U.S. patent application number 11/119990 was filed with the patent office on 2005-09-22 for recombinant dna processes using a dntp mixture containing modified nucleotides.
This patent application is currently assigned to Sigma-Aldrich Co.. Invention is credited to Hernan, Ron, Li, Chuan, Opper, Kristen E., Snyder, Leanne M., Song, Keming, Uder, Stephanie, Ward, Brian.
Application Number | 20050208564 11/119990 |
Document ID | / |
Family ID | 26670194 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050208564 |
Kind Code |
A1 |
Ward, Brian ; et
al. |
September 22, 2005 |
Recombinant DNA processes using a dNTP mixture containing modified
nucleotides
Abstract
Amplification mixtures, kits, amplicons, kits and processes are
provided for amplifying a nucleic acid. In particular, provided are
processes which utilize an amplification mixture comprising a
polymerase, a deoxynucleotidetriphosphate (DNTP) mixture which
contains modified dNTPs, a first primer and a second primer.
Further provided are DNTP mixtures which contain modified dNTPs for
at least two of the four nucleotide triphosphates, which when
incorporated into a polynucelotide, impart resistance to enzymatic
degradation by an exonuclease at the sites of incorporation of the
modified dNTPs. Also provided are the amplicons and vectors which
incorporate the modified nucleotides.
Inventors: |
Ward, Brian; (St. Louis,
MO) ; Snyder, Leanne M.; (St. Louis, MO) ; Li,
Chuan; (St. Louis, MO) ; Song, Keming;
(Ballwin, MO) ; Opper, Kristen E.; (Collinsville,
IL) ; Uder, Stephanie; (Edwardsville, IL) ;
Hernan, Ron; (St. Louis, MO) |
Correspondence
Address: |
SENNIGER POWERS LEAVITT AND ROEDEL
ONE METROPOLITAN SQUARE
16TH FLOOR
ST LOUIS
MO
63102
US
|
Assignee: |
Sigma-Aldrich Co.
St. Louis
MO
|
Family ID: |
26670194 |
Appl. No.: |
11/119990 |
Filed: |
May 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11119990 |
May 2, 2005 |
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10002292 |
Nov 15, 2001 |
|
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6902914 |
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60325612 |
Sep 28, 2001 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12; 435/91.2 |
Current CPC
Class: |
C12N 15/66 20130101;
C12Q 1/6846 20130101; C12Q 1/6846 20130101; C12Q 2525/125 20130101;
C12Q 2521/319 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
We claim:
1. An amplicon comprising a double-stranded, amplified nucleic acid
fragment, the nucleic acid fragment comprising at least two
modified dNTPs incorporated into the amplicon, wherein the nucleic
acid fragment is resistant to enzymatic degradation by an
exonuclease at the site of incorporation of the at least two
modified dNTPs, a first terminus complimentary to a first ligation
site sequence, and a second terminus complimentary to a second
ligation site sequence.
2. The amplicon of claim 1 wherein the first and second ligation
site sequences are not identical to each other.
3. A vector comprising the amplicon of claim 1.
4. The amplicon of claim 2 further comprising a first adaptor
sequence comprising a nucleotide sequence encoding for at least one
epitope tag.
5. The amplicon of claim 4 wherein the at least one epitope tag
comprises c-myc, polyhistidine, polyarginine,
glutathione-S-transferase (GST) tag, HA epitope, V5, Xpress.TM.,
and FLAG.RTM. epitope.
6. The process of claim 5 wherein the at least one epitope tag
comprises the FLAG.RTM. epitope.
7. A vector comprising the amplicon of claim 6.
8. The amplicon of claim 1 wherein the first and second termini of
the amplicon are 3' termini.
9. The amplicon of claim 1 wherein the amplicon has blunt-ended
termini.
10. The amplicon of claim 1 wherein the amplicon further comprises
a first single stranded overhang sequence at one 5' terminus end
which is complimentary to the first ligation site sequence, a
second single stranded overhang sequence at a second 5' terminus
end which is complimentary to the second ligation site
sequence.
11. The amplicon of claim 10 wherein the first and second ligation
site sequences are not identical to each other.
12. The amplicon of claim 10, the amplicon further comprising 3'
termini which terminate at sites of incorporation of the modified
dNTPs.
13. The amplicon of claim 12 wherein the first ligation site
sequence is an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII, BamHI,
BglII, BsiWI, EagI, EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI, SalI,
XbaI, XhoI or XmaI recognition sequence, and the second ligation
site sequence is an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII, BamHI,
BglII, BsiWI, EagI, EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI, SalI,
XbaI, XhoI or XmaI recognition sequence.
14. The amplicon of claim 12 wherein the first ligation site
sequence is an EcoRI recognition sequence.
15. The amplicon of claim 12 wherein the first ligation site
sequence is an XbaI recognition sequence, and the second ligation
site sequence is a BamHI recognition sequence.
Description
[0001] This application is a division of U.S. patent application
Ser. No. 10/002,292, filed Nov. 15, 2001, which claims priority to
U.S. provisional patent application Ser. No. 60/325,612 filed Sep.
28, 2001, the entire contents of which are incorporated herein by
reference.
FIELD OF INVENTION
[0002] The present invention relates to recombinant DNA processes
using a deoxynucleotide triphosphate (DNTP) mixture containing
modified dNTPs, which when incorporated into a polynucleotide,
impart resistance against enzymatic degradation by an exonuclease
at the sites of incorporation of the modified dNTPs.
BACKGROUND OF THE INVENTION
[0003] Many widely known recombinant DNA techniques involve
replicating or amplifying DNA. One such example is the cloning of
an insert DNA into a target DNA fragment. During this procedure,
the target fragment is typically digested with a restriction enzyme
such as EcoRI. Similarly, the insert DNA, having the gene of
interest, is digested with the same enzyme. In one type of
restriction enzyme digestion, cleavage of both the target DNA and
insert DNA leaves overlapping 3' or 5' nucleotide fragments on each
end. These cohesive, overlapping fragments or "sticky ends" are
well-known properties of some restriction enzymes. Incubation of
the target and insert DNA together at an appropriate temperature
allows the insert DNA to noncovalently bind to the target DNA. The
target DNA and insert DNA are held together by hydrogen bonding of
the cohesive ends. Further incubation with an enzyme, such as DNA
ligase, results in ligation of the insert DNA to the target
nucleotide strand.
[0004] Another method of adding an insert nucleotide fragment into
a target DNA is known as blunt-end ligation. Digestion with some
restriction enzymes, such as SrfI (GCCC/GGGC), SmaI (CCC/GGG), or
Eco RV (GAT/ATC) do not leave any 3' or 5' overhanging nucleotides
at the enzyme splice site. These enzymes are known as "blunt-end"
enzymes due to this feature of their enzymatic activity. After
digestion, blunt-end restriction enzymes maintain single 5'
"terminal" phosphates on both sides of the restriction site. These
terminal 5' phosphates are required by DNA ligase for any
subsequent religation of the digested DNA sequence.
[0005] The ExoClone.TM.-PCR Cloning Kit (Sigma, St. Louis, Mo.)
utilizes a method by which multibase sticky end ligations between
PCR products and suitably cleaved plasmid DNA is accomplished.
