WO2021037368A1 - Procédés de production et de clonage d'adnc rapide - Google Patents

Procédés de production et de clonage d'adnc rapide Download PDF

Info

Publication number
WO2021037368A1
WO2021037368A1 PCT/EP2019/073112 EP2019073112W WO2021037368A1 WO 2021037368 A1 WO2021037368 A1 WO 2021037368A1 EP 2019073112 W EP2019073112 W EP 2019073112W WO 2021037368 A1 WO2021037368 A1 WO 2021037368A1
Authority
WO
WIPO (PCT)
Prior art keywords
primer
seq
nucleotide sequence
cell
vector
Prior art date
Application number
PCT/EP2019/073112
Other languages
English (en)
Inventor
Sonia BARBIERI
Sara RAVASIO
Original Assignee
Institute For Research In Biomedicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute For Research In Biomedicine filed Critical Institute For Research In Biomedicine
Priority to PCT/EP2019/073112 priority Critical patent/WO2021037368A1/fr
Priority to PCT/EP2020/073809 priority patent/WO2021037886A1/fr
Publication of WO2021037368A1 publication Critical patent/WO2021037368A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/107—RNA dependent DNA polymerase,(i.e. reverse transcriptase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid

Definitions

  • the present invention relates to the field of cDNA production and cloning of nucleic acids encoding a polypeptide of interest.
  • the present invention relates to isolation and cloning of antibodies, for example comprising cDNA production, amplification and cloning based on antibody mRNA of B cells and/or plasma cells.
  • B cell-mediated immune protection operates primarily via antibodies secreted by plasma cells.
  • immunological memory is represented by the presence of long-lived plasma cells and of memory B cells, a subset of B cells which can persist in the human body over decades and which can be reactivated by a secondary encounter with their cognate antigen, thus differentiating to plasma cells.
  • An antibody (Ab) also known as an immunoglobulin (Ig)
  • Ig immunoglobulin
  • Antibodies (Abs) aid in the clearance of pathogens by neutralization of soluble toxins, by activation of complement or by interacting with other immune cells. Accordingly, identification and production of antibodies, in particular human monoclonal antibodies, is of great interest.
  • immunoglobulin genes need to be cloned and expressed as recombinant proteins.
  • mAbs monoclonal antibodies
  • combinatorial display libraries such as phage display libraries
  • phage display libraries are high-throughput screening methods allowing generation of higher affinity Abs (Burton DR, Barbas CF III, Adv Immunol. (1994) 57:191-280, Hoogenboom HR., Methods Mol Biol. (2002) 178, 1-37).
  • Combinatorial display libraries are constructed from immunoglobulin (Ig) variable genes of immunized or infected individuals.
  • the resulting antibodies do not represent necessarily the natural antibody repertoire and it is unlikely that a given VH/VL pair went through a selection process. Accordingly, combinatorial display libraries do not allow characterization of the properly-paired antigen-specific B cell repertoire.
  • B cell immortalization (Traggiai et al., 2004, Nat Med 10:871-875, Kwakkenbos MJ et al., Immunol Rev. (2016)) and B cell culture (Jin A. et al, Nat Med. (2009)15:1088-92.; Corti D et al. Science (2011) 333:850-6.), Epstein-Barr virus (EBV) immortalization and/or cytokine stimulation are used.
  • EBV Epstein-Barr virus
  • Immortalized B cells are kept in culture for several days to allow B cell proliferation and secretion of antibodies in the supernatant. While B cell immortalization and B cell culture allow for the isolation of high affinity Abs, they require the cumbersome screening of significant numbers of cells (usually more than 1 ,000) to identify the very few B cells of interest (usually around 10).
  • the hybridoma method is one of the oldest methods for generating mAbs and is based on the fusion (hybridoma) of antibody-producing B cells with an immortal myeloma cell line in a selective medium where only the hybridoma cells can survive producing a specific mAb.
  • the antibody-producing B cells are isolated from mice and, therefore, murine antibodies are obtained.
  • administration of murine antibodies can result in a HAMA (human anti-murine antibody) response, which can be life-threatening and decrease the effectiveness of the treatment.
  • murine antibodies must be humanized or the hybridoma technique must be combined with transgenic humanized mouse strains (Zhang C. Methods Mol Biol. (2012) 901 :117-35).
  • production of human mAbs is based on single cell sorting of B cells by flow cytometry, single-cell reverse transcription (RT)-PCR to isolate the immunoglobulin (Ig) heavy and light chain variable genes from single B cells, amplification of the Ig genes by PCR, and subsequent Ig gene expression vector cloning for antibody production in vitro.
  • RT single-cell reverse transcription
  • composition “comprising” thus encompasses “including” as well as “consisting” e.g ., a composition “comprising” X may consist exclusively of X or may include something additional e.g., X + Y.
  • x in relation to a numerical value x means x ⁇ 10%, including, for example, x + 5% or x ⁇ 7%.
  • disease as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
  • treatment refers in particular to “prophylactic settings” (e.g., administration of a drug before diagnosis or “in advance") and may be used interchangeably with the term “prophylaxis”.
  • prevention refers in particular to "prophylactic settings” (e.g., administration of a drug before diagnosis or “in advance") and may be used interchangeably with the term “prophylaxis”.
  • subject or “patient” are used interchangeably herein to mean all mammals including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In some embodiments, the patient is a human.
  • binding and similar reference means in particular “specifically binding”, which does not encompass non-specific sticking.
  • peptide refers to peptides, polypeptides, oligopeptides, oligomers or proteins comprising at least two amino acids joined to each other preferably by a normal peptide bond, or, alternatively, by a modified peptide bond, such as for example in the cases of isosteric peptides.
  • peptide refers to peptides, polypeptides, oligopeptides, oligomers or proteins comprising at least two amino acids joined to each other preferably by a normal peptide bond, or, alternatively, by a modified peptide bond, such as for example in the cases of isosteric peptides.
  • peptide polypeptide
  • protein may be used interchangeably.
  • peptide may also include “peptidomimetics” which are defined as peptide analogs containing non-peptidic structural elements, which peptides are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide.
  • a peptidomimetic lacks classical peptide characteristics such as enzymatically scissile peptide bonds.
  • a peptide, polypeptide or protein can comprise amino acids other than the 20 amino acids defined by the genetic code in addition to these amino acids, or it can be composed of amino acids other than the 20 amino acids defined by the genetic code.
  • a peptide, polypeptide or protein in the context of the present invention can equally be composed of amino acids modified by natural processes, such as post-translational maturation processes, or by chemical processes, which are well known to a person skilled in the art.
  • a peptide, polypeptide or protein can be branched following an ubiquitination or be cyclic with or without branching. This type of modification can be the result of natural or synthetic post-translational processes that are well known to a person skilled in the art.
  • the terms "peptide”, “polypeptide”, and “protein” may also include modified peptides, polypeptides and proteins.
  • peptide, polypeptide or protein modifications can include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of a nucleotide or of a nucleotide derivative, covalent fixation of a lipid or of a lipidic derivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross- linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation or ubiquitination.
  • a peptide, polypeptide or protein is a "classical” peptide, polypeptide or protein.
  • a “classical” peptide, polypeptide or protein is typically composed of amino acids selected from the 20 amino acids defined by the genetic code, linked to each other by a normal peptide bond.
  • a polypeptide or a protein may comprise, for example, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or at least 150 amino acid residues.
  • the term "antibody” encompasses various forms of antibodies including, without being limited to, whole antibodies, antibody fragments (such as antigen binding fragments), human antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as the characteristic properties according to the invention are retained.
  • the antibody is a monoclonal antibody. In some embodiments, the antibody does not occur in nature, such as an engineered antibody.
  • antibody generally also includes antibody fragments. Fragments of the antibodies may retain the antigen-binding activity of the antibodies. Such fragments are referred to as "antigen-binding fragments". Antigen-binding fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Fragments of the antibodies can be obtained from antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by recombinant means, for example by cloning and expressing a part (fragment) of the sequences of the heavy and/or light chain.
  • antigen-binding fragment also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody.
  • scFv single-chain Fv fragments
  • an scFv may comprise the CDRs from an antibody as described herein.
  • heavy or light chain monomers and dimers include heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker.
  • Antibody fragments may be contained in a variety of structures known to the person skilled in the art.
  • antibody includes all categories of antibodies, namely, antigen binding fragment(s), antibody fragment(s), variant(s) and derivative(s) of antibodies.
  • Human and humanized antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374; Harrison, Charlotte (2014) The full repertoire of humanized antibodies. Nature Reviews Drug Discovery 13: 336). While the present invention includes methods for producing human antibodies, human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire ora selection of human antibodies in the absence of endogenous immunoglobulin production.
  • transgenic animals e.g., mice
  • Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., j. Mol. Biol. 227 (1992) 381 -388; Marks, J.
  • Human monoclonal antibodies may also be prepared by using improved EBV-B cell immortalization as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871 -5. Methods for producing humanized antibodies are well-known in the art and described, for example, in Safdari Y, Farajnia S, Asgharzadeh M, Khalili M. (2013) Antibody humanization methods - a review and update. Biotechnol Genet Eng Rev.29:175-86).
  • antibodies, or an antigen-binding fragments thereof typically comprises (at least) three complementarity determining regions (CDRs) on a heavy chain and (at least) three CDRs on a light chain.
  • complementarity determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains.
  • the CDRs of a heavy chain and the connected light chain of an antibody together form the antigen receptor.
  • the three CDRs (CDR1, CDR2, and CDR3) are arranged non-consecutively in the variable domain, namely, in the heavy chain variable region (VH) and in the light chain variable region (VL), respectively.
  • antigen receptors are typically composed of two variable domains (on two different polypeptide chains, i.e. heavy and light chain: VH and VL), there are typically six CDRs for each antigen receptor (heavy chain: HCDR1 , HCDR2, and HCDR3; light chain: LCDR1, LCDR2, and LCDR3).
  • a classical single antibody molecule has usually two antigen receptors and therefore contains twelve CDRs.
  • the CDRs on the heavy and/or light chain may be separated by framework regions, whereby a framework region (FR) is a region in the variable domain which is less "variable" than the CDR.
  • FR framework region
  • variable region i.e., the heavy chain variable region (VH) and/or the light chain variable region (VL)
  • VH heavy chain variable region
  • VL light chain variable region
  • the position of the CDRs can be defined according to the IMGT numbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012).
  • Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgM, IgE, i.e. an a, g, m or e heavy chain).
  • the antibody is of the IgG type.
  • antibodies may be of IgG1 , lgG2, lgG3 or lgG4 subclass, for example lgG1 .
  • Antibodies may have a k or a l light chain.
  • Antibodies may be provided in purified form.
  • the antibody will be present in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
  • Antibodies can be used alone, or in combination, as prophylactic or therapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool.
  • B lymphocyte and “B cell” are used herein interchangeably.
  • a "B cell” is a type of white blood cells of the lymphocyte subtype.
  • a major function of a B cell is to secrete antibodies. Accordingly, B cells belong to the humoral component of the adaptive immune system.
  • B cells can present antigens and secrete cytokines.
  • B cells express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a specific antigen, against which it will initiate an antibody response.
  • B cells include, for example, plasma cells and memory B cells.
  • plasma cell refers to all antibody secreting cells (ASCs) that are found in peripheral blood, bone marrow, tissues or body fluids, or are generated in vitro from B cells.
  • ASCs antibody secreting cells
  • B cells differentiate into plasma cells that produce antibody molecules closely modelled after the receptors of the precursor B cell.
  • T cell which usually occurs in germinal centers of secondary lymphoid organs like the spleen and lymph nodes
  • the activated B cell begins to differentiate into more specialized cells.
  • Germinal center B cells may differentiate into memory B cells or plasma cells.
  • Recently (e.g., 4-30 days) generated plasma cells may also be referred to as "plasma blasts".
  • plasma blast numbers peak usually at day 6 or 7 after immunization. After infection with acute viruses, such as influenza or dengue virus, the plasma blast numbers typically drop to baseline level for example within 2-3 weeks after the onset of disease.
  • memory B cells refers to a B cell sub-type, which is important in generating an accelerated and more robust antibody-mediated immune response in the case of re-infection (also known as a "secondary immune response"). Memory B cells usually went through a highly mutative and selective germinal center reaction.
  • a B cell (such as a plasma cell or a memory B cell) may be of any species.
  • the B cell is a mammalian B cell.
  • the B cell is a human B cell.
  • isolated B cell refers to a B cell, which is not part of a human or animal body.
  • an isolated B cell may be a plasma cell or a memory B cell.
  • An isolated B cell may be obtained, for example, from (isolated) peripheral blood mononuclear cells (PBMCs) by cell sorting.
  • markers for specific B cells may be used, for example, CD138 may be used to identify plasma cells and CD20, CD27 and/or CD40, may be used to identify memory B cells.
  • amplicon refers in general to a DNA or RNA molecule, which is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication.
  • PCR polymerase chain reactions
  • LCR ligase chain reactions
  • the term “amplification” refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon.
  • amplicon is used interchangeably with the terms "PCR product” and "amplified DNA”.
  • amplicon and “amplified DNA” refer to a population of (double stranded) DNA copies of a (single stranded) template DNA (e.g., single stranded first strand cDNA produced from a ribonucleic acid (RNA) template).
  • the degree of amplification, and thus the size of the produced population of DNA copies, will vary but in some instances is 5X amplification or more, where e.g. , “5X amplification or more” refers to the production of 5 or more dsDNAs from each single product nucleic acid molecule.
  • the degree of amplification achieved may exceed 5X amplification and may include, but is not limited to, e.g.
  • 10X amplification or more 100X amplification or more, 1000X amplification or more, 10,000X amplification or more, 100,000X amplification or more, 1,000,000X amplification or more, etc.
  • Measures of the degree of amplification need not necessarily be exact and may be based on the average or the approximate average of a sample, where e.g., 10X amplification refers to the production of 10 DNAs or approximately 10 DNAs on average from each single template DNA.
  • the degree of amplification and/or the size of the produced population of dsDNA copies may be indirectly quantified, e.g., where the amount of DNA present in the reaction following amplification is measured and the degree of amplification is extrapolated therefrom.
  • the degree of amplification and/or the size of the produced population of DNA copies may be directly quantified, e.g., by directly measuring the number of produced DNA copies, e.g., using quantitative sequencing methods or Bioanalyzer (e.g., Agilent Bioanalyzer).
  • Bioanalyzer e.g., Agilent Bioanalyzer
  • amplification will not refer to the production of single product nucleic acid, e.g., from a template nucleic acid.
  • Amplification will generally include the production of more than a small number of copies, e.g., more than a single copy, of DNA from a single template DNA, including but not limited to e.g., more than 2 copies, more than 3 copies, more than 4 copies, more than 5 copies, more than 10 copies, more than 15 copies, more than 20 copies, more than 30 copies, more than 100 copies, more than 1 ,000 copies, more than 10,000 copies, more than 100,000 copies, more than 1 ,000,000 copies, etc.
  • Amplification, according to the herein described methods may be exponential or approximately exponential.
  • oligonucleotide refers to an oligonucleotide used to prime an extension reaction (e.g., wherein one nucleic acid strand is "extended” starting from the primer, for example by use of a polymerase).
  • an "oligonucleotide” is a single-stranded multimer of nucleotides from 2 to 500 nucleotides, e.g. , 2 to 200 nucleotides. In some embodiments, oligonucleotides are 10 to 50 nucleotides in length, for example 20 to 40 nucleotides. Oligonucleotides may be synthetic or may be made enzymatically.
  • Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides or "RNA oligonucleotides”), deoxyribonucleotide monomers (i.e., may be oligodeoxyribonucleotides or "DNA oligonucleotides”) or combinations thereof. Oligonucleotides may be, for example, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up to 500 or more nucleotides in length.
  • cDNA refers to DNA having a nucleotide sequence, which is complementary to the nucleotide sequence of an RNA molecule, such as messenger RNA (mRNA).
  • cDNA may be synthesized from a single-stranded RNA (e.g., mRNA or microRNA) template.
  • a reverse transcription reaction may be performed, which may be catalyzed by the enzyme reverse transcriptase (RNA-dependent DNA polymerase).
  • cDNA derived from mRNA does usually not contain any introns and/or promoters.
  • nucleotide sequences are represented herein according to the lUPAC system and as outlined in: Nomenclature for incompletely specified bases in nucleic acid sequences. Recommendations 1984. Nomenclature Committee of the International Union of Biochemistry (NC-IUB). Proc Natl Acad Sci U S A. 1986;83(1):4-8. doi:10.1073/pnas.83.1 .4, which is incorporated herein by reference. Accordingly, nucleobases are represented by the first letters of their chemical names: [G]uanine, [C] osine, [A]denine, and [T]hymine.
  • W is A or T
  • S is C or G
  • M is A or C
  • K is G or T
  • R is A or G
  • Y is C or T
  • B is C, G, or T
  • D is A, G, or T
  • H is A, C, or T
  • V is A, C, or G
  • N is any nucleotide (A, C, G, or T).
  • the present invention provides a method for generation and cloning of a DNA molecule encoding a polypeptide of interest comprising the following steps: (1 ) providing mRNA encoding the polypeptide of interest;
  • step (1 ) (2) performing RACE reaction using the mRNA provided in step (1 ) as template to obtain a cDNA molecule encoding the polypeptide of interest;
  • step (3) amplifying the DNA molecule obtained in step (2) by PCR.
  • step (3) (4) cloning the amplicon obtained in step (3) into a vector by circular polymerase extension cloning (CPEC).
  • CPEC circular polymerase extension cloning
  • the present inventors have surprisingly found that generation and cloning of a DNA molecule using a combination of RACE (for DNA generation based on mRNA) and CPEC (for cloning of the DNA into a vector) is time and cost efficient and applicable for high-throughput approaches.
  • methods of the prior art are usually low-throughput, relatively expensive and/or laborious.
  • methods of the prior art usually require at least an additional PCR step.
  • the method of the invention allows for cloning of full-length antibodies, while prior art methods usually focus on the variable regions of antibodies only.
  • step (3) While in general cDNA obtained in RACE (step (2)) may be purified before it is amplified by PCR (step (3)), the present inventors have surprisingly found that purification of the cDNA obtained in step (2) is not required. Accordingly, purification of the obtained cDNA before step (3) may be omitted. In other words, no purification of the obtained cDNA may be performed between steps (2) and (3).
  • step (3) may follow directly upon step (2) (without any intermediate steps, in particular without any purification of the cDNA). That is, the cDNA molecule (or a solution or composition comprising the cDNA molecule), which is obtained in step (2) may be directly used as template for the PCR amplification of step (3).
