidine isothiocyanate) (U.S. Patent Application No. US 20070281313).
Thermal lysing includes heating and repeated freeze-thaw methods. In some embodiments, the thermal lysing comprises lysing for 10-100 minutes at a temperature between 20-100 centigrade. In some embodiments, temperature for thermal lysing can be any temperature between 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 30-80, 40-80, 50-80, 60-80 or 70-80° C. In some embodiments, temperature for thermal lysing is no less than 20, 30, 40 or 50° C. In some embodiments, temperature for thermal lysing is no more than 100, 90 or 80° C. In some embodiments, time for thermal lysing can be any period between 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, or 30-40 minutes. In some embodiments, time for thermal lysing is no less than 20, 30, 40, 50, 60, 70, 80, or 90 minutes. In some embodiments, time for thermal lysing is no more than 90, 80, 70, 60, 50, 40, 30, or 20 minutes. In some embodiments, temperature for thermal lysing varies over time. In some embodiments, the thermal lysing is maintained under a temperature at 30-60° C. for 10-30 minutes, followed by a temperature at 70-90° C. for 5-20 minutes.
In some embodiments, the thermal lysing is carried out in the presence of a lysing reagent. In the presence of a lysing reagent, time or temperature required for lysing can be reduced. A lysing reagent can break protein-protein, lipid-lipid and/or protein-lipid interactions, thereby promoting release of genomic DNA from a cell.
In some embodiments, the lysing reagent comprises a surfactant and/or a lyase. Surfactants can be categorized into ionic, amphoteric and non-ionic surfactants. Generally, lysing efficacies of amphoteric and nonionic surfactants are weaker than that of ionic surfactants. Exemplary surfactants include, but are not limited to, one or more of NP-40, Tween, SDS, GHAPS, TritonX-100, TritonX-114, EDTA, sodium deoxycholate, sodium cholate, and guanidine isothiocyanate. Those skilled in the art can select type and concentration of a surfactant based on practical need. In some embodiments, working concentration of a surfactant is 0.01%-5%, 0.1%-3%, 0.3%-2% or 0.5-1%.
Exemplary lyases can be proteinase K, pepsin, papain, etc., or any combination thereof. In some embodiments, working concentration of a lyase is 0.01%-1%, 0.02%-0.5%, 0.03%-0.2%, or 0.4-0.1%.
In the method provided herein, a lysate containing genomic DNA can be used directly in a reaction mixture. For example, a biological sample may be pre-treated by lysing to obtain a lysate, which is then mixed with other components of the reaction mixture. If needed, the lysate can be further processed so that the genomic DNA therein is isolated, and then the isolated genomic DNA is further mixed with other components of reaction mixture to provide a reaction mixture.
In some embodiments, a nucleic acid sample obtained through lysing can be amplified without being purified.
The present application further provides a simpler method, i.e., directly mixing a genomic DNA-containing cell with other components required for amplification to obtain a reaction mixture, in other words, genomic DNA in the reaction mixture is present within a cell. In such circumstances, the reaction mixture may further contain surfactants (such as, but not limited to, one or more of NP-40, Tween, SDS, TritonX-100, EDTA, and guanidine isothiocyanate) and/or lyase (e.g., one or more of Protease K, pepsin, and papain) capable of lysing the cell. In this way, cell lysing and genomic DNA amplification both occur in the same reaction mixture, which not only improves reaction efficiency, shortens reaction time, but also retains a fairly good amplification effect.
In certain embodiments, the method provided herein may further comprise placing the reaction mixture in a lysing thermal cycle program after completion of step (a) and prior to step (b), such that the cell is lysed and the genomic DNA is released. Those skilled in the art can select a suitable lysing thermal cycle program according to the lysate components contained in the reaction mixture, type of the cell, etc. Exemplary lysing thermal cycle program includes placing the reaction mixture at 50° C. for 3 minutes to 8 hours (e.g., any time period between 3 minutes to 7 hours, 3 minutes to 6 hours, 3 minutes to 5 hours, 3 minutes to 4 hours, 3 minutes to 3 hours, 3 minutes to 2 hours, 3 minutes to 1 hour, 3 minutes to 40 minutes, 3 minutes to 20 minutes; such as 10 minutes, 20 minutes, 30 minutes, etc.), then at 80° C. for 2 minutes to 8 hours (e.g., any time period between 2 minutes to 7 hours, 2 minutes to 6 hours, 2 minutes to 5 hours, 2 minutes to 4 hours, 2 minutes to 3 hours, 2 minutes to 2 hours, 2 minutes to 1 hour, 2 minutes to 40 minutes, 2 minutes and 20 minutes; such as 10 minutes, 20 minutes, 30 minutes, etc.). The lysing thermal program can be run for 1 cycle, or 2 or more cycles as needed, depending on specific lysing conditions.
ii. Primers
The reaction mixture further contains two different types of primer, of which the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, and the second type of primer comprises the common sequence but not the variable sequence. The variable sequence in the first type of primer can bind to genomic DNA template and a certain length of genomic template can be replicated under the action of a nucleic acid polymerase, to obtain an amplification product with a common sequence at its 5′ end and a genomic sequence at its 3′ end, which is also referred to as a semi-amplicon herein. The variable sequence in the first type of primer can also pair with and bind to a semi-amplicon and replicate using the semi-amplicon as a template, generating an amplification product with a common sequence at its 5′ end and a complementary sequence of the common sequence at its 3′ end, which is also referred to as full-amplicon herein. The second type of primer can bind to the complementary sequence of the common sequence at 3′ end of a full-amplicon, and thereby further replicates the full-amplicon, and greatly increases its number.
The common sequence in the present application refers to a specific sequence located at 5′ end of the first type of primer. Length of a common sequence can be, e.g., 10-30, 12-29, 15-28, 18-26, or 20-24 bases. In the present application, a suitable common sequence is selected, such that it substantially does not bind to genomic DNA, which results in amplification, and avoids polymerization between first type of primer and first type of primer or between first type of primer and second type of primer.
In certain embodiments, a common sequence only comprises three or two types of bases with poor ability of self-complementary pairing, and does not comprise the other one or two types of bases. In certain embodiments, the common sequence consists of three types of bases, G, A and T, i.e., the common sequence does not contain the C base. In certain embodiments, the common sequence consists of three types of bases, C, A and T, i.e., the common sequence does not contain the G base. In certain embodiments, the common sequence consists of two types of bases, A and T, A and C, A and G, T and C, or, T and G, i.e., the common sequence does not contain G and C at the same time. Without wishing to be bound by theory, it is believed that if a common sequence contains C or G base, primer-primer polymerization may happen, which generates polymers and thereby impairs the ability to amplify genomic DNA. Preferably, a common sequence does not have any self-pairing sequence, or any sequence that would cause primer-primer pairing, or multiple bases of the same type in succession.
In certain embodiments, a suitable base sequence of common sequence and proportion of each base thereof can be selected, to ensure that the common sequence itself does not undergo base pairing with genomic DNA template sequence or resulted in amplification.
In certain embodiments, the common sequence is selected from the group consisting of: SEQ ID NO: 1 (GTG AGT GAT GGT TGA GGT AGT GTG GAG), SEQ ID NO: 2 (GTGGAGTTAGTGAGTGTAATGGAT), SEQ ID NO: 3 (GGTTTGGTGTGGTGTGTGGTGGTG), SEQ ID NO: 4 (ACAACACTATCAATCCCTATCCTAC), SEQ ID NO: 5 (ATGGTAGTGGGTAGATGATTAGGT), SEQ ID NO: 6 (CATATCCCTATACCTAATACCATTAC).