Amplified DNA inserts are produced using specially designed primers
whose 5' ends are cohesive with EcoRI cleaved vectors, i.e., 5'
pAATTC. A modified mix containing thiodeoxyguanosine triphosphate
(sdGTP) and deoxyguanosine triphosphate (dGTP) is used to amplify
high GC and long (up to 4 kb) targets. Because exonuclease III
cleaves phosphorothioates extremely slowly, if at all, digestion
with exonuclease III exposes the bases of the 5' termini which are
cohesive with the EcoRI digested vectors. This method is limited to
cloning DNA into EcoRI cleaved vectors because the EcoRI
recognition site is the only commonly used restriction site whose
5' four base overhang is punctuated by a base, i.e., guanine, not
represented within the 5' PAATTC overhang.
[0006] Several methods have been devised for preferentially cloning
insert DNA fragments into target sequences in one orientation.
These methods are commonly known as directional cloning techniques
and have been devised to position genes in the correct 5'.fwdarw.3'
orientation. Directional cloning is commonly performed by digesting
the target nucleotide sequences with two different restriction
enzymes. This method results in a molecule with dissimilar DNA ends
at the target insertion site. The insert DNA is then digested with
the same two restriction enzymes thereby having two dissimilar DNA
ends that correspond to a specific orientation in the target
insertion site. By following this procedure, the insert DNA only
binds to the target sequence in one orientation.
[0007] Another method of directionally cloning an insert into a
target sequence uses an exonuclease, such as Exonuclease III, to
create the "sticky ends". See Kaluz, et al., Nucleic Acids
Research, 1992, 20:4369-4370; U.S. Pat. Nos. 5,580,759, 5,518,901,
5,688,669 and 5,744,306. In this method, insert DNA fragments are
digested with exonuclease III, a double strand specific exonuclease
that catalyzes the stepwise release of nucleotides from the 3'
hydroxyl termini of double stranded DNA, to produce cohesive ends.
Digestion with exonuclease III is performed at low temperatures for
very short times, usually 30-90 seconds, in order to prevent
excessive degradation. After a timed digestion, the insert
fragments have 5' overlapping nucleotide tails. These 5' nucleotide
tails are engineered so that the 5' ends hybridize in one
orientation upon base pairing to the target plasmid DNA molecule
thereby resulting in a relatively simple method of directional
cloning. While the use of exonuclease III provides a relatively
simple method for directional cloning, it may be difficult to
control the length of the generated cohesive ends which may be
critical when cloning small size insert DNA.
[0008] There are several drawbacks, however, with single
restriction enzyme digestion cloning methods and double restriction
enzyme digestion directional cloning methods. For instance,
digesting both the target DNA and insert DNA with restriction
enzymes can be time consuming and multiple enzyme digestions
increase the risk that either the target or insert DNA sequence
will be cleaved at an internal restriction site. It is preferable
that the sequence of the target DNA be known so that restriction
enzymes are not selected which would cleave the sequence at an
internal restriction site. Additionally, some restriction enzymes
cleave very poorly, or not at all, when their recognition sequence
is at or near the termini of a DNA strand.
[0009] Accordingly, it is desirable to formulate reagents and
components for use in cloning and other recombinant DNA
methodologies which could be utilized independent of the sequence
being replicated or amplified. Such formulations would obviate the
need for tedious control of restriction digestion and at the same
time have minimal impact on PCR amplification performance. The
provision of such process and reagent mixtures would avoid tedious
or expensive aspects of current directional cloning such as
multiple restriction enzyme digestion, addition of extra
nucleotides to the insert and/or the use of multiple primers or
linkers.
SUMMARY OF THE INVENTION
[0010] One aspect of the present invention, therefore, is the
provision of materials, processes and kits for synthesizing DNA
from a target nucleic acid. Generally, the present invention
relates to processes which incorporate modified deoxynucleotide
triphosphates (dNTPs) into a nucleic acid. The incorporation of
these modified dNTPs impart resistance against enzymatic
degradation by an exonuclease at the site of incorporation of the
modified dNTPs thereby protecting the amplified product from
complete degradation.
[0011] Briefly, the present invention is directed to a process for
directionally ligating a nucleic acid to an adaptor sequence. In
this process, an amplification mixture comprising a polymerase, a
dNTP mixture which comprises modified dNTPs for at least one of the
four dNTPs comprising dATP, dCTP, dGTP, dTTP and analogs thereof, a
first primer and a second primer are used. The first primer is
complimentary to a portion of the first strand of the target
nucleic acid and has a first terminus which is complimentary to a
first ligation site sequence of the adaptor sequence. The second
primer is complimentary to a portion of the second strand of the
target nucleic acid and has a second terminus which is
complimentary to a second ligation site sequence of the adaptor
sequence.
[0012] The amplification mixture is used to amplify a target
nucleic acid thereby producing an amplified product, or an
amplicon, from the target nucleic acid sequence. The primers anneal
to each strand of the nucleic acid and each primer is extended
using a polymerase and the dNTP mixture. During the extension step
of the PCR, the modified dNTPs are incorporated into the amplicons
in lieu of one of the non-modified dNTPs. The incorporation of the
modified dNTPs into the amplicon impart resistance against
enzymatic degradation by an exonuclease at the site of
incorporation of the modified dNTPs.
[0013] Each terminus of the amplicons is then digested with an
exonuclease. The amplicons are protected from enzymatic degradation
by the exonuclease at the sites of incorporation of the modified
dNTPs and preferably, the termini of the digested amplicons
terminate at the sites of incorporation of the modified dNTPs.
After digestion with the exonuclease, the digested amplicons
contain a single stranded overhang sequence at each terminus. The
first single stranded overhang sequence and the second single
stranded overhang sequence are complimentary to a first ligation
site sequence and a second ligation site sequence in the first and
second adaptor sequence, respectively.
[0014] The first or second single stranded overhang sequence of the
digested amplicons is directionally ligated to either the first or
second ligation site sequence of the adaptor sequence. In a
preferred embodiment, the first and second ligation site sequences
are restriction enzyme recognition sequences. After digestion with
the exonuclease, the digested amplicons have a single stranded
overhang sequences which are complimentary to either a first or
second ligation site sequence in the first or second adaptor
sequence. In one aspect of the present invention, the adaptor
sequence is a cloning vector and the digested amplicons are
directionally inserted into a cloning vector having a ligation site
which is flanked by the first and second ligation site sequences.
Preferably, at least 80% of the digested amplicons are inserted in
only one direction into the cloning vector.
[0015] Another aspect of the present invention is the provision of
a process for producing amplicons using the an amplification
mixture comprising a polymerase, a dNTP mixture containing modified
dNTPs for at least two of the four nucleotide triphosphates, a
first primer and a second primer. The amplicons produced using the
amplification mixture have a first and second termini complimentary
to either a first or second ligation site sequence, and are
resistant to exonuclease degradation at the sites of incorporation
of the modified dNTPs.
[0016] A further aspect of the present invention is the provision
of amplicons which have at least two modified dNTPs incorporated
therein. The incorporation of the modified dNTPs protects the
polynucleotide sequence by imparting resistance to enzymatic
degradation by an exonuclease at the sites of incorporation of the
modified dNTPs in the amplicon. Such amplicons have a first and
second termini complimentary to either a first or second ligation
site sequence of an adaptor sequence. Such amplicons have various
uses, such as for the synthesis of RNA molecules and in vitro
synthesis of proteins. Also provided are vectors containing an
amplicon resistant to enzymatic degradation by an exonuclease.
