U.S. patent application number 10/106774 was filed with the patent office on 2002-07-25 for blocker-aided target amplification of nucleic acids.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Liu, Weiwei, Su, Xing.
Application Number | 20020098510 10/106774 |
Document ID | / |
Family ID | 24966301 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098510 |
Kind Code |
A1 |
Su, Xing ; et al. |
July 25, 2002 |
Blocker-aided target amplification of nucleic acids
Abstract
The present invention describes a method for blocking an
unwanted sequence from been amplified, duplicated or reverse
transcribed by using a blocking molecule. Preferred embodiments of
the blocking molecule have sequences complimentary at least
partially to the unwanted sequence. The preferred blocking molecule
can be made of nucleic acids and analogues, for example, peptide
nucleic acid and locked nucleic acid.
Inventors: |
Su, Xing; (Cupertino,
CA) ; Liu, Weiwei; (Palo Alto, CA) |
Correspondence
Address: |
AFFYMETRIX, INC
ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3380 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC.
|
Family ID: |
24966301 |
Appl. No.: |
10/106774 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10106774 |
Mar 25, 2002 |
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09738035 |
Dec 14, 2000 |
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6391592 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12; 435/91.2 |
Current CPC
Class: |
C12Q 1/6848 20130101;
C12Q 1/6848 20130101; C12Q 2525/186 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
We claim:
1. A method for blocking amplification of unwanted nucleic acid
sequences during a nucleic acid amplification process comprising
steps of: a) providing nucleic acid samples; b) blocking said
unwanted nucleic acid sequences by adding blocking molecules to
said samples; and c) amplifying unblocked nucleic acid
sequences.
2. The method of claim 1, wherein said amplification process is
polymerase chain reaction.
3. The method of claim 1, wherein said blocking molecules are PNA,
having 3 to 10,000 bases.
4. The method of claim 1, wherein said blocking molecules are RNA,
having 3 to 10,000 bases.
5. The method of claim 1, wherein said blocking molecules are DNA,
having 3 to 10,000 bases, and with a 2',3'-dideoxyribonucleotide to
its 3' end.
6. The method of claim 1, wherein said blocking molecule are RNA,
having 3 to 10,000 bases, and with a 3'-deoxyribonucleotide to its
3' end.
7. The method of claim 1 further comprising the steps of:
fragmenting said nucleic acid samples wherein the nucleic acid is
DNA.
8. The method of claim 7, wherein said fragmenting step is carried
out by at least restriction enzymes.
9. The method of claim 8, wherein said amplification process is
PCR.
10. The method of claim 7, wherein said blocking molecules are PNA,
having 3 to 10,000 bases.
11. The method of claim 7, wherein said blocking molecules are RNA,
having 3 to 10,000 bases.
12. The method of claim 7, wherein said blocking molecules are DNA,
having 3 to 10,000 bases, and with a 2',3'-dideoxyribonucleotide to
its 3' end.
13. The method of claim 7, wherein said blocking molecules are RNA,
having 3 to 10,000 bases, and with a 3'-deoxyribonucleotide to its
3' end.
14. A method for blocking amplification of unwanted DNA sequences
during a DNA amplification process comprising steps of: a)
providing DNA samples for amplification; b) fragmenting said DNA
samples; c) attaching adapters to said fragmented DNA samples, said
adapters comprising a primer region; d) blocking said unwanted DNA
sequences by adding blocking molecules to said fragmented DNA
samples, said blocking sequences being specific for unwanted DNA
sequences; e) adding primers having complimentary sequences to said
primer region in said adapters; and f) amplifying unblocked,
fragmented DNA samples by an amplification process.
15. The method of claim 14, wherein said fragmenting step is
accomplished by restriction digestion enzymes.
16. The method of claim 14, wherein said fragmenting step is
accomplished by restriction digestion enzymes and said amplifying
step is accomplished by polymerase chain reaction.
17. The method of claim 16, wherein said fragmenting step is
accomplished by at least two restriction digestion enzymes.
18. The method of claim 14, wherein said digesting step is
accomplished by at least three restriction digestion enzymes.
19. The method of claim 14, wherein said adapters further
comprising an RNA polymerase promoter sequence.
20. The method of claim 14, wherein said blocking molecules are
PNA, having 3 to 10,000 bases.
21. The method of claim 14, wherein said blocking molecules are
RNA, having 3 to 10,000 bases.
22. The method of claim 14, wherein said blocking molecules are
DNA, having 3 to 10,000 bases, and with a
2',3'-dideoxyribonucleotide to its 3' end.
23. The method of claim 14, wherein said blocking molecules are
RNA, having 3 to 10,000 bases, and with a 3'-deoxyribonucleotide to
its 3' end.
24. The method of claim 7 wherein fragmenting said DNA sample is by
restriction enzymes; blocking unwanted DNA sequences is by peptide
nucleic acids complimentary to the unwanted DNA sequence; and
amplifying unblocked, fragmented DNA samples is by polymerase chain
reaction, using specific primers that are specifically designed to
amplify target DNA sequences.
25. A kit for blocking unwanted nucleic acid sequences to be
amplified in an amplification process comprising: a) a blocker
comprising at least one of the blocking molecules, selecting from
the group consisting of PNA, RNA, DNA with a
2',3'-dideoxyribonucleotide to its 3' end, and RNA with a
3'-deoxyribonucleotide to its 3' end, each having 3 to 10,000
bases; and b) at least a pair of primers.
26. The kit of claim 25 further comprising: an adapter, a
restriction enzyme; and a ligase.
27. The method of claim 1 wherein the nucleic acid is RNA and
further comprising: synthesizing cDNA sequences from said mRNA
samples by reverse transcription.