The 5′ end of the first type of primer is a common sequence, and the 3′ end is a variable sequence. A common sequence and a variable sequence may be directly adjacent, or may have a spacer sequence of one or more bases. The variable sequence in the present application refers to a base sequence with an unfixed sequence, for example, it may comprise a random sequence. A random sequence may comprise any nucleotide that can undergo base pairing with a naturally-occurring nucleic acid, such as four types of naturally-occurring bases of A, T, G, and C, as well as other nucleotide analogs and modified nucleotides known to those skilled in the art, as long as it can pair with genomic DNA and achieve amplification. Nucleotide sequence in a variable sequence may be in multiple possibilities of variation. Therefore, the first type of primer may comprise a set of primers with different sequences, wherein each primer has a common sequence at its 5′ end, and a variable sequence at its 3′ end. The common sequences in these primers are identical, but the variable sequences may vary.
A variable sequence can have a suitable length, e.g., 2-20 bases, 2-19 bases, 2-18 bases, 2-17 bases, 2-16 bases, 2-15 bases 2-14 bases, 2-13 bases, 2-12 bases, 2-11 bases, 2-12 bases, 2-11 bases, 2-10 bases, 2-9 bases, 2-8 bases, 3-18 bases, 3-16 bases, 3-14 bases, 3-12 bases, 3-10 bases, 4-16 bases, 4-12 bases, 4-9 bases, or 5-8 bases. In certain embodiments, the variable sequence is 5 bases in length. In certain embodiments, the variable sequence is 8 bases in length. Theoretically, if base at each position is randomly selected from the four types of bases, A, T, G, and C, a variable sequence with a length of 4 bases can generate 256 possible random sequences by combination, and a variable sequence with a length of 5 bases can generate 1024 possible random sequences by combination, and so forth. These variable sequences can complementarily pair with corresponding sequences at different positions in genomic DNA, and thereby replication is initiated at different positions in genomic DNA.
The variable sequences can be selected by a random approach, certain limiting conditions can also be further applied on the basis of the random approach, in order to eliminate some unwanted situations or to enhance matching degree with target genomic DNA. In certain embodiments, three or more base positions in the variable sequence consist of one or more types of bases selected from G, A, and T (i.e., not being C), or consist of one or more types of bases from C, A, and T (i.e., not being G), to avoid complementary pairing between variable sequence and common sequence. In some embodiments, when the common sequence does not contain G but contains C, three or more base positions in the variable sequence consist of one or more types of bases of C, A and T (i.e., not being G). In some embodiments, when the common sequence does not contain C but contains G, three or more base positions in the variable sequence consist of one or more types of bases of G, A and T (i.e., not being C). In some embodiments, when a common sequence contains neither C nor G, three or more base positions in a variable sequence consist of one or more types of bases selected from G, A and T (i.e., not being C) or consists of one or more types of bases of C, A and T (i.e., not being G). The three or more bases can be located at 3′ end of the variable sequence, or can be located in middle part of the variable sequence. The three or more bases may be successive or unsuccessive. For example, none of three adjacent bases at 3′ end of the variable sequence is C, or three bases at 3′ end of a variable sequence which are spaced from each other are not C, or certain two successive bases as well as another base spaced therefrom at 3′end of a variable sequence are not C. When the three base positions are successive, they can be in the following exemplary sequences: TTT, GGG, TTG, GAA or ATG.
In certain embodiments, the variable sequence is selected from the group consisting of: (N)nGGG, (N)nTTT, (N)mTNTNG, and (N)xGTGG(N)y, wherein N is any random nucleotide that can undergo base pairing with a naturally-occurring nucleic acid, n is a positive integer selected from 3-17, m is a positive integer selected from 3-15, and x and y are positive integers selected from 3-13, respectively. In certain embodiments, the variable sequence in the first type of primer may have one or more sequences of (N)nGGG, (N)nTTT, (N)mTNTNG, (N)xGTGG(N)y. In certain embodiments, a variable sequence is selected from the group consisting of SEQ ID NO: 7 (GGG), SEQ ID NO: 8 TTT), SEQ ID NO: 9 (NNNTNTNG), SEQ ID NO: 10 (NNNGTGGNN).
In certain embodiments, variable sequences that are more evenly distributed in genome and with a higher coverage can also be selected through statistical calculations, thereby increasing opportunity of recognition between the variable sequence and genomic DNA.
In certain embodiments, the first type of primer can comprise SEQ ID NO: 11 [GTGAGTGATGGTTGAGGTAGTGTGGAG], SEQ ID NO: 12 [GTGAGTGATGGTTGAGGTAGTGTGGAG GGG], SEQ ID NO: 13 [GTGAGTGATGGTTGAGGTAGTGTGGAG TTT], SEQ ID NO: 14 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG] or SEQ ID NO: 15 [GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN], wherein N is any nucleotide (e.g., A, T, G, C) that can undergo base pairing with a naturally-occurring nucleic acid.
The second type of primer in the reaction mixture comprises the common sequence but not the variable sequence. The 5′ and 3′ ends of the second type of primer may or may not contain other additional sequences. In certain embodiments, the sequence of the second type of primer consists of the common sequence of the first type of primer. In certain embodiments, the second type of primer, in a 5′ to 3′ orientation, comprises or consists of the sequence of SEQ ID NO: 1 [GTGAGTGATGGTTGAGGTAGTGTGGAG].
In some embodiments, concentration of the primer in the reaction mixture is 300 ng-1500 ng/μL. In some embodiments, concentration of the primer in the reaction mixture is 300 ng-1400 ng/μL, 300 ng-1200 ng/μL, 300 ng-1000 ng/μL, 300 ng-800 ng/μL, 300 ng-600 ng/μL, or 300 ng-400 ng/μL. In some embodiments, concentration of the primer in the reaction mixture is 500 ng-1400 ng/μL, 600 ng-1400 ng/μL, 800 ng-1400 ng/μL, 900 ng-1400 ng/μL, 1000 ng-1400 ng/0_, or 1200 ng-1400 ng/μL. In some embodiments, concentration of the primer in the reaction mixture is 400 ng-1400 ng/μL, 500 ng-1200 ng/μL, 600 ng-1000 ng/0_, or 700 ng-800 ng/μL.
iii. Other Components
The reaction mixture further comprises other components required for DNA amplification, such as nucleic acid polymerase, a mixture of nucleotide monomers, and suitable metal ions and buffer components required for enzymatic activity, and the like. For at least one or more types of these components, reagents known in the art can be used.
Nucleic acid polymerase in the present application refers to an enzyme capable of synthesizing a new nucleic acid strand. Any nucleic acid polymerase suitable for the method of the present application can be used. Preferably, DNA polymerase is used. In certain embodiments, the method of the present application uses a thermostable nucleic acid polymerase, such as those whose polymerase activity does not decrease or decrease by less than 1%, 3%, 5%, 7%, 10%, 20%, 30%, 40% or 50% at a temperature for PCR amplification (e.g., 95° C.). In certain embodiments, the nucleic acid polymerase used in the method of the present application has strand displacement activity. The “strand displacement activity” of the present application refers to an activity of nucleic acid polymerase that enables separation of a nucleic acid template from the complementary strand with which it pairs and binds, and where such separation performs in a 5′ to 3′ direction, and is accompanied with generation of a new nucleic acid strand that is complementary to the template. Nucleic acid polymerases with strand displacement ability and applications thereof are known in the art, see e.g., U.S. Pat. No. 5,824,517, which is incorporated herein by reference in its entirety. Suitable nucleic acid polymerases include, but are not limited to: one or more of Phi29 DNA polymerase, Bst DNA polymerase, Bst 2.0 DNA polymerase, Pyrophage 3137, Vent polymerase (e.g. Thermococcus litoralis Vent polymerase, Deep Vent polymerase, Vent(-exo) polymerase, Deep Vent(-exo) polymerase), TOPOTaq DNA Polymerase, 9° Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant (lacking 3′-5′ exonuclease activity), Phusion® High-Fidelity DNA polymerase, Taq polymerase, Psp GBD (exo-) DNA polymerase, Bst DNA polymerase (full-length), E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase.
