[63] Continuation of Ser. No. 84 1,320, Feb. 20, 1992,
abandoned.
[51] Int. Cl.6 C12P 19/34
[52] U.S. Cl 435/91.2; 536/24.3; 536/24.2
[58] Field of Search 435/91.2; 536/24.3,
536/24.2
[56]
References Cited
U.S. PATENT DOCUMENTS
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4,994,370 2/1991 Silva et al 435/6
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Jones et al., "Sequence Specific Generation of a DNA Panhandle Permits
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Primary Examiner Margaret Parr
Assistant Examiner Scott William Houtteman
Attorney, Agent, or Firm Klarquist Sparkman Campbell Leigh and Whinston
[57] ABSTRACT
Methods for amplifying DNA sequences of interest are disclosed. The methods
can be performed using only one primer and are also useful in cloning protocols
and for sequencing large DNAs. The methods comprise cleaving a sample DNA
using an agent, such as a restriction endonuclease, that produces discrete
DNA fragments; ligating the fragments to "adapter" polynucleotides
having a ligatable end and first and second self-complementary sequences
separated by a spacer sequence, thereby forming ligated duplexes; denaturing
the ligated duplexes to form templates annealing molecules of an oligonucleotide
primer to the templates, the primers being homologous to a primer target
site associated with the sequence of interest; extending the primers using
a DNA polymerizing agent to form duplex products; and denaturing the duplex
products. Subsequent multiple cycles of annealing primers, extending the
primers, and denaturing duplex products are usually performed so as to achieve
the desired degree of amplification. Sequencing of large DNAs is performed
using multiple rounds of DNA amplification, each round employing a primer
homologous with a primer target site in the sequence of interest previously
amplified. Cloning is facilitated by including a replication origin and
selectable marker in the adapters.
44 Claims, 14 Drawing Sheets
Lechner et al., "The Structure of Replicating Adenovirus 2 DNA Molecules,"
Cell 12: 1007-1020 (1977).
Gyllensten et al., "Generation of Single-Stranded DNA by the Polymerase
Chain Reaction and its Application to Direct Sequencing of the HLA-DQA
Locus," Proc. NatL Acad Sci. USA 85:7652-7656 (1988).
Frohman, et al., "Rapid Production of Full-Length cDNAs from Rare Transcripts:
Amplification Using a Single Gene- Specific Oligonucleotide Primer,"
Proc. Natl. Acad. Sci USA 85:8998-9002 (1988).
Jayaraman, et al., "Polymerase Chain Reaction-Mediated Gene Synthesis:
Synthesis of a Gene Coding for Isozyme c of Horseradish Peroxidase,"
Proc. NatL Acad. Sci USA 88:4084- 4088 (1991).
Nickerson, et al., "Automated DNA Diagnostics Using an ELISA-Based
Oligonucleotide Ligation Assay," Proc. NatL Acad. Sci USA 87:8923-8927
(1990).
Mueller et al., "In Vivo Footprinting of a Muscle Specific Enhancer
by Ligation Mediated PCR," Science 246:780-786 (1989).
Chao et al., "Sequence Conservation and Divergence of Hepatitis o Virus
RNA," Virology 178:384-392 (1990).
Cariello, et al., "Deletion Mutagenesis During Polymerase Chain Reaction:
Dependence on DNA Polymerase," Gene 99:105-108 (1991).
Shuldiner et al., "RNA Template-Specific PCR: An Improved Method that
Dramatically Reduces False Positives in RT- PCR," BioTechniques
11:760-763 (1991).
Buck et al., "A General Method for Quantitative PCR Analysis of mRNA
Levels for Members of Gene Families: Application to GABA-A Receptor Subunits,"
BioTechniques 11:636-639 (1991).
Roux et al., "A Strategy for Single Site PCR Amplification of dsDNA:
Priming Digested Cloned or Genomic DNA from an Anchor-Modified Restriction
Site and a Short Internal Sequence," BioTechniques 8:48-57 (1990).
Horton et al., "Gene Splicing by Overlap Extension: Tailor- Made Genes
Using the Polymerase Chain Reaction," BioTechniques 8:528-535
(1990).
BOOMERANG DNA AMPLIFICATION
This is a continuation of application Ser. No. 07/841,320, filed Feb. 20,
1992 abandoned.
The present invention pertains to recombinant DNA technology.
The current method of choice for amplifying specific target DNA sequences
is the Polymerase Chain Reaction (PCR) technique described generally in
Mullis et al., U.S. Pat. No. 4,683,195. General features of PCR are shown
schematically in FIG. 1. One begins with double-stranded DNA 10 containing
a sequence of interest 12. The sequence of interest 12 is flanked by "primer
larger' sequences 14, 16. Primers 18, 20 are added to the DNA 10 along with
a DNA polymerase and deoxyribonucleoside triphosphates. (Usually, a heat
stable DNA polymerase is employed to ensure that the polymerase activity
is not destroyed by the heating required for denaturation.) The primers
18, 20 are single-stranded DNA oligonucleotides having sequences complementary
to the primer target sequences 14, 16, respectively. The resulting mixture
is heated to denature the DNA 10. After denaturation, the mixture is cooled
sufficiently to allow the primers 18, 20 to anneal to the primer target
sequences 14, 16, respectively, forming primed duplexes 21, 22, respectively.