28. The method of claim 27, wherein the blocking molecules are
selected from the group consisting of PNA, RNA, DNA with a
2',3'-dideoxyribonucleo- tide to its 3' end, and RNA with a
3'-deoxyribonucleotide to its 3' end, each having 3 to 10,000
bases.
Description
TECHNICAL FIELD
[0001] The present invention is in the field of genetic analysis
for medical diagnosis, genetic variation research, or genetic
engineering. More specifically, the present invention is in the
field of nucleic acid amplification.
BACKGROUND
[0002] Many differences in living organisms, including biological
traits, characters or disease susceptibilities, are closely related
to their genetic variations. Therefore, it is desirable to
understand genetic variations of organisms so that useful
information can be obtained to help selecting organisms with
desirable traits or characters or predicting an organism's disease
susceptibility and thus providing proper treatments.
[0003] Most often, the study of genetic variations, for example the
study of genetic polymorphism, involves the analysis of nucleic
acid sequences in DNA or RNA. The sequences of interest may be low
in occurrence in nucleic acid samples. On the other hand,
undesirable sequences may have high occurrence in samples. Some of
these undesirable sequences are repetitive sequences. The high
occurrence of unwanted sequences may cause serious interference
when analyzing genetic variations because they can produce a
significant background noise in genetic detection. The problem
becomes more severe when an amplification process is employed to
increase the copy numbers of the sequences of interest because the
amplification process may amplify both interested sequences and
unwanted sequences indiscriminately. The present invention is
directed to decreasing the possibility of amplifying unwanted
sequences during an amplification process so that sequences of
interest can be amplified while unwanted sequences will not be
amplified, thus decreasing the background noise in genetic
variation analysis. The present invention is especially useful to
suppress the amplification of repetitive sequences.
SUMMARY OF THE INVENTION
[0004] According to the present invention, methods are provided to
block unwanted nucleic acid sequences from being amplified in a
nucleic acid amplification process by adding blocking molecules
that bind to the undesirable nucleic acids sequences and thus
preventing the amplification of undesired sequences in the process.
The methods can be used to block any undesirable sequences and are
especially useful for blocking repetitive sequences.
[0005] In one embodiment of the invention, a method for blocking
amplification of undesirable DNA sequences during a DNA
amplification process comprises the following steps: providing DNA
samples from cells or homogenized tissues; fragmenting the DNA by
restriction enzymes o DNase followed by end modification and
adapter ligation; blocking undesirable DNA sequences by peptide
nucleic acids having complimentary sequences to the undesirable DNA
sequences prior to or during amplifying the DNA samples by
polymerase chain reaction (PCR) with proper reagents, enzymes and
primers.
[0006] In another embodiment of the invention, a kit is constructed
to carry out the blocking method. The kit comprises Cot-1 cRNA
sequences as the blocking molecules, a restriction digestion
enzyme, an adapter comprising a primer sequence and a cohesive end
corresponding to the restriction site specified by the restriction
enzyme, a ligase, and corresponding primers.
DETAILED DESCRIPTION
A. General
[0007] The present invention relies on many patents, applications
and other references for details known to those of the art.
Therefore, when a patent, application, or other reference is cited
or repeated below, it should be understood that it is incorporated
by reference in its entirety for all purposes as well as for the
proposition that is recited.
[0008] As used in the specification and claims, the singular form
"a," "an," and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "an agent"
includes a plurality of agents, including mixtures thereof.
[0009] An individual is not limited to a human being but may also
be other organisms including but not limited to mammals, plants,
bacteria, or cells derived from any of the above.
[0010] Throughout this disclosure, various aspects of this
invention are presented in a range format. It should be understood
that the description in range format is merely for convenience and
brevity and should not be construed as an inflexible limitation on
the scope of the invention. Accordingly, the description of a range
should be considered to have specifically disclosed all the
possible subranges as well as individual numerical values within
that range. For example, description of a range such as from 1 to 6
should be considered to have specifically disclosed subranges such
as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6,
from 3 to 6 etc., as well as individual numbers within that range,
for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the
breadth of the range.
[0011] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques of organic chemistry,
polymer technology, molecular biology (including recombinant
techniques), cell biology, biochemistry, and immunology, which are
within the skill of the art. Such conventional techniques include
polymer array synthesis, hybridization, ligation, and detection of
hybridization using a label. Such conventional techniques can be
found in standard laboratory manuals such as Genome Analysis: A
Laboratory Manual Series (Vols. I-IV), Using Antibodies: A
Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A
Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all
from Cold Spring Harbor Laboratory Press), all of which are herein
incorporated in their entirety by reference for all purposes.
[0012] Additional methods and techniques applicable to array
synthesis have been described in U.S. Pat. Nos. 5,143,854,
5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,412,087,
5,424,186, 5,445,934, 5,451,683, 5,482,867, 5,489,678, 5,491,074,
5,510,270, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,
5,599,695, 5,624,711, 5,631,734, 5,677,195, 5,744,101, 5,744,305,
5,770,456, 5,795,716, 5,800,992, 5,831,070, 5,837,832, 5,856,101,
5,871,928, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,
5,981,956, 6,025,601, 6,033,860, 6,040,138, and 6,090,555, which
are all incorporated herein by reference in their entirety for all
purposes.
[0013] All publications and patent applications cited above are
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication or patent application
were specifically and individually indicated to be so incorporated
by reference. Although the present invention has been described in
some detail by way of illustration and example for purposes of
clarity and understanding, it will be apparent that certain changes
and modifications may be practiced within the scope of the appended
claims.
B. Definitions
[0014] Some definitions are recited below, other definitions can be
obtained from the U.S. patents and references cited herein.