  • amplified DNA obtained by PCR may be purified before CPEC (cloning; step (4))
  • the present inventors have surprisingly found that purification of the amplified DNA obtained in step (3) (amplicon) is not required for CPEC (step (4)). Accordingly, purification of the obtained PCR-amplified DNA (amplicon) before step (4) may be omitted. In other words, no purification of the obtained amplified DNA molecule (amplicon) may be performed between steps (3) and (4).
  • step (4) may follow directly upon step (3) (without any intermediate steps, in particular without any purification of the amplicon). That is, the amplicon (or a solution or composition comprising the amplicon), which is obtained in step (3) may be directly used for cloning by circular polymerase extension cloning (CPEC).
  • CPEC circular polymerase extension cloning
  • purification of DNA may be omitted after RACE (step (2); i.e. the obtained cDNA may be used directly (without purification) for PCR amplification in step (3)) and/or after PCR amplification (step (3); i.e. the obtained amplicon may be used directly (without purification) for CPEC in step (4)).
  • no purification of DNA is performed after RACE (step (2); i.e. the obtained cDNA is used directly (without purification) for PCR amplification in step (3)) and after PCR amplification (step (3); i.e. the obtained amplicon is used directly (without purification) for CPEC in step (4)).
  • no purification of the mRNA encoding the polypeptide provided in step (1), of the cDNA obtained in step (2) and/or of the amplified DNA molecule obtained in step (3) is performed.
  • no purification of the mRNA encoding the polypeptide provided in step (1 ), of the cDNA obtained in step (2) and of the amplified DNA molecule obtained in step (3) may be performed.
  • no purification may be performed on the mRNA provided in step (1 ) (for example, after cell lysis), no purification of DNA may be performed after RACE (step (2)) and after PCR amplification no purification of the amplicon obtained in step (3) may be performed.
  • step (2) may follow directly upon step (1)
  • step (3) may follow directly upon step (2)
  • step (4) may follow directly upon step (3). That is, the mRNA provided in step (1) may be used directly (without any purification, e.g. after cell lysis) in step (2) (RACE); the cDNA obtained in step (2) may be used directly (without purification) for PCR amplification in step (3); and the amplicon obtained in step (3) may be used directly (without purification) for CPEC in step (4).
  • Step (7) Providing mRNA of interest
  • mRNA messenger RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • mRNA may be obtained from single cells or from multiple cells, for example from cell cultures (e.g., cell lines) or tissues.
  • Cells single cells or multiple cells, e.g. tissue
  • PBMCs peripheral blood mononuclear cells
  • Various protocols for the isolation (extraction) of mRNA from cells are known in the art and kits for (m)RNA isolation from cells are commercially available (e.g., RNeasy Kit (Qiagen)).
  • the mRNA in step (1) is provided by cell lysis.
  • a lysis buffer may be used.
  • Cells may be contacted with a lysis buffer to obtain a cell lysate comprising mRNA encoding a polypeptide of interest.
  • a denaturing lysis buffer may be used.
  • RNA lysis buffers are commercially available, for example Buffer RLT (Qiagen) or RLA RNA Lysis Buffer (Promega).
  • the lysis buffer contains a (non-ionic) detergent, such as Triton X-100 or Igepal CA-630.
  • the lysis buffer may contain an RNase inhibitor.
  • the lysis buffer may comprise Triton X- 100.
  • the concentration of Triton X-100 in the lysis buffer does not exceed 0.2%, more preferably the concentration of Triton X-100 in the lysis buffer does not exceed 0.15%, even more preferably the concentration of Triton X-100 in the lysis buffer does not exceed 0.1 %, still more preferably the concentration of Triton X-100 in the lysis buffer does not exceed 0.5%, and most preferably the concentration of Triton X-100 in the lysis buffer does not exceed 0.03%.
  • the lysis buffer may comprise 0.02% Triton X-100.
  • the lysis buffer may comprise an RNase inhibitor, such as RiboLock RNase Inhibitor (ThermoFisher; e.g. 1 U/ml).
  • the mRNA provided in step (1 ) is purified.
  • Kits and reagents for mRNA purification are commercially available, for example TurboCapture mRNA Kit (Qiagen).
  • the mRNA provided in step (1) is not purified. Accordingly, cell lysates comprising mRNA encoding a polypeptide of interest may be directly used for the RACE reaction (step (2)).
  • a polypeptide of interest may be expressed by any cell (ubiquitously) or by one or more specific cell type(s) only.
  • mRNA encoding the polypeptide of interest may be found in cells (a single cell or multiple cells, such as cell culture or tissue) expressing the polypeptide of interest (or at least transcribing the respective gene). If the starting material, e.g. a tissue, contains various cell types and only some cells may potentially contain mRNA encoding the polypeptide of interest, it may be useful to select specifically cells containing mRNA encoding the polypeptide of interest for cell lysis/(m)RNA isolation.
  • the method of the invention may include a step of identifying cells, which (potentially) contain mRNA encoding the polypeptide of interest. This may be achieved by various methods, for example by using cell markers (or combinations of cell markers), such as CD molecules.
  • Cells, which (potentially) contain mRNA encoding the polypeptide of interest may be selected (e.g., separated from other cells) for cell lysis or (m)RNA isolation to obtain mRNA encoding the polypeptide of interest.
  • cells may be sorted to obtain cells containing an mRNA encoding the polypeptide of interest.
  • flow cytometry such as FACS (fluorescence- activated cell sorting) may be used.
  • memory B cells and/or plasma cells may be isolated from the blood, in particular from peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • FACS FACS based on CD markers (for example CD19 and CD27 for memory B cells; CD19, CD27hi and CD38 for plasma cells).
  • cells may be directly sorted into plates or tubes containing lysis buffer for cell lysis.
  • the obtained cell lysate may then be used directly for the RACE reaction (step (2)).
  • mRNA may also be obtained by in vitro RNA synthesis, such as in vitro transcription, wherein RNA polymerase is used to produce mRNA based on a DNA template.
  • Kits and systems for in vitro transcription are commercially available (for example “MAXIscriptTM SP6 Transcription Kit”, “MAXIscriptTM T7 Transcription Kit”, “MAXIscriptTM T3 Transcription Kit” (all ThermoFisher); “HiScribe T7 High Yield RNA Synthesis Kit” (New England BioLabs); “RiboMAXTM Large Scale RNA Production Systems” (Promega)).
  • Rapid amplification of cDNA ends is a technique used in molecular biology to obtain the full length sequence of an mRNA molecule.
  • RACE results in the production of a cDNA copy of the full-length mRNA, produced through reverse transcription.
  • Full-length mRNA includes, in addition to the coding sequence, 5' and 3' untranslated regions (UTRs) and the poly-A tail at the 3' end of the mRNA.
  • UTRs 5' and 3' untranslated regions
  • RACE reaction refer to a reverse transcription (reaction), wherein a full-length mRNA is transcribed into a cDNA.
  • RACE can be used to amplify unknown 5' (5 '-RACE) or 3' (3 '-RACE) parts of RNA molecules where a portion of the RNA sequence is known and targeted by a gene-specific primer.
  • the RACE reaction in step (2) is performed on the mRNA provided in step (1).
  • the RACE reaction in step (2) may be performed on mRNA obtained from a single cell. Thereby, mRNA from a single cell is transcribed into cDNA.
  • the RACE reaction in step (2) may be performed on mRNA obtained from multiple cells, for example, from a cell culture (e.g., a cell line) or from cells sorted as described above. While the mRNA provided in step (1) may be purified before the RACE reaction, the RACE reaction may also be performed directly on a cell lysate comprising the mRNA encoding the polypeptide of interest. Thereby, the length of the protocol and operator time are reduced.
  • RT reverse transcription
  • a primer and/or dNTPs are added in step (1 ), e.g. during cell lysis.
  • the reverse transcriptase and a primer are added in step (2), for example in a reaction mix. If various components are added at the same time, a reaction mix containing the components may be prepared.
  • the reaction mixture conditions sufficient for reverse transcriptase-mediated extension of a hybridized primer include bringing the reaction mixture to a temperature ranging from 4°C to 72° C, such as from 16°C to 70°C, e.g. , 37°C to 50°C, such as 40°C to 45°C, including 42° C.
  • the reverse transcriptase may be thermo-sensitive, i.e. not thermostable. A thermo- sensitive reverse transcriptase may become inactive at a temperature above its active temperature range.
  • thermos-sensitive reverse transcriptase may become inactive or demonstrate significantly reduced activity after being exposed to temperatures of 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher or 95°C or higher. Accordingly, such temperatures may be used to inactivate the enzyme in order to end the reverse transcription reaction.
  • reverse transcription may be performed at about 42°C.
  • the reverse transcription may be ended by enzyme inactivation at about 72°C.
  • the reverse transcriptase may be combined into the reaction mixture such that the final concentration of the reverse transcriptase is sufficient to produce a desired amount of the RT reaction product, e.g., a desired amount of cDNA.
  • the reverse transcriptase is present in the reaction mixture at a final concentration of from 0.1 to 200 units/mL (U/mL), such as from 0.5 to 100 U/mL, such as from 1 to 50 U/mL, including from 5 to 25 U/mL, e.g., 15 to 20 U/mL.
  • U/mL units/mL
  • the reverse transcriptase is present in the reaction mixture at a final concentration of from 0.1 to 200 units/mL (U/mL), such as from 0.5 to 100 U/mL, such as from 1 to 50 U/mL, including from 5 to 25 U/mL, e.g., 15 to 20 U/mL.
  • dNTPs may be added to the mRNA template.
  • each of the four naturally-occurring dNTPs (dATP, dGTP, dCTP and dTTP) is added to the reaction mixture.
  • dATP, dGTP, dCTP and dTTP may be added to the reaction mixture such that the final concentration of each dNTP is from 0.01 to 100 mM, such as from 0.1 to 10 mM, including 0.5 to 5 mM (e.g., 1 mM).
  • the primer used in the RACE reaction may be an oligo-(dT) primer.
  • An oligo-(dT) primer is a (DNA) oligonucleotide containing a stretch of thymidine (T) nucleotides (desoxythymidine), which is capable of binding to the poly-A-tail of an mRNA molecule.
  • T thymidine
  • deoxythymidine thymidine
  • An oligo-(dT) primer may comprise a continuous stretch of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more thymidine (T) nucleotides.
  • the oligo-(dT) primer may comprise up to 5 additional nucleotides at its 3' end, for example including the sequence "VN", wherein N may be any nucleotide and V may be either A, C or G.
  • the oligo-(dT) primer may comprise any additional nucleotide sequence (or no additional nucleotide(s)).
  • the oligo-(dT) primer may be represented by the following general formula (I):
  • [X] represents any (DNA) nucleotide or any (DNA) nucleotide sequence
  • T is (desoxy)thymidine n may be an integer from 5 to 50, in particular from 10 to 30, e.g. from 15 to 25;
  • V is either A, C or G; and N may be any (DNA) nucleotide.
  • the oligo-(dT) primer comprises the nucleotide sequence as set forth in SEQ ID NO: 1 : oligo dT primer:
  • the oligo-(dT) primer may comprise any additional nucleotide sequence (or no additional nucleotide(s)) at its 5' end.
  • the oligo-(dT) primer may comprise a predetermined "universal" sequence, as described below in the context of the TSO, at its 5' end. Such a predetermined ("universal") sequence may be useful in later steps, in particular if the nucleotide sequence encoding the polypeptide of interest is unknown.
  • the oligo-(dT) primer may be added to the mRNA encoding the polypeptide of interest in step (2) of the method of the invention.
  • the mRNA may be provided in step (1) by cell lysis and an oligo-(dT) primer and/or dNTPs may be added before or during cell lysis.
  • the oligo-(dT) primer may be added to the mRNA before addition of reverse transcriptase and/or a template-switching oligonucleotide (TSO).
  • RNA encoding polypeptide of interest In addition to the template (mRNA encoding polypeptide of interest), the enzyme reverse transcriptase, a primer (such as an oligo-(dT) primer), and, optionally, dNTPs, further components may be useful in a RACE reaction. Those components may be included in a reaction mixture or added separately to the template. Examples of such components include buffer components that establish an appropriate pH, salt concentration (e.g., KCI concentration), metal cofactor concentration (e.g., Mg 2+ or Mn 2+ concentration), for the reverse transcription reaction to occur.
  • buffer components that establish an appropriate pH, salt concentration (e.g., KCI concentration), metal cofactor concentration (e.g., Mg 2+ or Mn 2+ concentration), for the reverse transcription reaction to occur.
  • nuclease inhibitors e.g., an RNase inhibitor and/or a DNase inhibitor
  • additives for facilitating amplification/replication of GC rich sequences e.g., GC-MeltTM reagent (Clontech Laboratories, Inc.
  • betaine e.g., betaine, DMSO, ethylene glycol, 1 ,2-propanediol, or combinations thereof
  • molecular crowding agents e.g., polyethylene glycol, or the like
  • enzyme-stabilizing components e.g., DTT present at a final concentration ranging from 1 to 10 mM (e.g., 5 mM)
  • any other reaction mixture components useful for facilitating reverse transcriptase reactions.
  • the following components may be added for reverse transcriptase reaction to the template (i.e., to the mRNA encoding the polypeptide of interest), for example in a reaction mixture, in addition to the enzyme reverse transcriptase, a primer (such as an oligo-(dT) primer), and, optionally, dNTPs:
  • - buffer such as a buffer appropriate for the reverse transcriptase used (e.g., as supplied by the manufacturer of the reverse transcriptase);
  • nuclease inhibitor such as an RNase inhibitor
  • enzyme-stabilizing component such as DTT (dithtoth reitol);
  • water such as DNase-free and/or RNase-free water.
  • the only further component added to the reaction mix or to the template is a template-switching oligonucleotide (TSO). If a reaction mix is prepared, the reverse transcriptase may be included in the reaction mix. In some embodiments, no MnCk is added/used in step (2).
  • TSO template-switching oligonucleotide
  • the following components may be added to the mRNA encoding the polypeptide of interest (template), for example in a reaction mix: a reverse transcriptase, an oligo-(dT) primer, a template-switching oligonucleotide (TSO), and optionally, dNTPs.
  • template for example in a reaction mix: a reverse transcriptase, an oligo-(dT) primer, a template-switching oligonucleotide (TSO), and optionally, dNTPs.
  • RNA transcriptase-RACE reverse transcriptase-RACE
  • reverse transcription of the template mRNA starts with an oligo(dT) primer, as described above, which anneals to the poly-A-tail at the 3' end of the mRNA.
  • the terminal transferase activity of the reverse transcriptase adds a few additional nucleotides (mostly deoxycytidine) to the 3' end of the newly synthesized cDNA strand.
  • TSO template switching oligonucleotide
  • TSO template switching oligonucleotide
  • the deoxycytidine nucleotides at the 3' end of the cDNA strand function as a TSO-anchoring site.
  • the reverse transcriptase Upon base pairing between the TSO and the appended deoxycytidine stretch, the reverse transcriptase "switches" template strands, from cellular RNA to the TSO, and continues replication to the 5' end of the TSO. Thereby, the resulting cDNA contains the complete 5' end of the transcript, and a "universal" predetermined sequence (complementary to the "universal" predetermined DNA-sequence included in the TSO).
  • template switching oligonucleotide refers to an oligonucleotide utilized in a template switching reaction, including the production of a cDNA from the template mRNA.
  • production of the cDNA may utilize template switching and the ability of certain reverse transcriptases to "template switch" as described above.
  • the TSO may be added to the mRNA template (e.g., included in a reaction mix) at a concentration sufficient to readily permit template switching of the reverse transcriptase from the mRNA template to the TSO.
  • the template switching oligonucleotide may be added (e.g., to the reaction mixture) at a final concentration of from 0.01 to 100 mM, such as from 0.1 to 10 mM, such as from 0.5 to 5 mM, including 0.75 to 2 mM (e.g., 1 mM).
  • the template switching oligonucleotide may include one or more nucleotides (or analogs thereof) that are modified or otherwise non-naturally occurring.
  • the template switching oligonucleotide may include one or more nucleotide analogs (e.g., LNA, FANA, 2'- O-Me RNA, 2'-fluoro RNA, or the like), linkage modifications (e.g., phosphorothioates, 3 '-3 ' and 5' - 5' reversed linkages), 5' and/or 3' end modifications (e.g.
  • isomeric nucleotides may be incorporated into the TSO, e.g. to improve cDNA yield.
  • modified bases such as iso-dC and iso-dG, may be appended to the 5' end of the TSO.
  • the TSO includes a modification that prevents the reverse transcriptase from switching from the TSO to a different template nucleic acid after synthesizing the complement of the 5' end of the TSO (e.g., a predetermined "universal" nucleotide sequence of the TSO).
  • Useful modifications include, but are not limited to, an abasic lesion (e.g., a tetrahydrofuran derivative), a nucleotide adduct, an iso-nucleotide base (e.g., isocytosine, isoguanine), and any combination thereof.
  • the TSO comprises a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • a locked nucleic acid (LNA) is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge "locks" the ribose in the V-endo (North) conformation.
  • LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired.
  • LNA nucleotides hybridize with DNA or RNA according to Watson-Crick base-pairing rules. The locked ribose conformation enhances base stacking and backbone pre-organization, thereby increasing the hybridization properties (melting temperature) of an oligonucleotide.
  • the reverse transcriptase (such as MMLV RT) may have terminal transferase activity, i.e. the reverse transcriptase may be capable of catalyzing the addition of deoxyribonucleotides to the 3' hydroxyl terminus of a RNA or DNA molecule. Accordingly, when the reverse transcriptase reaches the 5' end of the mRNA template, the reverse transcriptase may be capable of incorporating one or more additional nucleotides at the 3' end of the nascent cDNA strand not encoded by the mRNA template.
  • the reverse transcriptase when the reverse transcriptase has terminal transferase activity, the reverse transcriptase may be capable of incorporating 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additional nucleotides at the 3' end of the nascent cDNA strand. All of the nucleotides may be the same (e.g., creating a homonucleotide stretch at the 3' end of the nascent strand) or one or more of the nucleotides may be different from the other(s) (e.g., creating a heteronucleotide stretch at the 3' end of the nascent cDNA strand).
  • the terminal transferase activity of the reverse transcriptase results in the addition of a homonucleotide stretch of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the same nucleotides (e.g., all dCTP, all dGTP, all dATP, or all dTTP).
  • the reverse transcriptase may be an MMLV reverse transcriptase (MMLV RT).
  • MMLV RT incorporates additional nucleotides (predominantly deoxycytidine (dC; dCTP), e.g., three dCTPs) at the 3' end of the nascent cDNA strand.
  • a homonucleotide stretch (e.g., a homo-trinucleotide, such as C-C-C) may be added to the 3' end of the nascent cDNA strand.
  • additional nucleotides may be useful for enabling hybridization between a 3' hybridization domain of a TSO and the 3' end of the nascent cDNA strand, e.g., to facilitate template switching by the reverse transcriptase from the template mRNA to the TSO.
  • the TSO For hybridizing with these additional nucleotides added by the reverse transcriptase to the 3' end of the nascent cDNA strand, the TSO comprises at its 3' end a hybridization domain, which includes - at its 3' end - a stretch of nucleotides, which are capable of hybridizing with the additional nucleotides added by the reverse transcriptase to the 3' end of the nascent cDNA strand.
  • the nucleotides at the 3' end of the TSO are ribonucleotides (i.e., the 3' most nucleotides), while the other nucleotides of the TSO are deoxyribonucleotides.
  • the TSO may comprise DNA and RNA nucleotides.
  • the 2, 3, 4, or 5 (for example 3) nucleotides at the 3' end of the TSO are ribonucleotides, while all other nucleotides of the TSO (which are not located at the 3' end) are deoxyribonucleotides.
  • the TSO may comprise at its 3' end a homonucleotide stretch, for example a homo-trinucleotide, such as G-G-G, in particular rG-rG-rG (with "rG” representing a riboguanosine), which may be complementary to additional nucleotides of the 3' end of the nascent cDNA strand.
  • a homo-trinucleotide such as G-G-G, in particular rG-rG-rG (with "rG” representing a riboguanosine)
  • the TSO may comprise at its 3' end a hetero- trinucleotide comprising a cytosine nucleotide and a guanine nucleotide (e.g., an r(C/G)3 oligonucleotide), which may be complementary to the 3' end of the nascent cDNA strand.
  • a hetero- trinucleotide comprising a cytosine nucleotide and a guanine nucleotide (e.g., an r(C/G)3 oligonucleotide), which may be complementary to the 3' end of the nascent cDNA strand.
  • the TSO is a DNA oligo sequence that carries 3 riboguanosines (rGrGrG) at its 3' end.
