(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`19 November 2015 (19.11.2015)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
`
`WO 2015/173402 A1
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(51)
`
`International Patent Classification:
`CIZN 15/10 (2006.01)
`C12Q 1/68 (2006.01)
`
`(81)
`
`(21)
`
`International Application Number:
`
`PCT/EP2015/060777
`
`(22)
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`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
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`Filing Language:
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`Publication Language:
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`15 May 2015 (15.05.2015)
`
`English
`
`English
`
`Priority Data:
`141683136
`
`14 May2014 (14.05.2014)
`
`EP
`
`RUPRECHT-KARLS-UNIVERSITAT
`Applicants:
`HEIDELBERG [DE/DE]; Grabengasse 1, 69117 Heidel-
`berg (DE). DEUTSCHES KREBSFORSCHUNGSZEN-
`TRUM STIFTUNG DES OFFENTLICHEN RECHTS
`[DE/DE]; Im Neuenheimer Feld 280, 69120 Heidelberg
`(DE).
`
`Inventors: TURCHINOVICH, Andrey; Bellenstr. 26,
`68163 Mannheim (DE). SUROWY, Harald; Fahrbachweg
`4, 69126 Heidelberg (DE) BURWINKEL, Barbara;
`Montpellierstr. 13, 691 15 Heidelberg (DE).
`
`(74)
`
`Agent: ZWICKER, Jork; Radlkoferstr. 2, 81373 Munich
`(DE).
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`Published:
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`with international search report (Art. 21(3))
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`with sequence listing part ofdescription (Rule 5.2(a))
`
`(54) Title: SYNTHESIS OF DOUBLE—STRANDED NUCLEIC ACIDS
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`(57) Abstract: The present invention relates to a method for the synthesis of double—stranded nucleic acids from a Wide variety of
`samples and comprises the use of these nucleic acids for deep sequence analysis. Also, the present invention relates to specific re -
`agents used in the method of the present invention. Further, the invention relates to kits comprising reagents for the method of the
`invention and use of said kits.
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`
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`WO 2015/173402
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`PCT/EP2015/060777
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`Synthesis of double-stranded nucleic acids
`
`The present invention relates to a method for the synthesis of double—stranded nucleic acids
`
`from a wide variety of samples and comprises the use of these nucleic acids for deep sequence
`
`analysis. Also, the present invention relates to specific reagents used in the method of the present
`
`invention. Further, the invention relates to kits comprising reagents for the method of the invention
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`and use of said kits.Background of the Invention
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`Massive parallel sequencing (MPS) of nucleic acids requires the preparation of amplified
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`libraries where the region of the DNA to be sequenced is located between known 5’— and 3’— terminal
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`sequences. Current methods for MP8 libraries construction utilize either RNA or DNA adaptor
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`ligation to the 5’— and 3’- ends of the RNA or DNA samples. Ligation of adaptors is not only time
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`consuming but also a process of low efficiency that requires microgram inputs of nucleic acid samples.
