HK1193436B - Nucleic acid sample preparation methods and compositions - Google Patents

Description

Methods and compositions for preparing nucleic acid samples

This application claims priority to U.S. provisional application serial No. 61/427,321, filed on 27/2010, which is incorporated herein by reference in its entirety.

Technical Field

The present invention provides compositions and methods for preparing nucleic acid libraries (e.g., using whole genome amplification) in universal buffers, wherein purification of the nucleic acids is not required between or during steps. In certain embodiments, a small amount of starting nucleic acid (e.g., genomic DNA) is employed and the steps are accomplished in a single vessel. In some embodiments, the nucleic acid library can be subjected to a sequencing method or rolling circle amplification.

Background

In many research fields, such as gene diagnosis, cancer research or forensic medicine, the deficiency of genomic DNA can be a serious limiting factor in the type and number of gene tests that can be performed on a sample. One approach designed to overcome this problem is whole genome amplification. The goal is to amplify a limited DNA sample in a non-specific manner to produce a new sample that is indistinguishable from the original sample but has a higher DNA concentration. The goal of typical whole genome amplification techniques is to amplify a sample to the microgram level while respecting the original sequence characterization.

The first genome-wide amplification method was described in 1992 and is based on the principle of the polymerase chain reaction. Primer extension PCR technology (PEP) and Telenius and co-workers (Telenius et al, genomics.1992,13(3): 718-25; incorporated herein by reference) were developed by Zhang and co-workers (Zhang, L. et al, Proc. Natl. Acad. Sci. USA,1992,89: 5847-5851; incorporated herein by reference) and degenerate oligonucleotide primer PCR methods (DOP-PCR) were designed.

PEP involves a high number of PCR cycles, typically using Taq polymerase and a 15 base random primer that anneals at low stringency temperatures. While the PEP protocol has been improved in different ways, it still results in incomplete genome coverage, failing to amplify certain sequences, such as repetitive sequences. Failure to initiate and amplify regions containing repeated sequences can lead to incomplete characterization of the whole genome, as stable primer coverage across the length of the genome provides optimal characterization of the genome. This method also has limited efficiency for very small samples (e.g., single cells). Furthermore, the use of Taq polymerase means that the maximum product length is about 3 kb.

DOP-PCR is a method that typically uses Taq polymerase and a semi-degenerate oligonucleotide (e.g., CGACTCGAGNNNNNNATGTGG (SEQ ID NO:12), e.g., where N = A, T, C or G) that binds about one million sites within the human genome at low annealing temperatures. The first cycle is followed by a number of cycles with higher annealing temperatures, which only allow amplification of the labeled fragments in the first step. This results in incomplete characterization of the whole genome. Similar to PEP, DOP-PCR produces fragments of an average length of 400 and 500bp, with a maximum length of 3kb, although fragments of up to 10kb have been reported. On the other hand, as indicated for PEP, low input of genomic DNA (less than 1ng) reduces fidelity and genomic coverage (Kittler et al, anal. biochem.2002,300(2), 237-44).

Multiple displacement amplification (MDA, also known as Strand Displacement amplification; SDA) is an isothermal method based on the annealing of denatured DNA with random hexamers rather than PCR, followed by strand displacement synthesis at constant temperature (Blanco et al, 1989, J.biol.chem.264:8935-40, incorporated herein by reference). It has been applied to small genomic DNA samples, resulting in the synthesis of high molecular weight DNA with limited sequence characterization preference (Lizardi et al, Nature genetics1998,19, 225-232; Dean et al, Proc. Natl. Acad. Sci. U.S. A.2002,99, 5261-5266; both of which are incorporated herein by reference). As DNA is synthesized by strand displacement, an increasingly large number of priming events occur, forming a network of hyperbranched DNA structures. The reaction can be catalyzed by Phi29DNA polymerase or by large fragments of BstDNA polymerase. Phi29DNA polymerase has a proofreading activity that is as low as 1/100 below the error rate caused by Taq polymerase.

What is needed is a whole genome amplification method that does not require nucleic acid purification between or during steps, and/or can be accomplished in a single vessel.

Brief description of the invention

The present invention provides compositions and methods for preparing a nucleic acid library (e.g., a DNA library) in a universal buffer (e.g., using whole genome amplification, e.g., MDA), wherein nucleic acid purification is not required between or during steps. In certain embodiments, a small amount of starting nucleic acid (e.g., genomic DNA) is used (e.g., picogram or nanogram) and the steps are completed in a single vessel. In some embodiments, the nucleic acid library can be subjected to a sequencing method or rolling circle amplification.

In some embodiments, the invention provides a method of preparing a nucleic acid library in a universal buffer, the method comprising: a) adding a nucleic acid sample (e.g., genomic DNA) to a universal buffer, wherein the universal buffer comprises dntps and primers; b) contacting the universal buffer with a plurality of substantially purified enzymes, wherein the enzymes have polymerase activity, kinase activity, and phosphatase activity, and wherein the contacting is performed under conditions such that amplified nucleic acids are produced; c) treating the universal buffer (e.g., by mechanical, chemical, or enzymatic means) such that sheared amplified nucleic acids are produced; d) treating the universal buffer to inactivate the polymerase; and e) contacting the universal buffer with a ligase and a nucleic acid linker under conditions such that a nucleic acid library of ligated linkers is generated; wherein the above steps are accomplished in a universal buffer without the need for purification of the nucleic acid between or during some or all of the steps.

In particular embodiments, the amplified nucleic acid is produced by whole genome amplification (e.g., MDA). In further embodiments, some or all of the steps (e.g., steps a) -e)) are performed in a single vessel. In other embodiments, the method further comprises treating a universal buffer of the nucleic acid library comprising the adapter such that proteins and dntps are removed from the universal buffer. In specific embodiments, the treating comprises contacting the universal buffer with a protease and a phosphatase, or column purifying the universal buffer. In other embodiments, the linker comprises a hairpin primer, and wherein the linker-ligated nucleic acid library comprises a circular template.

In further embodiments, the method further comprises treating the universal buffer comprising the circular template with at least one exonuclease capable of digesting any non-circularized nucleic acids present. In some embodiments, the method further comprises heating a universal buffer comprising the exonuclease to inactivate the exonuclease. In further embodiments, the linker comprises a 3 'and/or 5' blocking group, and wherein the library of linked linkers comprises a terminally blocked template. In other embodiments, the method further comprises treating the universal buffer comprising the end-blocked template with at least one exonuclease capable of digesting any non-end-blocked nucleic acids present. In further embodiments, the method further comprises heating a universal buffer comprising the exonuclease to inactivate the exonuclease.