  • rGrGrG 3 riboguanosines
  • the complementarity between these consecutive rG bases and the 3' dC extension of the cDNA molecule empowers the subsequent template switching.
  • the 3' most rG may be replaced with a locked nucleic acid base (LNA) as described above.
  • the 3' hybridization domain of the TSO may vary in length, and in some instances ranges from 2 to 10 nucleotides in length, such as 3 to 7 nucleotides in length.
  • the TSO comprises a predetermined ("universal") nucleotide sequence, which can be arbitrarily selected and which can be useful in downstream steps.
  • the predetermined (“universal") nucleotide sequence may serve as template to incorporate (into the nascent cDNA strand) a primer binding site for a PCR primer in the PCR of step (3).
  • the predetermined (“universal") nucleotide sequence of the TSO enables its amplification based on the primer binding site, which is based on the predetermined nucleotide sequence.
  • the predetermined (“universal") sequence may be selected such that it provides optimal conditions for downstream applications, such as the PCR in step (3) or the CPEC cloning in step (4).
  • the predetermined ("universal") nucleotide sequence is a DNA sequence.
  • the predetermined ("universal") nucleotide sequence has a length of 15 to 30 nucleotides, for example 16 - 25 nucleotides, e.g. 20 - 23 nucleotides.
  • the predetermined ("universal") nucleotide sequence is located in the 5' portion, e.g. at the 5' end, of the TSO.
  • the TSO may comprise a nucleotide sequence as set forth in SEQ ID NO: 2: TSO (LNAcac primer): (SEQ ID NO: 2)
  • template switching oligonucleotides are described in WO 97/24455, the disclosure of which is herein incorporated by reference.
  • the only primers added and/or used in step (2) are an oligo-(dT) primer and a TSO as described above.
  • Reverse transcriptases capable of template-switching include, but are not limited to, retroviral reverse transcriptase, retrotransposon reverse transcriptase, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptase, and mutants, variants derivatives, or functional fragments thereof, e.g., RNase H minus or RNase H reduced enzymes.
  • the reverse transcriptase may be a Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT) or a Bombyx rnori reverse transcriptase (e.g., Bombyx mori R2 non- LTR element reverse transcriptase).
  • the reverse transcriptase is a Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT), for example an engineered Moloney Murine Leukemia Virus reverse transcriptase, such as SuperscriptTM II reverse transcriptase (InvitrogenTM; ThermoFisher), which is an engineered version of MMLV RT with reduced RNase H activity and increased thermal stability.
  • MMLV RT Moloney Murine Leukemia Virus reverse transcriptase
  • SuperscriptTM II reverse transcriptase InvitrogenTM; ThermoFisher
  • mRNA encoding a polypeptide of interest may be obtained by cell lysis, wherein the oligo-(dT) primer and dNTPs are added to the lysis buffer (or to a mixture containing the lysis buffer).
  • a reaction mix may be prepared, which comprises SuperscriptTM II reverse transcriptase (InvitrogenTM; ThermoFisher; e.g. at a final concentration of about 100 U), RNase inhibitor (e.g., at a final concentration of about 10 U), SuperscriptTM II first strand buffer (5x; InvitrogenTM; ThermoFisher; e.g.
  • DTT e.g., at a final concentration of about 5 mM
  • betaine e.g., at a final concentration of about 1 M
  • MgCl 2 e.g., at a final concentration of about 6 mM
  • TSO e.g., at a final concentration of about 1 mM
  • nuclease-free water to adjust the total volume.
  • a reaction mix may be prepared, which comprises SuperscriptTM II reverse transcriptase (InvitrogenTM; ThermoFisher; e.g. at a final concentration of about 100 U), RNase inhibitor (e.g., about 0.4 U/mL), SuperscriptTM II first strand buffer (5x; InvitrogenTM; ThermoFisher; e.g.
  • reaction mix may then be added to the mRNA obtained in step (1); for example the reaction mix may be added to the ceil lysate comprising the mRNA encoding a polypeptide of interest.
  • the reaction mix may be directly added to the cell lysate (samples containing the cell lysates) as obtained in step (1) (i.e., without purification of the mRNA).
  • the samples may then be incubated, for example in a thermal cycler, at about 42°C (for example for 30 min - 3 h, such as 1 h - 2 h, e.g. about 90 min).
  • the temperature may be increased to about 72 °C (e.g., for at least 5 min or 10 min, such as at least 15 min).
  • additional cycles e.g.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, such as 10) may optionally be performed, wherein the temperature is increased to about 50°C for about 2 min and, thereafter, decreased to 42°C for about 2 min. These additional cycles are not required for a successful reaction, but may give a marginal increment in yield.
  • step (3) of the inventive method for generation and cloning of a DNA molecule encoding a polypeptide of interest the cDNA molecule obtained by the reverse transcription in step (2) is amplified by polymerase chain reaction (PCR).
  • step (2) may be purified before it is amplified by the PCR in step (3) (for example, the cDNA may be separated from other components contained in the reaction mix of step (2) and/or in the cell lysate, such as the template RNA used in the reverse transcription of step (2)), purification of the cDNA obtained in step (2) is not essential for a successful PCR in step (3) (or the method of the invention in general). Rather, performing the PCR in step (3) directly (without any purification) on the reaction product of step (2) may save time and costs. Accordingly, the PCR in step (3) may be performed directly (without any purification) on the reaction product of step (2), which contains the cDNA.
  • PCR refers to a method of obtaining many copies of a DNA molecule (or a segment thereof). Thereby, the copies of the DNA molecule (or the segment thereof) are exponentially amplified to obtain large numbers of that particular DNA molecule or segment thereof.
  • PCR typically amplifies a specific region of a DNA molecule.
  • a PCR may comprise of a series of 10 - 50, such as 20 - 40 repeated temperature changes, also referred to as "thermal cycles", with each cycle usually comprising two or three discrete temperature steps.
  • the thermal cycles may be preceded by a single temperature step at a high temperature (e.g., >90 °C, such as about 94°C or about 98°C).
  • the thermal cycles may be followed by a "hold” at the end for final product extension (e.g., at about 72°C) or brief storage.
  • the temperatures used and the length of time they are applied in each cycle may depend on various parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature (T m ) of the primers.
  • the PCR (in step (3)) comprises the following sub-steps:
  • Initialization comprises heating the reaction chamber to a temperature of more than 90°C, such as 94-96 °C (or 98 °C), which may be held, for example, for 30 seconds - 10 minutes, for example 1 or 2 minutes, e.g., at 98°C.
  • Denaturation comprises heating the reaction chamber to more than 90°C, such as 94- 98 °C, e.g. for 5 - 50 seconds (such as 10 or 20 seconds). Thereby, DNA melting, or denaturation, of the double-stranded DNA template may occur.
  • Annealing comprises lowering the reaction temperature to the so-called “annealing temperature”, e.g. for 5 - 50 seconds (such as 20 or 30 seconds). Thereby, annealing of the primers to (each of) the single-stranded DNA templates is allowed and the polymerase may bind to the primer-template hybrid and begin DNA formation.
  • annealing temperature e.g. for 5 - 50 seconds (such as 20 or 30 seconds).
  • slow ramp annealing may be used, wherein the temperature is slowly lowered (following a "ramp") to achieve the annealing temperature (e.g., at a rate of 0.1 °C/sec, e.g. from 70°C to the annealing temperature).
  • the annealing temperature may vary depending on the primer sequences. Accordingly, a primer may be designed such that an envisaged annealing temperature can be achieved by methods well-known in the art.
  • Various primer design tools are available, for example, Primer-BLAST (Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012).
  • Primer-BLAST A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 13:134); PerlPrimer (Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR.
  • a typical annealing temperature may be about 3- 5 °C below the T m of the primers used.
  • the annealing temperature may be any temperature from 50°C to about 72 °C In some embodiments, the annealing temperature is higher than 60°C (e.g., between 60°C and 72°C), for example higher than 65°C (e.g., between 65°C and 72°C), such as about 68°C.
  • the DNA polymerase typically synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that are complementary to the template, for example in the 5'-to-3' direction.
  • the temperature at this step usually depends on the DNA polymerase used; for example for the DNA polymerase of Taq (Thermus aquaticus) a temperature from 70°C - 80°C, such as about 72°C may be used.
  • the time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. As a rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute; for example 10 - 20 s per kB may be used.
  • Final elongation This sub-step is optional and may ensure that any remaining single- stranded DNA is fully elongated.
  • Final elongation may be performed at a temperature of 70-74 °C, for example at about 72°C. This temperature may be applied, for example, for 1-15 minutes after the last PCR cycle, e.g. for about 2 or 5 minutes.
  • This sub-step is optional and may be employed for short-term storage of the PCR products.
  • the reaction chamber is cooled to 4-15 °C (e.g., about 4°C) for an indefinite time.
  • sub-steps (a), (e) and (f) are usually single steps (i.e., they are performed only once (if any) in a PCR)
  • sub-steps (b), (c) and (d) constitute a single "cycle" and are usually repeated. Multiple cycles may be required to amplify the DNA target to large numbers (e.g., millions) of copies. Under optimal conditions (i.e., if there are no limitations due to limiting substrates or reagents), at each extension/elongation step, the number of DNA target sequences is doubled.
  • a PCR reaction may comprise incubation at 98°C (sub-step (a), e.g.
  • PCR protocol may be particularly useful for amplification of immunoglobulin genes, for example with the exemplified PCR primers described elsewhere herein.
  • the PCR may be carried out in a total volume of 10-200 mL in small reaction tubes (for example, 0.2-0.5 mL volumes) in a thermal cycler.
  • the thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.
  • a DNA template that contains the DNA target region to amplify
  • an enzyme that polymerizes new DNA strands such as a DNA polymerase (for example heat-resistant Taq polymerase); two DNA primers that are complementary to the 3' ends of each of the sense and anti-sense strands of the DNA target; deoxynucleoside triphosphates (dNTPs also referred to as "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups); a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase; and, optionally, cations, such as magnesium (Mg) or manganese (Mn) ions (for example Mg 2+ ).
  • Mg magnesium
  • Mn manganese
  • reaction mix reaction mixture
  • reaction mixture may also include water (e.g. DNase RNase free water) to adjust the volume. Adequate portions of the reaction mix may be added to various template DNA samples to be amplified with the same reaction mix.
  • the DNA polymerase is a high-fidelity polymerase.
  • the fidelity of a DNA polymerase is the result of accurate replication of a desired template.
  • High- fidelity (HiFi) DNA polymerases couple low misincorporation rates with proofreading activity to obtain a reliable replication of the target DNA of interest, i.e. to avoid undesired PCR- induced mutations.
  • Various high fidelity polymerases are known in the art.
  • the DNA polymerase may be Q5 ® DNA polymerase, such as Q5 F-lot Start High-Fidelity DNA Polymerase.
  • a reaction mix may contain a DNA polymerase, such as Q5 Hot Start High- Fidelity DNA Polymerase (New England Biolabs; for example about 0.4U); suitable buffer (for example as supplied by the manufacturer of the DNA polymerase; such as 1 x Q5 reaction buffer), dNTPs (for example about 200 mM), at least two primers; water (such as DNase RNase free water) and, optionally, 0.5x Q5 High GC Enhancer (New England Biolabs).
  • a DNA polymerase such as Q5 Hot Start High- Fidelity DNA Polymerase (New England Biolabs; for example about 0.4U)
  • suitable buffer for example as supplied by the manufacturer of the DNA polymerase; such as 1 x Q5 reaction buffer
  • dNTPs for example about 200 mM
  • at least two primers for example about 200 mM
  • water such as DNase RNase free water
  • 0.5x Q5 High GC Enhancer New England Biolabs
  • several distinct primers may be included, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more primers.
  • two distinct DNA target regions are to be amplified in the same PCR approach four distinct primers (which are able to hybridize with four distinct DNA sequences; one primer pair for each of the two DNA target regions) may be used.
  • one primer is able to hybridize with both DNA target regions (e.g., the forward or the reverse primer)
  • three primers may be sufficient for amplification of two distinct DNA target regions.
  • a "primer pair” refers to (exactly) two primers, which are able to hybridize with the DNA template such that the DNA target sequence is amplified in the PCR. Accordingly, a primer pair typically hybridizes with the "ends" of the DNA target sequence comprised in the DNA template.
  • a primer pair typically consists of a forward primer and a reverse primer.
  • primer design tools are available, for example, Primer-BLAST (Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012). Primer-BLAST: A tool to design target- specific primers for polymerase chain reaction. BMC Bioinformatics.
  • PerlPrimer Marshall OJ. PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 2004 20(15):2471 -2472); or Primer3Plus (Schgasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and Rozen SG. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012 Aug 1;40(15):e115). In the method of the present invention even unknown nucleotide sequences can be obtained, amplified and cloned.
  • any mRNA may be transcribed into a cDNA by use of the oligo-(dT) primer as described above.
  • "universal" primers may be used for PCR amplification in step (3) (which are capable of hybridizing to the predetermined ("universal") sequences included in the cDNA due to the oligo-(dT) primer and the TSO).
  • one of the PCR primers may comprise or consist of a nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO.
  • a "respective" PCR primer may comprise (or consist of) a nucleotide sequence, which is comprised in the TSO sequence, such as SEQ ID NO: 3 (SEQ ID NO: 2 comprises SEQ ID NO: 3): (SEQ ID NO: 3)
  • At least a portion of the nucleotide sequence encoding the polypeptide of interest may be known.
  • one or both of the primers of the primer pair used for the PCR (step (3)) may be capable of hybridizing to the known nucleotide sequence.
  • the polypeptide of interest is an immunoglobulin (or a chain thereof)
  • the nucleotide sequence of the constant region may be known, while the nucleotide sequence of the variable region may be unknown.
  • one primer of the primer pair may comprise or consist of a nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO (as described above), while the other primer of the primer pair may hybridize with a specific known sequence.
  • one primer of the primer pair may comprise or consist of a nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO (as described above), while the other primer of the primer pair may comprise a nucleotide sequence, which is specific for an immunoglobulin constant region or a portion thereof, such as the "end" of the constant region.
  • end in the context of an immunoglobulin/antibody constant region refers to the most C-terminal 20 amino acids of a constant region of an antibody. Accordingly, an "end" of an immunoglobulin/antibody constant region (or of a complete immunoglobulin/antibody heavy or light chain) is intended to refer to the C-terminus of an immunoglobulin constant region.
  • an "end” (of an immunoglobulin/antibody constant region or of a complete immunoglobulin/antibody heavy or light chain) may refer to the at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 most C-terminal amino acids of an immunoglobulin constant region or, in more general, of an immunoglobulin (heavy or light) chain.
  • the forward primer and the (corresponding) reverse primer (of the same primer pair) used in the PCR reaction of step (3) each comprise a nucleotide sequence exhibiting very similar melting temperatures (T m ).
  • the melting temperature (T m ) refers to the temperature at which half of the primer-target nucleic acid duplexes remain hybridized and half of the duplexes dissociate into single strands. While it may be referred herein to the "melting temperature (T m ) of a primer", it is understood that any such expression usually means the temperature at which half of the primer-target nucleic acid duplexes remain hybridized and half of the duplexes dissociate into single strands (even if the target/template nucleic acid is not specifically mentioned).
  • the T m of a primer-target nucleic acid duplex may be experimentally determined or predicted as described in Sambrook and Russell, 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., Chapter 10, which is incorporated herein by reference.
  • Other more advanced models that depend on various parameters may also be used to predict the
  • tools for primer design also calculate/provide the melting temperature (T m ) of a designed primer, such that a primer can be designed such that it exhibits a certain T m .
  • tools for calculating the melting temperature (T m ) are known to the skilled person and include, for example, New England BioLabs Tm Calculator (URL: https://tmcalculator.neb. com/#!/main); Melting Temperature (Tm) Calculation (URL: http://www.biophp.org/minitools/melting_temperature/demo.php); Tm Tool (URL: https://www.dna.utah.edu/tm/; Owczarzy R. et al. (2008) Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cation. Biochemistry 47(19): 5336-5353).
  • An amplicon comprising - in both of its end regions (e.g., at both of its ends) - nucleotide sequences exhibiting very similar melting temperatures (T m ) may be useful for CPEC cloning in step (4).
  • very similar melting temperatures means that the difference between the melting temperatures of both primers (of the same primer pair) does not exceed 5°C, 4°C, 3°C, 2°C or 1 °C. As a general rule, the lower the difference between the melting temperatures of both primers (of the same primer pair), the better.
  • the melting temperature of the nucleotide sequences exhibiting very similar melting temperatures may be between 60°C and 72°C, for example about 61 °C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, or 71 °C, such as about 65°C or 68°C.
  • the higher the melting temperature the better.
  • the melting temperature may not exceed 72°C.
  • the nucleotide sequence exhibiting very similar melting temperatures may have a length of 15 - 35 nucleotides. In some embodiments, the nucleotide sequence exhibiting very similar melting temperatures has a length of 16 - 30, such as 18 - 25 nucleotides, e.g. 20 - 23 nucleotides.
  • one primer of a primer pair may comprise or consist of a nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO and the other primer of the primer pair may comprise a nucleotide sequence exhibiting very similar melting temperatures (T m ) as the nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO.
  • the other primer may also contain a predetermined "universal" sequence or a sequence specific for the nucleotide sequence encoding the polypeptide of interest as described above.
  • one primer of the primer pair may comprise or consist of a nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO (as described above), while the other primer of the primer pair may comprise (i) a nucleotide sequence, which is specific for an immunoglobulin constant region (or a portion thereof, such as the "end" of the constant region) and (ii) a nucleotide sequence exhibiting very similar melting temperatures (T m ) as the nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO.
  • T m very similar melting temperatures
  • primers comprising (i) a nucleotide sequence, which is specific for an immunoglobulin constant region (or a portion thereof, such as the "end" of the constant region, e.g. as defined above) and (ii) a nucleotide sequence exhibiting very similar melting temperatures (T m ) as the nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in the TSO, which may be used together with the primer comprising or consisting of SEQ ID NO: 3, may comprise or consist of SEQ ID NO: 4, 5, 6, 7, 8 or 9:
  • IgG primer (SEQ ID NO: 4)
  • IgA primer (SEQ ID NO: 5)
  • IgM primer (SEQ ID NO: 6)
  • IgE primer (SEQ ID NO: 7)
  • IgK primer (SEQ ID NO: 7)
  • Primers comprising or consisting of any one of SEQ ID NOs: 4, 5, 6 and 7 ("IgG primer”; "IgA primer”; “IgM primer”; “IgE primer”) are capable of hybridizing to a nucleotide sequence encoding the (end of) a heavy chain constant domain of the respective immunoglobulin isotype (IgG for SEQ ID NO: 4, IgA for SEQ ID NO: 5, IgM for SEQ ID NO: 6, and IgE for SEQ ID NO: 7).
  • Primers comprising or consisting of any one of SEQ ID NOs: 8 and 9 (“IgK primer”; “IgL primer”) are capable of hybridizing to a nucleotide sequence encoding the (end of) a light chain constant domain (kappa light chain for SEQ ID NO: 8; lambda light chain for SEQ ID NO: 9).
  • a primer comprising or consisting of SEQ ID NO: 4 (“IgG primer”) is capable of hybridizing to a nucleotide sequence encoding the (end of) a constant domain of an IgG heavy chain.
  • a primer comprising or consisting of SEQ ID NO: 5 (“IgA primer”) is capable of hybridizing to a nucleotide sequence encoding the (end of) a constant domain of an IgA heavy chain.