[0015] Analogue when used in conjunction with a biomonomer or a
biopolymer refers to natural and un-natural variants of the
particular biomonomer or biopolymer. For example, a nucleotide
analogue includes inosine and dideoxynucleotides. A nucleic acid
analogue includes peptide nucleic acids and linked nucleic acids.
The foregoing is not intended to be exhaustive but rather
representative. More information can be found in U.S. patent
application Ser. No. 80/630,427 which is hereby incorporated by
reference as stated above.
[0016] An array is an intentionally created collection of nucleic
acids which can be prepared either synthetically or
biosynthetically and screened for biological activity in a variety
of different formats (e.g., libraries of soluble molecules; and
libraries of oligos tethered to resin beads, silica chips, or other
solid supports). Additionally, the term "array" is meant to include
those libraries of nucleic acids which can be prepared by spotting
nucleic acids of essentially any length (e.g., from 1 to about 1000
nucleotide monomers in length) onto a substrate. Arrays are
described in more detail in the patents listed above.
[0017] Complementary or substantially complementary: Refers to the
hybridization or base pairing between nucleotides or nucleic acids,
such as, for instance, between the two strands of a double stranded
DNA molecule or between an oligonucleotide primer and a primer
binding site on a single stranded nucleic acid to be sequenced or
amplified. Complementary nucleotides are, generally, A and T (or A
and U), or C and G. Two single stranded RNA or DNA molecules are
said to be substantially complementary when the nucleotides of one
strand, optimally aligned and compared and with appropriate
nucleotide insertions or deletions, pair with at least about 80% of
the nucleotides of the other strand, usually at least about 90% to
95%, and more preferably from about 98 to 100%. Alternatively,
substantial complementarity exists when an RNA or DNA strand will
hybridize under selective hybridization conditions to its
complement. Typically, selective hybridization will occur when
there is at least about 65% complementarity over a stretch of at
least 14 to 25 nucleotides, preferably at least about 75%, more
preferably at least about 90% complementarity. See e.g., M.
Kanehisa Nucleic Acids Res. 12:203 (1984), incorporated herein by
reference.
[0018] Fragment or Sequence refers to a portion of a larger DNA
polynucleotide or DNA. A DNA molecule, for example, can be broken
up, or fragmented into, a plurality of fragments or sequences.
[0019] Genetic variation refers to variation in the sequence of the
same region between two or more organisms.
[0020] Hybridization refers to the process in which two
single-stranded polynucleotides bind non-covalently to form a
stable double-stranded polynucleotide; triple-stranded
hybridization is also theoretically possible. The resulting
(usually) double-stranded polynucleotide is a "hybrid." The
proportion of the population of polynucleotides that forms stable
hybrids is referred to herein as the "degree of hybridization."
Hybridizations are usually performed under stringent conditions,
for example, at a salt concentration of no more than 1 M and a
temperature of at least 25.degree. C. For example, conditions of 5X
SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a
temperature of 25-30.degree. C. are suitable for allele-specific
probe hybridizations. For stringent conditions, see, for example,
Sambrook, Fritsche and Maniatis. "Molecular Cloning A laboratory
Manual" 2.sup.nd Ed. Cold Spring Harbor Press (1989) which is
hereby incorporated by reference in its entirety for all purposes
above.
[0021] Nucleic acid refers to a polymeric form of nucleotides of
any length, such as oligonucleotides or polynucleotides, either
ribonucleotides, deoxyribonucleotides or peptide nucleic acids
(PNAs), that comprise purine and pyrimidine bases, or other
natural, chemically or biochemically modified, non-natural, or
derivatized nucleotide bases. The backbone of the polynucleotide
can comprise sugars and phosphate groups, as may typically be found
in RNA or DNA, or modified or substituted sugar or phosphate
groups. A polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. The sequence of
nucleotides may be interrupted by non-nucleotide components. Thus
the terms nucleoside, nucleotide, deoxynucleoside and
deoxynucleotide generally include analogs such as those described
herein. These analogs are those molecules having some structural
features in common with a naturally occurring nucleoside or
nucleotide such that when incorporated into a nucleic acid or
oligonucleoside sequence, they allow hybridization with a naturally
occurring nucleic acid sequence in solution. Typically, these
analogs are derived from naturally occurring nucleosides and
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be customized to
stabilize or destabilize hybrid formation or enhance the
specificity of hybridization with a complementary nucleic acid
sequence as desired. See U.S. Pat. No. 6,156,501 which is hereby
incorporated by reference in its entirety for all purposes.
[0022] Oligonucleotide or polynucleotide is a nucleic acid ranging
from at least 2, preferable at least 8, and more preferably at
least 15, 18, 20, 25, 30, 35 nucleotides in length or a compound
that specifically hybridizes to a polynucleotide. Polynucleotides
of the present invention include sequences of deoxyribonucleic acid
(DNA) or ribonucleic acid (RNA) or mimetics thereof which may be
isolated from natural sources, recombinantly produced or
artificially synthesized. A further example of a polynucleotide of
the present invention may be a peptide nucleic acid (PNA). See U.S.
Pat. No. 6,156,051 which is hereby incorporated by reference in its
entirety for all purposes. The invention also encompasses
situations in which there is a nontraditional base pairing such as
Hoogsteen base pairing which has been identified in certain tRNA
molecules and postulated to exist in a triple helix.
"Polynucleotide" and "oligonucleotide" are used interchangeably in
this application.
[0023] Polymerase Chain Reaction or PCR refers to the method to
amplify specific DNA sequences based on repeated cycles of
denaturation of double-stranded DNA, followed by oligonucleotide
primer annealing to the DNA template, and primer extension by a DNA
polymerase. Methods of PCR have been described in U.S. Pat. Nos.
