Patent Application
20150184214
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 17, 2014, is named 31918-US1_SL.txt and is 5,651 bytes in size.
In synthesizing oligonucleotide of length N, by-products of length N+/-1, N+/−2, etc., exist in abundance. This inherent error is directly related to the fidelity of the synthesis process. For applications where the incomplete products do not interfere with the result, oligonucleotides can be used as is. However, for fidelity-demanding applications, such as gene and genome assembly, purification is necessary to ensure the assembled genes or genomes are nearly error-free. Traditional oligonucleotide purification methods such as reverse phase and anion-exchange HPLC can separate full length (i.e., desired length) products from its N+/−1 and N+/−2 by-products, but efficiency decreases with increasing oligonucleotide length. The proposed method does not have the same constraints as traditional purification techniques.
The present invention is a method used for selection of oligonucleotides useful in particular applications developed by using certain advantageous properties of mRNA display. Briefly, mRNA display is a technique used for in vitro peptide synthesis and selection to create peptides or proteins that can bind to a desired target with high affinity or selectivity. The process results in translated peptides or proteins that are linked to their mRNA progenitor via a puromycin linkage. The complex then binds to an immobilized target in a selection step (affinity chromatography). The mRNA-protein fusions that bind well can then be reverse transcribed to cDNA and their sequence amplified via polymerase chain reaction. The result is a nucleotide sequence that encodes a peptide with high affinity for the molecule of interest.
The present invention uses certain aspects of mRNA display to select for or purify oligonucleotides that have the correct nucleotide sequence. The present invention takes advantage of the fact that a slight change in nucleotide sequence (addition, deletion, substitution, etc.) can cause large scale changes in the peptide expressed by the nucleotide sequence.
In one embodiment, the invention is a method of purifying a full-length target oligonucleotide from a pool further containing shorter or longer same-sequence oligonucleotide by-products, the method comprising: amplifying the pool to form a second pool of oligonucleotides; transcribing the second pool of oligonucleotides to form an RNA pool wherein the RNA molecules contain codons for at least one tag sequence; ligating the molecules of the RNA pool to puromycin; translating the puromycin-ligated RNA molecules to form a pool of chimeric molecules containing the RNA linked to a peptide expressed from the RNA; capturing and isolating the chimeric molecules that express the tag; reverse transcribing the RNA of the isolated chimeric moieties to form a pool of cDNA comprising the purified full-length oligonucleotide. Is some embodiments, the amplification uses at least two primers comprising a target binding site, and further comprising one or more of a promoter, an enhancer, a ribosome binding site, a translation initiation site and a sequence encoding at least one tag. In some embodiments, the first primer comprises promoter, enhancer, the ribosome binding site and the translation initiation site and the second primer comprises the sequence encoding at least one tag. In some embodiments, the amplification uses the second primer comprising the sequence encoding a first tag and a sequence encoding a second tag. In some embodiments, the second primer further comprises a sequence encoding a third tag. In some embodiments, the first primer comprises a target specific sequence conjugated to SEQ ID NO: 8. For example, the first primer comprises SEQ ID NO: 3. In some embodiments, the second primer comprises a target specific sequence conjugated to SEQ ID NOs: 9 or 10. For example, the second primer comprises SEQ ID NO: 6 or 7. In some embodiments the codons of the first tag sequence are in frame with the full-length oligonucleotide and out of frame with shorter and with longer same-sequence oligonucleotide by-products. In other embodiments, the codons of the second tag sequence are in frame with −1 shorter and with +2 longer same-sequence oligonucleotide by-products and out of frame with the full-length oligonucleotide. In other embodiments, the codons of the third tag sequence are in frame with −2 shorter and with +1 longer same-sequence oligonucleotide by-products and out of frame with the full-length oligonucleotide. In some embodiments the capturing is performed with a tag specific binding agent, for example, an antibody. In some embodiments, capturing comprises capturing of the first tag sequence. In some embodiments, capturing comprises capturing of the second tag sequence. In some embodiments, capturing comprises capturing of the third tag sequence. In some embodiments, the method further comprises amplifying the pool of cDNA comprising the purified full-length oligonucleotide. In some embodiments, the method further comprises repeating one or more cycles of steps of amplifying, transcribing, ligating the molecules to puromycin, translating, capturing and isolating, and reverse transcribing using the amplified pool of cDNA as the second pool of oligonucleotides.