  • a primer comprising or consisting of SEQ ID NO: 6 (“IgM primer”) is capable of hybridizing to a nucleotide sequence encoding the (end of) a constant domain of an IgM heavy chain.
  • a primer comprising or consisting of SEQ ID NO: 7 (“IgE primer”) is capable of hybridizing to a nucleotide sequence encoding the (end of) a constant domain of an IgE heavy chain.
  • a primer comprising or consisting of SEQ ID NO: 8 (“IgK primer”) is capable of hybridizing to a nucleotide sequence encoding the (end of) a constant domain of a kappa light chain.
  • a primer comprising or consisting of SEQ ID NO: 9 (“IgL primer”) is capable of hybridizing to a nucleotide sequence encoding the (end of) a constant domain of a lambda light chain.
  • Each of those primers (comprising or consisting of any one of SEQ ID NOs 4, 5, 6, 7, 8, or 9) may be used together (i.e., in the same primer pair) with the primer comprising or consisting of SEQ ID NO: 3.
  • the concentration of each primer used in the PCR does not exceed 1 mM, for example the concentration of each primer used in the PCR does not exceed 0.9 or 0.8 mM.
  • the concentration of each primer used in the PCR may be in the range from 0.1 to 1 mM, preferably from 0.15 to 0.9 mM, more preferably from 0.2 to 0.8 mM, even more preferably from 0.25 to 0.75 mM.
  • a primer comprising or consisting of SEQ ID NO: 3 may be used at a concentration of 0.5 to 1 mM, such as 0.75 mM.
  • a primer comprising or consisting of SEQ ID NO: 4 may be used at a concentration of 0.25 to 0.75 mM, such as 0.5 mM.
  • a primer comprising or consisting of SEQ ID NO: 5 may be used at a concentration of 0.25 to 0.75 mM, such as 0.5 mM.
  • a primer comprising or consisting of SEQ ID NO: 6 may be used at a concentration of 0.25 to 0.75 mM, such as 0.5 mM.
  • a primer comprising or consisting of SEQ ID NO: 7 may be used at a concentration of 0.25 to 0.75 mM, such as 0.5 mM.
  • a primer comprising or consisting of SEQ ID NO: 8 may be used at a concentration of 0.1 to 0.5 mM, such as 0.25 mM.
  • a primer comprising or consisting of SEQ ID NO: 9 may be used at a concentration of 0.1 to 0.5 mM, such as 0.25 mM.
  • a primer specific for an immunoglobulin heavy chain constant region such as a primer comprising or consisting of any one of SEQ ID NOs 4, 5, 6, and/or 7
  • a primer specific for an immunoglobulin light chain constant region such as a primer comprising or consisting of SEQ ID NO: 8 and/or 9
  • the corresponding primer comprising (or consisting of) a nucleotide sequence which is comprised in the TSO sequence (such as the primer comprising or consisting of SEQ ID NO: 3).
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 8; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 9, may be combined together in a single approach (comprising, for example 0.75 mM primer comprising or consisting of SEQ ID NO: 3, 0.5 mM primer comprising or consisting of SEQ ID NO: 4, 0.25 mM primer comprising or consisting of SEQ ID NO: 8, and 0.25 mM primer comprising or consisting of SEQ ID NO: 9).
  • separate PCRs may be performed to amplify DNA encoding immunoglobulin heavy and light chains or for distinct immunoglobulin isotypes.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 8; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 9, may be combined together in a single approach (comprising, for example 0.75 mM primer comprising or consisting of SEQ ID NO: 3, 0.5 mM primer comprising or consisting of SEQ ID NO: 5, 0.25 mM primer comprising or consisting of SEQ ID NO: 8, and 0.25 mM primer comprising or consisting of SEQ ID NO: 9).
  • separate PCRs may be performed to amplify DNA encoding immunoglobulin heavy and light chains or for distinct immunoglobulin isotypes.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 8; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 9, may be combined together in a single approach (comprising, for example 0.75 mM primer comprising or consisting of SEQ ID NO: 3, 0.5 mM primer comprising or consisting of SEQ ID NO: 6, 0.25 mM primer comprising or consisting of SEQ ID NO: 8, and 0.25 mM primer comprising or consisting of SEQ ID NO: 9).
  • separate PCRs may be performed to amplify DNA encoding immunoglobulin heavy and light chains or for distinct immunoglobulin isotypes.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 8; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 9, may be combined together in a single approach (comprising, for example 0.75 mM primer comprising or consisting of SEQ ID NO: 3, 0.5 mM primer comprising or consisting of SEQ ID NO: 7, 0.25 mM primer comprising or consisting of SEQ ID NO: 8, and 0.25 mM primer comprising or consisting of SEQ ID NO: 9).
  • separate PCRs may be performed to amplify DNA encoding immunoglobulin heavy and light chains or for distinct immunoglobulin isotypes.
  • primers capable of hybridizing to a nucleotide sequence encoding the (end of) a heavy chain constant region (e.g., as defined above) of different immunoglobulin isotypes may be combined in one approach.
  • the primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7, may be combined together in a single approach.
  • primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 4; (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 5; and/or (iii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 6; and/or (iv) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 7, may be combined together in a single approach.
  • primers capable of hybridizing to a nucleotide sequence encoding the (end of) a light chain constant region (e.g., as defined above) of kappa or lambda type may be combined in one approach.
  • the primer pairs (i) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 8; and/or (ii) primer comprising or consisting of SEQ ID NO: 3 and primer comprising or consisting of SEQ ID NO: 9, may be combined together in a single approach.
  • DNA encoding immunoglobulin heavy and light chains may be amplified in the same or in distinct PCR approaches.
  • the method of the invention comprises a single PCR (only). Accordingly, no further PCR (for example in the context of the RACE reaction or to further prepare the amplicons for cloning, e.g. into a vector) may be performed in addition to the above described single PCR.
  • the PCR product or a portion thereof may be subjected to gel electrophoresis for visualization and/or for purification.
  • this step is not required for the method of the invention. Rather, the PCR product may be directly used for cloning (CPEC) in step (4) without any purification of the PCR product.
  • step (4) of the method for generation and cloning of a DNA molecule encoding a polypeptide of interest the amplicon (i.e., the PCR product) obtained in step (3) (i.e., the amplified DNA molecule encoding the polypeptide of interest) is cloned into a linearized vector by circular polymerase extension cloning (CPEC).
  • CPEC circular polymerase extension cloning
  • CPEC is known in the art and described in detail, for example, in Quan J, Tian J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc. 2011 Feb;6(2):242-51 and in Quan J, Tian J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One. 2009 Jul 30;4(7):e6441, the disclosure of which is incorporated by reference herein.
  • CPEC is a sequence-independent cloning method, i.e. it can be used for cloning of DNA inserts having an unknown sequence. While other sequence-independent cloning methods require generating complementary single-stranded overhangs between the insert and the vector, a time-consuming and expensive process, CPEC eliminates this requirement by using the polymerase extension mechanism to extend double-stranded overlapping insert and vector to form a complete plasmid. Polymerase extension is the basis of the polymerase chain reaction (PCR) used for amplification of DNA sequences. The same principle is also used for CPEC.
  • PCR polymerase chain reaction
  • CPEC in contrast to PCR, no primers are added in CPEC, but instead the insert and the vector share overlapping sequences on both ends, which "prime” each other. Accordingly, CPEC extends overlapping regions between the insert and vector fragments to form a complete circular plasmid (thus, the name “Circular Polymerase Extension Cloning”). Accordingly, CPEC is differs from a standard PCR (only) in that no primers (and "template”) are added. Instead, the linearized vector and the insert are used in CPEC.
  • CPEC In CPEC, after denaturation and annealing, the insert and the vector hybridize (atthe overlapping sequences) and extend using each other as a template to form a complete double-stranded plasmid, leaving only one nick in each strand. Moreover, in contrast to a standard PCR, CPEC requires only very few cycles (re-amplifications of a given template sequence).
  • PCR-derived mutations are not propagated in CPEC to the same extent as one would anticipate for other sequence-independent cloning methods, such as SOEing (splice by overlap extension).
  • SOEing splice by overlap extension
  • CPEC is free of restriction digestion, ligation or single-stranded homologous recombination.
  • SLIC sequence and ligase independent cloning
  • Gibson assembly CPEC is standardized, scar-less, and sequence-independent.
  • CPEC is advantageous in that, since there is no exonuclease chew-back, small sequence fragments can be assembled directly without a preliminary SOEing step, there is no ATP addition step (unlike SLIC), there is only a single enzyme (polymerase) required (unlike Gibson), and since the CPEC assembly reaction occurs at higher temperatures than either SLIC or Gibson, stable secondary structures at the ends of assembly pieces are relatively less of a concern.
  • CPEC is a single-tube, one-step reaction that normally takes 5-10 min to complete for everyday laboratory cloning. CPEC offers significant benefits by combining simplicity, efficiency, versatility and cost-effectiveness in one method. In summary, CPEC is a simple, efficient and economical circular DNA assembly and cloning method.
  • the insert (such as the amplified DNA encoding the polypeptide of interest obtained in step (3)) and the linearized vector are first heat-denatured. Thereafter, the resulting single strands anneal with their overlapping ends and extend using each other as a template to form double-stranded circular plasmids.
  • all overlapping regions between insert(s) and the vector may be unique and designed to have similar melting temperatures (Tm).
  • the CPEC (in step (4)) comprises the following sub-steps:
  • Initialization comprises heating the reaction chamber to a temperature of more than 90°C, such as 94-96 °C (or 98 °C), which may be held, for example, for 30 seconds - 10 minutes, for example 1 or 2 minutes, e.g., at 98°C.
  • (b) Denaturation comprises heating the reaction chamber to more than 90°C, such as 94- 98 °C, e.g. for 5 - 50 seconds (such as 10 or 20 seconds). Thereby, DNA melting, or denaturation, of the double-stranded DNA insert and vector may occur.
  • Annealing comprises lowering the reaction temperature to the so-called “annealing temperature”, e.g. for 5 - 50 seconds (such as 20 or 30 seconds). Thereby, annealing of the overlapping sequences of the insert and vector is allowed.
  • “slow ramp annealing” may be used, wherein the temperature is slowly lowered (following a "ramp") to achieve the annealing temperature (e.g., at a rate of 0.1 °C/sec, e.g. from 70°C to the annealing temperature).
  • the annealing temperature may vary depending on the overlapping sequences of vector and insert.
  • the annealing temperature may be any temperature from 50°C to about 72 °C.
  • the annealing temperature is higher than 55°C (e.g., between 55°C and 72°C), for example higher than 60°C (e.g., between 60°C and 72°C), such as about 65°C.
  • the DNA polymerase typically synthesizes a new DNA strand complementary to the DNA template.
  • the temperature at this step usual ly depends on the DNA polymerase used; for example for the DNA polymerase of Taq (Thermus aquaticus) a temperature from 70°C - 80°C, such as about 72°C may be used.
  • the time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. As a rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute, for example 10 - 20 s per kB may be used.
  • Final elongation This sub-step is optional and may ensure that any remaining single- stranded DNA is fully elongated.
  • Final elongation may be performed at a temperature of 70-74 °C, for example at about 72°C. This temperature may be applied, for example, for 1-15 minutes after the last cycle, e.g. for about 2 or 5 minutes.
  • This sub-step is optional and may be employed for short-term storage of the CPEC products.
  • the reaction chamber is cooled to 4-15 °C (e.g., about 4°C) for an indefinite time.
  • sub-steps (a), (e) and (f) are usually single steps (i.e., they are performed only once (if any) in CPEC), while sub-steps (b), (c) and (d) (denaturation, annealing and elongation) constitute a single "cycle" and may be repeated.
  • 1 - 30 cycles (of sub-steps (b), (c) and (d)), such as 10 or 20 cycles, may be used.
  • CPEC may comprise incubation at 98°C (sub-step (a), e.g. for about 1 min); 20 cycles with about 10 sec at 98°C (sub-step (b)), about 20 sec at 65°C (sub-step (c)) and about 5 min at 72°C (sub-step (d)); and final extension of about 2 min at 72°C (sub-step (e)).
  • a CPEC protocol may be particularly useful for cloning of immunoglobulin genes, for example as described herein.
  • CPEC may be carried out in a total volume of 10-200 mL in small reaction tubes (for example, 0.2-0.5 mL volumes) in a thermal cycler.
  • the thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.
  • a (double stranded) DNA molecule to be inserted into a vector also referred to as "insert”
  • a linearized vector including nucleotide sequences overlapping with nucleotide sequences comprised in the insert an enzyme that polymerizes new DNA strands, such as a DNA polymerase (for example heat- resistant Taq polymerase); deoxynucleoside triphosphates (dNTPs also referred to as "deoxynucleotide triphosphates", nucleotides containing triphosphate groups); a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase; and, optionally, cations, such as magnesium (Mg) or manganese (Mn) ions (for example Mg 2+ ) ⁇
  • the components may be mixed in a "reaction mix” (reaction mixture), which may also include water (e.
  • the DNA polymerase is a high-fidelity polymerase.
  • the fidelity of a DNA polymerase is the result of accurate replication of a desired template.
  • High- fidelity (HiFi) DNA polymerases couple low misincorporation rates with proofreading activity to obtain a reliable replication of the target DNA of interest, i.e. to avoid undesired PCR- induced mutations.
  • Various high fidelity polymerases are known in the art.
  • the DNA polymerase may be Q5 ® DNA polymerase, such as Q5 Hot Start High-Fidelity DNA Polymerase.
  • a reaction mix may contain a DNA polymerase, for example a Q5 ® DNA polymerase, such as Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs; for example about 0.4U); suitable buffer (for example as supplied by the manufacturer of the DNA polymerase; such as 1 x Q5 reaction buffer), dNTPs (for example in the range from 100 to 200 mM, e.g. 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mM), optionally, water (such as DNase RNase free water) and, optionally, 0.5x or 1 x Q5 High GC Enhancer (New England Biolabs).
  • a DNA polymerase for example a Q5 ® DNA polymerase, such as Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs; for example about 0.4U)
  • suitable buffer for example as supplied by the manufacturer of the DNA polymerase; such as 1 x Q5 reaction buffer
  • the linearized vector (for example about 100 - 200 ng/25 mL, such as 100 ng/ 25 mL) may also be included in the reaction mix or it may be added separately.
  • about 20 pi of the reaction mix may be added to about 5 mI of the PCR product obtained in step (3) (also referred to as "amplicon” or "insert”; e.g. without any purification step after the PCR of Step (3)).
  • the final vector concentration may be in the range of 1 - 20 ng/mL, e.g. 4 - 10 ng/mL, such as 4 ng/mL.
  • the insert-to-vector molar ratio may be in the range of 0.5 : 1 to 3 : 1, such as in the range of 1 : 1 to 2 : 1 .
  • CPEC may be used to clone one (single) or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) inserts into a vector.
  • a single insert may be cloned into a single vector.
  • the amplified DNA encoding the polypeptide of interest (“amplicon”) obtained in the PCR of step (3) may be purified before CPEC in step (4) (e.g., the amplicon may be separated from other components, such as other DNA, contained in the reaction mix of step (3)), purification of the amplified DNA (amplicon) obtained in step (3) is not required for successful CPEC cloning (or for the method of the invention in general). Rather, performing the CPEC in step (4) directly (without any purification) on the reaction product of step (3), which contains the amplified DNA encoding the polypeptide of interest ("amplicon”), may save time and costs. Accordingly, CPEC in step (4) may be performed directly (without any purification) on the reaction product of step (3), which contains the amplified DNA encoding the polypeptide of interest ("amplicon").
  • the vector for CPEC may be any linearized vector, which comprises, e.g. at both termini of the linearized vector, nucleotide sequences, which overlap with nucleotide sequences comprised in the insert.
  • Each of the "overlapping" nucleotide sequences may be "unique", i.e. they occur only once in the linearized vector (and/or in the insert).
  • the linearized vector for CPEC in step (4) may comprise:
  • first nucleotide sequence e.g., included in one terminus of the linearized vector
  • first nucleotide sequence e.g., included in one terminus of the amplicon obtained in step (3)
  • the linearized vector for CPEC in step (4) may comprise:
  • any vector may be used in CPEC, as long as it is (i) linearized and (ii) contains two sequences overlapping with two sequences comprised in the insert. Typically, each of the two "overlapping" sequences is located at one (and the other) of the termini of the linearized vector and insert, respectively.
  • located "at a terminus" refers to sequences constituting the terminus (i.e., no further - “more terminal” nucleotides/sequences are present in the vector/insert) or sequences comprised in the terminus (e.g., with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides/base pairs located "more terminal” in the vector/insert).
  • the "overlapping" sequences constitute the termini of the linearized vector and of the insert.
  • one or both of the termini of the vector and/or insert includes up to 3 or 5 "more terminal" nucleotides/base pairs.
  • the vector may be an expression vector, e.g., for expression in mammalian cells.
  • the CPEC vector is usually a recombinant DNA molecule, i.e. a nucleic acid molecule which does not occur in nature.
  • the vector may comprise heterologous elements (i.e., sequence elements of different origin in nature).
  • the vector may comprise a multi cloning site, a heterologous promotor, a heterologous enhancer, a heterologous selection marker (to identify cells comprising said vector in comparison to cells not comprising said vector) and the like.
  • a vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence.
  • Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc.
  • a storage vector is a vector which allows the convenient storage of a nucleic acid molecule.
  • An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.
  • an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence.
  • a cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector.
  • a cloning vector may be, e.g., a plasmid vector or a bacteriophage vector.
  • a transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors.
  • a vector in the context of the present application may be a plasmid vector.
  • a commercially available vector may be used.
  • suitable vectors are commercially available.
  • Such vectors for immunoglobulin cloning may comprise complete (or partial) constant regions, such that only cloning of variable regions may be required.
  • DNA molecules encoding full-length (complete) immunoglobulin chains may be cloned into vectors.
  • An example of a commercially available vector for immunoglobulin cloning, wherein the constant regions are included in the vector, is the vector AbVec2.0-IGHG1 (Addgene plasmid # 80795; Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H. 2008. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J Immunol Methods 329(1 -2):112-24).
  • This vector is designed for expression of secretory immunoglobulin heavy chains, human IgGI isotype, and the insert (cloned gene) is under the control of a CMV promoter and ampicillin is used as selectable resistance.
  • the vector may already contain such "overlapping" sequences (e.g., if the primers in the PCR in step (3) were designed such that they "overlap” (are complementary to) vector sequences) and a convenient restriction site (e.g., located between the two "overlapping" sequences in the vector).
  • a convenient restriction site e.g., located between the two "overlapping" sequences in the vector.
  • PCR may be used to (i) linearize the vector and/or (ii) to append sequences "overlapping" with sequences at the termini of the insert to the termini of the vector.
  • primers may be used, which are designed to introduce overlapping regions with the inserts. Thereby, cloning sites can be selected most flexible.
  • the linearized vector for CPEC in step (4) may be prepared by PCR using primers designed to introduce nucleotide sequences into the vector, which are overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)).
  • the primer pair for preparing the CPEC vector may be designed such that the primers comprise nucleotide sequences, which are complementary to nucleotide sequences comprised in the primer pair used in the PCR of step (3) to obtain the insert.
  • the primer pair used in the PCR of step (3) to obtain the insert may include sequences exhibiting very similar melting temperatures.
