WO2001032931A9 - Method and compositions for selectively inhibiting amplification of sequences in a population of nucleic acid molecules - Google Patents
Method and compositions for selectively inhibiting amplification of sequences in a population of nucleic acid moleculesInfo
- Publication number
- WO2001032931A9 WO2001032931A9 PCT/US2000/041798 US0041798W WO0132931A9 WO 2001032931 A9 WO2001032931 A9 WO 2001032931A9 US 0041798 W US0041798 W US 0041798W WO 0132931 A9 WO0132931 A9 WO 0132931A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- population
- primer
- blocking primer
- acid molecules
- Prior art date
Links
Classifications
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- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
-
- 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
Definitions
- the invention relates to methods and compositions for selectively inhibiting amplification of undesired sequences in a population of nucleic acid molecules.
- RNAs corresponding to different genes can be present in different levels in cells. For example, transcripts from as few as 10- 15 genes may represent 10-15% of cellular mRNA by mass. In addition to these highly abundant transcripts, another 1000-2000 genes encode moderately abundant transcripts, which can account for up to 50% of cellular mRNA mass. Transcripts from the remaining genes fall into the low abundance class. Because many genes are identified by isolating complementary DNA (cDNA) corresponding to an RNA sequence, a significant problem can arise because of differences in the levels at which specific RNAs are present in cell types. The most abundant sequences can be repeatedly sampled, while the lowest abundance class may be rarely, if ever, sampled. Several normalization and subtractive hybridization protocols have been developed to help overcome this problem. These techniques can be technically difficult to perform, and they can fail to detect cDNAs corresponding to rare transcripts. SUMMARY OF THE INVENTION
- the invention is based in part on the discovery of a method for easily and inhibiting the amplification of repetitive nucleic acid sequences in a population of nucleic acids.
- the invention features a method of selectively inhibiting amplification of a target nucleic acid in a population of nucleic acid molecules.
- the method includes providing a population of nucleic acid molecules and contacting the population of nucleic acid molecules with at least one blocking primer to form an annealed blocking primer- template complex that includes the blocking primer and a complementary target sequence in the population of nucleic acid molecules.
- the blocking primer cannot be extended with a polymerase.
- An extendable primer is also contacted with the population of nucleic acid molecules under conditions that allow for formation of an annealed extendable primer- template complex.
- the extendable-primer template complex with a polymerase.
- the polymerase does not extend the extended blocking primer template complex.
- amplification of the target nucleic acid can be selectively inhibited.
- FIG. 1 is a schematic illustration comparing the structure of a peptide nucleic acid (PNA) and a deoxyribonucleic acid (DNA) molecule.
- PNA peptide nucleic acid
- DNA deoxyribonucleic acid
- FIG. 2 is a schematic illustration showing the alignment of the PNA oligonucleotides and restriction fragments of ISGF-3B cDNA.
- FIG. 3 is a representation of a graph showing the amounts of amplified sequence (y-axis) as a function of the size of the sequence product (x-axis) in the presence of various amounts of PNA oligomers.
- Amplification of a specific nucleic acid in a population has been inhibited by including a non-extendable blocking primer in the amplification.
- the non-extendible blocking primer hybridizes to the target nucleic acid but is not extended by a polymerase.
- the blocking primers described herein can also be used to inhibit replication and or transcription of desired RNA sequences.
- a preferred blocking primer is a peptide nucleic acid (PNA) oligomers.
- PNA oligomers are analogs of DNA in which the phosphate backbone is replaced with a pepti de-like backbone.
- the structure of PNAS and conventional nucleic acids are shown schematically in FIG. 1.
- the achiral backbone of a the PNA oligomer is made from N-(2-aminoethyl)-glycine units linked by amide bonds. The backbone is uncharged.
- PNA oligomers can be synthesized using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. Bioorg. Med. Chem. 4: 5-23, and Perry-O'Keefe, ⁇ t al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
- PNAs have been shown to bind to their complementary nucleic acid sequences with greatly improved affinity and specificity compared to DNA.
- thermal stability of a PNA/DNA or PNA/RNA duplex is essentially independent of the salt concentration in the hybridization solution, and can be used under low-salt conditions that cause the target nucleic acid sequence to unwind.
- PNA oligomers lack a phosphate backbone, PNA-oligomers also block the extension of cDNA synthesis mediated by reverse transcriptase, and thus inhibit the first strand cDNA synthesis from the respective mRNA.
- the blocking primers can be used to selectively inhibit amplification of a target nucleic acid in a population of nucleic acids by annealing the blocking primer to a target nucleic acid in the population of nucleic acids. Annealing of the blocking primer to the template results in formation of a double-stranded blocking primer- template complex that includes the blocking primer and the region of the target nucleic acid to which the blocking primer is bound.
