JP2003534772A - Methods and compositions for selectively inhibiting sequence amplification in a population of nucleic acid molecules - Google Patents

Abstract

  (57) [Summary] Disclosed are methods and compositions for selectively inhibiting amplification of a desired sequence in a population of nucleic acid sequences. The method comprises the steps of: providing a population of nucleic acid molecules; contacting the population of nucleic acid molecules with at least one first blocking primer to form an annealed blocking primer-template complex; The first blocking primer is complementary to the target nucleic acid in the population of nucleic acid molecules; contacting the population with at least one extendable primer to form an annealed extensible primer-template complex. And elongating the annealed extensible primer-template complex with a polymerase, wherein the polymerase does not extend the extended blocking primer-template complex, whereby the target nucleic acid Process that selectively inhibits amplification. To, a method is provided.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to methods and compositions for the selective amplification of unwanted sequences in a population of nucleic acid molecules.

BACKGROUND OF THE INVENTION It is believed that about 10,000-20,000 genes should be expressed in living cells depending on the particular cell type. RNAs corresponding to different genes may be present at different levels in cells. For example, transcripts from as few as 10-15 genes can account for 10-15% of cellular mRNA in mass. In addition to these highly abundant transcripts, an additional 1000-2000 genes encode moderately abundant transcripts. It can account for up to 50% of cellular mRNA mass. Transcripts from the rest of the genes fall into a low frequency class.

Since many genes are identified by isolating complementary DNA (cDNA) corresponding to RNA, significant problems arise due to differences in the levels at which a particular RNA is present in a cell type. obtain. The most abundant sequences can be present in the sample in an iterative manner, while the minimal frequency classes, if at all, can only be sampled slightly.

Several normalization and subtractive hybridization protocols have been developed to help overcome this problem. These technologies are
It can be technically difficult to carry out, and they can fail to detect the cDNA corresponding to the rare transcript.

SUMMARY OF THE INVENTION The invention is based, in part, on the discovery of methods 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 comprises the steps of 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, the complex comprising: Including a blocking primer and a complementary target sequence in the population of nucleic acid molecules. This blocking primer cannot be extended with a polymerase.

The extendable primer is also contacted with the population of nucleic acid molecules under conditions that allow the formation of annealed extendable primer-template complexes. This extendable primer-template complex involves a polymerase. But,
The polymerase does not extend the extended blocking primer-template complex. Therefore, the amplification of the target nucleic acid can be selectively inhibited.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references cited herein are hereby incorporated by reference in their entireties. In case of conflict, the present specification, including definitions, will control. In addition, the material,
The methods and examples are present for purposes of illustration only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION Amplification of specific nucleic acids in a population was inhibited by the inclusion of non-extending blocking primers in the amplification. The non-extending blocking primer is
It hybridizes to the target nucleic acid but is not extended by the 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) oligomer. PNA oligomers are analogs of DNA in which the phosphate backbone is replaced by a peptide-like backbone. The structures of PNA and conventional nucleic acids are shown schematically in FIG. The non-chiral PNA oligomer backbone is made up of N- (2-aminoethyl) glycine units linked by amide bonds. Four standard monomers, A, C, G and T, are attached to the backbone by a methylene carbonyl bond, where B is a base. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described below: Hyrup
, Et al. , 1996. Moorg. Med. Chem. 4: 5-23, a
nd Perry-O'Keefe, et al. , 1996. Proc. Na
tl. Acad. Sci. USA 93: 14670.

[0012] PNAs have been shown to bind to their complementary nucleic acid sequences with much improved affinity and specificity compared to DNA. Furthermore, PNA / DNA or PNA
The thermal stability of the / RNA duplex is essentially independent of salt concentration in the hybridization solution, and can be used under low salt conditions where unfolding of the target nucleic acid sequence occurs. PNA oligomers have no phosphate skeleton,
The A oligomer also blocks reverse transcriptase-mediated elongation of cDNA synthesis and thus inhibits first strand canonical cDNA synthesis from the respective mRNA.

The blocking primer can be used to selectively inhibit the amplification of target nucleic acids in a population of nucleic acids by allowing the blocking to anneal to the target nucleic acids in the population of nucleic acids. Annealing of the blocking primer to the template results in the formation of double-stranded blocking primer-template complex. The complex comprises the blocking primer and the region of the target nucleic acid bound between the blocking plies.