In some embodiments, the universal buffer further comprises an emulsifier. In certain embodiments, the emulsifier is a polysorbate (e.g., Tween20, Tween40, Tween60, or Tween 80). In other embodiments, the universal buffer further comprises TRIS (hydroxymethyl) aminomethane (TRIS). In further embodiments, the universal buffer further comprises a divalent metal cation. In further embodiments, the universal buffer further comprises an inorganic salt. In a further embodiment, the inorganic salt is ammonium sulfate.

In further embodiments, the universal buffer further comprises poly a. In additional embodiments, the universal buffer further comprises an alpha-linked disaccharide. In some embodiments, the α -linked disaccharide comprises trehalose. In further embodiments, the universal buffer further comprises a reducing agent. In particular embodiments, the reducing agent further comprises Dithiothreitol (DTT). In certain embodiments, the universal buffer further comprises albumin or an albumin-like protein.

In some embodiments, the method further comprises, after step c), incubating the universal buffer such that phosphorylated blunt ends (and/or a-tailed ends) are produced in the sheared amplified nucleic acids. In other embodiments, the amount of genomic DNA is from 10pg to 50ng (e.g., 10pg to 50pg, 50pg to 1ng, or 1ng to 50 ng). In some embodiments, the adaptor-ligated nucleic acid library is subjected to a sequencing method or subjected to rolling circle amplification. In certain embodiments, the plurality of substantially purified enzymes comprises phi29 polymerase, klenow-polymerase, polynucleotide kinase, pyrophosphatase, or any combination thereof.

In some embodiments, the present invention provides a composition comprising at least four (or at least five, or at least six, or at least seven or at least eight) of the following: a) a buffer, b) an emulsifier, c) a divalent metal cation, d) an inorganic salt, e) poly A, f) an alpha-linked disaccharide, g) a reducing agent and h) albumin or an albumin-like protein.

In certain embodiments, the composition further comprises TRIS (hydroxymethyl) aminomethane (TRIS). In other embodiments, the emulsifier is a polysorbate. In some embodiments, the polysorbate is selected from: tween20, Tween40, Tween60 or Tween 80. In a further embodiment, the inorganic salt is ammonium sulfate. In a further embodiment, the α -linked disaccharide comprises trehalose. In some embodiments, the reducing agent comprises Dithiothreitol (DTT).

In further embodiments, the composition further comprises dntps and/or primers. In other embodiments, the composition further comprises a plurality of substantially purified enzymes, wherein the enzymes have polymerase activity, kinase activity, and phosphatase activity. In some embodiments, the plurality of substantially purified enzymes comprises Phi29 polymerase, klenow-polymerase, polynucleotide kinase, pyrophosphatase, ligase, or any combination thereof.

In some embodiments, the composition further comprises a nucleic acid linker. In certain embodiments, the linker comprises a hairpin primer. In other embodiments, the composition further comprises an exonuclease. In certain embodiments, the exonuclease is exonuclease III or exonuclease VII. In further embodiments, the composition further comprises a random primer. In other embodiments, the random primers are suitable for use in whole genome amplification methods, such as MDA.

Detailed description of the drawings

FIG. 1 shows an exemplary embodiment of a single-tube library preparation process in flow chart form. The specific details (enzyme/incubation temperature and time, etc.) listed are merely exemplary and may vary depending on the different types of libraries made.

FIG. 2 shows the results from example 1 and shows the ability to amplify ng levels of DNA to μ g levels within 30 minutes with phi29 polymerase/klenow exo-polymerase in Whole Genome Amplification (WGA). Fig. 2A shows the total yield of amplification products as measured by qPCR, and fig. 2B shows gel electrophoresis analysis of the amplification products.

Figure 3 shows the results from example 1 and shows the ability to sonicate WGADNA fragments to a length of several hundred base pairs in WGA buffer.

FIG. 4 shows the results from example 1 and shows the ability of phi29/klenow exo-enzyme mixture to blunt and tail DNA with DNA oligonucleotides in WGA buffer and analyzed by mass spectrometry. The results of sample analysis by ESI-TOF mass spectrometer with T5000 system showed sample analysis both before (fig. 4A) and after (fig. 4B) WGA enzyme addition.

FIG. 5 shows the results from example 1 and shows the ability of phi29/klenow exo-enzyme mixture to fill in terminal DNA with DNA oligonucleotides in WGA buffer and analyzed by mass spectrometry. The results of sample analysis by ESI-TOF mass spectrometer with T5000 system showed sample analysis both before (fig. 5A) and after (fig. 5B) WGA enzyme addition.

FIG. 6 shows the results from example 1 and shows the ability of polynucleotide kinases to phosphorylate the 5' end of DNA using DNA oligonucleotides in WGA buffer and analysis by mass spectrometry. FIG. 6 shows analysis of samples with oligonucleotides before (FIG. 6A) and after (FIG. 6B) addition of polynucleotide kinase.

FIG. 7 shows the results from example 1 and shows the ability to ligate DNA fragments with T4 ligase in WGA buffer using DNA oligonucleotides and gel electrophoresis analysis.

FIG. 8 shows the results from example 1 and shows the ability to ligate DNA fragments with T4 ligase and mass spectrometry analysis using DNA oligonucleotides in WGA buffer. Sample analysis with ligase (FIG. 8A) and without ligase (FIG. 8B) was processed with an ESI-TOF mass spectrometer.

FIG. 9 shows the results from example 1 and shows the ability to digest DNA with exonuclease III and exonuclease VII exonuclease and gel electrophoresis analysis in WGA buffer using DNA oligonucleotides.

Figure 10 shows the results from example 1 and shows the ability to digest DNA with exonuclease III and exonuclease VII exonuclease and mass spectrometry analysis using DNA oligonucleotides in WGA buffer. The exonuclease treated sample (FIG. 10B) and the ligation reaction not treated with exonuclease (FIG. 10B) were then loaded on an ESI-TOF mass spectrometer using a T5000 system.