4,683,195, 4,6983,202, and 4,800,159, which are all incorporated
herein by reference in their entirety for all purposes. Additional
information on PCR may be found in PCR Technology: Principles and
Applications for DNA Amplification (ed. H. A. Erlich, Freeman
Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and
Applications (eds. Innis, et al., Academic Press, San Diego,
Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991);
Eckert et al., PCR Methods and Applications 1, 17 (1991); and PCR
(eds. McPherson et al., IRL Press, Oxford). The specific reagents,
conditions and time can be shown in the above references or by
reviewing the package instructions on the products sold by ABI
(foster City, Calif.) or Roche Molecular Systems (Alameda,
Calif.).
[0024] Polymorphism refers to the occurrence of two or more
genetically determined alternative sequences or alleles in a
population. A polymorphic marker or site is the locus at which
divergence occurs. Preferred markers have at least two alleles,
each occurring at frequency of greater than 1%, and more preferably
greater than 10% or 20% of a selected population. A polymorphism
may comprise one or more base changes, an insertion, a repeat, or a
deletion. A polymorphic locus may be as small as one base pair.
Polymorphic markers include restriction fragment length
polymorphisms, variable number of tandem repeats (VNTR's),
hypervariable regions, minisatellites, dinucleotide repeats,
trinucleotide repeats, tetranucleotide repeats, simple sequence
repeats, and insertion elements such as Alu. The first identified
allelic form is arbitrarily designated as the reference form and
other allelic forms are designated as alternative or variant
alleles. The allelic form occurring most frequently in a selected
population is sometimes referred to as the wild type form. Diploid
organisms may be homozygous or heterozygous for allelic forms. A
diallelic polymorphism has two forms. A triallelic polymorphism has
three forms.
[0025] Primer is a single-stranded oligonucleotide capable of
acting as a point of initiation for template-directed DNA synthesis
under suitable conditions, e.g., buffer and temperature, in the
presence of four different nucleoside triphosphates and an agent
for polymerization, such as, for example, DNA or RNA polymerase or
reverse transcriptase. The length of the primer, in any given case,
depends on, for example, the intended use of the primer, and
generally ranges from 3 to 6 and up to 30 or 50 nucleotides. Short
primer molecules generally require cooler temperatures to form
sufficiently stable hybrid complexes with the template. A primer
needs not reflect the exact sequence of the template but must be
sufficiently complementary to hybridize with such template. The
primer site is the area of the template to which a primer
hybridizes. The primer pair is a set of primers including a 5'
upstream primer that hybridizes with the 5' end of the sequence to
be amplified and a 3' downstream primer that hybridizes with the
complement of the 3' end of the sequence to be amplified.
[0026] Probe: A probe is a surface-immobilized molecule that can be
recognized by a particular target. Examples of probes that can be
investigated by this invention include, but are not restricted to,
agonists and antagonists for cell membrane receptors, toxins and
venoms, viral epitopes, hormones (e.g., opioid peptides, steroids,
etc.), hormone receptors, polypeptide, proteins, enzymes, enzyme
substrates, cofactors, drugs, lectins, sugars, nucleic acids,
oligosaccharides, and monoclonal antibodies.
[0027] Single Nucleotide Polymorphism or SNP occurs at a
polymorphic site occupied by a single nucleotide, which is the site
of variation between allelic sequences. This site of variation is
usually both preceded by and followed by highly conserved sequences
e.g., sequences that vary in less than {fraction (1/100)} or
{fraction (1/1000)} members of the populations of the given allele.
A SNP usually arises due to the substitution of one nucleotide for
another at the polymorphic site. These substitutions include both
transitions (i.e. the replacement of one purine by another purine
or one pyrimidine by another pyrimidine) and transversions (i.e.
the replacement of a purine by a pyrimidine or vice versa). SNPs
can also arise from either a deletion of a nucleotide or from an
insertion of a nucleotide relative to a reference allele.
[0028] Substrate refers to a material or group of materials having
a rigid or semi-rigid surface or surfaces. In many embodiments, at
least one surface of the solid support will be substantially flat,
although in some embodiments it may be desirable to physically
separate synthesis regions for different compounds with, for
example, wells, raised regions, pins, etched trenches, or the like.
According to other embodiments, the solid support(s) will take the
form of beads, resins, gels, microspheres, or other geometric
configurations.
[0029] Blocker or blocking compound refers to a molecule that has 2
components, it hybridizes to its complementary sequences (targets),
and the hybrids prevent the complemetary sequences (targets) from
being used as templates for sequence synthesis. Complementary in
this situation is not strictly interpreted as nucleic acid sequence
homology, but includes those molecules that bind in a similar
manner as ligands and antiligands, targets and receptors,
antibodies and their antigens (which can be nucleic acids, proteins
or other molecules), and any molecule that stereochemically
recognizes another.
C. The Methods
[0030] One aspect of the present invention provides a novel method
to prohibit undesirable nucleic acid sequences from being amplified
in an amplification process, preferably in a PCR process.
Consequently, the concentration of desirable nucleic acids is
increased relative to the undesirable nucleic acids. One essential
part of the invention is to use blocking molecules to block the
undesirable sequences so that synthesis of the complimentary
sequence by enzymatic means or other chemical means, which use the
blocked sequences as template, can not be completed. At the same
time, the sequences that do not contain the undesirable regions are
not blocked and thus can be synthesized completely. In the process
of amplification, the complete sequences can be used as template
for the next round of synthesis while the incomplete sequences can
not. The net result after a number of cycles of amplification is
that synthesis of undesirable sequences is reduced and synthesis of
target sequences is carried out normally. Therefore, the present
invention allows selective prohibition of amplification of certain
nucleic acids sequences. This invention can be used in any
amplification process that requires an existing nucleic acid
sequence as a template and is not limited to PCR.