In other embodiments, the invention is a kit for purifying a full-length target oligonucleotide from a pool further containing shorter or longer same-sequence oligonucleotide by-products, the method comprising a pair of primers wherein the first primer comprises a target binding site, a promoter, an enhancer, a ribosome binding site and a translation initiation site and the second primer comprises the sequence encoding at least one tag. In some embodiments, the kit further comprises one or more of the following: reagents for DNA amplification, reagents for DNA transcription, reagents for nucleic acid ligation, puromycin, reagents for RNA translation, at least one tag-binding agent specific for the at least one tag and reagents for RNA reverse transcription. In some embodiments, the second primer comprises the sequence encoding a first tag; and further comprises a sequence encoding a second tag. In some embodiments, the second primer further comprises a sequence encoding a third tag. In some embodiments, the first primer comprises a target specific sequence conjugated to SEQ ID NO: 8 and the second primer comprises a target specific sequence conjugated to SEQ ID NOs: 9 or 10. In some embodiments, the first primer comprises SEQ ID NO: 3 and the second primer comprises SEQ ID NOs: 6 or 7.
Definitions
The term “amplification” refers to the production of a plurality of nucleic acid molecules from a target nucleic acid. Amplification can be carried out by any method generally known in the art, such PCR, RT-PCR, Isothermal Amplification, Ligase Chain Reaction (LCR), Polymerase Ligase Chain Reaction, Gap -LCR, Repair Chain Reaction, 3SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Qb-amplification. In some amplification methods, primers hybridize to specific sites on the target nucleic acid molecules in order to provide an initiation site for extension by a polymerase.
The term “complementary” refers to the ability to form favorable thermodynamic stability and specific pairing between the bases of two nucleotides in a nucleic acid at an appropriate temperature and ionic buffer conditions. This pairing is dependent in part, on the hydrogen bonding properties of each nucleotide. Oligonucleotides, e.g., primers for amplification of target nucleic acids can be both fully complementary over their entire length with a target nucleic acid molecule or “partially complementary” wherein the primer contains some bases non-complementary to the corresponding base in a target nucleic acid.
The term “detecting” means assessing the presence or absence of a target nucleic acid in a sample.
The term “enriched” refers to any method of treating a sample comprising a target nucleic acid that allows one to separate the target nucleic acid from at least a part of other material present in the sample. “Enrichment” can thus be understood as a production of a higher relative amount of target nucleic acid over other material. The terms “purify” or “purified” can be used interchangeably with the terms “enrich” or “enriched.”
The term “excess” refers to a larger quantity or concentration of a certain reagent as compared to another reagent.
The term “hybridize” refers to the base-pairing between different nucleic acid molecules consistent with their nucleotide sequences. The terms “hybridize” and “anneal” can be used interchangeably.
The terms “nucleic acid” or “polynucleotide” can be used interchangeably and refer to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or an analog thereof, i.e., polymers including one or more synthetic or modified subunits. Exemplary modifications include methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, and the like), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids and the like). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Typically, the nucleotide monomers are linked via phosphodiester bonds, although synthetic forms of nucleic acids can comprise other linkages (e.g., peptide nucleic acids as described in Nielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be single or double-stranded and is not limited to any particular length.
The term “nucleotide” (unless otherwise specified) in addition to referring to the naturally occurring ribonucleotides or deoxyribonucleotides refer to related structural variants thereof, including derivatives and analogs, that are functionally equivalent with respect to the particular context in which the nucleotide is being used (e.g., hybridization to a complementary base), unless the context clearly indicates otherwise.