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`In addition, the resulting cDNA libraries are contaminated with adaptors cross— and self—ligation by—
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`products and require additional purification steps both before and after pre—amplification. More than
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`a decade ago, Clontech Laboratories described a method that harnesses the template switching activity
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`of the Moloney murine leukemia virus reverse transcriptase (MMLV —RT) to attach adaptors of choice
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`to the 5’—end of cDNA generated from of poly(A) tailed mRNA molecules. At the same time, a 3’—
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`adaptor sequence was incorporated into poly(dT) reverse transcription primer. This principle, named
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`SMART, is currently used in an Illumina Ultra Low RNA sequencing kit (Clontech) to generate full
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`length cDNA copies of mRNA molecules from a single cell. However, the method still requires
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`subsequent to the template synthesis (1) fragmentation of amplified cDNA, (2) ligation of platform-
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`specific 5’/3’—end adaptors and (3) pre—amplification of adaptors—ligated DNA fragments. Although
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`the SMART method is capable of preparing cDNA for sequencing from single—cell amounts of RNA,
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`it is time consuming, expensive and restricted to mRNA sequencing. So far, the approach of using
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`template switching activity of MMLV—RT has not been yet applied to sequence (1) RNA molecules
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`other than long RNAs and (2) any DNA molecules. The present invention describes a method to
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`generate ready—to—sequence double or single stranded DNA, preferably DNA libraries from picogram
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`(pg) amounts of either RNA or DNA molecules in a time frame of only a few hours. Small (<150 bp)
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`RNAs or DNAs (e.g. miRNA (microRNAs), piRNAs (piwiRNAs), degraded or bisulfite—converted
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`DNA) can be used as an input directly. However, long RNA or DNA has to be first fragmented by a
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`corresponding approach (e.g. sonication for DNA or Mg2+ incubation for RNA). The method of the
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`invention provides several advantages, which include a dramatic reduction in time required to provide
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`ready to sequence DNA, which may be based on DNA or RNA, the method is drastically cheaper than
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`any of the prior art methods. Current commercial kits for cDNA library preparation for next generation
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`sequencing of RNA and DNA are priced between $200 and $500 per samples depending on the
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`application, type of the kit and brand of the supplier. The rough estimates of the costs required for a
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`single DNA library preparation using the method of the invention is at least 20—fold lower, and the
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`method of the invention will permit sequencing of nucleic acids from sources from which sequencing
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`10
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`was impossible before due to the minimal amounts of DNA and/or RNA that could be obtained from
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`the sample. Examples of those include: DNA and RNA from small (diagnostic) amounts of liquid and
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`solid biopsies, targeted compartments of the cells (e. g. micronuclei, endoplasmic reticulum), fossils,
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`remnants of the extinct organisms, and forensics samples containing minute and highly fragmented
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`DNA molecules. The present invention is based in part on the discovery that DNA can also serve as
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`a substrate for a reverse tran scriptase.
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`Summary of the Invention
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`In a first aspect the present invention provides a method for the synthesis of double stranded
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`nucleic acid with a defined 3’ and 5’ terminal nucleotide sequence from a sample comprising single
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`stranded nucleic acid comprising the steps of:
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`a)
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`providing a sample comprising single stranded or double stranded nucleic acid, optionally
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`denaturing the double stranded nucleic acid;
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`b)
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`adding at least 5, preferably between 10 and 50 consecutive nucleotides to the 3—terminus of
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`the single stranded or double stranded nucleic acid,
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`c)
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`hybridizing a priming oligonucleotide complementary to the added nucleotide sequence and
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`synthesizing a cDNA or cRNA with a template dependent DNA or RNA polymerase to
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`generate a double stranded nucleic acid,
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`d)
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`hybridizing a template switching oligonucleotide (TSO) to said double stranded nucleic acid,
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`and
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`e)
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`extending the 3’ end of the cDNA or cRNA strand to synthesize a double stranded nucleic
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`acid, wherein one strand of the nucleic acid comprises the priming oligonucleotide, and a
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`cDNA or a cRNA that is complementary to the single stranded nucleic acid and to the template
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`switching oligonucleotide.