Detailed description of the invention

The present invention provides compositions and methods for preparing nucleic acid libraries (e.g., using whole genome amplification) in universal buffers, wherein purification of the nucleic acids is not required between or during steps. In certain embodiments, a small amount of starting nucleic acid (e.g., genomic DNA) is employed and the steps are accomplished in a single vessel. In some embodiments, the nucleic acid library is subjected to a sequencing method or rolling circle amplification.

Existing methods for preparing DNA libraries typically utilize physical methods for DNA shearing such as sonication, nebulization, and the like. This is followed by selection of fragments of appropriate length and then an enzymatic step (involving ligation of DNA linkers) to prepare the sample for sequencing. These methods require large amounts of starting material (e.g., several μ g), large amounts of time (many hours), and purification of DNA between steps. The use of a process that does not require purification between individual steps and that includes a whole genome amplification step would allow for a simpler one-tube (onetube) process. Conversely, this would allow the creation of a DNA library from a significantly smaller number of starting templates with much reduced time and effort. Such methods are provided by the present invention. The present invention provides a fast and simple method without the need for purification between steps, which can be performed in a single tube/container to create a DNA library from a small number of starting DNA templates for DNA sequencing or other applications.

In certain embodiments, the invention creates libraries of DNA templates using whole genome amplification (using enzymes such as phi29 polymerase and kleowexo-polymerase), physical DNA fragmentation methods (e.g., sonication), end repair/tailing-on reactions (using enzymes such as phi29 polymerase, kleowexo-polymerase, and polynucleotide kinase), ligation with DNA linkers (using enzymes such as T4 ligase), and exonuclease treatment (using enzymes such as exonuclease III and exonuclease VII). In certain embodiments, the invention requires less time (e.g., 30 minutes to 1 hour), less starting materials, and less handling/delivery time to create a DNA library. In a specific embodiment, the method of the invention is integrated in an automated microfluidic/robotic system.

In some embodiments, the resulting DNA library is subjected to sequencing techniques. Based on the sequencing method, the DNA library is created using appropriate linkers. Exemplary sequencing techniques are described below.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing techniques and dye terminator sequencing techniques, as well as "next generation" sequencing techniques. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because RNA is less stable in cells and is more susceptible to nuclease attack experimentally.

Chain terminator sequencing utilizes sequence-specific termination of DNA synthesis reactions using modified nucleotide substrates. Extension is initiated at a specific site on the template DNA by using a short radioactive or otherwise labeled oligonucleotide primer complementary to the template at that region. The oligonucleotide primers are extended with a DNA polymerase, standard four deoxynucleotide bases and a low concentration of single stranded terminator nucleotides (most commonly dideoxynucleotides). The reaction was repeated in four separate tubes with each base in turn as a dideoxynucleotide. The restriction incorporation of chain terminating nucleotides by the DNA polymerase produces a series of related DNA fragments that terminate only at the positions where the specific dideoxynucleotides are used. For each reaction tube, the fragments were separated by size by slab polyacrylamide gel electrophoresis or by capillary electrophoresis with viscous polymer. The sequence was determined by reading the lanes that produced the visual marker from the labeled primer as you scanned from top to bottom of the gel.

Alternatively, dye terminator sequencing tags the terminator. Complete sequencing can be accomplished in a single reaction by labeling each dideoxynucleotide chain terminator with a separate fluorescent dye that fluoresces at a different wavelength.

A group of methods known as "next generation sequencing" techniques have emerged as alternatives to Sanger sequencing and dye terminator sequencing (volekering et al,ClinicalChem.,55641-658,2009, Maclean et al.,NatureRev.Microbiol.,7287-296; each of which is incorporated herein by reference in its entirety). Next Generation Sequencing (NGS) methods share the common features of massively parallel, high-throughput strategies with the goal of lower cost compared to older sequencing methods. The NGS method can be roughly classified into a method requiring template amplification and a method not requiring template amplification. Methods requiring amplification include pyrosequencing (e.g., GS20 and GSFLX) marketed by Roche as the 454 technology platform, the Solexa platform marketed by Illumina, and the supportedoligonucleotideliganded detection (solid) platform marketed by applied biosystems. Non-amplification methods, also known as single molecule sequencing, are exemplified by the HeliScope platform marketed by Helicos BioSciences and the new platforms marketed by VisiGen, Oxford Nuclear technologies Ltd and Pacific biosciences, respectively.

In pyrosequencing (Voelkerding et al),ClinicalChem.,55641-658,2009, Maclean et al,NatureRev.Microbiol.,7287-296; U.S. Pat. nos. 6,210,891; U.S. Pat. nos. 6,258,568; each incorporated herein by reference in its entirety), template DNA fragmentation, end repair, linker ligation and ligation by ligationMicrobeads loaded with oligonucleotides complementary to the linkers capture a single template molecule for clonal amplification in situ. Individual microbeads loaded with a single template type are regionalized into water-in-oil microbubbles and the templates are clonally amplified using a technique known as emulsion PCR. The emulsion is broken after amplification and the microbeads are deposited into individual wells of a picotiter plate that acts as a flow cell during the sequencing reaction. Ordered, iterative introduction of each of the four dNTP reagents occurs in the flow cell in the presence of a sequencing enzyme and a luminescent reporter such as a fluorochrome enzyme. With the addition of an appropriate dNTP to the 3' end of the sequencing primer, the resulting ATP production results in a burst of luminescence within the well, which is recorded with the CCR camera. It is possible to achieve a read length of 400 bases or more, and 1 × 10⁶One sequence reads, which results in sequences of up to 5 hundred million base pairs (500 Mb).

On the Solexa/Illumina platform (Voelkerding et al),ClinicalChem.,55641-658,2009, Maclean et al,NatureRev.Microbiol.,7287-296, U.S. Pat. No. 6,833,246; U.S. patent No. 7,115,400; U.S. patent No. 6,969,488; each of which is incorporated herein by reference in its entirety), sequencing data is generated in the form of shorter length reads. In this method, single-stranded fragmented DNA ends are repaired to produce 5 'phosphorylated blunt ends, followed by Klenow-mediated addition of a single a base to the 3' end of the fragment. The addition of a facilitates the addition of T-overhang linker oligonucleotides that are subsequently used to capture template-linker molecules on the surface of a flow cell interspersed with oligonucleotide anchors (anchors). The anchors are used as PCR primers, but due to the length of the template and its similarity to other adjacent anchor oligonucleotides, extension by PCR results in "arch bridging" of molecules hybridized to adjacent anchor oligonucleotides to form a bridge-type structure on the flow cell surface. These DNA loops are denatured and cleaved. The forward strand was then sequenced with a reversible dye terminator. The sequence of the incorporated nucleotide was determined by detecting the incorporated fluorescence, each fluorescence removed and blocking prior to the next cycle of dNTP addition. The sequence read length ranged from 36 nucleotides to over 50 nucleotides, with each analysis run totalingOutputting more than one billion pairs of nucleotides.