The term “oligonucleotide” refers to a nucleic acid that includes at least two nucleic acid monomer units (e.g., nucleotides). An oligonucleotide typically includes from about six to about 175 nucleic acid monomer units, more typically from about eight to about 100 nucleic acid monomer units, and still more typically from about 10 to about 50 nucleic acid monomer units (e.g., about 15, about 20, about 25, about 30, about 35, or more nucleic acid monomer units). The exact size of an oligonucleotide will depend on many factors, including the ultimate function or use of the oligonucleotide. Oligonucleotides are optionally prepared by any suitable method, including, but not limited to, isolation of an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99, 1979); the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucci et al. (J. Am. Chem. Soc. 103:3185-3191, 1981); automated synthesis methods; Maskless Array Synthesis as disclosed in Singh-Gasson et al., Nature Biotechnology, 17: 974-978, 1999, or the solid support method of U.S. Pat. No. 4,458,066, or other methods known to those skilled in the art.
The terms “shorter or longer same-sequence oligonucleotide” or “shorter or longer same-sequence oligonucleotide by-product” are used interchangeably to refer to an oligonucleotide that has the same sequence as the target or desired oligonucleotide except, typically as a result of an imperfect in vitro synthesis process, is missing one or more nucleotides or includes additional one or more nucleotides that were erroneously incorporated.
The term “codon” refers to a sequence of three nucleotides in a DNA or RNA molecule that forms a unit of genetic code, i.e., translation of the nucleic acid sequence into an protein sequence.
The term “primer” refers to a polynucleotide capable of acting as a point of initiation of template-directed nucleic acid synthesis when placed under conditions in which polynucleotide extension is initiated (e.g., under conditions comprising the presence of requisite nucleoside triphosphates (as dictated by the template that is copied) and a polymerase in an appropriate buffer and at a suitable temperature or cycle(s) of temperatures (e.g., as in a polymerase chain reaction)). To further illustrate, primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes (e.g., nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), etc.). A primer is typically a single-stranded oligonucleotide (e.g., oligodeoxyribonucleotide). The appropriate length of a primer depends on the intended use of the primer but typically ranges from 6 to 40 nucleotides, more typically from 15 to 35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template for primer elongation to occur. In certain embodiments, the term “primer pair” means a set of primers including a 5′ sense primer (sometimes called “forward”) that hybridizes with the complement of the 5′ end of the nucleic acid sequence to be amplified and a 3′ antisense primer (sometimes called “reverse”) that hybridizes with the 3′ end of the sequence to be amplified (e.g., if the target sequence is expressed as RNA or is an RNA). A primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in ELISA assays), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available.
The term “quantifying” as used herein relates to the determination of the amount or concentration of a target nucleic acid present in a sample.
The term “target nucleic acid” is used herein to denote a nucleic acid in a sample which is to be analyzed, i.e. the presence, amount or nucleic acid sequence in a sample is to be determined. The target nucleic acid may be any type of DNA or RNA, a genomic sequence, or a specific gene, or any other fragment thereof.
The term “positive selection” means a purification step wherein the specific binding partner or antibody binds to the desired moiety, allowing the desired moiety to be recovered from a pool containing both the desired and undesired moieties. The term “negative selection” means a purification step wherein the specific binding partner or antibody binds to an undesired moiety and allows its removal, thereby partially or completely purifying the pool of moieties left behind after the removal of the undesired moiety.
The present invention provides a method for separating full length oligonucleotides from oligonucleotides containing errors, such as those that are N+/−1 or N+/−2, etc. To overcome the difficulty in separating full length products, oligonucleotides are translated into their peptide counterparts using mRNA display methods. An RNA-peptide chimera is generated, with the translated peptide covalently linked to its encoded nucleic acid. In the present invention, the mRNA includes nucleotide sequences that encode two specific tags: a tag expressed by in-phase oligonucleotides (called FLAG) and a tag expressed by oligonucleotides that are one step out of phase (called Strep). In some embodiments, no tag is expressed by oligonucleotides that are two steps out of phase. In other embodiments, an additional (third) tag is expressed by oligonucleotides that are two steps out of phase.
When sequences encoding a specific peptide tag are added to the 3′ end of the oligonucleotide, only oligonucleotides that are in-frame will translate the “correct” peptide tag (i.e. full length products and rare N+/−3, N+/−6, etc., by-products). During affinity purification of the “correct” peptide tag, followed by amplification of the nucleic acid of the RNA-peptide chimera, enrichment of the desired nucleotide sequences is achieved. Synthesis errors such as N+/−1 and N+/−2 would lead to translation out-of-frame and will translate the “incorrect” peptide tag or no tag. Such synthesis products are removed by the affinity purification.