In certain embodiments, the reaction mixture contains one or more of Thermococcus litoralis Vent polymerase, Deep Vent polymerase, Vent(-exo) polymerase, or Deep Vent(-exo) polymerase. In certain embodiments, the reaction mixture contains Thermococcus litoralis Vent polymerase. Thermococcus litoralis Vent polymerase refers to a natural polymerase isolated from Thermococcus litoralis. In certain embodiments, the reaction mixture contains Deep Vent polymerase. The Deep Vent polymerase refers to a natural polymerase isolated from Pyrococcus species GB-D. In certain embodiments, the reaction mixture contains Vent(-exo) polymerase. Vent(-exo) polymerase refers to an enzyme resulted from D141A/E143A gene engineering of Thermococcus litoralis Vent polymerase. In certain embodiments, the reaction mixture contains Deep Vent(-exo) polymerase. Deep Vent (-exo) polymerase refers to an enzyme resulted from D141A/E143A gene engineering of Deep Vent polymerase. The various Vent polymerases in the present application are commercially available, e.g., from New England Biolabs Company.
A reaction mixture can also comprise suitable metal ions required for exerting enzymatic activity of nucleic acid polymerase (e.g., Mg2+ ions in suitable concentration (e.g., at a final concentration of about 1.5 mM to about 8 mM), a mixture of nucleotide monomers (e.g., dATP, dGTP, dTTP, and dCTP), bovine serum albumin (BSA), dTT (e.g., at a final concentration of about 2 mM to about 7 mM), purified water, and the like.
In certain embodiments, the reaction mixture can also further comprise a pH regulator, such that pH value of the reaction mixture is maintained between 7.0-9.0. Suitable pH regulators may include, e.g, Tris HCl and Tris SO4. In certain embodiments, a reaction mixture can also further comprise one or more types of other components, e.g., DNase inhibitor, RNase, SO42−, Cl−, K+, Ca2+, Na+, and/or (NH4)+, and the like.
One of the features of the method provided herein is that the reaction mixture is provided prior to the step (b) and the step (c). As the preparation of the reaction mixture is fully completed before the thermal cycle program is performed, the reaction mixture can react according to predetermined settings upon entrance of the thermal cycle program, with no need to further open the lid or add any component, and thereby avoids contamination and improves reaction efficiency. In some embodiments, after the step (a) is completed, there is no need to further add reactants such as enzymes, primers and dNTPs to the reaction mixture. In some embodiments, providing of the reaction mixture is completed before the step (b). In some embodiments, no reactants (e.g., enzymes, primers, and dNTPs) are further added to the reaction mixture after initiation of the step (b). In some embodiments, the step (a) precedes the step (b) and the step (c).
Step (b): Placing in the First Thermal Cycle Program
The method provided herein comprises step (b): placing the reaction mixture in the first thermal cycle program, such that the variable sequence of the first type of primer can pair with the genomic DNA and amplify the genomic DNA to obtain a genomic amplification product, wherein the genomic amplification product comprises the common sequence at its 5′ end and comprises complementary sequence of the common sequence at its 3′ end.
“Amplification” in the present application means addition of nucleotides complementary to a nucleic acid template at the 3′ end of a primer under the action of a nucleic acid polymerase, whereby to synthesize a new nucleic acid strand that is base-complementary to the nucleic acid template. Suitable methods for amplifying nucleic acids may be used, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), or other suitable amplification methods. These methods are all known in the art, see e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202, as well as Innis et al., “PCR protocols: a guide to method and applications” Academic Press, Incorporated (1990) and Wu et al. (1989) Genomics 4:560-569, all of these literatures and patents are incorporated herein by reference in their entirety.
During the process of amplification, the reaction mixture is placed in a suitable thermal cycle program, such that DNA template double strands are unwound into single strands, primers hybridize with template single strands, and primers elongate. Thus, a thermal cycle program typically comprises: a denaturing or melting temperature at which DNA template double strands are unwound into single strands; an annealing temperature at which a primer specifically hybridizes with a single-strand DNA template; and an elongation temperature at which DNA polymerase adds nucleotides complementary to DNA template bases at the 3′ end of a primer, so that the primer elongates, and a new DNA strand that is complementary to the DNA template is obtained. The newly synthesized DNA strand can serve as a new DNA template in the next reaction cycle, for a new cycle of DNA synthesis.
In step (b) of the method of the present application, the reaction mixture is placed in a first thermal cycle program such that the variable sequence of the first type of primer in the reaction mixture can bind to the genomic DNA through base pairing, and that genomic DNA is replicated under the action of a nucleic acid polymerase.
In the first thermal cycle program, first the reaction mixture is placed in a thermal program capable of opening the double strands of the genomic DNA (step (b1)). To ensure that genomic DNA double strands are completely unwound into single strands (i.e., denaturing/melting), high reaction temperatures such as 90° C.-95° C. can be used and a long reaction time can be maintained. In certain embodiments, the thermal program in step (b1) comprises reacting at a temperature between 90° C.-95° C. for 1-10 minutes.
Next, the reaction mixture is placed in a thermal program that enables binding of the first type of primer to the single-strand DNA template (step (b2)). In this thermal program, the variable sequence in the first type of primer binds to complementary sequences at different positions in genomic DNA through base complementarity (i.e., annealing), and thereby replications are initiated at different positions in genomic DNA. Due to the diversity of variable sequences in the first type of primer, wherein differences exist with regard to both base ratio and sequence, the optimal binding temperature for each variable sequence to genomic DNA also varies greatly. Thus, at a given annealing temperature, it is possible that only some of the primers can bind to genomic DNA well, while the binding of the others to genomic DNA may not be ideal. In certain embodiments, the step (b2) comprises a program of placing the reaction mixture in more than one temperature, to facilitate sufficient binding of the first type of primer to the DNA template. For example, DNA denatured reaction mixture can be rapidly cooled to a low temperature, such as about 5° C.-10° C., followed by allowing the reaction mixture to react for a suitable period at different annealing temperatures respectively, by means of gradient heating, whereby to ensure that as many primers as possible pair with genomic DNA. In certain embodiments, step (b2) comprises allowing reacting for a suitable period (e.g., 3-50 s) at a first annealing temperature between 5-10° C. (e.g., 10° C.), allowing reacting for a suitable period (e.g., 3-50 s) at a second annealing temperature between 25-30° C. (e.g., 30° C.), and allowing reacting for a suitable period (e.g., 3-50 s) at a third annealing temperature between 45-50° C. (e.g., 50° C.).
It is well known in the art that annealing temperature of a primer is generally no more than 5° C. lower than Tm value of a primer, and an excessively low annealing temperature will lead to primer-primer non-specific binding, whereby resulting in primer aggregation and nonspecific amplification products. Therefore, low temperatures such as 5° C.-10° C. will not usually be used as primer annealing temperature. However, it is unexpectedly found by the inventors, that even if gradient heating starts from a low temperature (e.g., 5° C.-10° C.), pairing between primers and genomic DNA can still maintain good specificity, and amplification results still retain very low variability, indicating accurate and reliable amplification results. Meanwhile, since annealing temperatures for primers cover circumstance of low temperature, binding of wider range of primer sequences to genomic DNA is ensured, whereby better genomic coverage and amplification depth are provided.