  • the primer pair for preparing the CPEC vector may then include nucleotide sequences, which are complementary to nucleotide sequences exhibiting very similar melting temperatures of the step (3) (insert PCR) primer pair.
  • the primer pair for preparing the CPEC vector may be designed such that a restriction site, for example a blunt-end restriction site, is introduced into the vector, e.g.
  • no essential vector elements may be located between the two nucleotide sequences, which are overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)) in the (circularized) vector.
  • a BmgBI restriction site may be introduced. BmgBI is a restriction endonuclease recognizing the internal sequence "CACGTC” and cutting after "CAC” leaving a blunt end.
  • the primers for preparing the CPEC vector may contain (i) a nucleotide sequence capable of hybridizing with the vector sequence (i.e., the vector as provided as starting material, e.g. an unmodified vector); (ii) a nucleotide sequence overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)); and (iii) optionally, a (blunt-end) restriction site or a part thereof (in the latter case, one of the primers may contain a part of the restriction site and the other primer of the primer pair may contain at least the remaining portion of the restriction site).
  • primers may be designed to include at least two parts, each hybridizing to one end of the two neighboring fragments of the vector to be joined. If an additional short sequence needs to be inserted between two existing fragments (such as the restriction site as described above), it can be simply included in the primer design between the two overlapping regions.
  • the overlapping sequences between the vector and the insert may be selected such that all overlapping regions share very similar melting temperatures (Tm).
  • the forward primer and the (corresponding) reverse primer (of the same primer pair) used in the PCR reaction for preparation of the CPEC vector may comprise a nucleotide sequence exhibiting very similar melting temperatures (T m ).
  • tools for primer design e.g., as described above also calculate/provide the melting temperature (T m ) of a designed primer, such that a primer can be designed such that it exhibits a certain T m .
  • T m melting temperature
  • tools for calculating the melting temperature (T m ) include, for example, New England BioLabs Tm Calculator (URL: https://tmcalculator.neb. com/#!/main); Melting Temperature (Tm) Calculation (URL: http://www.biophp.org/minitools/melting_temperature/demo.php); Tm Tool (URL: https://www.dna.utah.edu/tm/; Owczarzy R. et al. (2008) Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cation. Biochemistry 47(19): 5336-5353).
  • very similar melting temperatures means that the difference between the melting temperatures of both primers (of the same primer pair) does not exceed 5°C, 4°C, 3°C, 2°C or 1 °C. For example, the difference between the melting temperatures of both primers (of the same primer pair) does not exceed 3°C or 2°C. As a general rule, the lower the difference between the melting temperatures of both primers (of the same primer pair), the better. If the overlapping sequences exhibit very similar melting temperatures, mis- hybridization may be reduced or eliminated and highest cloning efficiency and accuracy may be ensured.
  • the melting temperature of the nucleotide sequence exhibiting very similar melting temperatures may be between 60°C and 72°C, for example about 61 °C, 62°C, 63°C, 64°C, 65 °C, 66°C, 67°C, 68°C, 69°C, 70°C, or 71 °C, such as about 65°C or 68°C.
  • the higher the melting temperature the better.
  • the melting temperature may not exceed 70°C or 72°C.
  • the length of the overlapping region is of secondary consideration and may be dictated by the Tm.
  • the nucleotide sequence exhibiting very similar melting temperatures may have a length of 15 - 35 nucleotides. In some embodiments, the nucleotide sequence exhibiting very similar melting temperatures has a length of 16 - 30, such as 18 - 25 nucleotides, e.g. 20 - 23 nucleotides.
  • standard PCR primer selection rules and software can be applied to facilitate the design process (Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).
  • primers for preparing a CPEC vector based on the exemplified vector AbVec2.0-IGHG1 as described above which comprise (i) a nucleotide sequence, which capable of hybridizing with the vector sequence; (ii) a nucleotide sequence exhibiting a very similar melting temperature (T m ); and (iii) a blunt-end restriction site (for BmgBI), may comprise or consist of SEQ ID NO: 10 or 11 :
  • CarpRev primer (Italic: full BmgBI restriction site, underlined: nucleotide sequence exhibiting a very similar Tm): (SEQ ID NO: 11)
  • exemplified primers comprise sequences, which are complementary to sequences contained in the exemplified primers used in the insert-PCR (step (3)) described above (primers comprising any one of SEQ ID NO: 3 - 9). These complementary sequences are shown underlined in the above sequences of the "PracFwd primer” and the "CarpRev primer”.
  • the obtained linearized vector with the additional sequences may be either directly used for CPEC or purified before use.
  • standard gel purification e.g. with commercially available kits, such as NucleoSpin ® Gel and PCR clean-up (Macherey-Nagel)
  • the vector may be sequenced before it is used in CPEC.
  • the primers may be designed such that they include an additional restriction as described above, which may be also useful to remove undesired sequences in a vector.
  • this nucleotide sequence (encoding a constant region) may be removed, if the insert (i.e. the amplicon obtained in step (3)) comprises a nucleotide sequence encoding a full-length immunoglobulin chain (including the constant regions).
  • the newly introduced restriction site may optionally be used to digest the vector, e.g. after bacterial plasmid amplification.
  • the vector obtained by PCR may be ligated (using, e.g. a ligase) and propagated via bacteria in order to obtain larger amounts of the vector, e.g. for several CPEC reactions.
  • bacteria may be transformed by methods known in the art (e.g., chemically competent bacteria or by electroporation).
  • bacteria may be grown in large scale.
  • the plasmid (“empty" vector) may be extracted by methods known in the art.
  • the plasmid ("empty" vector) amplified by bacterial transformation may then be linearized (e.g. after bacteria! amplification or (directly) before its use in a CPEC reaction) by use of the restriction site (e.g., by BmgBI digestion).
  • plasmids ("empty" vector) extracted from the bacteria may then be sequenced.
  • a vector for CPEC may comprise (or consist of) a nucleotide sequence according to SEQ ID NO: 12.
  • a (linearized) vector for CPEC can be stored for a long time (e.g., at least one year), for example at -20 °C.
  • the vector may be used in many different approaches for the method of the present invention, as CPEC is sequence-independent. The only requirement is that appropriate primers (containing sequences overlapping/complementary to those of the vector) are used in the PCR step (3). Accordingly, the method of the present invention can be carried out without preparation of the CPEC vector (because the CPEC vector may be already ready-to-use).
  • the finished CPEC reaction product i.e., the vector containing the DNA insert which encodes the polypeptide of interest
  • the finished CPEC reaction product has a size of no more than 25 kb, preferably no more than 20 kb, more preferably no more than 15 kb, even more preferably no more than 10 kb, such as 8.4 kb.
  • the finished CPEC reaction product i.e., the vector containing the DNA insert which encodes the polypeptide of interest
  • the finished CPEC reaction product can be stored, e.g. at -20°C, for example for at least one year.
  • the CPEC reaction product can be directly used (i.e., without any purification) for many applications, such as transformation of bacteria.
  • the vector containing the DNA insert may optionally be used in a variety of further methods known in the art.
  • the encoded polypeptide of interest may be expressed and, optionally, characterized.
  • bacteria may be transformed with the CPEC product.
  • a portion e.g., a few mL, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mL
  • a portion e.g., a few mL, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mL
  • the finished CPEC reaction product may be loaded on an (agarose) gel, e.g. together with a DNA ladder and/or an empty vector control, for gel electrophoresis to assess whether CPEC reaction was successful.
  • This step is optional.
  • the CPEC reaction product (the vector with the insert) may be sequenced.
  • the vector containing the DNA insert (encoding the polypeptide of interest) may be purified, e.g. gel-purified.
  • the CPEC reaction product can be directly used (i.e., without any purification).
  • the CPEC reaction product i.e., the vector containing the DNA insert encoding the polypeptide of interest
  • the CPEC reaction product may be (directly) used for transformation.
  • electroporation or chemically competent cells may be used.
  • competent cells with transformation efficiencies greater than 1 x 108 c.f.u. (colony-forming units) per mg e.g., GC5 competent cells, Genesee Scientific
  • a number of methods can be used to determine the presence of inserts in bacterial colonies grown on a culture plate, including colony PCR (e.g., as described in Quan J, Tian J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc. 2011 Feb;6(2):242-51 , which is incorporated herein by reference), restriction mapping or direct sequencing.
  • expression plasmids containing immunoglobulin heavy and light chains may be transformed, for example, into competent E. coli. Transformed bacteria may then be screened for inclusion of plasmids encoding heavy and light chains, e.g. by colony PCR (and/or by sequencing).
  • the polypeptide of interest may be expressed based on the vector containing the DNA insert (encoding the polypeptide of interest).
  • cells such as mammalian cells
  • cells may be transfected (e.g., stably or transiently) with the vector containing the DNA insert (encoding the polypeptide of interest), for example (directly) with the CPEC reaction product or with (purified) plasmids obtained from bacteria.
  • the polypeptides of interest are immunoglobulin chains, heavy and light chain pairs (e.g., derived from a single immortalized B cell) may be co-transfected into mammalian cells (such as HEK293 cells) to express recombinant antibodies.
  • DNA molecule(s) each encoding a polypeptide of interest may be cloned into a vector.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more DNA molecule(s) (each) encoding a polypeptide of interest may be cloned into a (single) vector.
  • a single or two DNA molecule(s) (each) encoding a (single) polypeptide of interest may be cloned into a vector.
  • a single DNA molecule encoding a single polypeptide of interest may be cloned into a single vector.
  • a single DNA molecule (or each single DNA molecule) may encode one or more polypeptides of interest.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptide(s) of interest may be comprised in a (single) "DNA molecule encoding a polypeptide of interest".
  • a "DNA molecule encoding a polypeptide of interest" encodes exactly one or two polypeptide(s) of interest.
  • a polypeptide of interest may be any polypeptide.
  • the polypeptide of interest may be a full-length polypeptide (or protein; i.e., a "complete" polypeptide or protein) or a fragment thereof. Fragments of polypeptides/proteins may have a minimum length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids, such as 125, 150, 175, 200, 225, 250, 275, 300 or more amino acids.
  • full-length may have a minimum length of at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids, such as 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more amino acids.
  • the present invention also provides a method for producing a protein/polypeptide of interest or a fragment thereof comprising the following steps:
  • An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.
  • an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence.
  • Examples of cells which may be used for transfection (step (b)) include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells or plant cells or prokaryotic cells, including E coli.
  • the cells are mammalian cells, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293T cells, PER.C6 cells, NSO cells, human liver cells, myeloma cells or hybridoma cells.
  • the cell may be transfected with the expression vector encoding the protein/polypeptide of interest or the fragment thereof.
  • transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, e.g. into eukaryotic or prokaryotic cells.
  • RNA e.g. mRNA
  • transfection encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g.
  • the introduction is non-viral.
  • the cell may be transfected stably or transiently with the expression vector encoding the protein/polypeptide of interest or the fragment thereof.
  • the cells are stably transfected with the expression vector encoding the protein/polypeptide of interest or the fragment thereof.
  • the cells are transiently transfected with the expression vector encoding the protein/polypeptide of interest or the fragment thereof.
  • the protein of interest may be composed of more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) distinct polypeptide chains.
  • antibodies usually comprise a heavy chain and a light chain (e.g., exactly two distinct polypeptide chains).
  • the cell may be co-transfected with multiple (e.g., exactly two) distinct vectors, each encoding a (single) distinct polypeptide chain (wherein the protein of interest comprises the polypeptide chains encoded by the co-transfected vectors).
  • the protein of interest is an antibody
  • a vector encoding the heavy chain of the antibody and a vector encoding a light chain of the antibody may be co-transfected (into the same cell).
  • a vector encoding the heavy chain of an antibody and a vector encoding the light chain of the same antibody are co-transfected.
  • the cell may heterologously express the protein/polypeptide of interest.
  • the cell may be of another species than the protein/polypeptide of interest (e.g., CHO cells expressing human antibodies).
  • the cell type of the cell does not express the protein/polypeptide of interest in nature (even if the cell is derived from the same species as the protein/polypeptide of interest).
  • a cell may be used for expression, which does not express antibodies in nature (e.g., in a physiological situation in a living human or animal).
  • the host cell may impart a post-translational modification (PTM; e.g., glycosylation) on the polypeptide/protein of interest that is not present in their native state.
  • PTM post-translational modification
  • Such a PTM may result in a functional difference (e.g., reduced immunogenicity).
  • polypeptide/protein of interest may have a post-translational modification, which is distinct from the naturally produced polypeptide/protein of interest.
  • Harvesting of the protein/polypeptide may be achieved by methods known in the art. Selection of an appropriate method may depend on the expected localization of the protein/polypeptide of interest. For example, if the protein/polypeptide of interest is secreted, it may be found in the supernatant. In other cases, cell lysis may be required.
  • proteins/polypeptides of interest or fragments thereof, which are obtained by the method as described above.
  • such recombinantly produced polypeptides/proteins may differ from polypeptides/proteins obtained by prior art methods in that they also include their leader sequence.
  • the polypeptide of interest comprises a variable region of an immunoglobulin heavy chain (VH) or a variable region of an immunoglobulin light chain (VL).
  • VH immunoglobulin heavy chain
  • VL immunoglobulin light chain
  • the term "variable region" denotes each of the pair of light and heavy chains which is involved directly in binding an antibody to the antigen.
  • a variable region typically comprises complementarity determining regions (CDRs), which may be separated by framework regions.
  • CDRs complementarity determining regions
  • the CDRs of a heavy chain and the connected light chain of an antibody together form the antigen receptor.
  • the three CDRs (CDR1, CDR2, and CDR3) may be arranged non-consecutively in the variable domain.
  • antigen receptors may be composed of two variable domains (on two different polypeptide chains, i.e. heavy and light chain)
  • there may be six CDRs for each antigen receptor (heavy chain: CDRH1 , CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3).
  • Framework regions are usually less "variable" than the CDRs.
  • a variable region (or each variable region, respectively) may be composed of four framework regions, separated by three CDRs.
  • the polypeptide of interest does not comprise a constant region.
  • the constant region may be comprised in the vector.
  • Vectors for expressing immunoglobulins, which contain constant regions are known in the art and examples thereof are described elsewhere herein.
  • the polypeptide of interest which comprises a variable region, may further comprise an immunoglobulin (Ig) constant region or a portion thereof.
  • the heavy chain constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes.
  • Heavy chains g, a and d have a constant region composed of three tandem (in a line) Ig domains (CH1 , CH2 and CH3), and a hinge region for added flexibility; heavy chains m and e have a constant region composed of four immunoglobulin domains.
  • the light chain constant region (CL) may be K or l.
  • a polypeptide of interest may comprise the variable domain VH and the CH1 domain (and optionally the CH2 domain).
  • Antibodies may be of any isotype (e.g ., IgA, IgG, IgM, IgE i.e. an a, g, m or e heavy chain).
  • the antibody is of the IgG type.
  • antibodies may be IgG 1 , lgG2, lgG3 or lgG4 subclass, for example lgG1 .
  • Antibodies may have a k or a l light chain.
  • the polypeptide of interest comprises a full-length heavy chain or a full-length light chain of an antibody.
  • a full-length heavy chain includes the complete variable region VH as well as the complete constant region including all constant domains (three or four, depending on the isotype).
  • a full-length light chain includes the complete variable region VL as well as the complete constant region CL.
  • the primer hybridizing in step (3) of the above-described method (PCR) to the 3' end of the nucleotide sequence encoding the polypeptide of interest may comprise a nucleotide sequence complementary to a nucleotide sequence encoding a fragment of an immunoglobulin constant region included in the immunoglobulin constant region or the portion thereof of the polypeptide of interest.
  • said primer in step (3) of the method may comprise a nucleotide sequence complementary to the nucleotide sequence encoding the C-terminus of an immunoglobulin constant region (or of the immunoglobulin heavy or light chain).
  • the present invention also provides a method for generation and cloning of a DNA molecule encoding an immunoglobulin heavy or light chain comprising the following steps:
  • step (2) performing RACE reaction using the mRNA provided in step (1) as template to obtain a cDNA molecule encoding the immunoglobulin heavy or light chain or a fragment thereof comprising at least the variable region of the immunoglobulin heavy chain (VH) or light chain (VL), respectively;
  • step (3) (4) cloning the amplicon obtained in step (3) into a linearized vector by circular polymerase extension cloning (CPEC).
  • CPEC circular polymerase extension cloning
  • steps (1 ) - (4) of this method correspond to steps (1 ) - (4), respectively, of the more general method for generation and cloning of a DNA molecule encoding a polypeptide of interest as described above.
  • the polypeptide of interest is in this case an immunoglobulin heavy or light chain.
  • the fragment of the immunoglobulin heavy or light chain may further comprise an immunoglobulin constant region or a portion thereof (e.g., the mRNA and or the DNA may encode full-length heavy or light chains).
  • the mRNA in step (1 ) is provided by lysis of a B cell.
  • the B cell may be a plasma cell or a memory B cell.
  • B cells may be isolated from (isolated) peripheral blood mononuclear cells (PBMCs).
  • PBMCs may be extracted, for example, from (isolated) whole blood using ficoll, a hydrophilic polysaccharide that separates layers of blood, and gradient centrifugation.
  • B cells may be obtained from PBMCs, for example by cell sorting.
  • markers for specific B cells may be used.
  • CD138 may be used to identify plasma cells and CD20, CD27 and/or CD40, may be used to identify memory B cells.
  • the mRNA provided in step (a) may be obtained from a single cell (such as a single B cell) or from a single cell line or cell clone (such as a single B cell line or B cell clone).
  • Single B cells or B cell lines/B cell clones typically express only one single (type of) antibody. Accordingly, in such a situation by the mRNA provided in step (a) (in particular in step (1) of the above-described method) encodes only a single heavy chain sequence and a single light chain sequence.
  • step (c)) includes the same heavy and light chain combination as expressed by the B cell (or B cell line/B cell clone), from which the mRNA was isolated.
  • the present invention also provides a method for identifying the sequence of an antibody comprising the following steps:
  • the present invention also provides an antibody obtained by the above-described method.
  • such recombinantly produced antibodies may differ from antibodies obtained by prior art methods in that they also include, for example, the constant region from the "original" antibody (e.g., as expressed by the B cells from which mRNA is provided in step (1)).
  • the antibody is isolated including its leader signal and constant region.