The primed duplexes 21, 22 are capable of being enzymatically extended.
Since the polarity of each primer 18 is opposite the polarity of the other
primer 20, replication of the sequence of interest 12, beginning from the
3' end of each primer 18,20, will occur on both target strands 12a, 12b,
respectively, of the sequence of interest 12. (In FIG. 1, the arrows 23,
24 denote the replication direction of primed duplexes 21 and 22.) During
a "cycle" of replication, a strand complementary to each strand
12a, 12b of the sequence of interest is synthesized, wherein each strand
12a produces a complementary strand 12b (along with primer target 16) and
each strand 12b produces a complementary strand 12a (along with primer target
14). After each cycle of replication, the reaction mixture is heated to
denature the newly synthesized strands from their complementary parent strands.
This cycle is repeated as many times as necessary to obtain the desired
quantity of DNA of the sequence of interest 12. During each cycle of replication,
primers anneal not only to the strands from the original sequence of interest,
but also to strands produced by each round of replication. Thus, the number
of copies of the sequence of interest 12 substantially doubles during each
cycle. After multiple cycles, a large amount of the DNA from the sequence
of interest 12 is produced that can be sequenced, cloned, or visualized
on a gel.
Although PCR empowers users to amplify nucleic acid sequences exponentially,
it has certain drawbacks. For example, replication from each primer must
proceed in the direction of the primer on the complementary strand. Thus,
only sequences located between primer target sequences can be amplified
by PCR. However, it is often necessary or desirable to amplify sequences
located outside a region flanked by primer target sequences.
Another disadvantage of PCR is that it requires two primers, thereby requiring
that the practitioner have a detailed knowledge of sequences found in two
separate regions near the sequence of interest. This information is not
always available or readily obtainable.
The present invention, termed "Boomerang
DNA Amplification" (BDA), provides an alternative DNA amplification
method to PCR. A key advantage of BDA is that DNA amplification can be performed
using only one primer. As a result, the DNA that is amplified using BDA
is not limited to a region of the DNA situated between two primers. Thus,
BDA allows extremely long DNA sequences to be quickly determined by performing
a "round" of BDA on each of a series of overlapping regions in
the DNA. BDA can also be conveniently used for cloning DNA.
The BDA method begins with cleaving a sample DNA so as to form discrete
linear duplex fragments having ligatable ends (wherein the term "duplex"
denotes complementary sequences of DNA hydrogen-bonded to each other in
a standard Watson-Crick manner as known in the art.) Preferably, such cleavage
is performed using a restriction endonuclease that generates discrete fragments
of the DNA having what are known in the art as "sticky ends."
The agent used to cleave the DNA is selected such that, among the various
duplex fragments of DNA produced thereby, at least one of the fragments
will comprise a sequence of interest (SOI) and a primer target site associated
therewith.
The sequence of the SOI need not be known beforehand. The sequence of the
primer target site must be at least partially known, as determinable from
other data such as an amino-acid sequence of the corresponding protein or
from sequencing studies of regions of the DNA beginning at locations upstream
of the primer target site. Knowing at least a portion of the primer target
site permits an appropriate primer, homologous to the primer target site,
to be prepared for use in BDA. The primer target site can be located within
a SOI or flanking the SOI.
Because the fragments containing the SOI are linear duplexes, the SOI in
such fragments comprise a first region (in this case, a first "strand")
and a second region (a second "strand") complementary to the first
region.
The duplex fragments are ligated to "adapter" molecules. Adapters
are polynucleotides (either single-stranded or double-stranded) containing
internal sequences complementary to each other that are capable of annealing
to each other to form a duplex under appropriate conditions. Single-stranded
adapters have a single-stranded loop on a first end and an opposing second
end ligatable to the fragments of cleaved sample DNA. Double-stranded adapters
contain internal sequences complementary to each other, preferably located
at the ends of the adapters. At least one end of double-stranded adapters
is ligatable to cleaved sample DNA. Ligation of adapters is performed under
"ligation conditions" wherein an adapter is coupled to each end
of the duplex fragments, thereby forming templates usable for BDA. Usually,
a DNA ligase is used. As used herein, a "BDA template" is defined
generally as a DNA sequence that comprises at least a primer sequence and
an adapter sequence.
Oligonucleotide primers homologous to the primer target site are added to
the BDA templates. Because the primers bind only to BDA templates possessing
a primer target site, only such templates will be amplified in the BDA reaction.
A DNA polymerizing agent such as a DNA polymerase is also added along with
the usual dNTPs in a suitable buffer. Preferably, the DNA polymerase is
thermostable (to denaturation temperatures) so that all the required enzymatic
activity can be added to the BDA reaction at one time.