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`In a second aspect the present invention provides a priming oligonucleotide comprising the
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`following sequence elements:
`
`wherein
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`3 , 'Wm—X'Yn'z 1 o'Qt'ZZs—S ‘ ,
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`at each instance is independently selected from dA, dG, dC, dT and dU;
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`is selected from dA, dG, dC, dT, dU, rA, 1G, rC, rT and rU;
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`is a polynucleotide of at least 10 nucleotides length, wherein 80% or more of the sequence is
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`composed of an identical nucleotide or dinucleotide selected from dA, dG, dC, dT, dU, rA, rG,
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`rC, rT, rU, AC, AG, AT, AU, CA, CG, CT, CU, GA, GC, GT, GU, TA, TC, TG, TU, AA, CC,
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`GG, TT, UU, UA, UC, UG, and UT, wherein the other at most 20% or less of the sequence is
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`composed of nucleotides or dinucleotides that are different from the major nucleotide or
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`dinucleotide and also selected from dA, dG, dC, dT, dU, rA, rG, rC, rT, rU, AC, AG, AT, AU,
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`CA, CG, CT, CU, GA, GC, GT, GU, TA, TC, TG, TU, AA, CC, GG, TT, UU, UA, UC, UG,
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`and/or UT, with the proviso that X is different from the nucleotide or dinucleotide that
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`constitutes the majority of Y;
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`is a sequence of consecutive degenerate (wobble) DNA bases, preferably selected from N, V,
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`H, D, B and J, wherein N is the product of the incorporation of a nucleotide from an equimolar
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`mixture of dA, dT, dC and dG; B is the product of the incorporation of a nucleotide from an
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`equimolar mixture of dT, dC and dG; D is the product of the incorporation of a nucleotide
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`from an equimolar mixture of dA, dT and dG; H is the product of the incorporation of a
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`nucleotide from an equimolar mixture of dA, dT and dC; V is the product of the incorporation
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`of a nucleotide from an equimolar mixture of dA, dC and dG, J is the product of the
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`incorporation of a nucleotide from amixture of (0—100% dA) to (0—100% dG) to (0—100% dC)
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`to (0—100% dT) to (0—100% dU) to (0-100% rA) to (0-100% rG) to (0—100% rC) to (0-100%
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`W X Y
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`10
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`20
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`25
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`rT) to (0-100% rU);
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`Z1
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`30
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`Z2
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`is a polynucleotide of at least 5 nucleotides length of defined sequence, wherein the sequence
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`is different from Wm—X—Yn, preferably the sequence is also different from Q 7225;
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`is a polynucleotide of at
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`least 5 nucleotides length of defined sequence, wherein the
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`sequence is different from Wm—X—Yn—Zlo-Qt;
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`is an integer of 0 to 6, i.e. 0, l, 2, 3, 4, 5 or 6;
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`is an integer of 10 to 100, ifY is selected from dA, dG, dC, dT, dU, rA, rG, rC, rT, and rU, an
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`integer of 5 to 50, if Y is selected from AC, AG, AT, AU, CA, CG, CT, CU, GA, GC, GT,
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`GU, TA, TC, TG, TU, AA, CC, GG, TT, UU, UA, UC, UG and UT;
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`is O or 1;
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`is O or 1; and
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`is an integer of 0 to 6, i.e. 0, l, 2, 3, 4, 5 or 6.
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`In a third aspect
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`the present
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`invention provides a template switching oligonucleotide
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`comprising the following sequence elements
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`10
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`5 9 'Xp'Y—Qt'Zq'Ar'3 ’
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`wherein
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`X
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`t—<
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`N
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`is a chemical group selected from the group consisting of amino, biotin, glycerol, cholesterol,
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`digoxigenin, fluoro residue or nucleotide derivatives including abasic nucleotides, dideoxy—
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`ribonucleotides, 3’—deoxynucleotides, 2’—deoxyinosine, 2’—deoxyuridine;
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`is a known oligonucleotide sequence;
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`is a sequence of consecutive degenerate (wobble) DNA bases, preferably selected from N, V,
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`H, D, B and J, wherein N is the product of the incorporation of a nucleotide from an equimolar
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`mixture of dA, dT, dC and dG; B is the product of the incorporation of a nucleotide from an
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`equimolar mixture of dT, dC and dG; D is the product of the incorporation of a nucleotide
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`from an equimolar mixture of dA, dT and dG; H is the product of the incorporation of a
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`nucleotide from an equimolar mixture of dA, dT and dC; V is the product of the incorporation
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`of a nucleotide from an equimolar mixture of dA, dC and dG, J is the product of the
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`incorporation of a nucleotide from arnixture of (0—100% dA) to (0—100% dG) to (O—lOO% dC)
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`to (0—100% dT) to (0—100% dU) to (0—100% 1A) to (0—100% 1G) to (0—100% rC) to (0—100%
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`rT) to (0-100% rU);
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`is a ribonucleotide selected from the group consisting of AMP, CMP, GMP, TMP and UMP,
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`is a chemical group selected from the group consisting of amino, biotin, glycerol, cholesterol,
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`digoxigenin, phosphate, fluoro residue or nucleotide derivatives including abasic nucleotides,
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`dideoxy—ribonucleotides, 3’—deoxynucleotides, 2’—deoxyinosine, 2’—deoxyuridine;
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`is an integer of 0 to 6, i.e. 0, l, 2, 3, 4, 5 or 6;
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`is an integer of 0 to 10, i.e. 0, l, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
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`is an integer of at least 1; and
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`r
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`is an integer of 0 to 10, i.e. 0, l, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
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`In a fourth aspect the present invention provides a nucleic acid comprising the priming
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`oligonucleotide of the second aspect of the invention.