[0031] Other suitable amplification methods include the ligase
chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4,560 (1989)
and Landegren et al., Science 241, 1077 (1988)), transcription
amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173
(1989)), self-sustained sequence replication (Guatelli et al.,
Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)) and nucleic acid based
sequence amplification (NABSA). The latter two amplification
methods include isothermal reactions based on isothermal
transcription, which produce both single-stranded RNA (ssRNA) and
double-stranded DNA (dsDNA) as the amplification products in a
ratio of about 30 or 100 to 1, respectively.
[0032] The blocking effect of the present invention is based on
template-dependent duplication of nucleic acids. Therefore, the
present invention is suitable for blocking nucleic acid
amplification in transcription amplification, self-sustained
sequence replication, and nucleic acid based sequence
amplification.
[0033] The present invention may also be used to block unwanted
mRNA from being transcripted in in vitro reverse transcription of
mRNA. Preferably, the blocking should target the sequences near the
3' end of mRNA. More preferably, the blocking molecule may have
complimentary sequences to the sequences immediately nest to the 3'
poly(A) end of the unwanted mRNA molecule.
[0034] Nucleic acid amplification is frequently used in genetic
analysis, especially in gene expression monitoring and genotyping.
In one embodiment, the present invention is used to provide a
suitable sample preparation method for the detection of SNP. One of
the preferred embodiments of the detection of SNP is to hybridize
nucleic acid samples to a plurality of polynucleotide probes, or
arrays. Ideally, such arrays are immobilized on a solid
substrate.
[0035] Substrates having a surface to which arrays of
polynucleotides are attached are referred to herein as "biological
chips". The substrate may be, for example, all types of silicon,
fused silica or glass, and can have the thickness of a microscope
slide or glass cover slip, or thinner or thicker. Substrates that
are transparent to light are useful when the assay involves optical
detection, as described, e.g., in U.S. Pat. No. 5,545,531, the
disclosure of which is incorporated herein. Other substrates
include Langmuir Blodgett film, germanium,
(poly)tetrafluorethylene, polystyrene, gallium arsenide, gallium
phosphide, silicon oxide, silicon nitride, and combinations
thereof. More information about substrates can be found in the
array patents that are incorporated by reference above.
[0036] In the embodiment wherein arrays of nucleic acids are
immobilized on a surface, the number of nucleic acid sequences may
be selected for different applications, and may be, for example,
about 50, 100, 400, 500, 750, 1000, 2,000, 5,000, 10.sup.5,
10.sup.6, 10.sup.7, or 10.sup.8. In one embodiment, the surface
comprises at least 100 probe nucleic acids each preferably having a
different sequence, each probe contained in an area of less than
about 0.1 cm.sup.2, or, and each probe nucleic acid having a
defined sequence and location on the surface. In one embodiment, at
least 400, 1,000, 5,000, 10,000, 100,000 or more different nucleic
acids are provided on the surface, wherein each nucleic acid is
contained within an area less than about 10.sup.-3 cm.sup.2, as
described, for example, in U.S. Pat. No. 5,510,270. Additional
information may be found in the array patents referred elsewhere in
this application, which are incorporated for all purposes.
[0037] Arrays of nucleic acids for use in gene expression
monitoring and genotyping are described in PCT WO 98/15151, and
U.S. Pat. Nos. 6,040,138, 6,033,860, 5,871,928, 5,800,992,
6,027,880, 6,027,894, 5,968,740, 5,925,525, 5,858,659, 5,710,000,
5,974,164, 5,856,104 and 5,795,716 each of which is hereby
incorporated by reference for all purposes. In one embodiment,
arrays of nucleic acid probes are immobilized on a surface, wherein
the array comprises more than 100 different nucleic acids and
wherein each different nucleic acid is localized in a predetermined
area of the surface, and the density of the different
oligonucleotides is greater than about 60 different
oligonucleotides per 1 cm.sup.2.
[0038] Methods for screening using arrays of polymers, such as
nucleic acids, immobilized on a solid substrate, are disclosed, for
example, in U.S. Pat. No. 5,510,270, the disclosure of which is
incorporated herein. In this method, an array of diverse nucleic
acids is formed on a substrate. The fabrication of arrays of
polymers, such as nucleic acids, on a solid substrate, and methods
of use of the arrays in different assays, are described in: U.S.
Pat. Nos. 5,143,854, 5,242,979, 5,252,743, 5,324,663, 5,384,261,
5,405,783, 5,412,087, 5,424,186, 5,445,934, 5,451,683, 5,482,867,
5,489,678, 5,491,074, 5,510,270, 5,527,681, 5,550,215, 5,571,639,
5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,677,195, 5,744,101,
5,744,305, 5,753,788, 5,770,456, 5,831,070, 5,856,011, 5,858,695,
5,861,242, 5,871,928, 5,874,219, 5,858,837,5,919523, 5,925,525,
5,959,098, 5,968,740, 5,981,185, 6,013,440, 6,022,963, 6,027,880,
6,040,138, 6,045,996, and 6,083,697 all of which are incorporated
by reference in their entirety for all purposes. The above
disclosures describe various methods of fabricating nucleic acid
arrays, including spotting pre-made probes onto a solid support and
synthesizing probes directly onto the support. Any of the arrays
and methods of manufacturing arrays disclosed in the above
references are suitable for use in the presently claimed
invention.
[0039] Methods for labeling nucleic acids can be found in U.S.
patent application Ser. No. 08/882,649, filed Jun. 25, 1997, hereby
incorporated by reference in its entirety, and in commercial
products such as those sold by Enzo Biochem.