After primer annealing thermal program, the reaction mixture is placed in a thermal program that enables elongation of the first type of primer that binds to a single-strand DNA template under the action of the nucleic acid polymerase, to produce an amplification product (step (b3)). The elongation temperature is usually related to the optimum temperature for DNA polymerase, for which those skilled in the art can make specific selection according to specific reaction mixture. In certain embodiments, the DNA polymerase in the reaction mixture may have strand-displacement activity, such that if during elongation, the primer encounters a primer or amplicon that binds to the downstream template, the strand-displacement activity of the DNA polymerase can enable separation of the downstream-binding primer from the template strand, thereby ensuring that the elongating primer continues to elongate, so that longer amplification sequences are obtained. DNA polymerases with strand-displacement activity include, but are not limited to, e.g., phi29 DNA polymerase, T5 DNA polymerase, SEQUENASE 1.0 and SEQUENASE 2.0. In certain embodiments, the DNA polymerase in the reaction mixture is a thermostable DNA polymerase. Thermostable DNA polymerases include, but are not limited to, e.g., Taq DNA polymerase, OmniBase™ Sequence enzyme, Pfu DNA polymerase, TaqBead™ Hot Start polymerase, Vent DNA polymerase (e.g., Thermococcus litoralis Vent polymerase, Deep Vent polymerase, Vent (-exo) polymerase and Deep Vent (-exo) polymerase), Tub DNA polymerase, TaqPlus DNA polymerase, Tfl DNA polymerase, Tli DNA polymerase, and Tth DNA polymerase. In certain embodiments, the DNA polymerase in the reaction mixture may be a DNA polymerase that is thermostable and has strand-displacement activity. In certain embodiments, the DNA polymerase in the reaction mixture is selected from the group consisting of: one or more of Phi29 DNA polymerase, Bst DNA polymerase, Pyrophage 3137, Vent polymerase (e.g., Thermococcus litoralis Therm polymerase, Deep Vent polymerase, Vent(-exo) polymerase, Deep Vent(-exo) polymerase), TOPOTaq DNA polymerase, 9° Nm polymerase, Klenow Fragment DNA polymerase I, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, T7 phase DNA polymerase variant (lacking 3′-5′ exonuclease activity), Phusion® High-Fidelity DNA polymerase, Taq polymerase, Bst DNA polymerase (full length), E. coli DNA polymerase, LongAmp Taq DNA polymerase, OneTaq DNA polymerase. In certain embodiments, step (b3) comprises allowing reacting at an elongation temperature between 60-90° C. (e.g., 65-90° C., 70-90° C., 75-90° C., 80-90° C., 60-85° C., 60-80° C., 60-75° C., or 60-70° C.) for 1-15 minutes (e.g., 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-14, 3-14, 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, or 13-14 minutes). In certain embodiments, step (b3) comprises allowing reacting at 70° C. for 2 minutes.
After the primer extension program, the reaction mixture is placed in a thermal program capable of denaturing the amplification product into single strands (step (b4)). The temperature in this step may be similar to that in step (b1), but the reaction time is shorter. In certain embodiments, step (b4) comprises allowing reacting at a temperature of 90-95° C. for 10-50 s. At this point, DNA single strands in the reaction mixture contain not only the original genomic DNA single strands, but also newly synthesized DNA single strands resulted from amplification, both of which can serve as DNA templates in the next cycle, bind to primers and initiate the next cycle of replication.
Steps (b2) to (b4) are then repeated to a designated first cycle number to obtain genomic amplification product. The first cycle number should be at least 2. In the first cycle, the sequence at 3′ end of the variable sequence of the first type of primer is elongated, and the obtained amplification product has a common sequence at its 5′ end and a complementary sequence of the genomic template single-strand sequence at its 3′ end; such amplification products are also known as semi-amplicon. In the second cycle, the previous amplification products themselves can also serve as DNA templates to bind to the variable sequences in the first type of primers. The primer extends toward 5′ end of the amplification product under the action of nucleic acid polymerase until replication of the common sequence at 5′ end of the amplification product is completed, thereby obtaining a genomic amplification product having a common sequence at its 5′ end and a complementary sequence of the common sequence at its 3′end; such amplification product is also referred to as full-amplicon.
In certain embodiments, the first cycle number is controlled within a suitable range to ensure sufficient amplification products for use in subsequent reactions without compromising the accuracy of amplification products due to excessive number of cycles. For example, the first cycle number is at least 3, at least 4, at least 5, or at least 6, and preferably no more than 8, no more than 9, no more than 10, no more than 11, or no more than 12. If the cycle number is too low, few amplification products are obtained, and thus, to obtain sufficient amplification products, it is necessary to increase cycle number in the next step of amplification (i.e., step (c)), which will reduce the accuracy of amplification results. While, if cycle number is too high, sequence variation may occur during genomic DNA amplification, resulting in a templates bias in the next step of amplification (i.e., step (c)), leading to an inaccurate final amplification result.
In certain embodiments, in step (b), a step (b4′) is further comprised after step (b4), wherein the reaction mixture is placed in a suitable thermal program enabling hybridization of the 3′ end and the 5′ end of the genomic amplification product to form a loop structure, or enabling binding of the 3′ end of the genomic amplification product to a primer. It was previously considered that step (b4′) is capable of protecting 3′ end of a full amplicon, and thereby avoids head-to-tail polymerization between full amplicons, and consequently avoids conjunction of two sequences which are originally not adjacent in a genome. This will help improve the accuracy of amplification result.
In certain embodiments, the method directly proceeds to step (b5) after step (b4), without undergoing other steps (e.g., step (b4′)). In this way, full amplicons have not been subject to particular steps to avoid head-to-tail polymerization, and thus, theoretically, such amplification result should be somewhat defective with regard to accuracy. However, unexpectedly, in the method of the present application, even without a particular step after step (b4), which enables full amplicons to loop or enables binding of 3′ end to a primer, the final amplification result still has considerably high accuracy, which is comparable to the effect of the method which employs step (b4′). This simplifies reaction steps while still retaining specificity of reactions.
In step (b), not only the first type of primer but also the second type of primer exists in the reaction system. The second type of primer contains the common sequence in the first type of primer. Since the common sequence is substantially not complementary to genomic sequences, if specificity of amplification reaction is high enough, the second type of primer will not directly pair with genomic DNA and initiate replication of genomic DNA in step (b). However, when step (b) has undergone two cycles, full amplicons with a complementary sequence of the common sequence at its 3′ end begin to emerge in the reaction mixture, and 3′ end of such full amplicons can pair with the second type of primer through base-pairing, and thus might cause replication of the full amplicons by the second type of primer starting in step (b) (e.g., starting from the third cycle). This might interfere replication of genomic DNA by the first type of primer, causing insufficient amplification of genomic DNA by the first type of primer, by which desired coverage of genomic DNA is not reached. In addition, when the first type of primer and the second type of primer exist in the reaction system at the same time, the first type of primer and the second type of primer might undergo a template-independent primer-primer amplification reaction, resulting in generation of primer polymer. However, surprisingly, despite the presence of these uncertainties described above, the inventors of the present disclosure unexpectedly found that when both the first type of primer and the second type of primer exist in the reaction mixture at the same time and both are capable of amplification reaction, the first type of primer seems not being interfered by the second type of primer, and still retains high specificity and broad coverage for genomic DNA amplification, and when comparing to the method where the first type of primer alone is used in step (b) and then the second type of primer is further added in step (c), the results obtained are overall comparable.
Step (c): Placing in a Second Thermal Cycle Program
The method provided herein further comprises step (c): placing the reaction mixture obtained from step (b) in a second thermal cycle program such that the common sequence of the second type of primer can pair with 3′ end of the genomic amplification product and amplify the genomic amplification product to obtain an expanded genomic amplification product.