Sequencing nucleic acid molecules using the SOLID technique (Voelkerding et al),ClinicalChem.,55641-658,2009, Maclean et al,NatureRev.Microbiol.,7287-296; U.S. Pat. nos. 5,912,148; U.S. patent No. 6,130,073; each of which is incorporated herein by reference in its entirety) also involves fragmentation of the template, ligation of oligonucleotide linkers, attachment of microbeads, and clonal amplification by emulsion PCR. Following this, the template-bearing microbeads are immobilized on the derivatized surface of the glass flow cell and annealed to primers complementary to the adaptor oligonucleotides. However, instead of using this primer for 3 'extension, it is used to provide a 5' phosphate group to ligate an interrogation (hybridization) probe comprising two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels. In the SOLiD system, the interrogation probe has 16 possible combinations of two bases at the 3 'end of each probe and one of four fluorescences at the 5' end. The fluorescence color and thus the characteristics of each probe correspond to the encoding scheme of the specified color space. Denaturation is followed by multiple rounds (typically 7 rounds) of probe annealing, ligation and fluorescence detection, and then a second round of sequencing with primers offset by one base relative to the original primers. In this way, the template sequence can be computationally reconstructed and the template bases interrogated twice, resulting in increased accuracy. The sequence reads are on average 35 nucleotides in length, outputting more than 40 hundred million bases in total per sequencing run.

In certain embodiments, nanopore sequencing is used (see, e.g., Astier et al, JAmChemSoc.2006Feb8;128(5):1705-10, incorporated herein by reference). The theory behind nanopore sequencing should exploit the events that occur when a nanopore is immersed in and a potential (voltage) is applied across a conducting fluid: weak currents due to ionic conduction through the nanopore can be observed under these conditions, and the amount of current is extremely sensitive to the size of the nanopore. If a DNA molecule passes through (or a portion of a DNA molecule passes through) a nanopore, it may cause a change in the intensity of the current passing through the nanopore, thereby allowing the sequence of the DNA molecule to be determined.

HeliScope from Helicos BioSciences (Voelkerding et al),ClinicalChem.,55641-658,2009, Maclean et al,NatureRev.Microbiol.,7287-296; U.S. patent No. 7,169,560; U.S. patent No. 7,282,337; U.S. patent No. 7,482,120; U.S. patent No. 7,501,245; U.S. patent No. 6,818,395; U.S. patent No. 6,911,345; U.S. patent No. 7,501,245; each of which is incorporated herein by reference in its entirety) is the first commercial single molecule sequencing platform. The method does not require clonal amplification. The template DNA is cleaved and polyadenylated at the 3' end, the final adenosine carrying a fluorescent label. The denatured polyadenylated template fragment was ligated to a poly (dT) oligonucleotide on the surface of the flow cell. The initial physical position of the captured template molecules is recorded by a CCD camera, followed by label cleavage and washing away. Sequencing was accomplished by adding polymerase and sequentially adding fluorescently labeled dNTP reagents. The incorporation event generates a fluorescent signal corresponding to the dNTP and the signal is captured by a CCD camera prior to each round of dNTP addition. Sequence read lengths range from 25-50 nucleotides, with an overall output of over 10 hundred million nucleotide pairs per analysis run.

Another exemplary nucleic acid sequencing method developed by StratosGenomics, inc. that is also optionally suitable for use in the present invention involves the use of xpandomers. The sequencing process typically includes providing a daughter strand produced by template-directed synthesis. The daughter strand typically includes a plurality of coupled subunits in a sequence corresponding to a contiguous nucleotide sequence of part or all of the target nucleic acid in which individual subunits comprise a tether (tether), at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond is cleaved to yield an Xpandomer that is longer than the length of the plurality of subunits of the daughter strand. Xpandomers typically comprise a tether and a reporter element for resolving genetic information in a sequence corresponding to all or part of a contiguous nucleotide sequence of a target nucleic acid. The reporter element of the Xpandomer is then detected. Additional details regarding Xpandomer-based methods are described, for example, in U.S. patent publication No. 20090035777 entitled "high throughput nucleic acid sequencing by extension (highthroughput nucleic acid sequencing), filed on 19.6.2008, which is incorporated herein in its entirety.

Other novel single molecule sequencing methods include real-time sequencing by synthesis using the VisiGen platform (Voelkerding et al),ClinicalChem.,55641-658,2009; U.S. patent No. 7,329,492; U.S. patent application serial No. 11/671956; U.S. patent application serial No. 11/781166; each of which is incorporated herein by reference in its entirety), wherein strand extension of the immobilized primer DNA template is performed using a fluorescently modified polymerase and a fluorescent acceptor molecule, resulting in detectable Fluorescence Resonance Energy Transfer (FRET) by the addition of nucleotides.

Another real-time single molecule sequencing system developed by Pacific biosciences (Voelkerding et al),ClinicalChem.,55641-658,2009, Maclean et al,NatureRev.Microbiol.,7287-296; U.S. patent No. 7,170,050; U.S. patent No. 7,302,146; U.S. patent No. 7,313,308; U.S. Pat. nos. 7,476,503; all of which are incorporated herein by reference) utilize a material having a diameter of 50-100nm and containing about 20 zeptoliters (10x 10)^-21L) reaction wells of the reaction system. The sequencing reaction is carried out by using the fixed template, the modified phi29DNA polymerase and the fluorescence-labeled dNTP with high local concentration. High local concentration and continuous reaction conditions allow real-time capture of the incorporation events by fluorescence signal detection using laser excitation, optical waveguides and CCD cameras.

In certain embodiments, Single Molecule Real Time (SMRT) DNA sequencing using zero mode waveguide holes (ZMWs), developed by PacificBiosciences, or similar methods are applied. With this technique, DNA sequencing is performed on SMRT chips that each contain thousands of zero mode waveguide wells (ZMWs). ZMWs are tens of nanometer diameter holes made in a 100nm metal film deposited on a silicon dioxide substrate. Each ZMW becomes a vector providing exactly 20 zeptoliters (10)^-21Liter) nanophotonic visualization cell of the detection volume. In this volume, the activity of a single molecule can be detected in a background of thousands of labeled nucleotides.