[0017] A further aspect of the present invention is the provision
of a kit for directionally ligating a nucleic acid to an adaptor
sequence. These kits contain a dNTP mixture containing modified
dNTPs for at least two of the four nucleotide triphosphates which
during the extension step of the PCR, are incorporated into the
amplification product in lieu of one of the non-modified dNTPs, and
a set of instructions for using the dNTP mixture to directionally
ligate a target nucleic acid to an adaptor sequence.
[0018] This dNTP mixture protects nucleotides from enzymatic
degradation by an exonuclease thus increasing the efficiency and
the ease of the cloning process. A further aspect of the present
invention is to provide the dNTP mixture containing modified dNTPs
for at least two of the four nucleotide triphosphates, which during
the extension step of the PCR, are incorporated into the
amplification product in lieu of one of the non-modified dNTPs.
[0019] Other objects and features will be in part apparent and in
part pointed out hereinafter.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a flow chart schematically illustrating the
directional cloning strategy in the present invention.
[0021] FIG. 2 is a photograph of a DNA agarose gel illustrating
that product quality was independent of dNTP mixture, i.e., the
product from a PCR reaction using unmodified dNTPs did not differ
substantially from a reaction in which alpha thiophosphorano dATPs
(sdATP) and dGTPs (sdGTP) were substituted for a fraction of
unmodified dATP and dGTPs. The dNTP mixture was prepared as further
described in Example 1.
[0022] FIG. 3 contains graphs summarizing the amplification and
protection yields from a .sup.32PdNTP containing PCR. Yields were
measured by TCA precipitation. The top panel shows overall yields
of amplification (incorporation) products for all enzymes
normalized to Taq/sdNTP. The middle panel shows yields of sdNTP
products normalized to dNTP. Lower panel shows that while all
products were degraded from approximately 50% or less (+Exo/-Exo)
for all the deoxynucleotide amplifications, addition of ExoIII to
the thiodeoxypurine (sdPuTP) containing reactions resulted in
protections ranging from approximately 80 to 250%.
[0023] FIG. 4 is a graph illustrating that nucleotide protections
that are greater than 100% could occur if the polymerase is active
enough during ExoIII degradation to incorporate additional label
potentially resulting in blunt-ended PCR products.
[0024] FIG. 5 contains graphs illustrating that re-incorporation
during ExoIII digestion replaces an insignificant portion
(approximately 5-15%) of the nucleotides removed by ExoIII. These
data show it is safe to use the dNTP nucleotide mix with most
thermostable DNA polymerases.
[0025] FIG. 6 contains graphs illustrating titration results for
the four thionucleotides. The top panel illustrates that PCR is
tolerant of thionucleotides in the order G>A>T=C. The lower
panel (+ExoIII) reveals that the amplicons are preferentially
protected from ExoIII digestion in the order G>A>T>C.
[0026] FIG. 7 is a graph illustrating the PCR reaction
concentration ratios of alpha thiophosphano dATP (sdATP) to alpha
thiophosphano dGTP (sdGTP) utilized to optimize modified dNTP
incorporation.
[0027] FIG. 8 contains graphs illustrating that PCR product yield
begins to decrease at concentrations of thiodeoxypurine above 0.3
mM. The lower panel reports the occupation of purine sites by
thiodeoxypurine monophosphate (sdPuMP) as a function of dPuTP
concentration.
[0028] FIG. 9 contains graphs illustrating titration results for
the four phosphoborane nucleotides (bdNTP, N=A,C,G,T). These data
show that the PCR yields are negatively impacted by phosporoboranes
in the order G<C<T<A and that ExoIII protection increases
in the order G>A>C=T.
[0029] FIG. 10 is a graph PCR illustrating dPu occupation as a
function of alpha boranophosphano dATP (bdATP) and alpha
boranophosphano dGTP (bdGTP) concentration utilized in a PCR to
optimize modified dNTP incorporation.
[0030] FIG. 11 contains graphs illustrating verification of the
optimal bdATP/bdGTP ratio (bottom panel) established in FIG. 10.
The useful (high protection levels) and unimpeding (little or no
effect on PCR yield) concentration of bdPuTP range from
approximately 0.1 to 0.3 mM bdPuTP (top panel).
[0031] FIG. 12 is a diagram illustrating the primer possibilities
utilized to confirm the directional cloning results.
[0032] FIG. 13A is a photograph of a DNA agarose gel illustrating
that amplification across the insert yielded amplicons that were
larger than from pUC19 and the same as an amplification product
from a previously prepared construct containing an identical insert
(lanes 1-4 vs. 5 and 6 respectively).
[0033] FIG. 13B are photographs of agarose gels which demonstrate
that the use of primer set EcopUC/Lambda R and HindpUC/Lambda L
(FIG. 12) produced no PCR products while primer sets EcopUC/LambdaL
and HindpUC/Lambda R did, thus demonstrating that all of the clones
produced using EcopUC/LambdaL and HindpUC/Lambda R were
directionally inserted.
[0034] FIG. 14 is a photograph of a DNA agarose gel illustrating
the expected "insert" restriction fragment generated by digesting
pBX with BamHI/XbaI.
[0035] FIG. 15 is a graph illustrating the expected overhang length
as a function of modified nucleotide concentration. Clearly,
phosphoborano nucleotides can be used at higher cooccupation rates
without impacting PCR performance.
[0036] FIG. 16 is a photograph of a DNA agarose gel illustrating
the resulting ligation product PCR results. All ligation reactions
produced a relatively high molecular weight PCR product while only
EcopUC/lambda L and HindpUC/lambda R produced products that
co-migrated with the authentic clone amplification products.
[0037] FIG. 17 is a graph illustrating the fraction duplex and the
frequency of mutation as a function of sequence position. This
graph demonstrates that there is no correlation between mutational
frequency and degree of single stranded overhang.
[0038] FIG. 18 is a photograph of an agarose gel which demonstrates
that the the exonuclease digested amplicons are self-ligatable. The
restriction enzyme fragment is an EcopUC/HindpUC amplified from
pUC19 (i.e., amplified across MCS), cut with BamHI (lane B), EcoRI
(lane E), HindIII (lane H), or SAlI (lane S), XbaI (lane X). Lane U
is the fragment which was not digested with ExoIII.
[0039] FIG. 19 illustrates the nucleotide sequences for the adaptor
sequences for making linear DNA in vitro transcription/translation
constructs.
[0040] FIG. 20 is a photograph of an agarose gel which demonstrates
the detection of ligation between PCR amplicon and adaptors. Lane 1
and 2 are exonuclease III treated PCR products for gene p53 and
IkB, respectively. Lane 3 and 4 contain amplicons from ligated
p53-adaptor and IkB-adaptor constructs using primers specific for
the adaptor region.
DETAILED DESCRIPTION
[0041] All publications, patents, patent applications or other
references cited in this application are herein incorporated by
reference in their entirety as if each individual publication,
patent, patent application or reference are specifically and
individually indicated to be incorporated by reference.