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`In a fifth aspect the invention provides a kit comprising
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`a)
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`a reagent capable of adding nucleotides to the 3—terminus of the single stranded nucleic acid,
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`preferably an enzyme, more preferably a poly(A)—polymerase or terminal transferase (TT), and
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`optionally a blocking nucleotide preferably 3d—NTP, 3—Me—NTP and ddNTP
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`b)
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`c)
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`d)
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`a reverse transcriptase enzyme,
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`the priming oligonucleotide according to the second aspect, and
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`a template switching oligonucleotide according to the third aspect.
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`In a sixth aspect the present invention provides an array comprising at least one nucleic acid
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`comprising the priming oligonucleotide of the fourth aspect of the present invention.
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`In a seventh aspect the present invention provides the use of said kit and the use of the
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`synthesized double—stranded nucleic acid in personalized medicine; therapy monitoring; prediction,
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`prognosis, early detection of human or animal disease or forensic science analysis of nucleic acid
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`sequences of vim ses, bacteria, animals or plants or cells derived therefrom.
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`List of Figures
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`In the following, the content of the figures comprised in this specification is described. In this
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`context please also refer to the detailed description of the invention above and/or below.
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`Figure 1: Schematic representation of cDNA preparation methods using a combination of
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`polyA(dA) tailing and template switching capacity of MMLV-RT. Briefly, short single stranded
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`RNA or DNA fragments are polyadenylated or polydeoxyadenylated with either poly(A) polymerase
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`or terminal deoxytransferase. Then, a complementary DNA strand synthesis is carried out in the
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`presence of anchored poly(dT) oligonucleotide containing a custom 3’-adaptor sequence. Optionally,
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`the oligonucleotide comprises three different nucleotides at its 3’ prime end, i.e. C, G, or A (=V in the
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`schematic representation of Figure 1). When reverse transcriptase reaches the 5’ end of the RNA (or
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`DNA) template, the enzyme’s terminal transferase activity adds additional nucleotides (predominantly
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`dC) that are not encoded by the template. On the next step, the template switching oligonucleotide
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`containing three terminal rG nucleotides and custom 5’—adaptor sequences is added to the RT reaction
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`and serves as second template for the reverse transcriptase. The complementary interaction of the
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`three consecutive rG nucleotides at the 3’—end of the T80 and the dC—rich extended sequence of the
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`cDNA are thought to promote template switching. The second cDNA strand is generated during the
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`first cycle of the standard PCR reaction from a forward primer which is either fully or partially
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`complementary to the 3’-terminus of the first cDNA strand. Furthermore, the reverse primer used for
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`the PCR amplification of the cDNA (together with forward primer) is either fully or partially
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`complementary to the 3’ —terminus of the second cDNA strand.