Certain embodiments of the present invention utilize an oligonucleotide that is flanked by amplification sequences on either end. The sequences required for transcription and translation, the sequence encoding the peptide purification tag and an optional poly-A tail are included in the amplification primers. An example of the resulting amplification product is illustrated in
As shown in
An overview of the present method is found in
Looking to the individual steps of
Instead of using primers with target-hybridizing regions, Step 1 could also be performed by ligating adaptors on the ends of the desired nucleic acid sequence(s). Such adaptors would contain all the elements that were included in the primers as described above, i.e., the sequences necessary for transcription and translation and tag-coding sequences, as well as the optional poly-A tail. After adaptor ligation, the ligated product could be amplified via PCR (e.g., with primers that specifically hybridize to adaptor sequences) and then subjected to the remaining steps (2-7) of the present method.
After annealing of the primers to the desired nucleic acid sequence, amplification such as in situ PCR is performed, resulting in an amplified, double-stranded PCR product containing the untranslated regions, a start codon, the desired sequence, and a tag-coding region, e.g., FLAG region.
In step 2, after amplification the double-stranded products are transcribed to give the mRNA product. For example, the dsDNA library can be transcribed into mRNA enzymatically using T7 polymerase using methods well known in the art.
After the mRNA is generated from the transcription step, the mRNA is conjugated to puromycin, for example, by ligation to an oligonucleotide containing a puromycin at its terminus. The puromycin linker can be formed in any variety of ways. A standard protocol for formation of puromycin oligonucleotides can be found in Liu et al., Meth. Enzym., 318: 268-293 (2000), the disclosure of which is incorporated by reference. Further, puromycin oligonucleotides are commercially available (e.g., Gene Link, Sigma-Aldrich, etc.).
The RNA is ligated to the 3′-puromycin oligonucleotide to form translation templates (see
The translation templates are then used in a translation step (see step 4,
In vitro translation systems will invariably result in a pool of RNA-peptide chimerae wherein a subpopulation contains the desired nucleotide sequences (resulting in an appropriate expression of the tag), and other subpopulations where the nucleotide sequence is incorrect (e.g., N−1, N−2) resulting in inappropriate expression of the tag.
This mixed pool of chimerae is then purified (see Step 5,
Another embodiment of the present invention is a two-tag system for both positive selection of appropriate “correct” sequences and negative selection against inappropriate “incorrect” sequences. As an example, the experimental layout of a two-tag strategy using FLAG tag for positive selection and Strep tag for negative selection is described. It is not necessary in a one- or two-tag system to use FLAG and Strep tags. Other tags against which specific binders can be generated would be useful in this invention as well. In such a system, one tag would be expressed if the sequence remains properly in-frame, whereas a second tag would be expressed if the sequence is out of frame (for example, if the nucleotide sequence being transcribed and translated is N+/−1 or N+/−2). It is noted that the N−1 oligonucleotide will be in the same frame express the same tag as the N+2 oligonucleotide. The N−2 oligonucleotide will be in the same frame express the same tag as the N+1 oligonucleotide. For negative selection, after translation, antibodies raised against the second tag could be used to specifically bind the chimerae where the peptide is out of frame and remove these chimerae from the pool. The purified pool of chimera could then be subjected to an antibody raised against the first tag (positive selection) to bind the desired chimerae and allow for washing away of the rest.
Further, it is possible to employ a three-tag system for even greater purification, wherein the first tag indicates in-frame sequence, the second tag indicates a first out-of-frame sequence (e.g., N−1 or N+2) and a third tag indicates a second out-of-frame sequence (e.g. N−2 or N+1). In such a system, antibodies against the second and third tags can be used to remove the two types of out-of-frame chimerae and an antibody against the first tag can be used for the final purification of the in-frame chimerae.
As is readily apparent, the use of more than one tag may not necessarily mean that the system uses “negative selection.” For example, some applications may be designed to positively select for N+/−1 or N+/−2 sequences if it is desired to isolate and purify such sequences.