Since the genomic amplification product obtained from step (b), i.e. the full amplicon, has a complementary sequence to the common sequence at 3′ end, it can be complementary to the common sequence of the second type of primer; under the action of nucleic acid polymerase, the second type of primer extends and full length of the full amplicon is replicated.
In the second thermal cycle program, the reaction mixture is first placed in a thermal program capable of opening DNA double strands (step (c1)). The DNA double strands herein mainly refers to the double strands of genomic amplification product (i.e., full amplicon) obtained from step (b). Although original genomic DNA still exists in the reaction mixture at this point, the original genomic DNA is not DNA template to be amplified in step (c), since the second type of primer substantially does not bind to the genomic DNA. A higher reaction temperature such as 90° C.-95° C. can be used for reaction for a suitable period as long as the full-amplicon double strands to be amplified can be denatured into single strands. In certain embodiments, the thermal program in step (c1) comprises allowing reacting for 10-30 s (e.g., 20 s) at a melting temperature between 90-95° C. (e.g., 94° C.). In certain embodiments, a step to placing the reaction mixture in a thermal program capable of opening DNA double strands and allowing reacting for sufficient time, is further comprised after the first thermal cycle program ends but before the second thermal cycle program starts. This will help ensure complete denature of template DNA double-strands into single strands.
After step (c1), the reaction mixture is placed in a thermal program that enables binding of the second type of primer to single-strands of the genomic amplification product obtained from step (b). On the basis of base composition in the second type of primer, Tm value of the second type of primer can be calculated and a suitable annealing temperature for the second type of primer can be determined based on this Tm value. In certain embodiments, thermal program in step (c2) comprises allowing reacting for 10-30 s (e.g., 15 s) at an annealing temperature between 45-65° C. (e.g., 58° C.). In certain embodiments, the second type of primer is SEQ ID NO: 1 and the thermal program in step (c2) comprises allowing reacting for 10-30 s at 58° C. In certain embodiments, the annealing temperature in step (c2) is higher than that in step (b2). In step (c2), the reaction mixture may still contain the first type of primer that did not undergo reaction in step (b), and variable sequences of these first type of primers may pair with the DNA single-strand templates obtained from step (c1), resulting in incomplete amplification sequences. When annealing temperature in step (c2) is higher than that suitable for the first type of primer, binding of the first type of primer with single-strand DNA template can be reduced or avoided, thereby selectively allowing amplification of the second type of primer.
After completion of primer annealing, the reaction mixture is placed in a thermal program that enables elongation of the second type of primer that binds to single strands of the amplification product, under the action of the nucleic acid polymerase. In certain embodiments, the thermal program in step (c3) comprises allowing reacting for 1-15 minutes (e.g., 2 minutes) at an elongation temperature between 60-80° C. (e.g., 72° C.).
Steps (c1) to (c3) can be repeated to a second cycle number to obtain the desired expanded genomic amplification product. During this process, the genomic amplification product obtained in step (b) is further replicated and amplified, the number of which is greatly increased, in order to provide sufficient genomic DNA sequences for subsequent studies or operations. In certain embodiments, the second cycle number in the step (c4) is greater than the first cycle number in the step (b5). In certain embodiments, the second cycle number is controlled within a suitable range such that it can provide sufficient amount of DNA without compromising the accuracy of amplification due to excessive number of cycles. In certain embodiments, the second cycle number is 15-30 cycles (e.g., 15-30, 15-28, 15-26, 15-24, 15-22, 15-20, 15-18, 15-17, 16-30, 17-30, 18-30, 20-30, 22-30, 24-30, 26-30, 28-30 cycles).
In certain embodiments, step (c) further comprises placing the reaction mixture in the same thermal program as that in step (c3) (e.g. 72° C.) and allowing reaction for a suitable period (e.g., 5 minutes) after the second thermal cycle program. The reaction mixture is then placed at a temperature of 4° C. to terminate reaction. In certain embodiments, the reaction mixture is placed at a temperature of 4° C. to terminate reaction directly after completion of the reaction of step (c).
In certain specific embodiments, the present application also provides a method for amplifying genome of a cell comprising:
(a) providing a reaction mixture, wherein the reaction mixture comprises DNA of said genome DNA, a first type of primer, a second type of primer, a mixture of nucleotide monomers, and a nucleic acid polymerase, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and wherein the second type of primer comprises the common sequence but not the variable sequence;
(b) placing the reaction mixture in a first thermal cycle program such that the variable sequence of the first type of primer can pair with the DNA of the genome and amplify the DNA of the genome to obtain a genomic amplification product, wherein the genomic amplification product comprises the common sequence at its 5′ end and comprises complementary sequence of the common sequence at its 3′ end; wherein the first thermal cycle program comprises:
(b1) allowing reacting at a first denaturation temperature between 90-95° C. for 1-10 minutes;
(b2) allowing reacting at a first annealing temperature between 5-10° C. for 3-50 s, at a second annealing temperature between 25-30° C. for 3-50 s, and at a third annealing temperature between 45-50° C. for 3-50 s;
(b3) allowing reacting at a first elongation temperature between 60-90° C. for 1-15 minutes;
(b4) allowing reacting at a first melting temperature between 90-95° C. for 10-50 s;
(b5) repeating steps (b2) to (b4) to 6-9 cycles;
(c) placing the reaction mixture obtained from step (b) in a second thermal cycle program, such that the common sequence of the second type of primer can pair with 3′ end of the genomic amplification product and amplify the genomic amplification product to obtain an expanded genomic amplification product, wherein the second thermal cycle program comprises:
(c1) allowing reacting at a second denaturation temperature between 90-95° C. for 1-10 minutes;
(c2) allowing reacting at a second melting temperature between 90-95° C. for 10-30 s;
(c3) allowing reacting at a fourth annealing temperature between 45-65° C. for 10-30 s;
(c4) allowing reacting at a second elongation temperature between 60-80° C. for 1-15 minutes;
(c5) repeating steps (c2) to (c4) for 5-30 cycles;
(d) obtaining amplification product from the step (c);
wherein the reaction mixture is provided prior to the step (b) and the step (c).
In certain embodiments, genomic DNA in the reaction mixture in step (a) is present within a cell, i.e., the reaction mixture contain cells in which the genomic DNA to be amplified is contained. In certain embodiments, the reaction mixture in step (a) contains cells and further comprises components capable of lysing cells, such as surfactant and/or lyase, etc. Suitable surfactants, such as one or more of NP-40, Tween, SDS, TritonX-100, EDTA, and guanidine isothiocyanate, can be used. Suitable lyases, such as one or more of Protease K, pepsin and papain, can also be selected. In such embodiments, the method of amplifying cell genome as described above further comprises placing a reaction mixture in a lysing thermal cycle program after step (a) and prior to step (b) (e.g., placing a reaction mixture at 50° C. for 20 minutes, then at 80° C. for 10 minutes), to allow lysing of the cell and release of the genomic DNA. In this way, the entire amplification reaction actually involves allowing cell lysing and genome amplification to occur in the same reaction mixture, and is achieved by placing a reaction mixture in different thermal cycle programs. This greatly simplifies the procedure, avoids the risk of contamination brought by multiple sample manipulations, and also achieves good amplification specificity and low variability concerning amplification result.
In certain embodiments, the first type of primer comprises or consists of SEQ ID NO: 11, 12, 13, 14, and/or 15, wherein the common sequence comprises or consists of SEQ ID NO: 1.
Advantages
The method provided by the present application has several advantages over the methods in prior art.