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`Figure 2: To construct DNA libraries suitable for Illumina MiSeq or HiSeq platforms we have
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`used adaptor sequences from the N EBnext Small RNA Sequencing Kit (New England Biolabs). The
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`sequence corresponding to the 5’—adaptor was incorporated into the TSO and the 3’—adaptor sequence
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`was used to design a terminal tag of poly(dT) primer (Figure 2A). Either 1 ng or 5 pg of 22 nt RNA
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`and DNA as inputs for the DNA library preparation were used (Figure 2B). The efficacy of cDNA
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`synthesis was equal for DNA and RNAs. When using 1 ng of nucleic acids, a single PCR product
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`appeared after 17 PCR pre-amplification cycles (1/100 cDNA to PCR dilution). When 5 pg of nucleic
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`acids were used as an input, the amount of PCR cycles required to pre— amplify cDNA increased to 26.
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`When a lO/lOO cDNA to PCR dilution was used, the amount of cycles necessary to generate DNA
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`libraries decreased proportionally (data not shown). The only contaminating by—product in the reaction
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`were the excess of PCR primers, most of which can be removed by column purification. Sanger
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`sequencing has further confirmed that cDNA prepared from synthetic short DNA was pure (data not
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`shown).
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`Figure 3: Critical parameters of the DNA library preparation protocol 1. Primarily, the
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`poly(A) tailing reaction is critical for the optimal yield of cDNA. Too long poly(A) tails will
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`eventually decrease the effective concentration of poly(dT) primer, which will not only decrease the
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`amount of cDNA but also lead to a smear of larger by—products on the gel since the poly(dT) primer
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`will hybridize to various sites within the poly(A) tail. Figure 3A shows in the upper panel an
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`electropherogram obtained after 3% agarose gel electrophoresis of 1 ng cel—miR—39 which was
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`poly(A) tailed using different incubation times and concentrations of ATP. The lower panel shows an
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`electropherogram obtained after 3% agarose gel electrophoresis of DNA libraries generated from 1
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`ng of corresponding poly(A)
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`tailed cel—miR—39 using 100 nM ILPdTPo.
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`In Figure 3B an
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`electropherogram obtained after 3% agarose gel electrophoresis of DNA libraries generated from 1
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`ng of cel-miR-39 (poly(A) tailed for 10 min using different concentrations of ATP) using either 100
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`nM one—base anchored polydT primer (ILPdTPo) or 100 nM two—base anchored polydT primer
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`(ILPcTPt)
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`is
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`shown. Figure 3C shows electropherograms obtained after 3% agarose gel
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`electrophoresis of DNA libraries generated from 1 ng of cel—miR—39 (poly(A) tailed for 10 min using
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`0.] mM ATP) using MMLV—RT of different brands and using either 1 uM or 0.] uM of TSOS. Upper
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`figure: PCR amplification of cDNAs was performed for 17 cycles. Lower figure: PCR amplification
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`of cDNAs was performed for 21 cycles. 10 min poly(A)-tailing time and the 0.1 mM of final ATP
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`gave decent results for 22 nt RNA cloning. Secondly, the supplier and the brand of MMLV-RT
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`appeared to be critical for the sensitivity of the approach. Thus, out of 6 commercial MMLV—RTs
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`SuperScribe II (Invitrogen), SMARTScribe RT (Clontech) and SMART RT (Clontech) were most
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`efficient in providing the detectable amounts of cDNA after pre—amplification with the current
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`protocol, while the SuperScribe lll (lnvitrogen), Multiscribe RT (Applied Biosystems) and M—MLV
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`from NEB required 4 additional cycles of pre—amplification for a DNA library to be visible on agarose
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`gel (Figure 3C). This phenomenon can be explained by the fact that different MMLV—RT variants
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`might possess different RNAse H and terminal transferase activities (the latter is thought to facilitate
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`10
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`the template switching reaction). Thus the selection of an RT with RNAse H activity is preferred.