As is also apparent, the purification step of the present invention can be carried out with antibodies selective for the respective tags; however, the present invention should not be limited to only antibody-based purification. Any binding partner that has a specific binding affinity for the respective tag to the essential exclusion of other tags is potentially useful for the purification step of the inventive method. Examples of such binding partners include, but are not limited to, antibodies, polyclonal antibodies, monoclonal antibodies, antibody fragments, peptides and any other moiety which acts as a specific binder against the respective tag.
After the purification step, the isolated and purified chimera is subjected to reverse transcription to yield a cDNA (see step 6,
Primer design according to the present invention is illustrated on
In some embodiments, primers utilize one or more of the sequences listed in Table 1. The forward primers listed in Table 1 contain the necessary elements such as the promoter sequence, the Shine-Dalgarno sequence, a start codon, and the tags, in this case a FLAG tag and a Strep tag. Segments 1A and 1B are examples of the primer regions specific for the target sequence. It is understood that any other target can be used and a suitable target-specific region of the primer designed therefor. The sequence denoted as MRD_LP is a primer containing a sequence-specific region and a region containing the sequence elements necessary for the steps of the inventive method, i.e., transcription, translation and optionally, reverse transcription. LP is the sequence conjugated to the target-specific sequence.
As shown in Table 1, MRD_RP1 is a primer region used for a two tag method, and includes both a FLAG tag (which would be expressed properly if translation is in-frame) and a Strep tag (which would be expressed properly if translation is one nucleotide out of frame). In this example, the Strep tag is used for negative selection (i.e., those chimerae that have a properly expressed Strep tag are removed from the chimera pool) and the FLAG tag is used for positive selection (i.e., those chimerae that have a properly expressed FLAG tag are the desired in-frame chimerae and are selected). MRD_PR2 is a shorter primer congaing on the FLAG tag. RP1 and RP2 are sequences conjugated to the target-specific sequences.
It is understood that one of skill in the art can utilize the artificial sequences described herein, e.g., LP, RP1 and RP2 in combination with other target-specific sequences in place of sequences 1A and 1B to form forward and reverse primers. It is further understood that the artificial sequences LP, RP1 and RP2 can be modified as long as they contain the essential elements enabling transcription, translation, and reverse transcription as described herein.
Primers were designed for purposes of adding the necessary elements such as the promoter sequence, the Shine-Dalgarno sequence, a start codon, and the tags, in this case a FLAG tag and a Strep tag. Table 1 below lists the sequences used in these primers. Segments 1A and 1B show the primer regions specific for the target sequence. The sequence denoted as MRD_LP is a primer region used in primers designed for in-frame (N) translation. To test the method of the invention, out-of-frame sequences were also designed. MRD_LP+1 is a primer region used for detection of translation that is one nucleotide out of frame; MRD_LP+2 is a primer region used for detection of translation that is two nucleotides out of frame. The out-of-frame regions are modified by having an additional cytosine (MRD_LP+1) or two additional cytosines (MRD_LP+2) inserted into the primer sequence after the start codon.
As further seen in Table 2, MRD_RP1 is a primer region used for a two tag method, and includes both a FLAG tag (which would be expressed properly if translation is in-frame) and a Strep tag (which would be expressed properly if translation is one nucleotide out of frame). In this example, the Strep tag is used for negative selection (i.e., those chimerae that have a properly expressed Strep tag are removed from the chimera pool) and the FLAG tag is used for positive selection (i.e., those chimerae that have a properly expressed FLAG tag are the desired in-frame chimerae and are selected). MRD_PR2 is a shorter primer containing the FLAG tag.
Step 1: PCR
A reaction mixture was assembled containing template DNA, polymerase buffer, Hot-Start polymerase, forward and reverse primers, MgCl2 and dNTPs.
Cycling conditions were as follows: Step 1: 15 minutes at 95° C.; Step 2: 1 minute at 95° C.; Step 3: 1 minute at 58°; Step 4: 1 minute at 72°; Step 5: Go back to step 2 and repeat 17 times; Step 6: 10 minutes at 72°; Step 7: Finish and hold at 3.5°. As the primers incorporate the elements necessary for transcription, translation and purification, the amplification products of the in situ PCR are now ready to undergo the remaining steps of the process.