DEFINITIONS AND ABBREVIATIONS
[0042] To facilitate understanding of the invention, a number of
terms are defined below:
[0043] The "amplification" of nucleic acids refers to the
replication of one to many additional copies of a nucleic acid
sequence by a method catalyzed by an enzyme. Preferably, it is
carried out using polymerase chain reaction (PCR) technologies. A
variety of amplification methods are known in the art and are
described, inter alia, in U.S. Pat. Nos. 4,683,195 and 4,683,202
and in PCR Protocols: A Guide to Methods and Applications, ed.
Innis et al., Academic Press, San Diego, 1990. In PCR and other
primer extension methodologies, a primer refers to a short
oligonucleotide of defined sequence that is annealed to a DNA
template to provide an initiation site for a polymerase as in the
polymerase chain reaction.
[0044] A "polymerase" is a catalyst, usually a protein enzyme, for
forming an extension of an oligonucleotide along a DNA template
where the extension is complimentary to the template. A
"polymerase" is an enzyme that is capable of incorporating
nucleoside triphosphates to extend a 3' hydroxyl group of a nucleic
acid molecule, if that molecule has hybridized to a suitable
template nucleic acid molecule. Polymerase enzymes are discussed in
Watson, J. D., In: Molecular Biology of the Gene, 3rd Ed., W. A.
Benjamin, Inc., Menlo Park, Calif. (1977), which reference is
incorporated herein by reference, and similar texts.
[0045] Exonuclease activity is, under the conditions of the
reactions contemplated herein, the catalytic control or cleavage of
nucleotides beginning at the end of a nucleic acid. A "3' to 5'
exonuclease", or an enzyme having "3' to 5' exonuclease activity,"
begins the removal or cleavage of nucleotides at the 3' terminus of
a nucleic acid and proceeds towards the 5' end. A "5' to 3'
exonuclease", or an enzyme having "5 to 3' exonuclease activity",
begins the removal or cleavage of nucleotides at a 5' terminus of a
nucleic acid and proceeds toward the 3' end. There are enzymes that
contain either 5' to 3' exonuclease activity or 3' to 5'
exonuclease activity, or both activities in conjunction with
polymerase activities that could be used in the processes disclosed
herein.
[0046] "Complimentary" refers to the natural association of nucleic
acid sequences by base-pairing (A-G-T pairs with the complimentary
sequence T-C-A). Complementarity between two single-stranded
molecules may be partial, if only some of the nucleic acids pair
are complimentary, or complete, if all bases pair are
complimentary.
[0047] The term "recognition sequence" refers to a particular
sequence which a protein, chemical compound, DNA, or RNA molecule
(e.g., restriction endonuclease, a modification methylase, or a
recombinase) recognizes and binds. For example, a restriction
enzyme recognition sequence is a nucleic acid sequence which a
restriction enzyme will recognize, bind and cleave.
[0048] An "adapter sequence" is any duplex nucleotide sequence
suitable for cohesive ligation to one of the ends of an exonuclease
digested amplification product. Such adaptor sequences can result
from enzymatic (in vivo or in vitro) or synthetic sources and can
include but not be limited to cloning vehicles (plasmids, vectors,
BACs etc.), amplification products and sequences prepared by non
enzymatic means such as solid phase DNA synthesis.
[0049] The term "cloning vector" refers to a plasmid, virus,
retrovirus, bacteriophage, cosmid, artificial chromosome (bacterial
or yeast), or nucleic acid sequence which is able to replicate in a
host cell which may contain an optional marker suitable for use in
the identification of transformed cells, e.g., tetracycline
resistance or ampicillin resistance. Optionally, a cloning vector
may possess the features necessary for it to operate as an
expression vector.
[0050] The term "dNTP" refers to deoxynucleoside triphosphates. The
purine bases (Pu) include adenine(A), guanine(G) and derivatives
and analogs thereof. The pyrimidine bases (Py) include cytosine
(C), thymine (T), uracil (U) and derivatives and analogs thereof.
Examples of such derivatives or analogs, by way of illustration and
not limitation, are those which are modified with a reporter group,
biotinylated, amine modified, radiolabeled, alkylated, and the like
and also include phosphorothioate, phosphite, ring atom modified
derivatives, and the like. The reporter group can be a fluorescent
group such as fluorescein, a chemiluminescent group such as
luminol, a terbium chelator such as N-(hydroxyethyl)
ethylenediaminetriacetic acid that is capable of detection by
delayed fluorescence, and the like.
[0051] The term sdNTP represents a thio deoxynucleotide wherein
N=G, A, T or C.
[0052] The term bdNTP represents a borano deoxynucleotide wherein
N=G, A, T, or C.
[0053] The term bdPuTP represents a borano-substituted purine.
[0054] The term bdPyTP represents a borano-substituted
pyrimidine.
[0055] The term dPu represents a deoxypurine.
[0056] The term dPy represents a deoxypyrimidine.
[0057] The procedures disclosed herein which involve the molecular
manipulation of nucleic acids are known to those skilled in the
art. See generally Fredrick M. Ausubel et al. (1995), "Short
Protocols in Molecular Biology," John Wiley and Sons, and Joseph
Sambrook et al. (1989), "Molecular Cloning, A Laboratory Manual,"
second ed., Cold Spring Harbor Laboratory Press as incorporated
herein by reference.
[0058] Accordingly, the present invention provides materials,
processes and kits for directionally ligating a nucleic acid to an
adaptor sequence. The processes and kits utilize an. amplification
mixture including a polymerase, a deoxynucleotide triphosphate
(dNTP) mixture comprising modified dNTPs, a first primer and a
second primer. A portion of the first and second primers are
complimentary to the first and second strand of the double stranded
target nucleic acid, respectively. The first primer has a first
terminus which is complimentary to a first ligation site sequence
of the first adaptor sequence and the second primer has a second
terminus which is complimentary to a second ligation site sequence
of a second adaptor sequence. The primers are extended using the
dNTP mixture containing the modified dNTPs, which during the
extension step of the PCR, are incorporated into the amplification
product in lieu of one of the non-modified dNTPs. Amplicons are
produced which contain the modified and non-modified dNTPs. The
amplicons have a first and second terminus which are complimentary
to the first ligation site sequence of a first adaptor sequence and
the second ligation site sequence of a second adaptor sequence,
respectively. The amplicons are contacted with an exonuclease which
enzymatically degrades the amplicon by removing the non-modified
nucleotides at the termini of the amplicons thereby exposing the
sequences complimentary to either the first ligation site sequence
of the first adaptor sequence and the second ligation site sequence
of the second adaptor sequence. The first terminus of the digested
amplicon is then ligated to the first ligation site sequence of the
first adaptor sequence, and preferably, the second terminus of the
digested amplicon is also ligated to the second ligation site
sequence of the second adaptor sequence.
[0059] The present invention relates to processes for directionally
ligating a nucleic acid to an adaptor sequence which utilize an
amplification mixture to amplify a target nucleic acid thereby
producing an amplified product, or an amplicon, from the target
nucleic acid sequence. Preferably, the amplification mixture
comprises a polymerase; a dNTP mixture comprising modified dNTPs
for at least one of the four nucleotide triphosphates, i.e., dATP,
dGTP, dCTP, dTTP and analogs thereof; a first primer; and a second
primer. The amplicon is digested with an exonuclease and ligated to
a first adaptor sequence. In one embodiment, the adaptor sequence
is a cloning vector. Accordingly, one aspect of the present
invention is directed to processes for cloning a nucleic acid into
a cloning vector.