Because ZMWs are sequenced by synthesis, they provide a window for viewing DNA polymerases. In each chamber, a single DNA polymerase molecule is attached to the bottom surface such that it remains permanently within the detection volume. The phosphate-linked nucleotides, each type of which is labeled with a differently-stained fluorophore, are then introduced into the reaction solution at high concentrations, which facilitates speed, accuracy and throughput of the enzyme. Due to the small size of the ZMWs, only a small fraction of the time nucleotides occupy the detection volume even at such high, biologically relevant concentrations. Furthermore, the access to the detection volume is fast, lasting only a few microseconds, due to the very short distance that the diffusion must carry the nucleotides. The result is a very low background.

Procedures and systems for such real-time sequencing applicable to the present invention are described in, for example, U.S. patent No. 7,405,281 entitled "fluorescent nucleotide analogs and their uses" (fluorescent nucleotide analogs) granted to Xu et al on 29 th 2008, U.S. patent No. 7,315,019 entitled "optically limited array and its uses" (arraysoftical compositions and uses) granted to Turner et al on 1 st 1 th 2008, U.S. patent No. 7,313,308 entitled "optical analysis of molecules" (optical analysis of molecules) "granted to Turner et al on 25 th 12 th 2007, U.S. patent No. 7,302,146 entitled" apparatus and method for molecular analysis "(apparatus and method for monitoring optical analysis of molecules" (applied optical analysis of molecules) "granted to luminescence analysis of molecules" (applied luminescence analysis of molecules) "and U.S. patent No. 7,302,146 issued to Turner et al on 27 th 11 th 35 th 2007" apparatus and method for molecular analysis of molecules "(applied luminescence analysis of molecules) granted to luminescence analysis of molecules" on 1 st 7,170,050) on 1 th month "and method for monitoring signals from luminescence analysis of molecules" (applied luminescence analysis of molecules) on 3. and simultaneous detection of luminescence of molecules "detection of luminescence of cells" (applied by luminescence analysis of luminescence system on 3. on 3 th 1 st 3 and detection of luminescence molecules "simultaneous detection of luminescence of cells" detection of cells "by luminescence detection system (applied to monitor luminescence detection of luminescence of cells" and detection of luminescence of cells "simultaneous detection of cells" on luminescence of cells "on blood, U.S. patent publication No. 20080206764 entitled "flow cell system for single molecule detection" filed by Williams et al at 26.10.2007, U.S. patent publication No. 20080199932 entitled "active surface coupled polymerases" filed by Hanzel et al at 26.10.2007, U.S. patent publication No. 20080199874 entitled "controlled strand shearing of small circular DNA (controllabelstronsmini chemi cledinna)" filed by Otto et al at 11.2.2008, U.S. patent publication No. 20080199874 entitled "article having deposited thereon a localized molecule filed by Rank et al at 26.10.2007, U.S. patent publication No. 54 entitled" filed by Rank et al at 26.10.2007, article having deposited thereon a localized molecule thereon and method of producing the same (american patent publication No. 3631 entitled "real-time reaction reduction by analysis of light emission interaction of light emission molecules" (filed by american patent publication No. 3631 filed by mitrell analysis of light emission interaction of light emission molecules) "filed by mitrell et al at 26.10.10.3.3.3.3.3. 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united states patent publication No. 3,32 (united states patent publication No. 3,32) filed by lungma US patent publication No. 20080050747 entitled "articles having localized molecules deposited thereon and methods of producing the same" (Articeshaft containment and colloidal silica dispersed sheets) filed by Korlach et al at 14.8.2007, US patent publication No. 20080032301 entitled "articles having localized molecules deposited thereon and methods of producing the same" (Articeshaft containment and colloidal silica dispersed sheets) filed by Rank et al at 29.2007, US patent publication No. 20080032301 filed by Lundqis and colloidal silica et al at 9.2007 "method and system for simultaneously monitoring optical signals from multiple sources in real time" (US patent publication No. 2006 filed 2006 for sensing optical signals-biological filtration and colloidal silica dispersed sheets) and method for monitoring optical signals from multiple sources "(US patent publication No. 3 filed 2006 for sensing biological filtration and colloidal silica dispersed sheets)" filed by Korlach et al at 14.2007, US patent publication No. 3 filed by Biochemical filtration and colloidal silica dispersed sheets "method for monitoring optical signals from multiple sources in real time" and method for monitoring biological samples deposited thereon (US patent publication No. 3 filed by Articola filtration and colloidal silica dispersed sheets) "filed by Korlach et al at 14.2007, US patent publication No. 3" method for monitoring optical signals filed by Nonsylurea dispersed sheets "(US patent publication No. 3 filed by Arnica) and colloidal silica dispersed sheets)" filed by Rank 2.3 "method for monitoring enzymes (US patent publication No. 3 filed by Biochemical synthesis and colloidal silica dispersed sheets)" filed by Nomex 2.32 for monitoring, U.S. patent publication No. 20070231804 to systems and compositions and uses thereof (Methods, systems and compositions for monitoring optical signals from multiple sources) U.S. patent publication No. 3 to Methods and systems for simultaneously monitoring optical signals in real time (Methods and systems for detecting optical signals from multiple sources) on day 9 of 2007, U.S. patent publication No. 2 to "a method and system for simultaneously monitoring optical signals in real time" (U.S. patent publication No. 20070206187 to Methods and systems for detecting optical signal from multiple sources) "on day 9 of 2007, U.S. patent publication No. 20070196846 to" a polymerase for nucleotide analog incorporation (polymerase for detecting optical signal incorporation) on day 21 of 2006) "U.S. patent publication No. 366726 to" a method and system for simultaneously monitoring optical signals from multiple sources and for simultaneously monitoring optical signals in real time "(U.S. patent publication No. 20070161017 to Methods and systems for detecting optical signals) on day 7 of 2006, U.S. patent publication No. 20070161017 to Methods and uses of optical analysis of optical signals from multiple sources" (U.S. patent publication No. 20070161017 to Methods and systems for simultaneously monitoring optical signals from multiple sources) on day 9 of humans) "on day 7 of luminescence, Us patent publication No. 20070134128 entitled "homogeneous surface for hybridization material substrate and method for making and using the same" filed by Korlach at 27.11.2007, us patent publication No. 20070128133 entitled "mitigating photodamage in analytical reaction (mititionophotomageinanalacts)" filed by Eid et al at 2.12.2005, us patent publication No. 20070128133 entitled "reactive surface, substrate and method for making the surface and substrate" (reactive surface, substratres of fluorescence emission process of producing the same) "filed by Roitman et al at 30.9.2005, us patent publication No. 32 entitled" fluorescent nucleotide analogue and use thereof (fluorescent nucleotide analogue and use thereof) (filed by fluorescence emission of fluorescence emission process of producing the same) "filed at 9.29.2005, us patent publication No. 3632 filed by Xu et al at 9.11.11.11.36, and us patent publication No. for monitoring luminescence signal of fluorescence nucleic acid analogue and its use in fluorescence emission process of DNA polymerase (fluorescent nucleotide analogue)" filed by Xu et al and method for inactivating luminescence probe molecules (fluorescent nucleotide analogue and protein molecules) filed by naturobacterium strain of emission process at 2008. 2005 ' I.Acad.Sci.U.S.A.105(4): 11761181; all of which are incorporated herein by reference in their entirety.