In one aspect, the present disclosure combines steps of nucleic acid pre-amplification, amplification, etc. into one reaction, under thermal cycle condition. This reaction reduces manual operation, and whole genome amplification of nucleic acid can be accomplished simply by placing nucleic acid into a PCR tube and performing specific program, the amplification products of which have a high degree of genome coverage and low amplification bias. Operations of reagent preparation, and liquid addition with open lid are eliminated, and risks of contamination resulted from experimental environment and operators are reduced, and meanwhile, the overall experimental time period is shortened, with the overall amplification time being only 2.5 hours.
In another aspect, the present disclosure can also combine steps of cell lysing, nucleic acid pre-amplification and amplification etc. into one reaction, under thermal cycle condition, which further reduces manual operations and eliminates the step in which cells are separately lysed, and further shortens experimental time and reduces risks of contamination.
The method of the present application also retains high accuracy and broad coverage of amplification, in addition to simplified operations. The method of the present application uses quasi-linear amplification technique to reduce sequence-dependent amplification bias. In pre-amplification, a focus is on amplification from original sample DNA template, and with a high genome coverage and a small amplification bias. Full amplicons generated during pre-amplification phase are amplified in large number during amplification phase. Amplification process by such technique has high yield, with a minimum initial template of a few picograms, and the results are reliable and reproducible.
Application
In certain embodiments, the product obtained by amplification using the method of the present application can be further used for sequencing, such as for whole genome sequencing. Due to the high requirement on initial amount of samples to be analyzed (more than 100 ng) by various sequencing analysis platforms such as Next Generation Sequencing (NGS), Microarray, and fluorescent quantitative PCR, etc., whole genome amplification is needed if sufficient nucleic acid material for analysis need to be obtained from a single human cell (about 6 pg) or a sample in a small initial amount. Genomic DNA in a biological sample (e.g., a single cell) can be amplified by the method of the present application, and the product obtained from amplification can be sequenced by a suitable sequencing method in the art. Exemplary sequencing methods include, sequencing by hybridization (SBH), sequencing by ligase (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, molecular beacons, pyrosequencing, fluorescent in situ sequencing (FISSEQ), fluorescence resonance energy transfer (FRET), multiplex sequencing (U.S. patent application Ser. No. 12/027,039; porreca et al. (2007) NAT. Methods 4:931), polymerized colony (POLONY) sequencing (U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944 and PCT/US05/06425); swing sequencing (PCT US05/27695), TaqMan reporter probe digestion, nanogrid rolling circle sequencing (ROLONY) (U.S. patent application Ser. No. 12/120,541), FISSEQ beads (U.S. Pat. No. 7,425,431), and allele-specific oligonucleotide ligation assay, etc.
In certain embodiments, sequencing of amplification products of the method herein can be accomplished by high-throughput method. High-throughput methods typically fragmentize nucleic acid molecules to be sequenced (e.g., by means of enzymatic cleavage or mechanical shearing, etc.), to form large amount of short fragments ranging from tens to hundreds of bp in length. By sequencing tens of thousands, hundreds of thousands, millions, tens of millions, or even hundreds of millions of such short fragments in parallel in one sequencing reaction, throughput of sequencing can be greatly increased and time required for sequencing can be shortened. The measured sequences of short fragments can be joined into a complete sequence after data processing via software. A variety of high-throughput sequencing platforms are known in the art, such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, and Polonator platform technology, and the like. A variety of light-based sequencing techniques are also known in the art, see, e.g., those described in Landegren et al. (1998) Genome Res. 8: 769-76, Kwok (2000) Pharmacogenomics 1: 95-100, and Shi (2001) Clin. Chem. 47: 164-172.
In certain embodiments, products obtained from amplification using the method of the present application can also be used to analyze genotypes or genetic polymorphisms in genomic DNA, such as single nucleotide polymorphism (SNP) analysis, short tandem repeat (STR) analysis, restriction fragment length polymorphism (RFLP) analysis, variable number of tandem repeats (VNTRs) analysis, complex tandem repeat (CTR) analysis, or microsatellite analysis and the like, see, e.g., Krebs, J. E., Goldstein, E. S. and Kilpatrick, S. T. (2009). Lewin's Genes X (Jones & Bartlett Publishers) for reference, which is incorporated herein by reference in its entirety.
In certain embodiments, amplification products obtained by the method of the present application can also be used for medical and/or diagnostic analysis. For example, a biological sample from an individual may be amplified using the method of the present application, and whether abnormalities such as mutations, deletions, insertions or fusion between chromosomes are present in gene or DNA sequence of interest in the amplification product can be analyzed, whereby to evaluate the risk of developing certain disease for the individual, the progression stage, genotyping and severity of the disease, or the likelihood that the individual respond to certain therapy. The gene or DNA sequence of interest can be analyzed using suitable methods known in the art, such as, but not limited to, nucleic acid probe hybridization, primer-specific amplification, sequencing a sequence of interest, single-stranded conformational polymorphism (SSCP), etc.
In certain embodiments, the methods of the present application can be used to compare genomes derived from different single cells, in particular different single cells from the same individual. For example, when differences exist between genomes of different single cells of the same individual, such as between tumor cells and normal cells, genomic DNA of different single cells can be amplified separately using the method herein, and the amplification product can be further analyzed, for example, analyzed and compared by sequencing, or subject to comparative genomic hybridization (CGH) analysis. See, Fan, H. C., Wang, J., Potanina, A., and Quake, S. R. (2011). Whole-genome molecular haplotyping of single cells. Nature Biotechnology 29, 51-57., and Navin, N., Kendall, J., Troge, J., Andrews, P., Rodgers, L., Mclndoo, J., Cook, K., Stepansky, A., Levy, D., Esposito, D., et al. (2011). Tumour evolution inferred by single-cell sequencing. Nature 472, 90-94, for reference, all of which are incorporated herein by reference in their entirety.
In certain embodiments, the methods of the present application can be used to identify haploid structures or haploid genotypes in homologous chromosomes. Haploid genotype refers to the combination of alleles at multiple loci that are co-inherited on chromosome of the same haplotype. A biological sample (e.g., a single cell from an individual's diploid) can be divided into enough portions so that DNA sequences on two homologous haplotypes are statistically separated into different portions. Each section is assigned as one reaction mixture, and each reaction mixture is subjected to DNA amplification by the method of the present application, and then the amplification product is subjected to sequence analysis and is aligned with a reference genomic sequence (e.g., publically available standard genomic sequence of humans, see, International Human Genome Sequencing Consortium, Nature 431, 931-945 (2004)), to identify single nucleotide mutations therein. If no reference genome sequence is readily available, a region of suitable length assembled from multiple fragment sequences of genome by means of de-novo genome assembly can also be used for comparison.
In certain embodiments, products obtained from amplification using the method of the present application can be further used for analysis such as gene cloning, fluorescence quantitative PCR and the like.
In certain embodiments, the method of the present application can also further comprise analyzing the amplification product to identify disease- or phenotype-associated sequence features. In some embodiments, analyzing the amplification product comprises genotyping of DNA amplicon. In some other embodiments, analyzing the amplification product includes identifying polymorphism of DNA amplicons, such as single nucleotide polymorphism (SNP) analysis. SNP can be detected by some well-known methods such as oligonucleotide ligation assay (OLA), single base extension, allele-specific primer extension, mismatch hybridization and the like. A disease can be diagnosed by comparison of SNP to those of known disease phenotypes.
In some embodiments, the disease- or phenotype-associated sequence features include chromosomal abnormalities, chromosomal translocation, aneuploidy, deletion or duplication of a part of or all chromosomes, fetal HLA haplotypes and paternal mutations.