  • Antibodies obtained with methods of the prior art usually include only either the leader or the constant region, but not both.
  • the expression level of IgM may be 10-100 times higher with the method of the present invention as compared to prior art methods using a "standard” leader and/or a "standard” constant region (e.g., which may be included in the vector for cloning/expression).
  • including the leader sequence and full constant region from the original antibody can reveal new antibody structures and sequences (like mutations, insertions and/or deletions).
  • vectors for example, expression vectors, obtained by the methods of the present invention.
  • a vector comprises a DNA insert encoding the polypeptide of interest as described above.
  • the present invention also provides a combination of a first and a second vector, wherein the first vector comprises a first DNA insert encoding an immunoglobulin heavy or light chain or a fragment thereof comprising at least the variable region of the immunoglobulin heavy chain (VH) or light chain (VL), respectively, as described above; and the second vector comprises a second DNA insert encoding the corresponding immunoglobulin heavy or light chain or a fragment thereof comprising at least the variable region of the immunoglobulin heavy chain (VH) or light chain (VL), respectively, as described above.
  • a vector is usually a recombinant nucleic acid molecule, i.e. a nucleic acid molecule which does not occur in nature.
  • the vector may comprise heterologous elements (i.e., sequence elements of different origin in nature).
  • the vector may comprise a multi cloning site, a heterologous promotor, a heterologous enhancer, a heterologous selection marker (to identify cells comprising said vector in comparison to cells not comprising said vector) and the like.
  • a vector in the context of the present invention is suitable for incorporating or harboring the desired nucleic acid sequence.
  • Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc.
  • a storage vector is a vector which allows the convenient storage of a nucleic acid molecule.
  • An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.
  • an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a (heterologous) promoter sequence.
  • a cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector.
  • a cloning vector may be, e.g., a plasmid vector or a bacteriophage vector.
  • a transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors.
  • a vector in the context of the present invention is usually a DNA vector, in particular a DNA plasmid.
  • a vector in the sense of the present application may comprise a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication.
  • a vector in the context of the present application may be a plasmid vector.
  • the present invention also provides a polynucleotide comprising the nucleotide sequence as set forth in SEQ ID NO: 12, or a sequence variant thereof having at least 50% sequence identity.
  • the polynucleotide may comprise a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 60% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 75% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 85% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 91 % sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 92% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 93% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 94% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 95% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 96% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 97% sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 98 % sequence identity with SEQ ID NO: 12.
  • the polynucleotide may comprise a nucleotide sequence having at least 99% sequence identity with SEQ ID NO: 12.
  • Said polynucleotide may be a vector as described above.
  • the nucleotide sequence of SEQ ID NO: 12 relates to a vector, for example for expression of immunoglobulins or fragments thereof, which is prepared for CPEC cloning as described elsewhere herein.
  • the present invention also provides cell or a cell line comprising the vector according the present invention as described above.
  • the cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells or plant cells or prokaryotic cells, including E coli.
  • the cells are mammalian cells, such as a mammalian cell line. Examples include human cells, CHO cells, HEK293 cells, PER.C6 cells, NS0 cells, human liver cells, myeloma cells or hybridoma cells.
  • the terms “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
  • the cell may be transfected with a vector according to the present invention, e.g., as described above, for example with an expression vector.
  • transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, e.g. into eukaryotic or prokaryotic cells.
  • RNA e.g. mRNA
  • transfection encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g.
  • the introduction is non-viral.
  • the cells of the present invention may be transfected stably or transiently with the vector according to the present invention.
  • the cells are stably transfected with the vector according to the present invention encoding the polypeptide of interest, such as an immunoglobulin (antibody).
  • the cells are transiently transfected with the vector according to the present invention encoding the polypeptide of interest, such as an immunoglobulin (antibody).
  • the cell may heterologously express the protein/polypeptide of interest.
  • the cell may be of another species than the protein/polypeptide of interest (e.g., CHO cells expressing human antibodies).
  • the cell type of the cell does not express the protein/polypeptide of interest in nature (even if the cell is derived from the same species as the protein/polypeptide of interest).
  • the protein/polypeptide of interest is an antibody (or a chain thereof)
  • a cell may be used for expression, which does not express antibodies in nature (e.g., in a physiological situation in a living human or animal).
  • the host cell may impart a post-translational modification (PTM; e.g., glycosylation) on the polypeptide/protein of interest that is not present in their native state.
  • PTM post-translational modification
  • Such a PTM may result in a functional difference (e.g., reduced immunogenicity).
  • polypeptide/protein of interest may have a post-translational modification, which is distinct from the naturally produced polypeptide/protein of interest.
  • a polynucleotide comprising any one of SEQ ID NOs 1 - 20 or a sequence variant thereof.
  • sequence variant refers to any alteration in a reference sequence, wherein a reference sequence may be any one of SEQ ID NO: 1 to SEQ ID NO: 20.
  • the sequence variant may be a functional sequence variant, i.e. a sequence variant maintaining the function of the reference sequence; for example, for a primer to specifically hybridize and prime a (specific) extension reaction (or, e.g., for an expression vector to provide the required functionality for expression of an insert).
  • a sequence variant may have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a reference sequence.
  • sequence variant includes in particular such variants that comprise mutations and/or substitutions in comparison to the reference sequence.
  • the term "primer” refers to an oligonucleotide used to prime an extension reaction (e.g., wherein one nucleic acid strand is “extended” starting from the primer, for example by use of a polymerase).
  • a primer usually "hybridizes” to a nucleic acid strand, which serves as “template” (or “target”) for the extension reaction.
  • template or “target” for the extension reaction.
  • one nucleic acid strand is “extended” starting from the primer, for example by use of a polymerase.
  • the newly synthesized nucleic acid strand (the nascent strand) is usually complementary to the template strand.
  • a primer specifically hybridizes to a region of a template nucleic acid with which the primer or other polynucleotide shares at least some complementarity. Whether a primer specifically hybridizes to a target nucleic acid is determined by such factors as the degree of complementarity between the primer and the target nucleic acid and the temperature at which the hybridization occurs, which may be informed by the melting temperature (Tm) of the primer.
  • Tm melting temperature
  • melting temperature (T m ) refers to the temperature at which half of the primer-target nucleic acid duplexes remain hybridized and half of the duplexes dissociate into single strands. While it may be referred herein to the “melting temperature (T m ) of a primer", it is understood that any such expression usually means the temperature at which half of the primer-target nucleic acid duplexes remain hybridized and half of the duplexes dissociate into single strands (even if the target/template nucleic acid is not specifically mentioned).
  • the T m of a primer-target nucleic acid duplex may be experimentally determined or predicted as described in Sambrook and Russell, 2001 , Molecular Cloning: A Laboratory Manual, 3rd ed. ; Cold Spring Harbor Press, Cold Spring Harbor N.Y., Chapter 10, which is incorporated herein by reference.
  • Other more advanced models that depend on various parameters may also be used to predict the T m of primer/target duplexes depending on various hybridization conditions.
  • tools for primer design also calculate/provide the melting temperature (T m ) of a designed primer, such that a primer can be designed such that it exhibits a certain T m .
  • tools for calculating the melting temperature (T m ) are known to the skilled person and include, for example, New England BioLabs Tm Calculator (URL: https://tmcalculator.neb. com/#!/main); Melting Temperature (Tm) Calculation (URL: http://www.biophp.org/minitools/melting_temperature/demo.php); Tm Tool (URL: https://www.dna.utah.edu/tm/; Owczarzy R. et al. (2008) Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cation. Biochemistry 47(19): 5336-5353).
  • the term "complementary” refers to a nucleotide sequence that base-pairs by non-covalent bonds to all or a region of a template/target nucleic acid.
  • adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) in DNA.
  • thymine is replaced by uracil (U).
  • U uracil
  • A is complementary to T and G is complementary to C.
  • A is complementary to U.
  • complementary may include situations, in which a nucleotide sequence is at least partially complementary (i.e., “full complementarity" for each base over the complete length of a nucleotide sequence may occur, but may be not required for a nucleotide sequence to be considered as “complementary”). Accordingly, the term “complementary” may encompass situations, wherein the duplexes are fully complementary such that every nucleotide in one strand is complementary to every nucleotide in the other strand in corresponding positions.
  • a nucleotide sequence may be partially complementary to a template/target, in which not all nucleotides are complementary to every nucleotide in the target nucleic acid in all the corresponding positions.
  • a primer may be fully (i.e., 100%) complementary to the target nucleic acid (or a portion thereof), or the primer and the target nucleic acid may share some degree of complementarity that is less than "full” (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%).
  • a primer comprising or consisting of SEQ ID NO: 1 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1) is usually an oligo-(dT) primer as described above.
  • Such a primer may be useful in the RACE reaction (step (2) of the method of the invention), for example alone or in combination with a primer comprising or consisting of SEQ ID NO: 2 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2).
  • a primer comprising or consisting of SEQ ID NO: 2 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2) is usually a template-switching oligo (TSO) as described above.
  • TSO template-switching oligo
  • Such a TSO may be useful in the RACE reaction (step (2) of the method of the invention), for example alone or in combination with a primer comprising or consisting of SEQ ID NO: 1 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 ).
  • a primer comprising or consisting of SEQ ID NO: 1 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 ).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 1 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1 ) and a second primer comprising or consisting of SEQ ID NO: 2 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2).
  • a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 1 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 1 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 2.
  • a primer pair may be useful in the RACE reaction (step (2) of the method of the invention).
  • a primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof may be a PCR primer for amplification of a DNA molecule obtained in a RACE reaction using a primer with a predetermined ("universal") sequence as described above. Therefore, such a primer may comprise or consist of a nucleotide sequence corresponding to the predetermined ("universal") sequence comprised in a primer used in a RACE reaction to obtain the DNA molecule to be amplified as described above.
  • Such a primer may be useful in the PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 4 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 4).
  • such a primer may be useful in the PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 5 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 5).
  • a primer comprising or consisting of SEQ ID NO: 5 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 5).
  • such a primer may be useful in the PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 6 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6).
  • a primer comprising or consisting of SEQ ID NO: 6 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6).
  • such a primer may be useful in the PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 7 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 7).
  • a primer comprising or consisting of SEQ ID NO: 7 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 7).
  • such a primer may be useful in the PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 8 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 8).
  • a primer comprising or consisting of SEQ ID NO: 8 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 8).
  • such a primer may be useful in the PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 9 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 9).
  • a primer comprising or consisting of SEQ ID NO: 9 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 9).
  • a primer comprising or consisting of SEQ ID NO: 4 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 4) may be useful for PCR amplification of an immunoglobulin heavy chain of the IgG type as described above.
  • Such a primer may be useful in a PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3).
  • a primer comprising or consisting of SEQ ID NO: 5 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 5) may be useful for PCR amplification of an immunoglobulin heavy chain of the IgA type as described above.
  • Such a primer may be useful in a PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3).
  • a primer comprising or consisting of SEQ ID NO: 6 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6) may be useful for PCR amplification of an immunoglobulin heavy chain of the IgM type as described above.
  • Such a primer may be useful in a PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3).
  • a primer comprising or consisting of SEQ ID NO: 7 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 7) may be useful for PCR amplification of an immunoglobulin heavy chain of the IgE type as described above.
  • Such a primer may be useful in a PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3).
  • a primer comprising or consisting of SEQ ID NO: 8 or a sequence variant thereof may be useful for PCR amplification of an immunoglobulin light chain of the kappa type as described above.
  • Such a primer may be useful in a PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3).
  • a primer comprising or consisting of SEQ ID NO: 9 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 9) may be useful for PCR amplification of an immunoglobulin light chain of the lambda type as described above.
  • Such a primer may be useful in a PCR (step (3) of the method of the invention), for example in combination with a primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3) and a second primer comprising or consisting of SEQ ID NO: 4 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 4).
  • a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 9
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 4.
  • a primer pair may be useful in the PCR (step (3) of the method of the invention).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3) and a second primer comprising or consisting of SEQ ID NO: 5 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 5).
  • a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%,
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 5.
  • a primer pair may be useful in the PCR (step (3) of the method of the invention).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3) and a second primer comprising or consisting of SEQ ID NO: 6 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 6).
  • a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 9
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 6.
  • a primer pair may be useful in the PCR (step (3) of the method of the invention).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3) and a second primer comprising or consisting of SEQ ID NO: 7 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 7).
  • a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%,
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 7.
  • a primer pair may be useful in the PCR (step (3) of the method of the invention).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3) and a second primer comprising or consisting of SEQ ID NO: 8 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 8).
  • a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%,
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 8.
  • a primer pair may be useful in the PCR (step (3) of the method of the invention).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3) and a second primer comprising or consisting of SEQ ID NO: 9 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 9).
  • a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 9
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 9.
  • a primer pair may be useful in the PCR (step (3) of the method of the invention).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 3 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3) and a second primer comprising or consisting of any one of SEQ ID NOs: 4 - 9 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 4 - 9).
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 3 and a second primer having a nucleotide sequence as set forth
  • a primer comprising or consisting of SEQ ID NO: 10 or a sequence variant thereof may be useful for CPEC (step (4) of the method of the invention), for example alone or in combination with a primer comprising or consisting of SEQ ID NO: 11 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 11).
  • Such a primer is described in more detail above and may comprise (i) a nucleotide sequence, which is capable of hybridizing with the vector sequence; (ii) a nucleotide sequence exhibiting a very similar melting temperature (T m ); and, optionally, (iii) a blunt-end restriction site or a portion thereof (for BmgBI).
  • a primer comprising or consisting of SEQ ID NO: 11 or a sequence variant thereof may be useful for CPEC (step (4) of the method of the invention), for example alone or in combination with a primer comprising or consisting of SEQ ID NO: 10 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 10).
  • Such a primer is described in more detail above and may comprise (i) a nucleotide sequence, which is capable of hybridizing with the vector sequence; (ii) a nucleotide sequence exhibiting a very similar melting temperature (T m ); and, optionally, (iii) a blunt-end restriction site or a portion thereof (for BmgBI).
  • the present invention also provides a primer pair comprising a first primer comprising or consisting of SEQ ID NO: 10 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 10) and a second primer comprising or consisting of SEQ ID NO: 11 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 11 ).
  • the primer pair comprising a first primer having a nucleotide sequence as set forth in SEQ ID NO: 10 and a second primer having a nucleotide sequence as set forth in SEQ ID NO: 11 .
  • a primer pair may be useful in CPEC (step (4) of the method of the invention).
  • a primer comprising or consisting of SEQ ID NO: 13 or a sequence variant thereof may be useful for sequencing a vector prepared for CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 12).
  • a primer comprising or consisting of SEQ ID NO: 14 or a sequence variant thereof may be useful for sequencing a vector prepared for CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 14).
  • a primer comprising or consisting of SEQ ID NO: 15 or a sequence variant thereof may be useful for sequencing a vector prepared for CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 15).
  • a primer comprising or consisting of SEQ ID NO: 16 or a sequence variant thereof may be useful for sequencing a vector prepared for CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 12).
  • a primer comprising or consisting of SEQ ID NO: 17 or a sequence variant thereof may be useful for sequencing a vector prepared for CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 17).
  • a primer comprising or consisting of SEQ ID NO: 18 or a sequence variant thereof may be useful for sequencing a plasmid containing an insert by CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 12) containing an exemplified insert as described herein.