[0060] The target nucleic acid is amplified using methods of
amplification known in the art. Any nucleic acid specimen can be
utilized as the starting nucleic acid template. Thus, the
amplification process may employ DNA or RNA, wherein DNA or RNA may
be double or single stranded. In the event that RNA is to be used
as a template, enzymes, and/or conditions optimal for reverse
transcribing the template to DNA known to those in the art would be
utilized. Preferably, the target nucleic acid is a double stranded
DNA.
[0061] Several in vitro amplification techniques may be modified
using methods known in the art, to use a DNA polymerase to
enzymatically synthesize a DNA from the target nucleic acid, e.g.,
synthesis of RNA from DNA by transcription is followed by reverse
transcription. Several suitable techniques include but are not
limited to, the polymerase chain reaction (PCR) method,
transcription-based amplification system (TAS), self-sustained
sequence replication system (3SR), ligation amplication reaction
(LAR), Q.beta. RNA replication system and run-off transcription. A
preferred method of amplification is PCR amplification which
involves an enzymatic chain reaction in which exponential
quantities of the target nucleic acid are produced relative to the
number of reaction steps performed. PCR amplification techniques
and many variations of the PCR are known and well documented. See
e.g., Saiki et al., Science 239: 487-491 (1988); U.S. Pat. Nos.
4,682,195, 4,683,202 and 4,800,159, which are incorporated herein
by reference.
[0062] Typically, the selected nucleic acid, preferably, double
stranded DNA, is denatured, thus forming single strands which are
used as templates. One oligonucleotide primer is substantially
complimentary to the negative (-) strand and another primer is
substantially complimentary to the positive (+) strand. DNA primers
are DNA sequences capable of initiating synthesis of a primer
extension product. Primers "substantially complimentary" to each
strand of the target nucleic acid sequence will hybridize to their
respective nucleic acid strands under favorable conditions known to
one skilled in the art, e.g., pH, salt concentration, cation
concentration, temperature.
[0063] The primers used herein are complimentary to the first and
second strand of the target nucleic acid to be amplified. The first
primer contains a first terminus complimentary to a portion of the
first strand of the target nucleic acid and complimentary to a
first ligation site sequence of the first adaptor sequence. The
second primer contains a second terminus complimentary to a portion
of the second strand of the target nucleic acid and complimentary
to a second ligation site sequence of the second adaptor sequence.
The sequences of the first and second ligation site sequences of
the first and second adaptor sequence are not identical and
therefore, the sequences complimentary to the first and second
ligation site sequences are not identical. It is not required that
the sequence complimentary to a portion of the target nucleic acid
and the sequence complimentary to the ligation site sequence of the
adaptor sequence be exclusive of one another. Accordingly, two
primers are utilized wherein the first terminus of the first primer
is complimentary to a first ligation site sequence of the first
adaptor sequence and the second terminus of the second primer is
complimentary to a second ligation site sequence in the second
adaptor sequence.
[0064] The first terminus of the first primer can be either a 3'
terminus or a 5' terminus. Likewise, the second terminus of the
second primer can be either a 3' terminus or a 5' terminus. In a
preferred embodiment, the first terminus of the first primer is a
5' terminus which is complimentary to the first ligation site
sequence in a first adaptor sequence and the second terminus of the
second primer is a 5' terminus which is complimentary to the second
ligation site sequence in the second adaptor sequence, wherein the
5' termini of the first and second primers are not identical.
[0065] It is preferred that the first primer's terminus which is
complimentary to the first ligation site sequence of the first
adaptor sequence be approximately between one and ten nucleotides
in length, more preferably two to seven nucleotides in length,
still more preferably two to five nucleotides in length, and most
preferably four nucleotides in length. It is also preferred that
the second primer's terminus which is complimentary to the second
ligation site sequence of the second adaptor sequence be
approximately between one to ten nucleotides in length, more
preferably two to seven nucleotides in length, still more
preferably two to five nucleotides in length, and most preferably
four nucleotides in length. In a preferred embodiment, the 5'
terminus of the first primer contains a four nucleotide sequence
complimentary to a first ligation site sequence in the first
adaptor sequence, and the 5' terminus of the second primer contains
a four nucleotide sequence complimentary to a second ligation site
sequence in the second adaptor sequence, wherein the 5' terminus of
the first primer is not identical to the 5' terminus of the second
primer.
[0066] Several methods are known in the art which are used to
produce 5' overhang sequences. For example, 5' overhang sequences
can be produced by a modification of the so called "exchange or
replacement labeling" reaction. See Sambrook et al., "Molecular
Cloning", Second Ed., page 5.39. This method utilizes a 3' to 5'
exonuclease bearing DNA polymerase (typically T4 DNA polymerase) to
burn back to the first occurrence of a single nucleotide included
in the reaction mix.
[0067] In a preferred embodiment, the 3' or 5' termini of the first
and second primers which are complimentary to the first and second
ligation site sequences of the first and second adaptor sequence,
respectively, are restriction enzyme recognition sequences. Many
restriction enzymes are known in the art, such as those available
from New England Biolabs, Beverly, Mass. Preferably, the 5' termini
of the first and second primers contain restriction enzyme
recognition sites corresponding to a restriction enzyme which
leaves a 5' overhang restriction site. The 5' termini of the first
and second primers contain restriction enzyme recognition sites
which are not identical to each other. While 5' overhang
restriction sites are preferred, the first and second primers
termini containing restriction enzyme recognition sites
corresponding to a restriction enzyme which leaves a 3' overhang
restriction site may also be used.
[0068] In a preferred embodiment, the 5' terminus of the first
primer sequence is an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII,
BamHI, BglII, BsiWI, EagI, EcoRI, HindIII, NcoI, NgoMIV, NheI,
NotI, SalI, XbaI, XhoI or XmaI recognition sequence and the 3'
terminus of the second primer sequence is an Acc65I, AflII, AgeI,
AcaI, ApoI, AvrII, BamHI, BglII, BsiWI, EagI, EcoRI, HindIII, NcoI,
NgoMIV, NheI, NotI, SalI, XbaI, XhoI or XmaI recognition sequence.
Preferably, the sequences of the 5' termini of the first and second
primer are not identical.
[0069] Annealing the primers to the denatured DNA templates is
followed by extension with an enzyme to result in newly synthesized
+ and - strands containing the target DNA sequence. This annealing
process consists of the hybridization of the primer to
complimentary nucleotides of the DNA sequence template in a
buffered aqueous solution. It is understood that the nucleotide
sequence of the primer need not be completely complimentary to the
portion of the DNA template in order to effectively anneal to the
DNA template. The mixture containing the DNA templates is then
heated to a temperature sufficient to separate the two
complimentary strands of DNA. In a preferred embodiment, the
mixture containing the DNA templates is heated to about 90 to
100.degree. C. from about 0.5 to 10 minutes, even more preferably
from 0.5 to 4 minutes to allow the DNA templates to denature and
form single strands. The reaction mix is next cooled to a
temperature sufficient to allow the primers to specifically anneal
to sequences flanking the gene or sequence of interest. Preferably,
the reaction mixture is cooled to 50 to 60.degree. C., for
approximately 0.5 to 5 minutes.