In some embodiments, the disease or phenotype may be beta-thalassemia, Down's syndrome, cystic fibrosis, sickle cell disease, Tay-Sachs disease, Fragile X syndrome, spinal muscular atrophy, hemoglobinopathy, Alpha-thalassemia, X-linked diseases (diseases dominated by genes on the X chromosome), spinal bifida, anencephaly, congenital heart disease, obesity, diabetes, cancer, fetal sex, and fetal RHD.
Kit
Another aspect of the application also provides a kit for genomic DNA amplification, wherein the kit comprises a mixture containing a first type of primer and a second type of primer, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and the second type of primer comprises the common sequence but not the variable sequence.
In certain embodiments, wherein the mixture further comprises a mixture of nucleotide monomers (e.g., dATP, dGTP, dTTP, and dCTP), dTT and Mg2+. In certain embodiments, Mg2+ concentration in the mixture is between 2 mmol-8 mmol/μL, dNTP concentration is between 1 mmol-8 mmol/μL and dTT concentration is between 2 mmol-7 mmol/μL. In certain embodiments, the mixture further comprises one or more components selected from the group consisting of: bovine serum albumin (BSA), a pH-regulator (e.g., Tris HCl), DNase inhibitor, RNase, SO42−, Cl−, K+, Ca2+, Na+, and/or (NH4)+ and the like. In certain embodiments, pH value range of the mixture is between 7.0-9.0.
In certain embodiments, the kit further comprises a nucleic acid polymerase, and the nucleic acid polymerase is not contained in the mixture of the first type of primer and the second type of primer. In such embodiments, the nucleic acid polymerase can be stored in a separate container, optionally forming a mixture with other components, or being present in a substantially pure form.
In certain embodiments, the mixture of first type of primer and second type of primer further comprises a nucleic acid polymerase. In certain embodiments, the mixture comprises: a first type of primer, a second type of primer, a mixture of nucleotide monomers, Mg2+, dTT, Tris HCl and a nucleic acid polymerase, and one or more components selected from the group consisting of: BSA, DNase inhibitor, RNase, SO42−, Cl−, K+, Ca2+, Na+, and (NH4)+, etc. In such embodiment, the mixture may contain all reactants required for amplification reaction except genomic DNA. When such mixture is used in the amplification reaction of the present application, reagents containing genomic DNA may be directly mixed with the mixture in the kit, and optionally, a proper amount of pure water may be added to obtain required reaction volume, then the reaction mixture in step (a) of the method of the present application can be obtained.
In certain embodiments, a kit further comprises a component capable of lysing a cell, such as one or more surfactants, and/or one or more lyases. Exemplary surfactants include, but are not limited to, one or more of NP-40, Tween, SDS, TritonX-100, EDTA, and guanidinium isothiocyanate. Exemplary lyases can be one or more of Protease K, pepsin, and papain. The component that lyses a cell can be stored separately in a separate container, or mixed with other components. In certain embodiments, a kit comprises a surfactant and a lyase, placed separately in different containers, or placed in the same container.
In certain embodiments, the mixture of first type of primer, second type of primer, and nucleic acid polymerase further comprises a surfactant and/or a lyase.
In certain embodiments, the kit comprises one container, wherein all of the reagents are contained. In certain embodiments, the kit comprises two containers, wherein one container stores components required in amplification reaction including nucleic acid polymerase, and the other container stores components required in cell lysing including lyase. In certain embodiments, the kit comprises four containers, wherein a first container stores nucleic acid polymerase, a second container stores components required in amplification reaction other than nucleic acid polymerase, a third container stores lyase, and a fourth container stores components required in cell lysing other than lyase.
Another aspect of the application also provides a kit for genomic DNA amplification, comprising a first type of primer and a second type of primer, and further comprises an instruction for users, said instruction records the following steps: mixing the first type of primer and the second type of primer in the same container before said amplifying, wherein the first type of primer comprises, in a 5′ to 3′ orientation, a common sequence and a variable sequence, wherein the common sequence consists of three or two types of bases selected from the group consisting of four types of bases: G, A, C and T, providing that the common sequence does not comprise G and C at the same time, and wherein the second type of primer comprises the common sequence but not the variable sequence. The first type of primer and the second type of primer in the kit may be placed separately in different containers, but the instruction may include the step of mixing the two in the same container before amplification.
Single-cell genomic DNA: Cultured human epidermal fibroblasts (AFP) in a good state were digested with trypsin and the digested cells were collected into a 1.5 ml EP tube. The collected cells were centrifuged and rinsed with 1×PBS solution. After rinsing, 1×PBS was added to suspend the cells. A portion of the cell-containing suspension was aspirated using a pipette, and single cells were picked using a mouth pipette under a 10× microscope, the volume of aspirated PBS solution not exceeding 1 microliter, and the picked single cells were transferred into a PCR tube containing 4 microliters of lysis buffer (containing Tris-Cl, KCl, EDTA, Triton X-100 and Qiagen Protease). After brief centrifugation, the PCR tube was placed on a PCR instrument where a lysing program was performed, and the specific program is shown in Table 1.
Positive control: standard genomic DNA was diluted to 30 pg/μl DNA solution with nuclease-free water, and 1 μl of the solution above was added to a PCR tube containing 4 μl cell lysis buffer. The standard genomic DNA was genomic DNA of human cells extracted previously.
Negative control: 5 μl of cell lysis buffer was added to a PCR tube.
The method of the present example is also referred to as three-step method herein, since it basically comprises three steps, namely, lysing cells, pre-amplification, and exponential amplification.
Human epidermal fibroblasts were isolated and lysed according to the method described in Example 1, to obtain single-cell genomic DNA. Single cell whole genome amplification kit of Jiangsu Yikon Genomics Co., Ltd. (Cat. No. YK001A/B) was used, and amplification was performed pursuant to its manufacturer's instructions. Specifically, a pre-amplification buffer and a pre-amplification enzyme mixture were mixed in a ratio of 30:1 to generate a pre-amplification mixture. 30 μl of pre-amplification mixture was added respectively to PCR tubes each containing samples to be amplified (genomic DNA, positive control and negative control obtained according to Example 1). The PCR tubes were placed into a PCR instrument for pre-amplification, and the pre-amplification program is shown in Table 2.
An amplification buffer and an amplification enzyme mixture were mixed in a ratio of 30:0.8 to generate an amplification mixture. 30 μl of the amplification mixture was added to a PCR tube where pre-amplification has completed, followed by exponential amplification, the program for which is shown in Table 3.
The method of the present example is also referred to as two-step method herein, basically comprising two steps, namely, lysing cells and amplification reaction.
Human epidermal fibroblasts were isolated and lysed according to the method described in Example 1, to obtain single-cell genomic DNA.
An amplification mixture was prepared, containing Na+, Mg2+, Cl−, Tris-Cl, TritonX-100, dNTP, Vent polymerase, primer of SEQ ID NO: 1, primer of SEQ ID NO: 12, and primer of SEQ ID NO: 13.
Primers used in this example were designed according to the following principles:
1. Common sequence of a primer only contains three types of bases with weak self-complementary pairing ability, such as G, A and T.
2. One or more of the three types of bases mentioned in 1 are used at 3′end of primer variable base sequence (three or more bases that are successive or unsuccessive), to ensure that phenomenon of self-complementary pairing or complementary pairing with 5′ end of a different primer will not occur to the 3′ end of the primer.
3. By statistical calculation of recognition sites of primer variable base sequences on genome, sequences that meet the above conditions above, are more evenly distributed on genome and with higher coverage, are selected, to increase opportunity of recognition between variable base sequence and genomic DNA.