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`Figure 4: Critical parameters for the cDNA library protocol 11. An electropherogram
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`obtained after 3% agarose gel electrophoresis of DNA libraries generated from 1 ng cel—miR—39
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`(poly(A) tailed for 10 min using 0.1 mM ATP) using different template switching oligonucleotides
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`(TSO) at a final concentration of 1 MM is shown. Upper figure: PCR amplification of cDNAs was
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`performed for 17 cycles. Lower figure: PCR amplification of cDNAs was performed for 2] cycles
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`(Figure 4A). The structure of TSO appears to be critical for the sensitivity and the performance of the
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`method. Both pure DNA and pure RNA TSO failed to yield any adequate amount of the targeted
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`cDNA after 17 cycles of pre-amplification PCR. This could be eXplained by the fact that a sequence
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`of three riboG has a much stronger affinity for the template switching than three deoxyriboG, while
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`the pure RNA oligonucleotide is prone to forming significant secondary structures that decrease the
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`availability of the 3’—terminus. Furthermore, when TSO with four instead of three terminal riboG
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`nucleotides was used, the yield of the cDNA was dramatically reduced (Figure 4A), presumably due
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`to the ability of four consecutive G to form quadrupleX structures. An option of blocking the terminal
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`3—OH group of the TSO to prevent its polyA tailing which might occur when poly(A) polymerase is
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`not completely deactivated was also tested. Although thermal deactivation of E. coli poly(A)
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`polymerase for 20 min at 65°C before the RT reaction was complete, the usage of 3—OH blocked TSO
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`would be mandatory in case that: (l) poly(A) tailing and the RT are performed simultaneously or (2)
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`poly(A) tailing of RNA cannot be heat inactivated. Surprisingly, blocking the 3-OH terminal of TSO
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`with either monophosphate or biotin abrogated the efficacy cDNA synthesis under the conditions used
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`(Figure 4A). Nevertheless, when 3—OH group of TSO was blocked with phosphate or dideoxycytidine
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`(ddC), similar amounts of cDN A product appeared four PCR cycles later.
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`In Figure 4B an
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`electropherogram obtained after 4% agarose gel electrophoresis (left) and A gilent Bioanalyser (right)
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`of DNA libraries generated from 1 ng cel—miR—39 (poly(A) tailed for 10 min using 0.1 mM ATP)
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`using 5’-end unblocked TSO3 or 5’-biotin-blocked TSOS is shown. Note the small fraction of the ~30
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`bp longer DNA libraries which likely correspond to the products of secondary template switching
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`events (white arrow).
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`Figure 5: Critical parameters of the DNA library preparation protocol III. Panel A:
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`Electropherograms obtained after 3% agarose gel electrophoresis of DNA libraries generated from 1
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`ng of different template cel—miR—39 oligos (RN As were poly(A) tailed for 10 min using 0.1 mM ATP;
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`DNAs were poly(dA) tailed for 30 min using 0.1 mM ATP) is shown. The efficacy of DNA library
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`synthesis from DNA templates containing 5’—biotin is dramatically lower as compared to the 5’—OH
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`or 5’—Phosphate templates. Panel B: An electropherogram obtained after 3% agarose gel
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`10
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`electrophoresis of DNA libraries generated from 1 ng of cel-miR-39 RNA (poly(A) tailed for 10 min
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`using 0.1 mM ATP) using either water or 20% DMSO (5% in final reaction) as a media for RT reaction
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`is shown. The addition of DMSO does not interfere with the efficacy of DNA library preparation.