The extendable primer also anneals to the population of nucleic acids to form an annealed extendable primer-template complex. An extendable primer means that the nucleotide can be incorporated at the end of the annealed primer in the presence of polymerase, nucleotide triphosphates and other cofactors. Since the blocking primer is not extendable, the polymerase does not extend the extended blocking primer-template complex. Therefore, amplification of target sequences that hybridize to the blocking primer can be selectively inhibited.

The specific target sequence to which the amplificate is amplified is determined by the binding specificity of the blocking primer. Therefore, any desired sequence can be selectively inhibited as long as a blocking primer can be designed that specifically binds to its target sequence. In some embodiments, the target nucleic acid is present in a relatively high copy number in the nucleic acid population. For example, blocking primers can be designed to hybridize to moderately or highly abundant (frequent) transcripts. Alternatively or additionally, blocking primers are
For example, it can be designed to hybridize to undesired sequences (since it has been previously characterized).

In various embodiments, the blocking primer is between 8 and 50 nucleotides in length, eg, the blocking primer is between about 10 and about 30.
Or between 13 and 30 nucleotides in length.

The blocking primer is from about 50 pmol / μl to about 700 pmol
/ Μl, for example, the blocking primer is about 100
It may be provided at a concentration of pmol / μl to about 500 pmol / μl, or the blocking primer may be about 250 pmol / μl to about 350 pmol / μl
It may be provided at a concentration of μl.

In a preferred embodiment, more than one blocking primer is used. In some embodiments, the blocking primer anneals to a region of the target nucleic acid that is physically bound to the binding region of the first blocking primer.
For example, multiple blocking primers can be designed to anneal to a particular RNA molecule. Preferably, one or more blocking primers anneal to the region complementary to the 3'side of the molecule of RNA.

In another embodiment, the additional blocking primer is from mRNA to c
It is designed to anneal to the target nucleic acid, which envisages the 5'region of the second strand synthesized in DNA-based amplification. For example, inhibition of specific mRNAs may be desired. To the sequence near the 3'end of the mRNA molecule, its first blocking primer anneals. The second blocking primer anneals to a sequence that binds to a sequence homologous to the 5'end of the RNA.

In a preferred embodiment, the blocking primer anneals to the target sequence near the end (eg, the 3 ′ end) of the nucleic acid molecule.

The blocking primer (s) of the invention may be used with a single extendable primer, and in some embodiments multiple extendable primers may be used.

Any desired population can be used as a source of population of nucleic acid molecules. Therefore,
The nucleic acid can be genomic DNA, cDNA or mRNA (eg, poly A + RNA). When RNA is used, it can be derived, for example, from plants, unicellular animals, multicellular animals, bacteria, viruses, fungi or yeast.

When poly A + containing RNA is used, the preferred blocking primer is
It contains an oligo dT sequence at its 5'end and at its 3'end the desired poly A +
It contains a sequence specific to the contained RNA.

In general, any nucleic acid polymerase capable of extending annealed extendable primers can be used. Suitable polymerases include, for example, DNA-dependent DN
A polymerase, RNA-dependent DNA polymerase (reverse transcriptase), DNA-dependent RNA polymerase, and RNA-dependent RNA polymerase.

Examples of DNA-dependent DNA polymerases include, for example, Bacillus
a DNA polymerase derived from stearothermophilus (Bst),
E. E. coli DNA polymerase I Klenow fragment, bacteriophage T4 and T7 DNA polymerase, and a DNA polymerase derived from: Thermus aquaticus (Taq), Pyrococ.
cus furosis (Pfu), and Thermococcus li
toralis (Vent). Bst DNA polymerase efficiently incorporates 3'-O-(-2-nitrobenzyl) -dATP into the growing DNA strand, is highly processable, is very stable, and has 3'-5 'exo. It has been shown to lack nuclease activity. The coding sequence for this enzyme has been determined. See US Pat. Nos. 5,830,714 and 5,814,506, which are incorporated herein by reference.

Examples of reversal enzymes include, for example, avian myeloblastosis virus (AMV), Moloney murine leukemia virus and human immunodeficiency virus-1 (HIV-1). The HIV-1 reverser enzyme is particularly preferred. This is because it is well characterized both structurally and biochemically. See, for example: Huang, et al. , Science 282: 166
9-1675 (1998).