[0070] Preferably, the enzyme catalyzing the extension reaction is
a polymerase, and more preferably, the polymerase used to amplify
the target nucleic acid is a thermostable polymerase. Heat-stable
(thermophilic) DNA polymerases are particularly preferred as they
are stable when PCR is conducted in a single solution in which the
temperature is cycled. Representative heat-stable polymerases are
the DNA polymerases isolated from Bacillus stearothermophilus
(Bio-Rad, Richmond, Calif.), Thermus thermophilus (FINZYME, ATCC
#27634), Thermus species (ATCC #31674), Thermus aquaticus strain TV
1151B (ATCC #25105) and Thermus filiformis (ATCC #43280), the
polymerase isolated from Thermus flavus (Molecular Biology
Resources; Milwaukee, Wis.). Particularly preferred is Taq DNA
polymerase available from a variety of sources including
PerkinElmer, (Boston, Mass.), Promega (Madison, Wis.) and
Stratagene (La Jolla, Calif.), and AmpliTaq.TM. DNA polymerase, a
recombinant Taq DNA polymerase available from PerkinElmer.
[0071] The polymerase extends the primer by incorporating dNTPs
from the dNTP mixture into the resulting polynucleotide, or
amplicon. Preferred polymerases incorporate the modified dNTPs
efficiently, base-specifically and independent of the surrounding
sequence context. In a preferred embodiment, the polymerase used to
amplify the target nucleic acid is Taq, REDTaq, AccuTaqLA,
AmpliTaq.TM., KlenTaqLA, Ultma, Pwo, or Pfu and more preferably,
Taq polymerase, REDTaq, AccuTaqLA and AmpliTaq.TM.. A combination
of two or more of the above polymerases such as, for example, a
combination of Taq and Pfu, may also be used in the processes of
the present invention. The temperature of the reaction mixture is
then set to the optimum for the DNA polymerase to allow DNA
extension to proceed.
[0072] The modified and unmodified dNTPs in the dNTP mixture are
incorporated into the resulting polynucleotide, or amplicon. For
use in a process of directionally ligating a nucleic acid to an
adaptor sequence, the dNTP mixture comprises modified dNTPs for at
least one of the four nucleotide triphosphates comprising dATP,
dGTP, dCTP, dTTP and analogs thereof. In one preferred embodiment,
the adaptor sequence is a cloning vector and the dNTP mixture for
use in a process of cloning a nucleic acid into a cloning vector
comprises modified dNTPs for at least two of the four nucleotide
triphosphates comprising dATP, dGTP, dCTP, dTTP and analogs
thereof. It will be appreciated that by virtue of the present
invention that all modified dNTPs which serve as a substrate for a
polymerase and protect nucleotides and/or the phosphodiester
linkages between the nucleotides from enzymatic degradation by an
exonuclease can be utilized in the dNTP mixture of the present
invention. Preferably, any molecular manipulation, e.g.,
modifications to the base, sugar and/or phosphate, which produces a
modified dNTP which serves as a substrate for a DNA polymerase and
protects nucleotides and/or the phosphodiester linkages between the
nucleotides from enzymatic degradation by an exonuclease can be
utilized in the dNTP mixture of the present invention.
[0073] Preferably, the polymerase incorporates the modified dNTPs
efficiently, base-specifically and independent of the surrounding
sequence context. It is also preferred that the modified dNTPs in
the dNTP mixture do not inhibit the enzyme catalyzed incorporation
of the modified and unmodified dNTPs into the nucleotide sequence
of the amplicon. Preferably, the modified dNTPs minimally affect
the efficiency of the incorporation of the modified and unmodified
dNTPs. In a preferred embodiment, the modified dNTPs reduce the
efficiency of the polymerase incorporation of modified and
unmodified dNTPs into the polynucleotide sequence of the amplicon
by approximately 60%, more preferably, by approximately 70%, even
more preferably by approximately 80%, and most preferably by
approximately 90%. In a preferred embodiment, the modified dNTPs
reduce the efficiency of the polymerase incorporation of modified
and unmodified dNTPs into the polynucleotide sequence of the
amplicon by less than 90%.
[0074] Preferably, the dNTP mixture comprises modified dNTPs
wherein each modified dNTP is a fraction of the total amount of the
particular deoxynucleotide triphosphate base in the dNTP mixture.
In a process for directionally ligating a nucleic acid to a first
adaptor sequence, the dNTP mixture contains modified dNTPs for at
least one of the four dNTPs comprising dATP, dGTP, dCTP, dTTP and
analogs thereof, preferably, the dNTP mixture contains modified
dNTPs for at least two of th four dNTPs comprising dATP, dGTP,
dCTP, dTTP and analogs thereof. dNTP.sub.1 represents the at least
one modified dNTP in the dNTP mixture, if two modified dNTPs are
present in the dNTP mixture, dNTP.sub.2 represents the second of
the at least two modified dNTPs in the dNTP mixture, if three
modified dNTPs are present in the dNTP mixture, dNTP.sub.3
represents the third of the at least two modified dNTPs in the dNTP
mixture, and if four modified dNTPs are present in the dNTP
mixture, dNTP.sub.4 represents the fourth of the at least two
modified dNTPs in the dNTP mixture. Preferably, the dNTP mixture
comprises modified dNTPs for at least two dNTPs, modified
dNTP.sub.1 and modified dNTP.sub.2. Each modified dNTP.sub.1 and
modified dNTP.sub.2 can be a modified dATP, modified dGTP, modified
dCTP or modified dTTP, provided that dNTP.sub.1 and dNTP.sub.2 are
not the same modified dNTP. The modified dNTPs are enzymatically
incorporated into the amplicons in lieu of one of the non-modified
dNTPs during primer extension. The modified dNTPs impart resistance
by blocking the action of the exonuclease, for example, exonuclease
III, or any other 3' to 5' exonuclease that cleaves normal
phosphodiester linkages between the nucleotides but to which the
modified dNTPs prove resistant.
[0075] In one embodiment, the dNTP mixture comprises three modified
dNTPs, dNTP.sub.1, dNTP.sub.2 and dNTP.sub.3, wherein each modified
dNTP is a fraction of the total amount of the particular
deoxynucleotide triphosphate base in the dNTP mixture. Each
modified dNTP.sub.1, dNTP.sub.2 and dNTP.sub.3 can be a modified
dATP, modified dGTP, modified dCTP or modified dTTP, provided that
dNTP.sub.1, dNTP.sub.2 and dNTP.sub.3 are not the same modified
dNTP. In another embodiment, the dNTP mixture comprises four
modified dNTPs, dNTP.sub.1, dNTP.sub.2, dNTP.sub.3 and dNTP.sub.4,
wherein each modified dNTP is a fraction of the total amount of the
particular deoxynucleotide triphosphate base in the dNTP mixture.
Each modified dNTP.sub.1, dNTP.sub.2, dNTP.sub.3 and dNTP.sub.4 can
be a modified dATP, modified dGTP, modified dCTP or modified dTTP,
provided that modified dNTP.sub.1, dNTP.sub.2, dNTP.sub.3 and
dNTP.sub.4 are not the same modified dNTP. The modified dNTPs are
enzymatically incorporated into the amplicons in lieu of one of the
non-modified dNTPs during primer extension. The modified dNTPs
impart resistance by blocking the action of the exonuclease, for
example, exonuclease III, or any other 3' to 5' exonuclease that
cleaves normal phosphodiester linkages between the nucleotides but
to which the modified dNTP proves resistant.