2 uL were taken from the 25 uL PCR reaction and run on a 4% agarose gel (
Lane 1 shows the product formed when the primers contain only the target specific elements without the transcription/translational elements or the tags included (primers 1A and 1B). In lanes 2-3, the “in-frame” primer is used as left primer (MRD_LP), while the right primer is varied and has both FLAG and Strep tags (MRD_RP1, lane 2) and only FLAG tags (MRD_RP2, lane 3). In lanes 4-5, the left primer is the “N+1” out-of-frame primer (MRD_LP+1), while the right primer is varied to have both tags (MRD_RP1, lane 4) or only FLAG tag (MRD_RP2, lane 5). In lanes 6-7, the left primer is the “N+2” out-of-frame primer (MRD_LP+2), while the right primer is varied to have both tags (MRD_RP1, lane 6) or just FLAG tag (MRD_RP2, lane 7).
As can be seen from
Step 2. mRNA Library
In this step, DNA was converted to RNA using in-vitro transcription kit from Promega (T7 RiboMAX in-vitro transcription kit) according to manufacturer's instructions.
The following T7 RiboMax (Promega) transcription reagents (50 uL) were assembled:
The mixture was incubated at 37° C. for 3 h. 8 uL of RQ DNase (1 U/uL) was added, and incubated at 37° C. for 1 hour. Purification was performed with RNeasy mini (Qiagen), with elution in 26 uL Qiagen RNeasy H2O (yield is generally ˜1000 ng/uL).
Step 3. Ligate Puromycin Spacer to mRNA.
Ligation of the puromycin linker (custom synthesis via Biosearch Technologies, Inc) to the transcribed RNA occurred via T4 RNA ligase, which allowed for the ligation of 2 single stranded RNA species. mRNA was heated at 75° C. for 1 min (then snap chilled) before assembling the ligase reaction. The following reagents (50 L) were assembled:
The mRNA was incubated at 15° C. for 2 h. Samples were purified using RNeasy column, eluted in 51 uL Qiagen RNeasy H2O (yield is generally ˜200 ng/uL).
Step 4. PURExpress Translation of mRNA-Spacer.
The RNA-puromycin construct was translated into RNA-puromycin-peptide fusion using New England Biolab's PureExpress In-vitro translation kit as directed. As translation of the RNA-puromycin construct proceeds, ribosome moves along the RNA template, and once it reaches the 3′ end of the template, the fused puromycin will enter the ribosome's A site and be incorporated into the nascent peptide. The mRNA-polypeptide fusion is then released from the ribosome, resulting the RNA-puromycin-peptide fusion or chimera. The following reagents (25 uL) were assembled:
Incubation occurred at 37° C. for 1 h.
Step 5. Purification
In-frame translated fusion products will correctly translate the affinity purification tag, in this case the FLAG tag, whereas any frameshift mistakes (i.e. N+/−1, N+/−2) will not. Purification in this case was done via the short peptide FLAG tag (DYKDDDDK (SEQ ID NO: 11)) and anti-FLAG antibody coated magnetic beads. All fusions with a correctly translated FLAG tag will bind to the anti-FLAG antibody that is conjugated to the magnetic bead, whereas frameshift mistakes will be washed away. Additionally, non-specific DNA is digested using Promega's RNase-free DNase RQ1. The beads with anti-FLAG antibody are commercially available from Sigma.
To perform the purification, the reaction was mixed with 200 uL of TBSTE [TBST (10 mM TriszHCl, pH 8.0, 150 mM NaCl, 0.02% Tween-20) with 2 mM EDTA]. 20 uL of anti-flag magnetic beads were added. This mixture was incubated at 4C for 1 h on a rotating platform. The magnetic beads were washed 3× with 300 uL TBSTE, then washed again 1× with 300 uL Qiagen RNeasy H2O. The resulting sample is then subjected to RQ1 treatment. RQ1 is a RNase-free DNase for removal of any subsisting DNA in the sample. To perform this, the sample was resuspended in 100 uL of RQ1 buffer, 10 uL of RQ1 was added, sample was incubated at 37° C. for 1 h. After incubation, the magnetic beads were washed 3× with 300 uL TBSTE, then washed 1× with 300 uL Qiagen RNeasy H2O.