A suitable DNA-dependent RNA polymerase can be used when an RNA product is desired. Preferred examples of these enzymes include, for example, E. RNA polymerase from E. coli [Yin, et al. , Science 270: 16
53-1657 (1995)] and bacteriophage T7, T3 and SP.
RNA polymerase derived from 6 may be mentioned. Alternatively, modified T7 R
NA polymerase functions as a DNA-dependent DNA polymerase.

Suitable RNA-dependent RNA polymerases include, for example, RNA-dependent RNA polymerases from the following virus families: Bromovirus, Tobamovirus, Tombs virus, Levivirus, Hepatitis C-like virus, and Picornavirus. See, for example: Huang et al. , S
science 282: 1668-1675 (1998); Lohmann.
et al. J. Virol. 71: 8416-8428 (1997);
Lohmann et al. , Virology 249: 108-118.
(1998), and O'Reilly and Kao, Virolog.
y 252: 287-303 (1998).

[0029]   The invention is further illustrated in the following non-limiting examples.

Example 1 Inhibition of cDNA Synthesis Using PNA Oligonucleotides To demonstrate inhibition of cDNA synthesis using PNA oligonucleotides, PNA oligomers specific for the transcription factor ISGF-3B mRNA were synthesized. . This synthesis yields a 15-mer 116-130 for the sense on the sense strand.
Position and included the 16-mer at positions 241-256 for the antisense strand. This PNA oligonucleotide was used to inhibit first strand cDNA synthesis of poly A + mRNA isolated from human MG-63 cells.

The two biotinylated 15-mer PNA oligomers were labeled with PE Biosystem.
ms (PE Biosystems, 500 Old Connecticu
t Path, Framingham, MA 01701). The sequence of the sense PNA oligomer is CAG TCT TGG CAC CTA (SEQ ID NO: 1) (positions 116-130), and the antisense PNA oligomer is CTG GTG AAC CTG CTC (SEQ ID NO: 2) (241-25).
6th place). Concentrations were adjusted to 100 pmol / μl in ddH ₂ O and stored in aliquots at -20 ° C until used.

To isolate poly A + RNA, human MG63 cells were lysed, and total cellular mRNA was removed by the Trizol lysis procedure (Life Technologies, Rockville) except that the genomic DNA digestion step with DN haze was omitted.
, MD). After quantification using a GeneQuant photometer, total RNA was analyzed for CPG streptavidin mRNA isolation kit (CPG,
Inc. , Lincoln Park, NJ) for direct poly A + isolation. The estimated total RNA yield from 6 T150 flasks of MG-63 cells was 580 μg. Poly A + was stored in aliquots at −20 ° C. in 70% ethanol until use.

To perform the synthesis of cDNA in the presence of PNA toxic primers, two aliquots of poly A + mRNA (about 230 μg total RNA) were precipitated, pooled and dissolved in 24 μl oligo dT solution (100 pmol / μl), and PC
Dispensed into 12 well strips of R thin-walled tubes. PNA (100 pmol
/ Μl) and DEPC-H ₂ O were added according to the table below.

Sample DEPC-H ₂ O Antisense PNA Sense PNA 1 and 2 10 μl 0 μl 0 μl 3 and 4 8 μl 1 μl 1 μl 5 and 6 6 μl 2 μl 2 μl 7 and 8 4 μl 3 μl 5 μl 5 μl 5 μl 4 μl 5 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 4 μl 5 μl .

The sample was heated to 70 ° C. in a thermocycler for 10 minutes and transferred to an ice bath for 2 minutes. The next steps and second strand synthesis were performed essentially as described below: Gubler et al. , Gene 25: 2
63, 1983. Quantitation of cDNA yield was performed using PicoGreen fluorescence measurement. The final cDNA concentration of all samples was 1 ng / ml with TE buffer.
Adjusted to μl and stored at −20 ° C. until use.

US Pat. No. 5,871,697 and Nat Biotechnol. 19
99 Aug; 17 (8): 798-803, a quantitative expression analysis (QEA) reaction was prepared for the sequence referred to as: d0hl (
172nt), d0p0 (71nt), d0y0 (370nt), mls0 (2
67nt), s0c0 (104nt), s0x1 (253nt), y0h0 (1
41 nt) and 10 m0 (349.8 nt). FIG. 2 shows ISGF-3B cd
The position of the PNA oligomer in the NA construct is shown ("PNA002" and "
PNA001 "). 10m0349.8 and d0y0 from this region
A sequence fragment designated -70 is also shown.