[0076] In a process for directionally ligating a nucleic acid to a
first adaptor sequence, the dNTP mixture contains modified dNTPs
for at least one of the four dNTPs comprising dATP, dGTP, dCTP and
dTTP. dNTP.sub.1 represents the at least one modified dNTP in the
dNTP mixture used specifically for directional ligation, and
dNTP.sub.1 can be a modified dATP, modified dGTP, modified dCTP or
modified dTTP. In a preferred embodiment, the dNTP mixture utilized
for directionally ligating a nucleic acid to an adaptor sequence
comprises two modified dNTPs, modified dNTP.sub.1 and dNTP.sub.2.
The modified dNTPs in the dNTP mixture are enzymatically
incorporated into the amplicons in lieu of one of the non-modified
dNTPs during primer extension. The modified dNTPs impart resistance
by blocking the action of the exonuclease, for example, exonuclease
III, or any other 3' to 5' exonuclease that cleaves normal
phosphodiester linkages between the nucleotides but to which the
modified dNTP proves resistant.
[0077] The dNTP mixture utilized in a process for directionally
ligating a nucleic acid to an adaptor sequence comprises at least
one modified dNTP comprising a modified dATP, modified dGTP,
modified dCTP or a modified dTTP. Preferably, the at least one
modified dNTP is a fraction of the total amount of the particular
deoxynucleotide triphosphate base in the dNTP mixture. Preferably,
the modified dNTPs are modified purines or pyrimidines, more
preferably, the modified dNTPs are alpha phosphate modified purines
or pyrimidines, even more preferably, alpha phosphate modified dATP
or dGTP, and most preferably, alpha phosphate modified dGTP. In a
preferred embodiment, the dNTP mixture utilized for directional
ligation comprises at least one modified dNTP wherein the modified
dNTPs are alpha boranophosphorano dNTPs or alpha thiophosphorano
dNTPs, more preferably, the modified dNTPs are alpha
boranophosphorano dNTPs. In another preferred embodiment, the
modified dNTPs in the dNTP mixture utilized for directional
ligation are alpha boranophosphorano dATPs, alpha boranophosphorano
dGTPs, alpha thiophosphorano dATPs or alpha thiophosphorano dGTPs,
more preferably, the modified dNTPs in the dNTP mixture are alpha
boranophosphorano dATPs, alpha boranophosphorano dGTPs, and most
preferably, the modified dNTPs are alpha boranophosphorano
dGTPs.
[0078] Preferred nucleotide boranotriphosphates, e.g.,
5'-alpha-boranotriphosphates and methods of synthesis are disclosed
in U.S. Pat. Nos. 5,260,427, 5,659,027 and 5,683,869, and preferred
nucleotide thiotriphosphates, e.g., 5'-alpha-thiotriphosphates and
methods of synthesis are disclosed in Labeit et al., Meth. Enzymol.
1987, 155: 166 and Nakamaye et al., Nucl. Acids. Res., 1988, 16:
9947, the entirety of which are incorporated herein by reference.
Once incorporated into the polynucleotide, thiophosphorodiester and
boranophosphodiester bonds are resistant to exonuclease
digestion.
[0079] Preferably, the dNTP mixture utilized to directionally
ligate a nucleic acid to a first adaptor sequence comprises
modified dNTPs for at least two of the four dNTPs comprising dATP,
dGTP, dCTP and dTTP which are preferably modified purines, modified
pyrimidines, or a combination of modified purines and pyrimidines.
More preferably, the modified dNTPs in the dNTP mixture comprise
alpha phosphate modified dNTPs, still more preferably, the modified
dNTPs in the dNTP mixture comprise alpha phosphate modified
purines, alpha phosphate modified pyrimidines or a combination of
alpha phosphate modified purines and alpha phosphate modified
pyrimidines, and most preferably, alpha phosphate modified dATP and
dGTP. In a preferred embodiment, the modified dNTPs in the dNTP
mixture comprise a combination of alpha boranophosphorano dNTPs and
alpha thiophosphorano dNTPs, more preferably, the modified dNTPs in
the dNTP mixture comprise alpha thiophosphorano dNTPs, and most
preferably, the modified dNTPs in the dNTP mixture comprise alpha
boranophosphorano dNTPs. In another preferred embodiment, the
modified dNTPs in the dNTP mixture comprise a combination of alpha
boranophosphorano dATP and dGTP and alpha thiophosphorano dATP and
dGTP, more preferably, the modified dNTPs in the dNTP mixture
comprise alpha thiophosphorano dATP and dGTP, and most preferably,
the modified dNTPs in the dNTP mixture comprise alpha
boranophosphorano dATP and dGTP.
[0080] The amount of the four nucleotide triphosphates in the dNTP
mixture is determined by the concentration of modified dNTPs
relative to the concentration of non-modified dNTPs. In a process
for directionally ligating a nucleic acid to a first adaptor
sequence, the dNTP mixture comprises modified dNTPs for at least
one of four dNTPs comprising dATP, dCTP, dGTP and dTTP. Preferably,
the amount of the four nucleotide triphosphates in the dNTP mixture
utilized for directional cloning is determined by the ratio
(R.sub.1) of the concentration of modified dNTP.sub.1 ([modified
dNTP.sub.1]) relative to the concentration of non-modified
dNTP.sub.1 ([dNTP.sub.1]) which is shown as follows:
R.sub.1=[modified dNTP.sub.1]/[dNTP.sub.1]
[0081] In a preferred embodiment, the ratio of the concentration of
modified dNTP.sub.1 relative to the concentration of non-modified
dNTP.sub.1 is less than 20, more preferably, the ratio is less than
9, and even more preferably, the ratio is less than 1.
[0082] Preferably, modified dNTP.sub.1 in the dNTP mixture utilized
in a process for directionally cloning a nucleic acid into a vector
are modified purines, and more preferably, alpha phosphate
substituted purines. In a preferred embodiment, the ratio of the
concentration of alpha phosphate substituted dNTP.sub.1 relative to
the concentration of non-modified dNTP.sub.1 is about 0.05 to 20,
more preferably, the ratio is about 0.05 to 10, and most
preferably, 0.05 to 4.
[0083] In a preferred embodiment, modified dNTP.sub.1 is alpha
thiophosphorano dATP, alpha thiophosphorano dGTP, alpha
boranophosphorano dATP or alpha boranophosphorano dGTP, more
preferably, dNTP.sub.1 is alpha boranophosphorano dATP or alpha
boranophosphorano dGTP, and most preferably, dNTP.sub.1 is alpha
boranophosphorano dGTP.