[0040] Accessing genetic information using high density DNA arrays
is further described in Chee, Science 274:610-614 (1996), the
disclosure of which is incorporated herein by reference.
[0041] One advantage of using high density arrays for genotyping is
the ability to interrogate SNP and polymorphism at multiple sites
or on different genes. Those skilled in the art will appreciate
that it is difficult to detect multiple SNPs by amplifying nucleic
acid with specific primers because the use of multiple specific
primers is costly and time consuming. On the other hand, random
priming or semi-random priming, or priming according to an adapter
sequence is cheap and simple to amplify multiple nucleic acid
sequences. However, because random priming or semi-random priming,
or priming according to an adapter sequence is not specific,
unwanted sequences may also been amplified. High concentration of
unwanted sequences can interfere detection of multiple SNPs. The
use of present invention will significantly decrease the
interference caused by the high concentration of unwanted sequences
produced during amplification of nucleic acids and thus enabling
the utilization of high density arrays to detect multiple gene
SNP.
[0042] Those skilled in the art will appreciate that there are many
ways to obtain appropriate nucleic acid samples for the purpose of
genetic research and analysis. Nucleic acid samples may be samples
derived from any number of sources including genomic DNA, cDNAs,
pools of fragments, cloned sequences, etc. Any suitable biological
sample can be used for assay of genomic DNA. Convenient suitable
tissue samples include whole blood, semen, saliva, tears, urine,
fecal material, sweat, buccal, skin and hair. Pure red blood cells
are not suitable. As those skilled in the art will appreciate, for
assays of cDNA or mRNA, the tissue sample must be obtained from an
organ in which the target nucleic acid is expressed, e.g., the
liver for a target nucleic acid of a cytochrome P450.
[0043] Although nucleic acid samples can be amplified without
specific treatments, as one of skill in the art will appreciate,
longer DNA fragments are more difficult to amplify with high
fidelity. Preferably, these samples are fragmented before
amplification. Any known method of fragmentation may be employed.
Various methods of fragmenting nucleic acids are known to those of
skill in the art. These methods may be, for example, either
chemical or physical in nature. Chemical fragmentation may include
partial degradation with a DNAse, partial depurination with acid,
restriction enzymes or other enzymes that cleave nucleic acid at
known or unknown locations. Physical fragmentation methods may
involve subjecting the nucleic acid to a high shear rate. High
shear rates may be produced, for example, by moving nucleic acid
through a chamber or channel with pits or spikes, or forcing the
nucleic sample through a restricted size flow passage, e.g., an
aperture having a cross-sectional dimension in the micron or
submicron scale. Combinations of physical and chemical
fragmentation methods may likewise be employed, such as
fragmentation by heat and ion-mediated hydrolysis. More information
regarding sample preparation can be found in U.S. patent
application Ser. Nos. 09/428,350, 60/105,867, 60/136,125,
60/162,739, 60/191,345 and 60/228,253, which are all incorporated
herein by reference in their entirety for all purposes.
[0044] Those of skill in the art will be familiar with the
digestion of nucleic acids with restriction enzymes. In a preferred
embodiment of the invention, particularly when genomic DNA is used
as the sample source, a combination of restriction enzymes is used,
as specific combinations of restrictions enzymes may result in a
larger percentage of genomic DNA fragments of suitable length for
amplification.
[0045] A specific restriction enzyme will typically cut the DNA at
a given recognition sequence, and that recognition sequence
statistically appears in the genomic DNA every X number of base
pairs, where X varies with the length of the given recognition
sequence (i.e., restriction enzymes that have a four-base
recognition site will cut more frequently than restriction enzymes
with a six- or eight-base recognition site). Thus, the combination
of restriction enzymes be used may be altered to produce fragments
in a desired range of sizes.
[0046] A fragmentation can involve several fragmentation methods.
For example, a fragmentation can start with a physical method and
be followed by an enzymatic digestion. The enzymatic digestion may
employ one enzyme or more than one enzymes.
[0047] It might be desirable to modify fragmented nucleic acid
samples. In one embodiment of the present invention, adapters are
attached to the fragments. Adapter sequences and their uses are
well known to those skilled in the art. Such information can be
found in Maniatis, et al., "Molecular Cloning: A Laboratory
Manual," 2.sup.nd Ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y. (1989) ("Maniatis et al."). An adapter can be
used as a complimentary sequence for a primer; as a label; to
introduce a special functional sequence, such as an RNA promoter
region or restriction site; or as separation elements or any
desired use. Typically adapters are short oligonucleotides of known
sequence between 5 and 20 bases in length, but they can be much
longer as desired for a particular application.
[0048] In the embodiment in which the DNA is fragmented with known
restriction enzymes, adapters may be designed to specifically
hybridize to the known overhangs, or cohesive ends, produced by the
specific restriction enzymes used. If these adapters will later be
used as primer sites for PCR, it may be desirable to design
adapters containing sequences that are known to be appropriate PCR
priming sequences. Alternatively, if a linear method of
amplification is to be used, such as that described in
International PCT Application WO 90/06995, one or more of the
adapters may also include a promoter sequence.
[0049] Alternatively, if methods of fragmentation are employed such
that the ends of the fragments are unknown, the ends of the
fragments may be filled in with the appropriate nucleotides, for
example, by the use of T4 DNA polymerase, and adapters may be
blunt-end ligated to the fragments. Methods of filling in DNA
overhangs are known to those of skill in the art. See, for example,
Ausubel, et al., (Eds), Current Protocols in Molecular Biology,
Section 3.5.9 and throughout. Blunt end hybridization is described
in, for example, Ausubel, et al., (Eds) (Sections 3.143.2 and
3.16.8). Of course this method may be employed even when the ends
of the fragments are known.