Step 6: RT Reaction
RNA was converted back to cDNA via Invitrogen SuperScript reverse transcriptase. Elution of the cDNA from the magnetic beads was done using mild denaturant (in this case 0.1N NaOH). Magnetic beads were discarded. To perform this step, the following protocol was used. Resuspend the magnetic beads with the following reagents (80 uL):
The above master mix was mixed with the beads and incubated at 37° C. for 30 min. Beads were then washed 2× with 300 uL TBSTE, and washed again 1× with 300 uL Qiagen RNeasy water.
The fusion products were then eluted with 0.1N NaOH by the following protocol: 1) Elute in 100 uL 0.1N NaOH; 2) Add 1 ul of 100× tRNA as carrier; 3) Elute at room temperature for 3 minutes; 4) Purify in Millipore column, 5000 rpm 1 min; 5) Ethanol precipitate (NaAc+EtOH) the flow through; 6) Resuspend in 100 uL H2O.
Step 7. Amplification.
The cDNA was amplified using Phusion polymerase from NEB to reconstitute the purified construct from step 1. Assemble the following reagents (400 uL):
Stepwise PCR was run to assess amplification quality after different cycles. The following conditions were used: Step 1: 1 minute at 98° C.; Step 2: 20 seconds at 95° C.; Step 3: 1 minute at 64° C.; Step 4: 30 seconds at 72° C.; Step 5: Go back to step 2 and repeat x times (depending upon which step in the PCR is desired for evaluation); Step 6: 2 minute at 72° C.; Step 7: hold at 3.5° C.
After in-vitro transcription/translation, purification and reverse transcription, a PCR cycle titration was performed. An electrophoresis gel separating products taken at various amplification steps is shown in
It should be noted that these lanes in
With sample 2, which represents the in-frame translation of the FLAG purification tag, PCR product is seen by cycle 12, whereas with sample 4/6, PCR product is seen by cycle 15, which represents approximately 8 fold enrichment. By 18 cycles, the difference between the in-frame and the out-of-frame products is readily visible.
Libraries were sequenced using the MiSEQ instrument. Oligonucleotide was purchased and evaluated for purity (percentage of full length constructs) both before and after undergoing the inventive method. It is well known that purchased oligonucleotides will contain a certain percentage of imperfectly formed moieties.
As can be seen from
It should be noted that the data used in
In this example, steps 1-4 are as described in Example 2 are performed using the two-tag primer (e.g., the products shown in lanes 2, 4, and 6 in
In this example, steps 1-4 are carried out as described in Example 2; however, the second primer contains a nucleotide sequence that is expressed as a third peptide tag when the transcription/translation is out of frame by two nucleotides. The peptide sequence is not important other than that antibodies must be available (or synthesized) that are specific for the peptide sequence. For purposes of this example, the third tag is denominated as TAG3.
In an example of the three tag system, after step 4, FLAG tag is expressed only in in-frame products (N), Strep tag is expressed in only those moieties that are one step out of frame and TAG3 is expressed only in those moieties that are two steps out of frame. Instead of step 5 having two substeps, as in Example 4, it would now have 3 substeps wherein, for example, the first step is to incubate the sample with anti-TAG3 antibody beads and remove them from the remaining sample, the second substep is to incubate with anti-Step antibody beads and remove them from the sample, and the third substep is to incubate the remaining sample with anti-FLAG beads, collect those beads and elute the desired FLAG-tagged product. It is also possible to do both negative collections of the Strep and TAG3 products through simultaneous incubation with anti-Strep and anti-TAG3 beads, thus only having two substeps.
While the invention has been described in detail with reference to specific examples, it will be apparent to one skilled in the art that various modifications can be made within the scope of this invention. Thus the scope of the invention should not be limited by the examples described herein, but by the claims presented below.
| Number | Date | Country | |
|---|---|---|---|
| 61922267 | Dec 2013 | US |