- An extendable primer is also annealed to the population of nucleic acids to form an annealed extendable primer-template complex.
- extendable primer is meant that the nucleotides can be incorporated onto the end of the annealed primer in the presence of a polymerase, nucleotide triphosphates, and other cofactors.
- the blocking primer is not extendable, the polymerase does not extend extended blocking primer template complex.
- amplification of the target sequence hybridizing to the blocking primer can be selectively inhibited.
- the particular target sequence whose amplification is amplified is determined by the binding specificity of the blocking primer.
- any desired sequence can be selectively inhibited, as long as it is possible to design a blocking primer that binds specifically to the target sequence.
- the target nucleic acid is present at relatively high copy number in the population of nucleic acids.
- the blocking primer can be designed to hybridize to moderately or highly abundant transcripts.
- the blocking primer can be designed to hybridize to a sequence whose amplification is otherwise not desired, e.g., because it has been previously characterized.
- the blocking primer is between 8 and 50 nucleotides in length, e.g., the blocking primer is between about 10 and 30 nucleotides or 13 and 30 nucleotides in length.
- the blocking primer can be provided at a concentration of about 50 picomoles/ ⁇ 1 to about 700 picomoles/ ⁇ 1, e.g. , the blocking primer can be provided at a concentration of about 100 picomoles/ ⁇ 1 to about 500 picomoles/ ⁇ 1 or about 250picomoles/ ⁇ 1 to about 350 picomoles/ ⁇ 1.
- more than one blocking primer is used.
- the blocking primer anneals to a region of a target nucleic acid that is physically linked to the binding region of a first blocking primer.
- multiple blocking primers can be designed to anneal to a specific RNA molecule.
- one or more of the blocking primers anneal to a region complementary to the 3' and of the RNA molecule.
- an additional blocking primer is designed to anneal to a target nucleic acid that corresponds to the 5' region of the second strand synthesized in a mRNA to cDNA based amplification. For example, inhibition of a specific mRNA may be desired.
- the first blocking primer anneals to a sequence near the 3' end of the mRNA molecule.
- the second blocking primer anneals to a sequence that binds to a sequence homologous to the 5' end of the RNA.
- the blocking primer anneals to a target sequence near the end (e.g., the 3' end) of a nucleic acid molecule.
- the blocking primer or primers of the invention can be used in conjunction with a single extendable primer, in some embodiments multiple extendable primers can be used.
- Any desired population can be used as the source of the population of nucleic acid molecules.
- the nucleic acid can be a genomic DNA, a cDNA, or an mRNA (such as polyA-i- RNA).
- RNA can be derived from, e.g., a plant, a single-celled animal, a multi-cellular animal, a bacterium, a virus, a fungus, or a yeast. If desired, the RNA can also be partitioned prior to use with a blocking primer
- a preferred blocking primer is one that includes at its 5 ' terminus an oligo dT sequence, and at its 3 ' terminus a sequence that is specific for a polyA+-containing RNA of interest.
- any nucleic acid polymerase that can extend the annealed extendable primer.
- Suitable polymerases include, e.g., DNA-dependent DNA polymerases, RNA- dependent DNA polymerases (reverse transcriptases), DNA-dependent RNA polymerases, and RNA-dependent RNA polymerases.
- DNA-dependent DNA polymerases include, e.g., the DNA polymerase from Bacillus stearothermophilus (Bst), the E.
- the coli DNA polymerase I Klenow fragment the bacteriophage T4 and T7 DNA polymerases, and those from Thermus aquaticus (Taq), Pyrococcus furiosis (Pfu), and Thermococcus litoralis (Vent).
- the Bst DNA polymerase has been shown to efficiently incorporate 3'-O- (-2-Nitrobenzyl)-dATP into a growing DNA chain, is highly processive, very stable, and lacks 3 '-5' exonuclease activity.
- the coding sequence of this enzyme has been determined. See U.S. Patent Nos. 5,830,714 and 5,814,506, incorporated herein by reference.
- reverse transcriptases examples include, e.g., reverse transcriptase from Avian Myeloblastosis Virus (AMV), Moloney Murine Leukemia Virus, and Human Immunodeficiency Virus- 1 (HIV-1). HIV-1 reverse transcriptase is particularly preferred because it is well characterized both structurally and biochemically. See, e.g., Huang, et al, Science 282: 1669-1675 (1998).
- a suitable DNA-dependent RNA polymerase can be used when an RNA product is desired.
- Preferred examples of these enzymes include, e.g., RNA polymerase from E. coli [Yin, et al, Science 270: 1653-1657 (1995)] and RNA polymerases from the bacteriophages T7, T3, and SP6.
- a modified T7 RNA polymerase functions as a DNA dependent DNA polymerase.