[0050] One essential step of the present invention is adding
blocking molecules to nucleic acid samples to prevent amplification
of an undesirable sequence. A typical blocking molecule is any
molecule that is capable of binding a specific region of an
undesirable nucleic acid sequence, thus interrupting the synthesis
of the undesirable nucleic acid sequence. Additionally, the
blocking molecule must not be capable of serving as starting
molecule of nucleic acid synthesis. Such blocking molecules may
comprise at least proteins, nucleic acids or their analogues.
[0051] In one embodiment of the present invention, peptide nucleic
acids are used as the blocking molecules. Unlike nucleic acids in
which nucleotides are linked with phosphodiester bonds, nucleotides
in peptide nucleic acids are linked with polyamide backbones. Given
the same sequences, a peptide nucleic acid sequence has a higher
affinity for the same complimentary nucleic acids sequence than a
normal nucleic acid sequence has. In addition, peptide nucleic
acids can not serve as a starting molecule, or a primer, for
nucleic acid polymerases. In other words, they are unextendable to
nucleic acid polymerases. Peptide nucleic acids that comprise a
polyamide backbone and the bases found in naturally occurring
nucleosides are commercially available. Those skilled in the art
will know how to synthesis peptide nucleic acids. Peptide nucleic
acid polymers with desired sequences are also commercially
available.
[0052] Similarly, locked nucleic acids are suitable blocking
molecules. Like peptide nucleic acids, locked nucleic acids have a
high affinity to nucleic acids and not extendable. In addition,
locked nucleic acid polymers with specific sequence are
commercially available.
[0053] In another embodiment, an end-modified nucleic acid sequence
is used as the blocking molecule. The purpose of end-modification
is to make the sequence unextendable to nucleic acid polymerases.
There are many ways to make such modifications. Preferably, if a
DNA sequence is used as the blocking molecule, the sequence is
modified by attaching a dideoxyribonucleotide to one end or both
ends of the sequence. Specifically, such modification could be
attaching a 2',3'-dideoxyribonucleotide to the 3' end of the
sequence, or a 2',5'-dideoxyribonucleotide to 5' end of the
sequence, or a 2',3'-dideoxyribonucleotide to the 3' end of the
sequence and a 2',5'-dideoxyribonucleotide to the 5' end of the
sequence. There are many ways to synthesize a DNA sequence.
Services to synthesize a specific DNA sequence are readily
available. DNA sequences can be synthesized from a DNA synthesizer
(ABI, Foster City, Calif.). Specific DNA sequences may also be
obtained by PCR. A person skilled in the art will know that
attaching a nucleotide to the end or ends of a DNA sequence can be
achieved either chemically or enzymetically. Methods to add a
nucleotide to the ends of a DNA sequence can be found in Sambrook,
Fritsche and Maniatis. "Molecular Cloning A laboratory Manual"
2.sup.nd Ed. Cold Spring Harbor Press (1989) which is hereby
incorporated by reference in its entirety for all purposes
above.
[0054] A sequence of RNA could be a good candidate for a blocking
molecule. It is known that an RNA-DNA hybrid has a higher melting
temperature than a DNA-DNA hybrid. In addition, RNA is a poor
primer for some commonly used DNA polymerases, such as Taq
polymerase used in PCR. Therefore, an unmodified RNA sequence can
be used as a blocking molecule, especially in a PCR process.
However, better blocking function might be achieved by modifying
the RNA sequence to make it unextendable. Such modification could
be, for example, adding a 3'-deoxyribonucleotide to the 3' end of
the RNA sequence, a 5'-deoxyribonucleotide to the 5' end of the RNA
sequence, or a 3'-deoxyribonucleotide to the 3' end and a
5'-deoxyribonucleotide to the 5' end of the RNA sequence. Methods
to modify RNA sequences can be found in, for example, Sambrook,
Fritsche and Maniatis. "Molecular Cloning A laboratory Manual"
2.sup.nd Ed. Cold Spring Harbor Press (1989) which is hereby
incorporated by reference in its entirety for all purposes
above.
[0055] RNA sequences can be obtained in many ways. For example,
those skilled in the art may obtain a corresponding DNA sequence
comprising an RNA polymerase promoter region. Then the DNA sequence
is amplified, for example, by PCR. Finally, the RNA sequence then
can be synthesized from the DNA sequence with an appropriate RNA
polymerase and reagents. The RNA polymerase could be either T3, T7
or SP6 RNA polymerase. The DNA sequence could be obtained
commercially, or by the methods mentioned above. If the DNA
sequence does not contain a promoter region, it is desirable to
ligate a promoter region to the DNA sequence. One skilled in the
art may also clone the DNA sequence into a cloning vector
comprising a promoter or promoters and produce a large quantity of
the vector that is later used as a template to produce RNA
transcripts. Detailed methods for synthesizing RNA can be found in
Sambrook, Fritsche and Maniatis. "Molecular Cloning A laboratory
Manual" 2.sup.nd Ed. Cold Spring Harbor Press (1989), Kwoh et al.,
Proc. Natl. Acad. Sci. USA 86, 1173 (1989), Guatelli et al., Proc.
Nat. Acad. Sci. USA, 87, 1874 (1990), which are hereby incorporated
by reference in its entirety for all purposes above.
[0056] The blocking molecules can be used in combination to block
multiple unwanted sequences. For example, several blocking
molecules are designed to target some most abundant repetitive
sequences, and these blocking molecules are used in one
amplification reaction to block those targeted repetitive
sequences. Preferably, 5%, 10%, 15% or 20% of the unwanted,
different sequences are blocked. More preferably, 25%, 30%, 35%,
40%, 45% or 50% of the unwanted, different sequences are blocked.