Examples

Example 1

Generation of Single tube DNA libraries

This example describes an exemplary single tube method for generating a DNA library from a small amount of starting material that does not require DNA purification between steps and employs a universal buffer. The process is generally outlined in figure 1. Briefly, the process starts with an aqueous sample, which is then subjected to a lysis/nucleic acid extraction protocol and eluted in universal Whole Genome Amplification (WGA) buffer. The composition of the buffer is shown in table 1:

TABLE 1

The sample was then heated at 95 ℃ for 1 minute to denature the genomic DNA and allow short random primer binding. After cooling to a suitable temperature (e.g., -37 ℃) a WGA enzyme mixture comprising phi29 polymerase, klenoxexo-polymerase, polynucleotide kinase and pyrophosphatase was added and incubated at 37 ℃ for 30 minutes to amplify the genomic material.

The sample is then physically sheared (e.g., by sonication) and allowed to incubate at 30 ℃ for an additional 30 minutes to blunt the ends of the freshly sheared molecules. After incubation at elevated temperature to inactivate the polymerase (e.g., 10 minutes at 75 ℃) DNA linkers (in this example hairpin oligonucleotides as described below) are added, along with T4 ligase and ATP. The reaction was allowed to incubate at 25 ℃ for an appropriate amount of time (blunt ends completed in-15 minutes, longer end-plus-a tail reaction). In this example, a single-stranded circular DNA molecule is generated. Thus, exonuclease is then added and incubated at 37 ℃ to remove any non-circularised DNA present. It is noteworthy that if this example forms non-circularized templates for different sequencing techniques, the exonuclease may be altered and some 5 'and/or 3' blocking agents included on the linker to achieve the same result. The exonuclease is then heat inactivated at 95 ℃ and the sample is treated with other clearing reagents, including proteinase K to remove enzyme/protein components and phosphatase to remove unused dNTPs (although other clearing procedures such as those with resins may also be used). After a suitable incubation time and temperature and inactivation of the scavenging enzyme with elevated temperature, the reaction proceeds through a final scavenging/size selection process (e.g., bind elution and flow through the resin).

FIG. 2 shows the yield obtainable with WGA starting with 1ng of Klebsiella pneumoniae (Kp) genomic DNA using the method described above. The yield of Kp specific DNA was over 2.5 μ g (2500 fold amplification) showing a typical paste-like (smear) pattern seen in WGA reactions on gel electrophoresis with appropriate amounts of total DNA.

The materials and methods used to generate the data of fig. 2 are as follows. 1ng of Klebsiella pneumoniae (Klebsiella pneumoniae) ((ii))K.pneumoniae) (Kp) purified genomic DNA was used as starting material. The buffer, 100 units of Phi29 and 40 units of klenow-in table 1 were used in WGA using the following reaction conditions: heating to 95 deg.C for 1 min before adding enzyme in a total volume of 100. mu.l, and coolingThe sample was brought to 4 ℃ and then held at 37 ℃ for 30 minutes followed by 75 ℃ for 10 minutes after adding the enzyme and mixing. Figure 2A shows the total yield of amplification products as measured by qPCR, and figure 2B shows gel electrophoresis analysis of the amplification products visualized with 1% agarose, ethidium bromide UV light.

Whole genome DNA (20. mu.l) prepared as in FIG. 2 was sonicated for an appropriate amount of time in a thin-walled PCR tube floating in a 4 ℃ water bath using a cup horn (cuphorn) sonicator (Misonix3000, power class 10 (full power about 200 w)). A10. mu.l aliquot of these reactions was then loaded onto a 1% EtBr/agarose gel and run at 100V for 45 minutes with a UV light source. Figure 3 shows the effect of different amounts of sonication time on WGADNA size in a9 buffer. After 5 minutes most of the DNA is in the range of a few hundred bp, which is a reasonable range for many sequencing techniques (although further sonication can further reduce its size if smaller fragments are required).

Next, the "rxn pre" oligonucleotides (shown below, at a final concentration of 1. mu.M each) were hybridized to each other and mixed with the buffer in Table 1 and 100 units of phi29 and 40 units of klenow-O-. The reaction was then incubated at 30 ℃ for 30 minutes and then at 75 ℃ for 10 minutes. The samples were then analyzed by ESI-TOF mass spectrometry using a T5000 system before (fig. 4A) and after (fig. 4B) addition of WGA enzyme. The sequence of the end-filled oligonucleotides is shown below with the addition of non-template a (the major product).

Before Rxn

Top (topstrand): CATGCGGATGCAGAGGAGGACGACTCTGATGTCT (SEQIDNO:1)

Bottom strand (bottomstrand): GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATGTGT (SEQIDNO:2)

Rxn rear (plus A as shown)

Winding: CATGCGGATGCAGAGGAGGACGACTCTGATGTCTTCATTGCA

(SEQIDNO:3)

And (3) chain descending: GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATGA (SEQIDNO:4)

Shown in figure 4 is the enzyme used for WGA in a9 buffer to end-fill and add an a tail to the DNA oligonucleotide used for the sheared DNA model when incubated at 30 ℃ for 30 minutes. In this part of the example, which hybridizes using a set of oligonucleotides with both 5 'and 3' overhangs, this allows WGA enzyme to cut off the 3 'overhang and instead fill in the 5' overhang resulting in a blunt-ended DNA molecule, which may then have non-template a added. After incubation at 30 ℃ for 30 minutes, the samples were heated at 75 ℃ for 10 minutes and analyzed using an ESI-TOF mass spectrometer (T5000 system).