In a typical BDA "cycle," the resulting mixture is heated to a
temperature suitable to denature the BDA templates, then cooled to a range
typical of "hybridizing conditions" to allow complementary sequences
to anneal to each other, such as the primers to anneal to the primer target
sites on the BDA templates. Each primer is then "extended" under
DNA replication conditions in which the DNA polymerizing agent is active
and dNTPs are incorporated into a primer-extension I product, complementary
to the BDA template, that grows from and includes the primer. Thus, primer
extension forms a duplex on the BDA template. In order to proceed further
with BDA, each such primer extension product must have incorporated sequences
complementary to at least a portion I of each of the first and second self-complementary
sequences of an adapter. Preferably, particularly when using single-stranded
adapters, primer extension is allowed to proceed past a sequence on the
BDA template that is complementary to the primer target site. After primer
extension, the duplex products are denatured.
Typically, multiple such "cycles" are performed until the desired
amount of SOI DNA is produced. A "round" of BDA is comprised of
one or more cycles all employing the same primer. Afterward, the DNA is
typically size fractionated on a gel. The amplified DNA can then be used
for sequencing, cloning, or other use. As described in further detail herein,
BDA cloning is an example wherein a round typically comprises only one cycle.
For sequencing a large DNA, multiple "rounds" of BDA can be performed,
wherein each round is directed to amplifying a particular segment of the
DNA, preferably in a sequential segment-by-segment manner ("walking"
down the DNA). Each such round comprises a number of cycles . sufficient
to achieve the desired amount of amplification. The DNA obtained in each
round is sequenced using conventional methods. In each round, the primer
target site for use in the subsequent round is obtained from the sequence
information obtained using DNA amplified in the preceding round. As a result,
the primer target site used in the subsequent round is located downstream
of the primer target site used in the preceding round and different primers
are used in each round. Also, the DNAs amplified in each round overlap,
thereby allowing registration of sequences of the DNAs amplified in several
rounds. Such registration permits accurate sequences of very long DNAs to
be determined.
BDA can also be used for cloning a DNA sequence of interest. In such a method,
adapters are ligated to compatible DNA fragments at least some of which
contain an SOI and a primer target site. The adapters include an origin
of replication and a selectable marker. Also, in each cycle, primer extension
is performed for a time sufficient to produce primer extension products
that extend along the entire BDA template, thereby forming duplexes that
include the SOI, the origin of replication, and the selectable marker. Subsequent
treatment using a single-strand-specific endonuclease will not degrade the
duplexes but will damage other DNAs present. Subsequent transformation of
susceptible host cells results in cloning of the duplexes.
FIG. 1 schematically depicts the amplification
of DNA using the prior art PCR method.
FIG. 2A schematically shows a single-stranded
(or "panhandled") adapter molecule according to the present invention
usable for performing Boomerang DNA Amplification (BDA).
FIG. 2B schematically shows a double-stranded
adapter molecule according to the present invention usable for performing
BDA.
FIG. 2C schematically shows a way in which double-stranded
adapters and single-stranded adapters can be synthesized.
FIG. 3A schematically shows beginning steps
in a BDA process according to the present invention, wherein sample DNA
containing a sequence of interest is cleaved using a restriction endonuclease
and "panhandled" adapters are attached to the resulting fragments
of the sample DNA, thereby forming closed-loop structures.
FIG. 3B is a continuation of FIG.
3A showing further steps in a BDA process using panhandled adapters,
wherein a primer is annealed to each closed-loop structure that contains
a primer target sequence and subsequent primer extension results in duplication
of at least a portion of the sequence of interest.
FIG. 3C is a continuation of FIG.
3B showing steps in a subsequent cycle of DNA amplification via BDA
using panhandled adapters.
FIG. 3D is a continuation of FIG.
3C summarizing repeated cycles of DNA amplification in a BDA process
using panhandled adapters and the type of DNA product formed therefrom.
FIG. 4 schematically shows how a sequence of
interest can be amplified using BDA and two non-homologous primers, thereby
generating two different overlapping portions of the sequence of interest.
FIG. 5A schematically shows beginning steps
in a BDA process according to the present invention, wherein sample DNA
containing a sequence of interest is cleaved using a restriction endonuclease
and double-stranded adapters are attached to the resulting fragments of
sample DNA.
FIG. 5B is a continuation of FIG.
5A showing further steps in a BDA process employing double-stranded
adapters.
FIG. 5C is a continuation of FIG.
5B showing further steps in a BDA process employing double-stranded
adapters.
FIG. 6 schematically illustrates a method by
which BDA can be used to produce a clonable vector of a sequence of interest
without the need to perform repeated cycles of DNA replication.
FIG. 7 schematically illustrates the production
of BDA adapters from a recombinant plasmid, pIR8, as described in Example
1.
FIG. 8 schematically illustrates BDA products
generated from a closed-loop structure comprising a sequence of interest
to which BDA adapters have been ligated, with particular emphasis on the
location of certain restriction endonuclease cleavage sites useful for ascertaining
which BDA products were formed, as detailed in Examples
44-52.
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