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`Figure 6: DNA libraries preparation from human RNA and DNA. Panel A: An
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`electrophero gram obtained after 4% agarose gel electrophoresis (left) of DNA libraries generated from
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`1 ng of control cel—miR—39 (C39R) and 1 ng ofpoly(A) enriched RNA isolated from U208 cells which
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`was fragmented by incubation with magnesium ions for 10 min (R10) was shown. In addition, one
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`DNA library was generated from R10 sample which was not pre—treated with T4 PNK before poly(A)
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`tailing (—RNK labeled). The number of PCR cycles used for the pre-amplification of the cDNA
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`libraries and the concentration of poly(dT) reverse primer (ILPdTPo) are indicated below the
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`electrophero gram for each sample. DNA libraries which were sequenced on Illumina MiSeq were cut
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`from the agarose gel, isolated by PureLink Gel Purification kit and analyzed by Agilent Bioanalyser
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`High Sensitivity DNA chips (right). Panel B: Left: An electropherogram obtained after 3% agarose
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`gel electrophoresis of DNA libraries generated from approximately 3 ng of bisulfite—converted DNA
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`from U208 cells was shwon (Figure 6B). In addition, one DNA library was generated from B sample
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`which was pre—treated with T4 PNK before poly(dA) reaction (+RNK labeled). In negative control
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`library, 1 “L on water was used (H20). Right: Agilent Bioanalyser electropherogram showing gel
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`purified DNA libraries generated from 1 ng of poly(A) enriched RNA from U208 cells which was
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`fragmented by incubation with magnesium ions for 5 min (R5), bisulfite-converted DNA (B) and 1
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`ng of control cel—miR—39 RNA (C39R). Panel C: Electropherogram obtained after 4% agarose gel
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`electrophoresis (left) and Agilent Bioanalyser (right) of DNA libraries generated from approximately
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`150 pg of human blood plasma DNA isolated from two healthy donors (D1 and D11). In control
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`experiments either water (H20) or 1 ng of synthetic cel—miR—39 DNA (C39D) were used. The number
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`of PCR cycles used for the pre—amplification of the cDNA libraries and the concentration of poly(dT)
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`reverse primer (ILPdTPo) are indicated below the electrophero gram for each sample. Panel D:
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`Electropherogram obtained after 4% agarose gel electrophoresis of DNA libraries generated from
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`approximately 200 pg of human blood plasma RNA isolated from two healthy donors (RI and RH).
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`In control experiments either water (H20) or 1 ng of synthetic cel—miR—39 RNA (C39R) were used.
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`In addition, DNA libraries were generated from circulating RNA samples which were not pre—treated
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`with T4 PN K before poly(A) tailing (—RNK labeled). The number of PCR cycles used for the pre—
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`amplification of the cDNA libraries and the concentration of poly(dT) reverse primer (lLPdTPo) are
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`indicated below the electropherogram for each sample. DNA libraries from circulating plasma RNA
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`of both individuals were purified from agarose gel, however, only RI library was sequences on
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`lllumina MiSeq.
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`Figure 7: Accuracy of the index read for multiplexed sample libraries in one sequencing
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`lane depending on the sequence of the priming oligonucleotide. Shown are the respective ratios of
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`index reads that were identified with zero errors and one error, depending on the sequence composition
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`of the priming oligonucleotide used to generate the double stranded nucleic acid, more precisely on
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`the portion complementary to the poly(A)tail created in the prior step of the method. Eight DNA
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`fragment libraries were generated in parallel from identical (1 ng) amounts of the input source material
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`(human genomic DNA), using four different priming oligonucleotides as shown and two replicates
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`for each oligonucleotide. The eight resulting libraries were pre—amplified with primers as appropriate
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`for multiplexed sequencing on current Illumina sequencer systems, each reverse primer including a
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`different index sequence that is used to determine the library of origin for an identified read. The used
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`index sequences correspond to Illumina index sequences 1—8 and each index sequence differs in at
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`least 3 positions from all others. Equimolar amounts of each library were pooled and single—end
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`sequenced on one lane with 70 cycles for read#l and 6 cycles for the index read on an lllumina MiSeq
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`system. For each of the eight libraries with different index sequences, the corresponding number of
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`index read sequences containing no errors or one error were recorded. Depicted are the mean
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`frequency values of index sequence reads with 0 or 1 error of the two libraries generated with each of
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`the four different types of priming oligonucleotides, respectively, with error bars denoting the
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`difference of the means to the values of the single libraries. Evidently, using the priming
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`oligonucleotide “20G” allows a considerably increased accuracy of index read sequence compared to
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`the “30A” priming oligonucleotide, while maintaining the same efficiency of DNA fragment library
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`generation.