Next, the "rxn pre" oligonucleotides (shown below, at a final concentration of 1. mu.M each) were hybridized to each other and mixed with the buffer in Table 1 above and 100 units of phi29 and 40 units of klenow-O-. The reaction was then incubated at 37 ℃ for 5 minutes and then at 75 ℃ for 10 minutes. The samples were then analyzed by ESI-TOF mass spectrometry with a T5000 system before (fig. 5A) and after (fig. 5B) addition of WGA enzyme. The sequence of the end-filled oligonucleotides depicted in the right part of the figure, with blunt ends (main product), is shown below.

Before Rxn

Winding: CATGCGGATGCAGAGGAGGACGACTCTGATGTCT (SEQIDNO:1)

And (3) chain descending: GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATGTGT (SEQIDNO:2)

Rxn rear (plus A as shown)

Winding: CATGCGGATGCAGAGGAGGACGACTCTGATGTCTTCATTGC (SEQIDNO:5)

And (3) chain descending: GCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATG (SEQIDNO:6)

Shown in figure 5 is that the enzyme used for WGA in the buffer of table 1 end-blunted but did not add an a tail to the DNA oligonucleotide used for the sheared DNA model when incubated at 37 ℃ for 5 minutes. In particular, when hybridizing, the above set of oligonucleotides with both 5 'and 3' overhangs is used, which allows WGA enzyme to cleave off the 3 'overhangs and instead fill in the 5' overhangs to give blunt-ended DNA molecules. After 5 min incubation at 37 ℃, the samples were heated at 75 ℃ for 10 min and analyzed with an ESI-TOF mass spectrometer (T5000 system).

Next, two complementary oligonucleotides are hybridized to each other to give a blunt-ended duplex without 5 'phosphate or 3' phosphate. The duplexes were mixed with the buffer of table 1 and 5 units of polynucleotide kinase and incubated at 30 ℃ for 30 minutes and then at 75 ℃ for 10 minutes. The samples were then analyzed by ESI-TOF mass spectrometry using a T5000 system.

Oligonucleotide # 1: 5' TGCGGATGCAGAGGAGGATGACTCTGATGTCT (SEQ ID NO:7)

Oligonucleotide # 2: 5' AGACATCAGAGTCATCCTCCTCTGCATCCGCA (SEQIDNO:8)

In figure 6 it is shown that the polynucleotide kinase will phosphorylate the DNA in the buffer of table 1. In particular, when hybridizing, the above set of oligonucleotides having blunt ends and no 5' phosphate is used. After incubation with polynucleotide kinase at 37 ℃ for 30 min, the reaction was heated to 75 ℃ for 10 min and analyzed with an ESI-TOF mass spectrometer (T5000 system). FIG. 6 shows analysis of samples with oligonucleotides before (FIG. 6A) and after (FIG. 6B) addition of polynucleotide kinase.

Next, two complementary oligonucleotides, each with a 5' overhang, are hybridized together. Hairpin oligonucleotides with complementary 5' overhangs are also hybridized separately. These oligonucleotides were then mixed with 1000 sticky end ligation units of T4DNA ligase in the buffer of Table 1. The reaction was then incubated at 16 ℃ for an appropriate amount of time (30 min, 60 min, 120 min) and subsequently at 75 ℃ for 10 min. Samples (including the hairpin-only control, the insert-only control, and the hairpin + insert-but ligase-free control) were then loaded on a 1% agarose gel and visualized with ethidium bromide and a UV light source.

Inserted oligonucleotide:

5'-P-GAAGCATGCGGATGCAGAGGAGGACGACTCTGATGTCTTCATTGC(SEQIDNO:9)

5'-P-GAAGGCAATGAAGACATCAGAGTCGTCCTCCTCTGCATCCGCATG(SEQIDNO:10)

hairpin structure oligonucleotide

5'-P-CTTC TCTCTCTCttttcctcctcctccgttgttgttgttGAGAGAGA(SEQIDNO:11)

The complementary 5' overhang is bolded, the complementary stem (stem) of the hairpin is underlined, and the lower case hairpin bases indicate unpaired bases.

FIG. 7 shows that ligation can be performed by gel electrophoresis using T4DNA ligase and ATP in the buffer in Table 1. In particular, a set of oligonucleotides having 5 '"sticky ends" on both ends of a duplex DNA molecule and a hairpin oligonucleotide having a complementary 5' "sticky end" overhang are used when hybridizing. The ligation was performed in the buffer of Table 1 with ATP added at 16 ℃. For the analysis, gel electrophoresis was used and it was shown that after 30 minutes the reaction was completed yielding mainly products migrating to-120 bp, minor products migrating to-75 bp and starting material without "insert" (which migrated to-50 bp) (note: hairpin oligonucleotides are not visible on the gel despite their high concentration, since they are oligonucleotides of a significantly single stranded part, which allows minimal ethidium bromide intercalation).

Next, the same two complementary oligonucleotides, each with 5' overhangs, were hybridized together (SEQ9 and SEQ 10). Hairpin oligonucleotides with complementary 5' overhangs also hybridized separately (SEQ ID NO: 11). These oligonucleotides were then mixed with 1000 sticky end ligation units of T4DNA ligase in the buffer of Table 1. The reaction was then incubated at 16 ℃ for an appropriate amount of time 30 minutes followed by incubation at 75 ℃ for 10 minutes. The sample (including hairpin + insert but no ligase control) was then loaded on an ESI-TOF mass spectrometer using a T5000 system.