- Suitable RNA-dependent RNA polymerases include, e.g., RNA-dependent RNA polymerases from the viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picomaviruses.
- Example 1 Inhibition of cDNA synthesis using PNA oligonucleotides
- PNA oligomers specific for transcription factor ISGF-3B mRNA were synthesized.
- the synthesized included a 15-mer at position 116-130 for sense for the sense strand and a 16 mer at position 241-256 for the antisense stand.
- the PNA oligonucleotides were used to inhibit the first strand cDNA synthesis of poly A+ mRNA isolated from human MG-63 cells.
- the two biotinylated 15-mer PNA-oligomers were purchased from PE Biosystems (PE Biosystems, 500 Old Connecticut Path, Framingham, MA 01701).
- the sequence of the sense PNA-oligomer was CAG TCT TGG CAC CTA (SEQ ID NO: 1) (position 116-130), and antisense PNA-oligomer CTG GTG AAC CTG CTC (SEQ ID NO:2) (position 241-256).
- the concentrations were adjusted to 100 pmoles/ ⁇ l with ddH2 ⁇ and stored in aliquots at -20°C until used.
- RNA was processed directly for poly A+ isolation using a CPG-Strepavidin mRNA isolation kit (CPG, Inc., Lincoln Park, NJ). The estimated yield of total RNA from 6 T150 flasks of MG-63 cells was 580 ⁇ g. Poly A+ was stored in aliquots at -20°C in 70% ethanol until used.
- FIG.2 shows the location of the PNA oligomers on the ISGF-3B cDNA construct (denoted as "PNA002" and "PNA001").
- sequence fragments denoted lOmO- 349.8 and dOyo-70 from this region A mixture of both sense and antisense IGSF-3B PNA-oligomers dose dependently and specifically inhibited the first strand cDNA synthesis of the IGSF-3B gene.
- the inhibitory effect on amplification of the 10m0-349.8 and dOyO fragments is shown in FIGS. 3A and 3B.
- the figures show that as increasing amounts of the PNA oligomers were added, the size of the peak corresponding to lOmO 349.8 (FIG. 3A) or dOyO 370 (FIG. 3B) decreased.
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- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2001535611A JP2003534772A (en) | 1999-11-02 | 2000-11-02 | Methods and compositions for selectively inhibiting sequence amplification in a population of nucleic acid molecules |
EP00991917A EP1226281A2 (en) | 1999-11-02 | 2000-11-02 | Method and compositions for selectively inhibiting amplification of sequences in a population of nucleic acid molecules |
AU36402/01A AU3640201A (en) | 1999-11-02 | 2000-11-02 | Method and compositions for selectively inhibiting amplification of sequences ina population of nucleic acid molecules |
CA002388619A CA2388619A1 (en) | 1999-11-02 | 2000-11-02 | Method and compositions for selectively inhibiting amplification of sequences in a population of nucleic acid molecules |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16294399P | 1999-11-02 | 1999-11-02 | |
US60/162,943 | 1999-11-02 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2001032931A2 WO2001032931A2 (en) | 2001-05-10 |
WO2001032931A3 WO2001032931A3 (en) | 2002-05-23 |
WO2001032931A9 true WO2001032931A9 (en) | 2002-11-14 |
Family
ID=22587772
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/041798 WO2001032931A2 (en) | 1999-11-02 | 2000-11-02 | Method and compositions for selectively inhibiting amplification of sequences in a population of nucleic acid molecules |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1226281A2 (en) |
AU (1) | AU3640201A (en) |
WO (1) | WO2001032931A2 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1253205A1 (en) * | 2001-04-24 | 2002-10-30 | LION Bioscience AG | Method of blocking amplification of selected sequences |
WO2003093502A2 (en) * | 2002-04-30 | 2003-11-13 | Gnothis Holding Sa | Multiplexed determination of nucleic acid polymorphisms |
WO2005010209A2 (en) * | 2003-07-24 | 2005-02-03 | Qiagen Gmbh | Method for the reverse transcription and/or amplification of nucleic acids |
CN105209642A (en) * | 2013-03-15 | 2015-12-30 | 卡耐基华盛顿学院 | Methods of genome sequencing and epigenetic analysis |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9211979D0 (en) * | 1992-06-05 | 1992-07-15 | Buchard Ole | Uses of nucleic acid analogues |
-
2000
- 2000-11-02 WO PCT/US2000/041798 patent/WO2001032931A2/en not_active Application Discontinuation
- 2000-11-02 EP EP00991917A patent/EP1226281A2/en not_active Withdrawn
- 2000-11-02 AU AU36402/01A patent/AU3640201A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO2001032931A2 (en) | 2001-05-10 |
AU3640201A (en) | 2001-05-14 |
WO2001032931A3 (en) | 2002-05-23 |
EP1226281A2 (en) | 2002-07-31 |
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