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`Figure 8: The advantage of controllable vs. non-controllable polynucleotide tailing on DNA
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`and RNA templates.
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`An example demonstrating beneficial effects of controllable poly(A)- and poly(dA)—tailing on
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`the yield of CDNA generated from synthetic cel—miR—39 DNA (left) and cel—miR—39 RNA (right).
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`Controllable poly(A)— and poly(dA)—tailing allows more efficient production of libraries using the
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`same concentration of the RT primer, and/or when the concentration of ATP in the solution is
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`suboptimal. If the ratio of ATP (or dATP) to RNA (or DNA) template is higher than optimal, than
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`long (>300 nt) tails would result. Long polynucleotide tails decrease the effective concentration of
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`poly(dT) primer what decrease the yield of the library and produce a smear of larger by—products on
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`the gel since the excess of poly(dT) primer hybridizes to a site within the large poly(A) tail. A:
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`Electropherogram of 3% agarose gel electrophoresis of DNA libraries obtained after poly(dA)—tailing
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`of 1 ng cel—miR—39 DNA template and using 10 nM poly(dT) reverse primer (ILPdTPo) either in the
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`presence (C) or absence (NC) of the blocking ddATP nucleotide (dATP/ddATP ratio 1/50). Note,
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`significantly higher yield of the library after controllable poly(dA)—tailing is achieved with the same
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`concentration of
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`the reverse primer. B: electropherogram obtained after 3% agarose gel
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`electrophoresis of DNA libraries obtained after poly(A)—tailing of 1 ng cel—miR—39 RNA template
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`either in the presence (C) or absence (NC) of the blocking 3d—ATP nucleotide (ATP/3d—ATP ratio
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`1/30). Note, the ratio of ATP to RNA template (1 mM ATP to 1 ng 22 nt template) was suboptimal.
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`Note, significantly higher yield of the library and absence of a smear of larger by—products is achieved
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`with controllable tailing.
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`Detailed Descriptions of the Invention
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`Before the present invention is described in detail below, it is to be understood that this
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`invention is not limited to the particular methodology, protocols and reagents described herein as these
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`may vary. It is also to be understood that the terminology used herein is for the purpose of describing
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`particular embodiments only, and is not intended to limit the scope of the present invention which will
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`be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms
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`used herein have the same meanings as commonly understood by one of ordinary skill in the art.
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`Several documents are cited throughout the text of this specification. Each of the documents
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`cited herein (including all patents, patent applications, scientific publications, manufacturer's
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`specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its
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`entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate
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`such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as
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`being “incorporated by reference
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`In the event of a conflict between the definitions or teachings of
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`such incorporated references and definitions or teachings recited in the present specification, the text
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`of the present specification takes precedence.
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`In the following, the elements of the present invention will be described. These elements are
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`listed with specific embodiments, however, it should be understood that they may be combined in any
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`manner and in any number to create additional embodiments. The variously described examples and
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`preferred embodiments should not be construed to limit the present invention to only the explicitly
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`described embodiments. This description should be understood to support and encompass
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`embodiments which combine the explicitly described embodiments with any number of the disclosed
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`and/or preferred elements. Furthermore, any permutations and combinations of all described elements
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`in this application should be considered disclosed by the description of the present application unless
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`the context indicates otherwise.
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`Definitions
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`In the following, some definitions of terms frequently used in this specification are provided.
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`These terms will, in each instance of its use, in the remainder of the specification have the respectively
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`defined meaning and preferred meanings.
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`As used in this specification and the appended claims, the singular forms "a", "an", and "the"
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`include plural referents, unless the content clearly dictates otherwise.
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`As used in this specification the term “nucleic acid” comprises polymeric or oligomeric
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`macromolecules, or large biologic