  • a primer comprising or consisting of SEQ ID NO: 19 or a sequence variant thereof may be useful for sequencing a plasmid containing an insert by CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 19) may be useful for sequencing a plasmid containing an insert by CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 12) containing an exemp
  • a primer comprising or consisting of SEQ ID NO: 20 or a sequence variant thereof may be useful for sequencing a plasmid containing an insert by CPEC as described herein; for example, the vector comprising or consisting of SEQ ID NO: 12 or a sequence variant thereof (e.g., sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 12) containing an exemplified insert as described herein.
  • the present invention also provides a set of primer pairs comprising:
  • a first primer pair comprising an oligo-(dT)-primer and a template-switching oligonucleotide (TSO), wherein the TSO comprises a predetermined nucleotide sequence having a length of 15 to 30 nucleotides;
  • a second primer pair comprising a forward primer and a reverse primer
  • the nucleotide sequence of the forward primer comprises the predetermined nucleotide sequence (e.g., as defined above for the TSO)
  • the reverse primer comprises a nucleotide sequence exhibiting very similar melting temperatures as the predetermined nucleotide sequence (e.g., as defined above for the TSO).
  • Such a set of primer pairs may be useful in the method of the present invention as described herein.
  • the first primer pair may be useful for a RACE reaction (step (2) of the method of the invention), while the second primer pair may be useful for a PCR reaction (step (3) of the method of the invention).
  • the primer pair useful in the RACE reaction step (2) of the method of the invention
  • the primer pair useful in the PCR step (3) of the method of the invention
  • the second primer pair of the set of primer pairs apply accordingly to the second primer pair of the set of primer pairs.
  • the predetermined nucleotide sequence may have a length of 16 - 25 nucleotides, such as 20 - 23 nucleotides.
  • the melting temperature of the predetermined nucleotide sequence may be at least 65°C, for example at least 68°C.
  • the TSO may comprise a locked nucleic acid (LNA) as described elsewhere herein.
  • the reverse primer of the second primer pair may comprise a nucleotide sequence, which is able to hybridize to an immunoglobulin constant region.
  • the nucleotide sequence, which is able to hybridize to an immunoglobulin constant region may be specific for an immunoglobulin heavy chain or for an immunoglobulin light chain.
  • the nucleotide sequence, which is able to hybridize to an immunoglobulin constant region may be specific for an immunoglobulin isotype, such as IgG.
  • set of primer pairs may optionally further comprise:
  • a third primer pair comprising primers for preparing a CPEC vector, wherein each of the primers of the third primer pair contains (i) a nucleotide sequence capable of hybridizing to the vector sequence (i.e., the vector as provided as starting material, e.g.
  • an unmodified vector (ii) a nucleotide sequence overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)); and (iii) optionally, a (blunt-end) restriction site or a part thereof (in the latter case, one of the primers may contain a part of the restriction site and the other primer of the primer pair may contain at least the remaining portion of the restriction site).
  • the nucleotide sequence overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)) contained in one of the primers of the third primer pair may be a nucleotide sequence complementary to a nucleotide sequence contained in the forward primer of the second primer pair; while the nucleotide sequence overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)) contained in the other of the primers of the third primer pair may be a nucleotide sequence complementary to a nucleotide sequence contained in the reverse primer of the second primer pair.
  • the nucleotide sequence overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)) contained in one of the primers of the third primer pair may be a nucleotide sequence complementary to the nucleotide sequence comprising the predetermined nucleotide sequence (e.g., as defined above for the TSO) contained in the forward primer of the second primer pair; while the nucleotide sequence overlapping/complementary to nucleotide sequences (at the termini) of the insert (the amplified DNA encoding the polypeptide of interest obtained in step (3)) contained in the other of the primers of the third primer pair may be a nucleotide sequence complementary to the nucleotide sequence exhibiting very similar melting temperatures as the predetermined nucleotide sequence (e.g., as described herein) contained in the reverse primer of the second primer pair. Accordingly, such primer introduce nucleotide sequences
  • Figure 1 shows for Example 1 the PCR results on a gel with three lanes (1, 2 and 3). While lane 1 shows the DNA ladder, lane 2 shows the PCR results on cDNA obtained from a single immortalized B cell and lane 3 shows the PCR results on cDNA obtained from 100 plasma cells.
  • the PCR yielded an amplicon of approximately 1500 base pairs (bp) for the heavy chain, including the entire V-D-J rearrangement and the whole constant region, as shown as the upper bands in lanes 2 and 3.
  • Figure 2 shows for Example 2 the PCR results on a gel with five lanes (1 , 2, 3, 4 and 5).
  • lane 1 shows the DNA ladder
  • lanes 2 and 4 show the PCR results on cDNA obtained from a single plasma cell
  • lanes 3 and 5 shows the PCR results on cDNA obtained from a single memory B cell.
  • separate PCRs were performed for heavy and light chains.
  • the same PCR program used in Example 2 corresponds to the PCR program used in Example 1 .
  • the higher and lower bands in Figure 2 represent the heavy and light chain PCR products from plasma cell (lanes 2 and 4) and memory B cell (lanes 3 and 5), respectively.
  • Figure 3 shows for Example 3 IgA, IgG and IgM distribution in one of the four expression plates.
  • Example 1 Immunoglobulin gene rescue by 5'RACE RT-PCR. cloning into CPEC vector and expression
  • memory B cells and plasma cells were isolated from peripheral blood mononuclear cells (PBMCs) and resuspended in MACS buffer (PBS 1 x, SIGMA; EDTA 2mM, Applichem; 1 % FBS, GIBCO).
  • Memory B cells were CD19+ and CD27+, while plasma cells IgA, IgM and IgE were CD19+, CD27hi, CD38+, and HLA DR+/-.
  • Single, two and three cells were sorted with a BD Sorter Aria in single wells of 384-well plate (twin.tec PCR plate 384, Eppendorf) containing 10, 7.5, or 5 mI of lysis buffer (0.02% Triton X-100, 1 U/mL RiboLock RNase Inhibitor from ThermoFisher). Plates were kept on ice during the procedure and sealed with AluminaSeal (Diversified Biotech). In other cases memory B cells and plasma cells were separated in two tubes of 500 uL of MACS buffer, 5 aliquots of 100 mL were distributed in LoBind Eppendorf tubes and centrifuged 5 minutes at 500 ref. Tubes were placed on ice, supernatant removed and cells resuspended in 10 mL/per 10-10000 of lysis buffer. Lysate was stored at -80 °C or used immediately.
  • cDNA was synthetized as described in steps 9 - 11 ("Reverse Transcription") of the protocol described by Picelli S, et al. (Picelli S, Faridani OR, Bjorklund AK, Winberg G, Sagasser S, Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014 Jan;9(1 ):171-81).
  • genomic DNA extraction and Phi29 amplification may be performed from 1 -10 cells, for example when proteins or antibodies with different structure/sequence are found, to track the stage at which they diversified or any genomic anomalies.
  • 20 mL of the cDNA solution in 0.5 mL Eppendorf tubes or 384 plates (e.g., as obtained in the RACE reaction) may be centrifuged at 1400 ref and 17 mL of the supernatant may be removed and placed in another 384 plate or Eppendorf tube and use. Plates, tubes and cDNA may be kept on ice during the procedure and sealed with AluminaSeal.
  • Genomic DNA from single cells or purified genomic DNA may be amplified using Repli-g Single Cell kit (QIAGEN) following manufacturer's instructions.
  • cDNA and genomic DNA may be stored at -80 °C or used immediately.
  • the primer mix used for this reaction contained isper primer, IgG primer, IgK primer for the immortalized B cells aliquots while for cDNA obtained from plasma cells a mix containing isper primer, IgG primer and IgL primer was used: ispcr primer: (SEQ ID NO: 3)
  • IgG primer (SEQ ID NO: 4)
  • IgK primer (SEQ ID NO: 8)
  • IgL primer (SEQ ID NO: 9)
  • the “ispcr primer” is a “universal” primer, i.e. a primer which is independent from the "target” sequence. This is achieved by designing the “ispcr primer” such that it comprises a sequence, which corresponds to a sequence comprised in the "LNAcac primer” used in the previous step for cDNA synthesis.
  • the sequence of the "ispcr primer”, which is included in the “LNAcac primer” is shown underlined (see above, “LNAcac primer” sequence). Accordingly, the “ispcr primer” can bind to all synthesized cDNA (independent of the encoded gene), because the respective sequence is included in each cDNA by use of the "LNAcac primer” during cDNA synthesis.
  • the respective reverse primer in contrast, is an immunoglobulin isotype specific primer, which binds to an immunoglobulin constant region, which is specific for an immunoglobulin isotype, such as the "IgG primer” for a heavy chain of an antibody of the IgG isotype or the "IgK primer” and “IgL primer” for the kappa and lambda light chain, respectively.
  • the primers used in the PCR amplify DNA sequences encoding heavy and light chains of IgG antibodies expressed by the respective immortalized B cells and plasma cells, independent from the sequences of the variable regions of the antibodies.
  • the reverse primers also contain a sequence exhibiting very similar melting temperatures T m as a sequence contained in the "ispcr primer", which is shown underlined in the above reverse primer sequences.
  • immunoglobulin genes from more than 100 cells were PCR amplified with isotype specific primers.
  • 2 mI cDNA were added to a final volume of 25 mI mix containing 1 x Q5 reaction buffer, 0.5x Q5 High GC Enhancer (New England Biolabs), 200 mM dNTPs (New England Biolabs), 0.4U Q5 polymerase (Q5 Hot Start High-Fidelity DNA Polymerase, New England Biolabs), 0.75 mM ispcr primer, 0.5 mM IgG primer, 0.25 mM IgK primer, and 0.25 mM IgL primer in DNase RNase free water (Invitrogen) in PCR tubes.
  • DNase RNase free water Invitrogen
  • Immunoglobulin DNA was amplified by 30 sec incubation at 98°C, 35 cycles with 10 sec at 98°C, 20 sec at 68°C, 2 min at 72°C, and final extension of 2 min at 72°C on TAdvanced Biometra thermocycler.
  • PCR results are shown in Figure 1 on a gel with three lanes (1, 2 and 3). While lane 1 shows the DNA ladder, lane 2 shows the PCR results on cDNA obtained from a single immortalized B cell and lane 3 shows the PCR results on cDNA obtained from 100 plasma cells.
  • the PCR reaction yielded an amplicon of approximately 1500 base pairs (bp) for the heavy chain, including the entire V-D-J rearrangement and the whole constant region, as shown in Figure 1 as the upper bands in lanes 2 and 3.
  • CPEC vector For circular polymerase extension cloning (CPEC), a CPEC vector was prepared.
  • a "CPEC vector” may be any linearized vector, wherein both termini of the linearized vector end with a specific sequence. Such specific sequences may be appended to both termini of the linearized vector by PCR.
  • AbVec2.0-IGHG1 Additional vector plasmid # 80795; Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H. 2008. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.
  • the vector was digested with restriction enzymes EcoRI and HindIII (New England Biolabs) according to the producer's manual, in order to remove the Ig gene signal peptide sequence and the constant region of the heavy chain present in the vector.
  • restriction enzymes EcoRI and HindIII New England Biolabs
  • the primers in the above-described PCR were designed such that the whole constant regions were amplified (and are, thus, contained in the amplicons to be inserted into the vector), no further constant region sequences are required in the vector.
  • CarpRev primer (SEQ ID NO: 11) These primers comprise sequences, which are complementary to sequences contained in the inserts (amplicons) due to the primers used in the PCR described above (ispcr primer, IgC primer, IgK primer and IgL primer). These complementary sequences are shown underlined in the above sequences of the "PracFwd primer” and the “CarpRev primer”.
  • the CarpRev primer comprises a BmgBI restriction site (shown in the above sequence in italics) and the first 3 nucleotides at the 5' terminus of PracFwd primer (GTC; shown in the above sequence in italics) represent a ("cut") part of a BmgBI restriction site.
  • BmgBI is a restriction endonuclease recognizing the internal sequence CACGTC and cutting after CAC leaving a blunt end.
  • the incorporation of a (blunt-end) restriction site enables re- circularization of the vector by ligase.
  • PCR amplification of the vector 10 ng of linearized vector were added to a final volume of 50 mI mix containing 1 x Q5 reaction buffer, 0.2x Q5 High GC Enhancer (New England Biolabs), 500nM dNTPs (New England Biolabs), 2U Q5 polymerase (Q5 Hot Start High- Fidelity DNA Polymerase, New England Biolabs), 0.5 mM PracFwd primer, 0.5 mM IgG CarpRev primer in DNase RNase free water (Invitrogen) in PCR tubes.
  • Vector was amplified by 30 sec incubation at 98°C, 20 cycles with 10 sec at 98°C, 20sec at 65°C, 2min at 72°C, and final extension of 2min at 72°C.
  • the PCR amplified vector was gel purified according to standard protocol (NucleoSpin ® Gel and PCR clean-up, Macherey-Nagel) and 1 pg of vector was digested in a mix containing 1 x NEBufferTM 3.1 , 1 U of BmgBI (New England Biolabs), in DNase RNase free water (Invitrogen) in Eppendorf tube and incubated 2 hours at 37°C. After digestion the plasmid was purified according to standard protocol (NucleoSpin ® Gel and PCR clean-up, Macherey- Nagel) and re-circularized by treating the digested vector with T4 ligase (New England Biolabs), according to the producer's manual.
  • NucleoSpin ® Gel and PCR clean-up, Macherey-Nagel 1 pg of vector was digested in a mix containing 1 x NEBufferTM 3.1 , 1 U of BmgBI (New England Biolabs), in
  • plasmids were sequenced by Sanger method at Microsynth AG, Balgach, Switzerland, using the following primers: 1 ABsense, 2ABsense, 3ABsense, 4ABsense and 5ABsense:
  • PCR products were cloned into CPEC vector by 1 min incubation at 98°C, 20 cycles with 10 sec at 98°C, 20sec at 65°C, 5min at 72°C, and final extension of 2min at 72°C.
  • expression plasmids containing immunoglobulin heavy and light chains were transformed into competent E. coli as described above for the CPEC vector. Bacteria were then screened for inclusion of heavy and light chains. To this end, single bacterial colonies were used to inoculated LB medium and screened by colony PCR, e.g. as described in Quan et al. (Quan J, Tian J. Circular polymerase extension cloning for high- throughput cloning of complex and combinatorial DNA libraries. Nat Protoc. 2011 Feb;6(2):242-51).
  • DNase RNase free water Invitrogen
  • Heavy and light chain pairs derived from the single immortalized B cell were co-transfected into HEK293 cells to express recombinant antibodies testing different conditions of DNA concentration (50 or 100 ng each chain), HEK293 cell number (0.7, 1 .4, 2.8x10 mL), culture volume (250 uL or 500 uL), number of expression days. All tested conditions resulted in expression of recombinant antibodies.
  • Expi293 cells were maintained in culture in flasks containing Expi293TM Expression Medium (GIBCO) and incubated in a 37°C incubator with 380% relative humidity and 8% C02 on an orbital shaker platform (Kunher incubator) until cultures reach a density of 3-5 x 10 6 viable cells/mL.
  • the day before transfection Expi293 cells were diluted with Expi293TM Expression Medium to a density of 1.5x10 6 /mL in a flask and put back into the incubator.
  • the day of transfection cells were diluted with Expi293TM Expression Medium to a density of 1.5x10 6 /mL in a flask and 800 mL were transferred to each well of a 96 square deepwell plates (PP-Masterblock, 2.0 mL, V-bottom 127, 8/86 mm, sterile, Greiner bio-one).
  • CPEC reaction i.e., the direct, unpurified CPEC product
  • 50 ng of each immunoglobulin chain, heavy and light purified plasmid after bacterial transformation
  • 100 ng of a mix of chains heavy and light purified plasmid after bacterial transformation
  • a mix of heavy and light chains from more than one immunoglobulin pair purified plasmid after bacterial transformation
  • Each plate was sealed with a sandwich covers consisting of a stainless steel cover, 0,2 m filter and microfiber inlays and a flexible silicone layer for sealing (Duetz Microflask system, Applikon Biotechnology) and plates were fixed with the clamp system (Applikon Biotechnology) in a Kunher incubator at 37°C with 380% relative humidity and 8% C02 on an orbital shaker platform running at 500 rpm for 3 days.
  • a sandwich covers consisting of a stainless steel cover, 0,2 m filter and microfiber inlays and a flexible silicone layer for sealing (Duetz Microflask system, Applikon Biotechnology) and plates were fixed with the clamp system (Applikon Biotechnology) in a Kunher incubator at 37°C with 380% relative humidity and 8% C02 on an orbital shaker platform running at 500 rpm for 3 days.
  • ELISA may be used for quantification of the expressed antibodies.
  • Example 2 was essentially carried out as Example 1 described above, however, memory B cells and plasma cells were isolated from frozen PBMC. While in Example 1 only IgC primer was used, also other Ig isotypes (IgA, IgG, IgM and IgE) were investigated in Example 2. To this end, 100x10 6 of total PBMC were processed as described in Example 1 and memory B and plasma cells were single sorted in a 384-well plate as described in Example 1 .
  • IgA, IgG, IgM and IgE Ig isotypes
  • RACE- ready cDNA from a single memory B cell and a single plasma cell was prepared as described in Example 1, followed by PCR.
  • the PCR differed from that of Example 1 in that a mix of ispcr, IgA, IgG, IgM and IgE primers was used to amplify the heavy chain, while for the light chains ispcr, IgK and IgL were used as described above.
  • IgA primer (SEQ ID NO: 5)
  • IgM primer (SEQ ID NO: 5)
  • IgE primer (SEQ ID NO: 7)
  • IgA, IgM and IgE primers were designed in a similar manner as described in Example 1 for the IgG primer, except that they are specific for other immunoglobulin isotypes as indicated (IgA, IgM and IgE, respectively, instead of IgG).
  • PCR results are shown in Figure 2 on a gel with five lanes (1, 2, 3, 4 and 5). While lane 1 shows the DNA ladder, lanes 2 and 4 show the PCR results on cDNA obtained from a single plasma cell and lanes 3 and 5 shows the PCR results on cDNA obtained from a single memory B cell. In contrast to Example 1, separate PCRs were performed for heavy and light chains. The PCR programs were the same as in Example 1 . The higher and lower bands in Figure 2 represent the heavy and light chain PCR products from plasma cell (lanes 2 and 4) and memory B cell (lanes 3 and 5), respectively.
  • Example 3 isolation of antibodies from plasma cells from a donor with disease symptoms
  • PBMC peripheral blood mononuclear cells
  • CD19+, CD27hi, CD38+, HLA DR+ plasma blasts and CD19+, CD27hi, CD38+, HLA DR- plasma cells were single sorted in two 384 well-plates, one for each phenotype, as described in Example 1.
  • cDNA production, PCR amplification and CPEC cloning was performed as described in Examples 1 and 2 with the primers for the constant regions of isotypes IgG, IgA, IgE and IgM as described above.
  • Example 2 Thereafter, 5 mL of CPEC reaction from each well of HLA DR+ plasma blasts were used for Expi293 transfection in 4x96 deep well plates as described in Example 1 .
  • the HLA DR- CPEC plate was stored at -80 °C for a week and used thereafter. After 3 days of expression, plates were tested for isotype identification by ELISA. To this end, each expression plate was screened for the presence of IgG, IgA and IgM.
  • NuncTM MaxiSorpTM ELISA plates were coated with 50 mL either with goat anti-human IgA-UNLB, or goat anti-human IgG-UNLB, or goat anti-human IgM-UNLB diluted in 1xPBS (SIGMA) at a final concentration of 10 pg/mL and incubated overnight at 4 °C (all antibodies used for coating were from SouthernBiotech). After coating plates were washed once with 200 mL of a solution made of 1 xPBS, 0.05% Tween20 (PBS-Tween20), and blocked with 200 mL of 1 xPBS/5% BSA (SIGMA) for one hour at room temperature.
  • 1xPBS 0.05% Tween20
  • 96-well expression plates were removed from the incubator, centrifuged 5 minutes at 500 ref. 100 mL of supernatant from each well was placed at the top of a round bottom 96-well cell culture plate and used for 1 :3 serial dilution in 1xPBS 5% BSA buffer. The remaining supernatant was placed in a new 96-deep well plate, sealed with Acetate foil (Sarstedt) for short term 4 °C storage.
  • IgA, IgG and IgM antibodies were quantified by ELISA using as standard 12 serial dilutions starting from a concentration of 2 pg/mL of human serum IgA, IgG and IgM, and following the expression screening protocol as described above. OD405nm was read with a Biotek plate reader and data were processed and analysed with Biotek Gen5 Data Analyzer Software.
  • Figure 3 illustrates IgA, IgG and IgM distribution in one of the four expression plates.
  • a total of 345 antibodies out of 360 wells screened were obtained from HLA DR+ plate, with an efficiency of 95%, and isotype distribution of 254 IgG (73%), 53 IgA (15%) and 38 IgM (11 %), while for HLA DR- a total of 260 antibodies out of 312 wells (81 %) was obtained with IgG: 164 (63%), IgA: 85 (33%) and IgM: 11 (4%).