4. The use ratio and composition of the three types of bases in primer common sequence are specially designed to ensure that the common sequence will not bind to genomic DNA and generate amplification. 60 μl of amplification mixture is added to PCR tubes of each sample to be amplified (genomic DNA, positive control and negative control obtained according to Example 1), respectively. The PCR tubes were placed into a PCR instrument for amplification, and the amplification program is shown in Table 4.
Gel Electrophoresis
5 microliters of unpurified amplification product from the three-step method of Example 2 and unpurified amplification product from the two-step method of Example 3 were taken, respectively, and were respectively added with 1 microliter of 6×DNA loading buffer (purchased from Beijing ComWin Biotech Co., Ltd., Cat. No. CW0610A) for sample loading. 1% agarose gel was used as the gel, and DM2000 (purchased from Beijing ComWin Biotech Co., Ltd., Cat. No. CW0632C) was used as the marker.
See
Purification Product
50 microliters of unpurified amplification products from the three-step method of Example 2 and from the two-step method of Example 3 were taken, and the amplification products were purified with a universal column purification kit (purchased from Beijing ComWin Biotech Co., Ltd., Cat. No. CW2301), the purification steps of which were performed in accordance with the kit instructions. 50 microliters of EB was used for elution. After purification was completed, 2 μl of the purified product was subject to concentration measurement using Nanodrop (AOSHENG, NANO-100). Results of concentration measurement are shown in Table 5.
Concentration measurement results show that: post-purification concentration of amplification products obtained by the two amplification methods were comparable, with no significant difference.
Detection of Disease-Causing Sites
20 pathogenic sites were randomly selected (see the table below for selected sites) and primers were designed. Selected pathogenic sites and their corresponding primers are shown in Table 6 and Table 7, respectively.
7 amplification products amplified according to Example 2 and 7 amplification products amplified according to Example 3 were randomly selected as template DNA, respectivey. The 20 pathogenic sites above were respectively amplified by PCR using dye-containing 2× Taq MasterMix (purchased from Beijing ComWin Biotech Co., Ltd., Cat. No. CW0682). Composition of amplification system is shown in Table 8, and amplification program is shown in Table 9.
Amplification results are shown in
Another 20 pathogenic sites were further randomly selected (see the table below for the selected sites) and primers were designed. The selected pathogenic sites and their corresponding primers are shown in Table 10 and Table 11, respectively.
3 samples amplified according to Example 2 (shown as 2_1, 2_3 and 2_7 in
Amplification results are shown in
q-PCR Detection Using Quality Detection Primers
4 samples amplified according to the three-step method of Example 2 (shown as 3-5, 3-6, 3-9, 3-10 in
Amplification results are shown in
Gene Sequencing
10 purified products amplified by the two-step method of Example 3 (shown as 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10 in
Results of the two-step method in Example 3 is shown in
In sequencing results, various indicator parameters of high-throughput sequencing results are also provided, as shown in
The copy-number variation coefficient can be used to compare the dispersion degree of sample copy-number after the sample is amplified by the two types of amplification methods. The average copy-number variation coefficient of all amplified samples using the two-step method of Example 3 was 0.1200, while that of all amplified samples using the three-step method of Example 2 was 0.1205. There was no significant difference between the copy-number variation coefficients of the two types of amplification methods. See
ADO Evaluation
150 SNP sites were randomly selected and corresponding primers were designed. See Table 15 for selected sites and corresponding primers.
To measure the efficiency of amplifying a given site and allelic dropout (ADO) in order to determine amplification efficiency and whether the amplification was successful, two amplification products (3-1 and 3-2) of the three-step method of Example 2, one amplification product (2-1) of the two-step method of Example 3, and 120 ng of DNA extracted from human epidermal fibroblasts (AFP) cells were randomly selected and subjected to multiplex PCR, respectively (see Table 15 for primers). Composition of amplification system is shown in Table 16, and amplification program is shown in Table 17.
The amplification products were constructed into a genomic library by means of fragmentation, and sequenced with hiseq2500 sequencer, with an average sequencing depth of 5 Mb (see
In addition, results of second-generation sequencing analysis show that a total of 23 homozygous loci were detected in multiplex PCR products using gDNA as starting material. Of these 23 homozygous loci, 23 and 22 were detected respectively in the two amplification products of the three-step method of Example 2; 21 were detected in amplification product from the two-step method of Example 3. There was no significant difference between ADO proportion of homozygous loci of amplification product of Example 2 and that of Example 3. See Table 18 for detailed data.
Second-generation sequencing analysis results showed that a total of 62 heterozygous loci were detected in multiplex PCR products using gDNA as starting material. Of these 62 heterozygous loci, 59 and 56 were detected respectively in the two samples of Example 2; 51 were detected in one sample of Example 3. There was no significant difference between ADO proportion of heterozygous loci of amplification product of Example 2 and that of Example 3. See Table 19 for detailed data.
The method of the present example is also referred to as one-step method herein, because lysing of cell, pre-amplification and exponential amplification were combined and completed within one step.
Human epidermal fibroblasts were isolated and lysed according to the method of Example 1 to obtain single-cell genomic DNA.
Amplification mixture was prepared, which contained Na+, Mg2+, Cl−, Tris-Cl, dNTP, TritonX-100, Vent polymerase, primer of SEQ ID NO:1, primer of SEQ ID NO:12, and primer of SEQ ID NO:13.
Gel Electrophoresis
5 microliters of unpurified amplification product from the three-step method of Example 2 and unpurified amplification product from the one-step method of Example 5 were taken, respectively, and were respectively added with 1 microliter of 6×DNA loading buffer (purchased from Beijing ComWin Biotech Co., Ltd., Cat. No. CW0610A) for sample loading. 1% agarose gel was used as the gel, and DM2000 (purchased from Beijing ComWin Biotech Co., Ltd., Cat. No. CW0632C) was used as the marker.
See
Purification Product
50 microliters of unpurified amplification products from the three-step method of Example 2 and from the one-step method of Example 5 were taken, and the amplification products were purified with a universal column purification kit (purchased from Beijing ComWin Biotech Co., Ltd., Cat. No. CW2301), the purification steps of which were performed in accordance with the kit instructions. 50 microliters of EB was used for elution. After purification was completed, 2 μl of the purified product was subject to concentration measurement using Nanodrop (AOSHENG, NANO-100). Results of concentration measurement are shown in Table 21.
Concentration measurement results show that: post-purification concentration of amplification products obtained by the two amplification methods after purification were comparable, with no significant difference.
Pathogenic Site Detection
Twenty pathogenic sites were randomly selected and primers were designed. The selected pathogenic sites and their corresponding primers are shown in Table 6 and Table 7 in Example 4, respectively.
Four samples amplified according to Example 2 and four samples amplified according to Example 5 were randomly selected as template DNA, respectively. Composition of amplification system and amplification program are as shown in Tables 8 and 9 in Example 4, respectively, except that the cycle numbers in
Amplification results are as shown in
Gene Sequencing
10 purified products amplified by the three-step method of Example 2 (shown as 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10 in
Results of the one-step method in Example 5 are as shown in
In sequencing results, various index parameters of high-throughput sequencing results are also provided, as shown in
The copy-number variation coefficient can be used to compare the dispersion degree of sample copy-number, after the sample is amplified by the two types of amplification methods. The average copy-number variation coefficient of all amplified samples using the one-step method of Example 5 was close to that of all amplified samples using the three-step method of Example 2. See
Although various aspects and embodiments have been disclosed by the present disclosure, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for the purpose of illustration only, and are not intended to limit the scope of the present disclosure. The actual scope of the protection of the present disclosure is governed by the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201510556237.2 | Sep 2015 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2016/097208 | 8/29/2016 | WO | 00 |