Most preferably, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of
the unwanted, different sequences are blocked.
[0057] The sequence of the blocking molecule can be determined
based on what sequence is desired to block. For example, if
repetitive sequences are to be blocked, the blocking molecule can
be designed to have complimentary sequences to the repetitive
sequences. Such sequences can be commercially ordered, chemically
synthesized or can be made with PCR, or cloning or other nucleic
acid synthesis methods. After necessary modification as mentioned
above, the sequences can be used as molecule to block repetitive
sequences. Similarly, a blocking molecule can be designed to have
complimentary sequences of any known sequences to block the
amplification of those sequences. In addition, a specific sample of
nucleic acids with unknown sequences can be isolated and be used as
blocking molecules after proper modifications, if the
amplification, duplication or reverse transcription of the
sequences is undesirable.
[0058] Suitable nucleic acid blocking molecules may have 3, 5, 10,
15, 20, 30, 35, 40, 50, or 75 bases to 500, 700, 800, 1,000, 5,000
or 10,000 bases or more. The preferred length of the nucleic acid
blocking molecule or its analogue is about 14 to 300 bases. Most
preferably, the molecule has about 20 to 100 bases. Those skilled
in the art will appreciate that the longer the molecule, the more
specific the blocking effect of the molecule. Therefore, short
blocking molecules may block a number of sequences, and the
blocking effect tends to be random. Consequently, the length of the
blocking molecule should be designed to achieve specific goals of
applications.
[0059] The effective incubation time to achieve blocking effect
depends on the length of the blocking molecule. Normally, several
seconds to several minutes of incubation time is sufficient.
Blocking can occur from 0.degree. C. to 90.degree. C. The suitable
temperature for blocking depends on both the length of the blocking
molecule and the nature of the molecule. For example, short
blocking molecules can be used in low temperature, and it might be
not effective at high temperature conditions. On the other hand,
blocking molecule made of a PNA can work well at high temperature.
The blocking can occur in different buffers with pH 5-10. Such
buffers can be TrisHCl, Tricine, TES, HEPES, MOPS, Phosphate,
acetate, citrate or any other common buffers used in chemical
reactions. More information of buffers can be found in Maniatis et
al., "Molecular Cloning: A Laboratory Manual," 2.sup.nd Ed. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0060] The preferred concentration to effect blocking function is 1
pM to 1 mM, more preferably, 1 nM to 100 uM, or most preferably, 10
nM to 10 uM. The blocking molecule can be dissolved in water, or
any buffer mentioned above, prior to use. Effective blocking can be
achieved with the presence of cations such as Na+, K+, Li+, NH4+,
Mg++, Mn++, or other cations present in amplification, duplication
or reverse transcription reaction buffers, and anions such as Cl--,
SO4-2, CO3-2, NO3- or other anions present in amplification,
duplication or reverse transcription reaction buffers, from 1 mM to
1M.
[0061] In the preferred embodiment of the invention, the blocking
effect is achieved under the conditions, such as temperature,
acidity, ionic strength and other parameters, similar to those of
specific amplification, duplication or reverse transcription
reactions. Preferably, the blocking molecule can be dissolved in
most commonly used buffers to dissolve nucleic acids. Such buffers
can be found in commonly used laboratory hand books, for example,
in Maniatis et al., "Molecular Cloning: A Laboratory Manual,"
2.sup.nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989).
[0062] The present invention can be applied to block unwanted
nucleic acid sequences in any amplification process that requires
an existing nucleic acids sequence as template. Normally, PCR is
the preferred amplification process used with the present
invention. Further, the present invention can be used in
combination with either random priming or specific priming. The
random primer used in random priming PCR may have six, seven,
eight, nine, ten, eleven, or more members. The random primer may
comprise some partially specific sequences to target interested
sequences, or they may be semi-random primers. The specific primers
could be designed from known sequences or from the adapters added.
The primers, either random or specific, may be attached with
additional adapters for specific purposes. For example, the adapter
may comprise a promoter region, a restriction site, a second primer
site, or a probe. See provisional application No. 60/172,340 which
is hereby incorporated by reference in its entirety for all
purposes.
[0063] For convenience, in one embodiment of the present invention,
the necessary reagents to carry out the present invention are
packed in a research kit. The kit comprises at least the blocking
molecule. Preferably, the kit comprises a solution containing the
blocking molecule, a solution of an adapter comprising a primer
sequence, a solution of a ligase, and a solution of corresponding
primers. More preferably, the kit can further comprise a solution
of restriction enzymes containing at least one restriction enzyme
to fragment the nucleic acid samples.
D. EXAMPLES
[0064] Reference will now be made in detail to illustrative
embodiments of the invention. While the invention will be described
in conjunction with the illustrative embodiments, it will be
understood that the invention is not so limited. On the contrary,
the invention is intended to cover alternatives, modifications and
equivalents, which may be included within the spirit and scope of
the invention.
[0065] For example, DNA molecules of Human Cot-1 fraction (BRL) are
blunt-ended and ligated to a T7 promoter, 100 ug RNA transcripts
are made from 0.1 ug of ligated Cot-1 DNA. DNA fragments of one kb
are isolated from agarose gel after digesting human DNA with Pvu II
restriction enzyme. Adapters containing T3 and Sp6 promoter sites
are ligated to the PvuII fragments. In a PCR reaction, use 1 ng of
ligated PvuII fragments as templates, use T3 and Sp6 sequences as
primer, add 1 ug Cot-1 RNA as a blocker. Other reagents are also
included as required for a PCR reaction. After 30 cycles of PCR
reaction, the PCR products are fragmented and end-labeled by
biotin. The labeled product is used for GeneChip hybridization for
genotyping assay.
* * * * *