FIG. 8 shows that ligation can be performed with T4DNA ligase and ATP in the buffer of Table 1 by mass spectrometry. The oligonucleotide set shown above, which has 5 '"sticky ends" on both ends of the duplex DNA molecule and a hairpin oligonucleotide with a complementary 5' "sticky end" overhang, is used when hybridizing. Ligation was performed in the buffer of Table 1 with ATP added at 16 ℃ for 30 minutes. For analysis, mixtures of hairpin structured "insert" oligonucleotides and "hairpin structured" oligonucleotides were analyzed with ESI-TOF mass spectrometry with (fig. 8A) or without (fig. 8B) addition of ligase. It shows that in the absence of ligase only the starting material is seen, but in the presence of ligase no insert oligonucleotide is seen and the presence of a high molecular weight product is seen, corresponding to the insert duplex molecule with the hairpin structure oligonucleotide attached at either end (the hairpin structure is still observed in the ligase addition reaction as it is 10 times the initial concentration of insert).

Next, two complementary oligonucleotides, each with a 5' overhang, were hybridized together (SEQ9 and SEQ 10). Hairpin oligonucleotides with complementary 5' overhangs were also hybridized separately (SEQ ID NO: 11). These oligonucleotides were then mixed with 1000 sticky end ligation units of T4DNA ligase in the buffer of Table 1. The reaction was then incubated at 16 ℃ for an appropriate amount of time 30 minutes and then at 75 ℃ for 10 minutes. The sample was then mixed with exonuclease III and exonuclease VII and incubated at 37 ℃ for 30 minutes. The sample and insert only control, hairpin only control and ligation not treated with exonuclease control were run on a 1% agarose gel and visualized with ethidium bromide and a UV light source.

FIG. 9 shows that exonucleases (specifically exonuclease II and exonuclease VII) are functional in the buffer of Table 1 and can be used to remove non-circular DNA products from the reaction. These reactions were performed using ligation reactions as run in FIGS. 8 and 9 and were subjected to exonuclease III and exonuclease VII for 30 minutes at 37 ℃. The sample was then analyzed by gel electrophoresis, which showed removal of non-cyclic products and retention of cyclic products.

Next, the same two complementary oligonucleotides, each with a 5' overhang, were hybridized together (SEQ9 and SEQ 10). Hairpin oligonucleotides with complementary 5' overhangs were also hybridized separately (SEQ ID NO: 11). These oligonucleotides were then mixed with 1000 sticky end ligation units of T4DNA ligase in the buffer in Table 1. The reaction was then incubated at 16 ℃ for an appropriate amount of time 30 minutes and then at 75 ℃ for 10 minutes. The reaction was then mixed with exonuclease III and exonuclease VII and incubated at 37 ℃ for 30 minutes. The sample (FIG. 10B) and ligation reaction not treated with exonuclease (FIG. 10B) were then loaded on an ESI-TOF mass spectrometer using a T5000 system.

FIG. 10 shows that exonucleases (specifically exonuclease II and exonuclease VII) are functional in the buffer of Table 1 and can be used to remove non-circular DNA products from the reaction. These reactions were performed using ligation reactions as run in FIGS. 8 and 9 and were subjected to exonuclease III and exonuclease VII for 30 minutes at 37 ℃. The sample was then analyzed with an ESI-TOF mass spectrometer, which showed removal of non-cyclic products and retention of cyclic products.

All publications and patents mentioned in this application are herein incorporated by reference. Various modifications and variations of the methods and compositions of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described with respect to specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the claims.

Claims (24)

1. A method of preparing a nucleic acid library in a universal buffer, the method comprising:

a) mixing a nucleic acid sample with a universal buffer, wherein the universal buffer comprises dntps and primers;

b) contacting the universal buffer with a plurality of purified enzymes, wherein the enzymes have polymerase activity, kinase activity, and phosphatase activity, and wherein the contacting is performed under conditions such that amplified nucleic acids are produced;

c) treating the universal buffer such that sheared amplified nucleic acids are produced;

d) treating the universal buffer to inactivate the polymerase; and

e) contacting the universal buffer with a ligase and a nucleic acid linker under conditions such that a library of nucleic acids linked to the linker is generated;

wherein the above steps are accomplished in the universal buffer without nucleic acid purification.

2. The method of claim 1, wherein the nucleic acid sample comprises genomic DNA, and wherein the amplified nucleic acid is produced by whole genome amplification.

3. The method of claim 1, wherein said steps a) -e) are performed in a single vessel.

4. The method of claim 1, further comprising treating the universal buffer of the nucleic acid library containing the adaptor such that proteins and dntps are removed from the universal buffer.

5. The method of claim 1, wherein the linker comprises a hairpin structured primer, and wherein the linker-ligated nucleic acid library comprises a circular template.

6. The method of claim 5, further comprising treating said universal buffer containing said circular template with at least one exonuclease capable of digesting any non-circularized nucleic acids present.

7. The method of claim 6, further comprising heating said universal buffer containing said exonuclease to inactivate said exonuclease.

8. The method of claim 1, wherein the universal buffer further comprises an emulsifier.

9. The method of claim 1, wherein the universal buffer further comprises TRIS (hydroxymethyl) aminomethane (TRIS).

10. The method of claim 1, wherein the universal buffer further comprises a divalent metal cation.

11. The method of claim 1, wherein the universal buffer further comprises an inorganic salt.

12. The method of claim 1, wherein the universal buffer further comprises poly a.

13. The method of claim 1, wherein the universal buffer further comprises an alpha-linked disaccharide.

14. The method of claim 1, wherein the universal buffer further comprises a reducing agent.

15. The method of claim 1, wherein the universal buffer further comprises albumin or an albumin-like protein.

16. The method of claim 1, wherein the amount of the nucleic acid sample is between 10pg and 50 ng.

17. A composition, comprising: a) a buffer, b) an emulsifier, c) a divalent metal cation, d) an inorganic salt, e) poly (A) f) an alpha-linked disaccharide, g) a reducing agent and h) albumin or an albumin-like protein; also included are purified enzymes, wherein the enzymes have polymerase activity, kinase activity, and phosphatase activity.

18. The composition of claim 17, further comprising TRIS (hydroxymethyl) aminomethane (TRIS).

19. The composition of claim 17, wherein the emulsifier is a polysorbate.

20. The composition of claim 19, wherein the polysorbate is selected from the group consisting of: tween20, Tween40, Tween60 or Tween 80.

21. The composition of claim 17, wherein the inorganic salt is ammonium sulfate.

22. The composition of claim 17, wherein the α -linked disaccharide comprises trehalose.

23. The composition of claim 17, wherein the reducing agent comprises Dithiothreitol (DTT).

24. The composition of claim 17, wherein the composition further comprises dntps and primers.