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Microbiology (AREA)
  • Peptides Or Proteins (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de génération et de clonage rapides d'une molécule d'ADN codant pour un polypeptide d'intérêt, tel qu'un anticorps. Le procédé combine RACE pour la génération d'ADNc avec un clonage d'extension par polymérase circulaire (CPEC) et est, ainsi, largement indépendant de la séquence. La présente invention concerne également des procédés de production et de séquençage d'une protéine d'intérêt, telle qu'un anticorps, ainsi que des amorces, des vecteurs et des cellules utiles dans de tels procédés.
PCT/EP2019/073112 2019-08-29 2019-08-29 Procédés de production et de clonage d'adnc rapide WO2021037368A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/EP2019/073112 WO2021037368A1 (fr) 2019-08-29 2019-08-29 Procédés de production et de clonage d'adnc rapide
PCT/EP2020/073809 WO2021037886A1 (fr) 2019-08-29 2020-08-26 Procédés de production et de clonage d'adnc rapide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2019/073112 WO2021037368A1 (fr) 2019-08-29 2019-08-29 Procédés de production et de clonage d'adnc rapide

Publications (1)

Publication Number Publication Date
WO2021037368A1 true WO2021037368A1 (fr) 2021-03-04

Family

ID=67810605

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/EP2019/073112 WO2021037368A1 (fr) 2019-08-29 2019-08-29 Procédés de production et de clonage d'adnc rapide
PCT/EP2020/073809 WO2021037886A1 (fr) 2019-08-29 2020-08-26 Procédés de production et de clonage d'adnc rapide

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/EP2020/073809 WO2021037886A1 (fr) 2019-08-29 2020-08-26 Procédés de production et de clonage d'adnc rapide

Country Status (1)

Country Link
WO (2) WO2021037368A1 (fr)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997024455A2 (fr) 1996-01-03 1997-07-10 Clontech Laboratories, Inc. PROCEDES ET COMPOSITIONS DE CLONAGE D'ADNc ENTIER
WO2015040401A1 (fr) * 2013-09-19 2015-03-26 Kymab Limited Production d'un vecteur d'expression et criblage cellulaire à haut rendement
WO2015058780A1 (fr) * 2013-10-25 2015-04-30 Biontech Ag Méthode et kit pour déterminer si un patient présente une réponse immunitaire
WO2015094861A1 (fr) * 2013-12-17 2015-06-25 Clontech Laboratories, Inc. Procédés d'ajout d'adaptateurs à des acides nucléiques et compositions pour leur mise en œuvre
WO2019060771A2 (fr) * 2017-09-22 2019-03-28 University Of Washington Marquage combinatoire in situ de molécules cellulaires
WO2019070655A1 (fr) * 2017-10-03 2019-04-11 Bristol-Myers Squibb Company Vecteurs pour le criblage de séquences de régions variables
EP3476944A1 (fr) * 2016-06-23 2019-05-01 Riken Rt-pcr en une étape avec commutation de matrice
EP3491128A1 (fr) * 2016-07-29 2019-06-05 New England Biolabs, Inc. Procédés et compositions destinés à prévenir la concatémérisation pendant la commutation de matrice

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3673064A4 (fr) * 2017-08-24 2021-05-26 Takara Bio USA, Inc. Procédés de production d'acides nucléiques à l'aide d'oligonucléotides modifiés par un stimulus

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997024455A2 (fr) 1996-01-03 1997-07-10 Clontech Laboratories, Inc. PROCEDES ET COMPOSITIONS DE CLONAGE D'ADNc ENTIER
WO2015040401A1 (fr) * 2013-09-19 2015-03-26 Kymab Limited Production d'un vecteur d'expression et criblage cellulaire à haut rendement
WO2015058780A1 (fr) * 2013-10-25 2015-04-30 Biontech Ag Méthode et kit pour déterminer si un patient présente une réponse immunitaire
WO2015094861A1 (fr) * 2013-12-17 2015-06-25 Clontech Laboratories, Inc. Procédés d'ajout d'adaptateurs à des acides nucléiques et compositions pour leur mise en œuvre
EP3476944A1 (fr) * 2016-06-23 2019-05-01 Riken Rt-pcr en une étape avec commutation de matrice
EP3491128A1 (fr) * 2016-07-29 2019-06-05 New England Biolabs, Inc. Procédés et compositions destinés à prévenir la concatémérisation pendant la commutation de matrice
WO2019060771A2 (fr) * 2017-09-22 2019-03-28 University Of Washington Marquage combinatoire in situ de molécules cellulaires
WO2019070655A1 (fr) * 2017-10-03 2019-04-11 Bristol-Myers Squibb Company Vecteurs pour le criblage de séquences de régions variables

Non-Patent Citations (48)

* Cited by examiner, † Cited by third party
Title
"Post-translational Covalent Modifications of Proteins", 1983, ACADEMIC PRESS
BOERNER, P. ET AL., J. IMMUNOL., vol. 147, 1991, pages 86 - 95
BRUGGEMANN, M. ET AL., YEAR IMMUNOL., vol. 7, 1993, pages 3340
BURTON DRBARBAS CF III, ADV IMMUNOL., vol. 57, 1994, pages 191 - 280
CARBONETTI S. ET AL., J IMMUNOL METHODS, vol. 448, September 2017 (2017-09-01), pages 66 - 73
CARBONETTI SARA ET AL: "A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 448, 26 May 2017 (2017-05-26), pages 66 - 73, XP085153334, ISSN: 0022-1759, DOI: 10.1016/J.JIM.2017.05.010 *
CARBONETTI SOLIVER BGVIGDOROVICH VDAMBRAUSKAS NSACK BBERGL EKAPPE SHISATHER DN: "A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning", J IMMUNOL METHODS, vol. 448, September 2017 (2017-09-01), pages 66 - 73, XP085153334, doi:10.1016/j.jim.2017.05.010
COLE ET AL.: "Monoclonal Antibodies and Cancer Therapy", 1985, ALAN R. LISS, pages: 77
CORTI D ET AL., SCIENCE, vol. 333, 2011, pages 850 - 6
HARRISON, CHARLOTTE: "The full repertoire of humanized antibodies", NATURE REVIEWS DRUG DISCOVERY, vol. 13, 2014, pages 336
HOOGENBOOM HR., METHODS MOL BIOL., vol. 178, 2002, pages 1 - 37
HOOGENBOOM, H. R.WINTER, G., MOL. BIOL., vol. 227, 1992, pages 381 - 388
JAKOBOVITS, A. ET AL., NATURE, vol. 362, 1993, pages 255 - 258
JAKOBOVITS, A. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 2551 - 2555
JIAYUAN QUAN ET AL: "Circular polymerase extension cloning of complex gene libraries and pathways", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 4, no. 7, 30 July 2009 (2009-07-30), pages e6441 - 1, XP002729117, ISSN: 1932-6203, DOI: 10.1371/JOURNAL.PONE.0006441 *
JIN A. ET AL., NAT MED., vol. 15, 2009, pages 1088 - 92
KAI DALLMEIER ET AL: "Simple and inexpensive three-step rapid amplification of cDNA 5' ends using 5' phosphorylated primers", ANALYTICAL BIOCHEMISTRY, vol. 434, no. 1, 1 March 2013 (2013-03-01), pages 1 - 3, XP055055903, ISSN: 0003-2697, DOI: 10.1016/j.ab.2012.10.031 *
KWAKKENBOS MJ ET AL., IMMUNOL REV., 2016
LEFRANC, M.-P. ET AL., NUCLEIC ACIDS RES., vol. 37, 2009, pages D1006 - D1012
MARKS, J. D. ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
MARSHALL OJ: "PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR", BIOINFORMATICS, vol. 20, no. 15, 2004, pages 2471 - 2472
OWCZARZY R. ET AL.: "Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cation", BIOCHEMISTRY, vol. 47, no. 19, 2008, pages 5336 - 5353, XP055040115, Retrieved from the Internet <URL:https://www.dna.utah.edu/tm> doi:10.1021/bi702363u
OWCZARZY R. ET AL.: "Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cation", ΒIOCHEMISTRY, vol. 47, no. 1 9, 2008, pages 5336 - 5353, XP055040115, Retrieved from the Internet <URL:URL:https://www.dna.utah.edu/tm> doi:10.1021/bi702363u
PICELLI SFARIDANI ORBJORKLUND AKWINBERG GSAGASSER SSANDBERG R: "Full-length RNA-seq from single cells using Smart-seq2", NAT PROTOC., vol. 9, no. 1, January 2014 (2014-01-01), pages 1 71 - 81, XP002742134, doi:10.1038/nprot.2014.006
PICELLI SIMONE ET AL: "Full-length RNA-seq from single cells using Smart-seq2", NATURE PROTOCOLS, NATURE PUBLISHING GROUP, GB, vol. 9, no. 1, 1 January 2014 (2014-01-01), pages 171 - 181, XP037004947, ISSN: 1754-2189, [retrieved on 20140102], DOI: 10.1038/NPROT.2014.006 *
PROC NATL ACAD SCI USA, vol. 83, no. 1, 1986, pages 4 - 8
QUAN JTIAN J: "Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries", NAT PROTOC., vol. 6, no. 2, February 2011 (2011-02-01), pages 242 - 51
QUAN JTIAN J: "Circular polymerase extension cloning of complex gene libraries and pathways", PLOS ONE, vol. 4, no. 7, 30 July 2009 (2009-07-30), pages e6441, XP002729117, doi:10.1371/journal.pone.0006441
RATTAN ET AL.: "Protein Synthesis: Post-translational Modifications and Aging", ANN NY ACAD SCI, vol. 663, 1992, pages 48 - 62
SAFDARI YFARAJNIA SASGHARZADEH MKHALILI M: "Antibody humanization methods - a review and update", BIOTECHNOL GENET ENG REV., vol. 29, 2013, pages 1 75 - 86
SAMBROOK, J.RUSSELL, D.W: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SCHOONBROODT SONIA ET AL: "Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library", NUCLEIC ACIDS RESEARCH, INFORMATION RETRIEVAL LTD, vol. 33, no. 9, 1 January 2005 (2005-01-01), pages E81 - 1, XP002581479, ISSN: 0305-1048, [retrieved on 20050519], DOI: 10.1093/NAR/GNI080 *
SEIFTER ET AL.: "Analysis for protein modifications and nonprotein cofactors", METH. ENZYMOL., vol. 182, 1990, pages 626 - 646, XP009082492, doi:10.1016/0076-6879(90)82049-8
SPIDEL JARED L ET AL: "Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 439, 24 September 2016 (2016-09-24), pages 50 - 58, XP029823318, ISSN: 0022-1759, DOI: 10.1016/J.JIM.2016.09.007 *
SPIDEL JLVAESSEN BCHAN YYGRASSO LKLINE JB: "Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells", J IMMUNOL METHODS, vol. 439, December 2016 (2016-12-01), pages 50 - 58, XP029823318, doi:10.1016/j.jim.2016.09.007
TIJSSEN: "Overview of principles of hybridization and the strategy of nucleic acid probe assays", 1993, ELSEVIER, article "Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes"
TILLER ET AL: "Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 329, no. 1-2, 31 October 2007 (2007-10-31), pages 112 - 124, XP022389335, ISSN: 0022-1759 *
TILLER T. ET AL., JOURNAL OF IMMUNOLOGICAL METHODS, vol. 329, 2008, pages 112 - 124
TILLER TMEFFRE EYURASOV STSUIJI MNUSSENZWEIG MCWARDEMANN H: "Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning", J IMMUNOL METHODS, vol. 329, no. 1-2, 2008, pages 112 - 24, XP022389335
TRAGGIAI EBECKER SSUBBARAO KKOLESNIKOVA LUEMATSU YGISMONDO MRMURPHY BRRAPPUOLI RLANZAVECCHIA A: "An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus", NAT MED., vol. 10, no. 8, 2004, pages 871 - 5, XP002385893
TRAGGIAI ET AL., NAT MED, vol. 10, 2004, pages 871 - 875
UNTERGASSER ACUTCUTACHE IKORESSAAR TYE JFAIRCLOTH BCREMM MROZEN SG: "Primer3--new capabilities and interfaces", NUCLEIC ACIDS RES., vol. 40, no. 15, 1 August 2012 (2012-08-01), pages e115
VAN DIJK, M. A.VAN DE WINKEL, J. G., CURR. OPIN. CHEM. BIOL., vol. 5, 2001, pages 368 - 374
VON BOEHMER LLIU CACKERMAN SGITLIN ADWANG QGAZUMYAN ANUSSENZWEIG MC: "Sequencing and cloning of antigen-specific antibodies from mouse memory B cells", NAT PROTOC., vol. 11, no. 10, October 2016 (2016-10-01), pages 1908 - 1923
WARDEMANN HYURASOV SSCHAEFER AYOUNG JMEFFRE ENUSSENZWEIG M: "Predominant autoantibody production by early human B cell precursors", SCIENCE, vol. 301, 2003, pages 1374 - 1377, XP009136339, doi:10.1126/science.1086907
WRAMMERT JSMITH KMILLER JLANGLEY WAKOKKO KLARSEN CZHENG N-YMAYS IGARMAN LHELMS C: "Rapid cloning of high-affinity human monoclonal antibodies against influenza virus", NATURE, vol. 453, 2008, pages 667 - 671, XP002524388, doi:10.1038/NATURE06890
YE JCOULOURIS GZARETSKAYA ICUTCUTACHE IROZEN SMADDEN T: "Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction", BMC BIOINFORMATICS, vol. 13, 2012, pages 134, XP021132324, doi:10.1186/1471-2105-13-134
ZHANG C., METHODS MOL BIOL., vol. 901, 2012, pages 11 7 - 35

Also Published As

Publication number Publication date
WO2021037886A1 (fr) 2021-03-04

Similar Documents

Publication Publication Date Title
AU2020213348B2 (en) Uniquely tagged rearranged adaptive immune receptor genes in a complex gene set
JP7357661B2 (ja) 試料に関連するポリヌクレオチドの同定
US20210172015A1 (en) Immune profiling by primer extension target enrichment
KR20180133840A (ko) 면역종양학을 위한 조성물 및 방법
Romão et al. Construction of high-quality camel immune antibody libraries
CN114685671B (zh) 特异性结合Taq DNA聚合酶的单克隆抗体及其应用
US20110053803A1 (en) Methods for creating antibody libraries
Duan et al. Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections
KR20110058861A (ko) 조류 유래 항체의 클로닝 방법
KR20200085781A (ko) 선택된 항체를 발현하도록 유전자 변형된 b 세포를 생산하기 위한 시스템 및 방법
TW202216754A (zh) 雙特異性抗體car細胞免疫療法
KR20230052291A (ko) Ceacam 양성 암을 치료하기 위한 조성물 및 방법
CN102325903A (zh) 得到免疫球蛋白编码核酸的方法
KR20230051677A (ko) 메소텔린 양성 암을 치료하기 위한 조성물 및 방법
WO2021037368A1 (fr) Procédés de production et de clonage d&#39;adnc rapide
US20090042740A1 (en) Novel Method for Cloning Variable Domain Sequences of Immunological Gene Repertoire
AU2013352067A1 (en) Preparation of gene-specific templates for the use in single primer amplification
TW202214846A (zh) 用於治療ｅｇｆｒ陽性癌症之組合物及方法
CA2970023C (fr) Procede de production d&#39;anticorps monoclonal de lapin recombine specifique d&#39;un allotype
RU2775914C2 (ru) Способ амплификации нуклеиновых кислот тяжелой и легкой цепи человеческих иммуноглобулинов для создания рекомбинантных антител, композиции праймеров (варианты)
EP1392866B1 (fr) Nouveau procede pour cloner les sequences a domaine variable du repertoire des genes immunologiques
WO2022238890A1 (fr) Procédé de production d&#39;anticorps à partir de cellules plasmatiques uniques
AU2002309777A1 (en) Novel method for cloning variable domain sequences of immunological gene repertoire

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19762129

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19762129

Country of ref document: EP

Kind code of ref document: A1