A mixture of both sense and antisense IGSF-3B PNA oligomers dose-dependently and specifically inhibited first strand cDNA synthesis of the IGSF-3B gene. The inhibitory effect on the amplification of the 10m0-349.8 and d0y0 fragments is shown in FIGS. 3A and 3B. These figures show that as the amount of PNA oligomer added increased, 10m0 349.8 (FIG. 3A) and d0y0 370 (
The size of the peak corresponding to FIG. 3B) was reduced. Since PNA was designed to inhibit the 10m0 349.8 subsequence located at the 5'end of the ISGF-3B gene, almost complete inhibition was inferred for this subsequence. Reversals of downstream fragments appear to be unaffected. This suggests that PNA oligomers specifically inhibit amplification of nucleic acid sequences.

[Brief description of drawings]

FIG. 1 is a schematic diagram comparing the molecular structures of peptide nucleic acid (PNA) and deoxyribonucleic acid (DNA).

FIG. 2 is a schematic diagram showing an alignment of PNA oligonucleotides and restriction fragments of ISGF-3B cDNA.

FIG. 3 is a representative example of a graph showing the amount of amplified sequence (y-axis) as a function of sequence product size (x-axis) in the presence of varying amounts of PNA oligomers. is there.

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Claims (20)

[Claims] 1. A method of selectively inhibiting amplification of a target nucleic acid in a population of nucleic acid molecules, the method comprising the steps of: providing a population of nucleic acid molecules; Contacting two first blocking primers to form an annealed blocking primer-template complex, wherein the first blocking primer is complementary to a target nucleic acid in the population of nucleic acid molecules; Contacting the population with at least one extendable primer to form an annealed extendable primer-template complex; and extending the annealed extendable primer-template complex with a polymerase Wherein the polymerase is the extended blocking primer template. Not extending the over preparative complex, thereby selectively inhibit amplification of the target nucleic acid, the process including, method. 2. The method of claim 1, wherein the target nucleic acid is present in high copy number in the population of nucleic acid molecules. 3. The method according to claim 1, wherein the blocking primer is a peptide nucleic acid. 4. The method of claim 3, wherein the blocking primer is between 8 and 50 nucleotides in length. 5. The method of claim 3, wherein the blocking primer is between about 10 and about 30 nucleotides in length. 6. The method of claim 3, wherein the blocking primer is between 13 and 20 nucleotides in length. 7. The method of claim 3, wherein the blocking primer is provided at a concentration of between about 50 pmol / μl and about 700 pmol / μl. 8. The method of claim 3, wherein the blocking primer is provided at a concentration of between about 100 pmol / μl and about 500 pmol / μl. 9. The method of claim 3, wherein the blocking primer is provided at a concentration of between about 250 pmol / μl and about 350 pmol / μl. 10. The method of claim 1, further comprising contacting the population of nucleic acid molecules with a second blocking primer. 11. The method of claim 1, further comprising contacting the population of nucleic acid molecules with a second extendable primer. 12. The method of claim 1, wherein the population of nucleic acid molecules is RNA. 13. The method of claim 12, wherein the population of RNA molecules is poly A + RNA. 14. The first extendable primer is a 3 ′ end oligo dT.
The method of claim 1, wherein the method comprises a sequence. 15. The method of claim 1, wherein the polymerase is an RNA-directed DNA polymerase. 16. The method of claim 1, further comprising contacting the population of nucleic acid molecules with a second blocking primer. 17. The method of claim 16, further comprising contacting the population of nucleic acid molecules with the third blocking primer. 18. A method of selectively inhibiting a target nucleic acid in a population of RNA molecules, the method comprising the steps of: providing a population of RNA molecules; contacting the population of nucleic acid molecules with a blocking primer. Contacting the population with an extendable primer having an extendable 3'terminal oligo dT sequence to form an annealed extendable primer-template complex; and the extendable primer complex. With an RNA-directed DNA polymerase, the polymerase not extending the extended blocking primer-template complex, thereby selectively inhibiting amplification of the target nucleic acid. Including the method. 19. The method of claim 18, wherein the blocking primer is a peptide nucleic acid. 20. The method of claim 18, further comprising a blocking primer.