[0084] In a preferred embodiment, dNTP.sub.1 in the dNTP mixture
utilized to directionally ligate a nucleic acid to a first adaptor
sequence are alpha thiophosphorano dNTPs, more preferably, alpha
thiophosphorano dATP or dGTP and most preferably, alpha
thiophosphorano dGTP. In one preferred embodiment, modified
dNTP.sub.1 is alpha thiophosphorano dATP and the ratio of the
concentration of alpha thiophosphorano dATP relative to the
concentration of non-modified dATP is 0.05 to 10, more preferably,
1.0 to 9, and most preferably, 1.5 to 5. More preferably, modified
dNTP, is alpha thiophosphorano dGTP and the ratio of the
concentration of alpha thiophosphorano dGTP relative to the
concentration of non-modified dGTP is 0.05 to 10, more preferably,
0.05 to 7, and most preferably, 1.0 to 3.0.
[0085] In a more preferred embodiment, dNTP, in the dNTP mixture
utilized to directionally ligate a nucleic acid to an adaptor
sequence are alpha boranophosphorano dNTPs, more preferably, alpha
boranophosphorano dATP or dGTP and most preferably, alpha
boranophosphorano dGTP. Preferably, modified dNTP.sub.1 is alpha
boranophosphorano dATP and the ratio of the concentration of alpha
boranophosphorano dATP relative to the concentration of
non-modified dATP is 0.05 to 10, more preferably, 0.05 to 4, and
most preferably, 0.01 to 2.0. In a more preferred embodiment,
modified dNTP.sub.1 is alpha boranophosphorano dGTP and the ratio
of the concentration of alpha boranophosphorano dGTP relative to
the concentration of non-modified dGTP is 0.05 to 20, more
preferably, 0.1 to 10, and most preferably, 0.1 to 5.
[0086] In a preferred embodiment, the dNTP mixture comprises
modified dNTPs for two of the four dNTPs, modified dNTP.sub.1 and
modified dNTP.sub.2. Preferably, the amount of the four nucleotide
triphosphates in the dNTP mixture is determined by the ratio
(R.sub.2) of the concentration of modified dNTP.sub.1 ([modified
dNTP.sub.1]) to the concentration of modified dNTP.sub.2 ([modified
dNTP.sub.2]) relative to the concentration of non-modified
dNTP.sub.1 ([dNTP.sub.1]) to the concentration of non-modified
dNTP.sub.2 ([dNTP.sub.2]) which is shown as follows:
R.sub.2=([modified dNTP.sub.1]/[modified
dNTP.sub.2])/([dNTP.sub.1]/[dNTP.- sub.2])
[0087] In a preferred embodiment, the ratio of the concentration of
modified dNTP.sub.1 to the concentration of modified dNTP.sub.2
relative to the concentration of non-modified dNTP, to the
concentration of non-modified dNTP.sub.2 is less than 51, more
preferably, the ratio is less than 27, and even more preferably,
the ratio is less than 13.
[0088] Preferably, the modified dNTP, and modified dNTP.sub.2 in
the dNTP mixture are modified purines and more preferably, alpha
phosphate substituted dNTPs. In a preferred embodiment, the ratio
of the concentration of alpha phosphate substituted dNTP.sub.1 to
the concentration of alpha phosphate substituted dNTP.sub.2
relative to the concentration of non-modified dNTP.sub.1 to the
concentration of non-modified dNTP.sub.2 is about 0.05 to 6.4, more
preferably, the ratio is about 0.1 to 3.2, even more preferably,
the ratio is about 0.2 to 1.6.
[0089] In a preferred embodiment, the two modified dNTPs in the
dNTP mixture are alpha thiophosphorano dNTPs, and more preferably,
alpha thiophosphorano dGTP and alpha thiophosphorano dATP .
Preferably, the ratio of the concentration of alpha thiophosphorano
dGTP to the concentration of alpha thiophosphorano dATP relative to
the concentration of non-modified dGTP to the concentration of
non-modified dATP is between 0.8 to 5.3, more preferably, the ratio
is between 0.17 to 2.7, even more preferably, the ratio is between
0.33 to 1.33, and most preferably, the ratio is about 0.66.
[0090] In another preferred embodiment, the modified dNTPs in the
dNTP mixture are alpha boranophosphorano dNTPs, and more
preferably, alpha boranophosphorano dGTP and alpha
boranophosphorano dATP . Preferably, the ratio of the concentration
of alpha boranophosphorano dGTP to the concentration alpha
boranophosphorano dATP relative to the concentration of
non-modified dGTP to the concentration of non-modified dATP is
between 0.05 to 6.4, more preferably, the ratio is between 0.1 to
3.2, even more preferably, the ratio is between 0.2 to 1.6, and
most preferably, the ratio is about 0.4.
[0091] In another embodiment, the dNTP mixture comprises three
modified dNTPs, dNTP.sub.1, dNTP.sub.2 and dNTP.sub.3 wherein each
modified dNTP is a fraction of the total amount of the particular
deoxynucleotide triphosphate base in the dNTP mixture. Each
dNTP.sub.1, dNTP.sub.2 and dNTP.sub.3 is a modified dATP, dCTP,
dGTP or dTTP, provided that dNTP.sub.1, dNTP.sub.2 and dNTP.sub.3
are not the same modified dNTP. The amount of the four nucleotide
triphosphates in the dNTP mixture comprising three modified dNTPs
is determined by the ratio (R.sub.3) of the product of the
concentration of modified dNTP.sub.1 ([modified dNTP.sub.1]), the
concentration of modified dNTP.sub.2 ([modified dNTP.sub.2]) and
the concentration of modified dNTP.sub.3 ([modified dNTP.sub.3])
relative to the product of the concentration of non-modified
dNTP.sub.1 ([dNTP.sub.1]), the concentration of non-modified
dNTP.sub.2 ([dNTP.sub.2]) and the concentration of non-modified
dNTP.sub.3 ([dNTP.sub.3]), which is shown as follows: 1 R 3 = ( [
modified dNTP 1 ] .times. [ modified dNTP 2 ] .times. [ modified
dNTP 3 ] ) / ( [ dNTP 1 ] .times. [ dNTP 2 ] .times. [ dNTP 3 ]
)
[0092] In order to determine the concentration of each modified
dNTP.sub.1, dNTP.sub.2, and dNTP.sub.3 in the dNTP mixture, an
acceptable ratio of each modified and non-modified dNTP.sub.1,
dNTP.sub.2, and dNTP.sub.3 is individually determined by titration.
An acceptable ratio is obtained when a compromise between
amplification toxicity is minimized and protection against
exonuclease digestion is maximized. In a preferred embodiment, this
is when PCR amplification yield and quality is identical to
reactions performed in the absence of any modified nucleotides
while exonuclease protection is essentially quantitative, i.e.,
exonuclease digestion can result in a mean overhang length of less
than 20 base pairs. It is possible however that a reduction in
amplification yield may need to be tolerated so that adequate
protection can be obtained. The type of experiment is exemplified
in the s-dNTP and b-dNTP titrations shown in FIGS. 6 and 9. See
"Quality Improvement through Planned Experimentation 2nd Edition",
Ronald Moen, Thomas Nolan, Lloyd Provost, McGraw Hill, N.Y., 1998,
incorporated herein by reference.
[0093] After the preferred concentration of each individual
modified and non-modified dNTP.sub.1, dNTP.sub.2, and dNTP.sub.3 is
determined, the total mixture of modified and non-modified
dNTP.sub.1, dNTP.sub.2, and dNTP.sub.3 is determined by titration.
The composition of the dNTP mixture containing modified dNTPs for
three of the four dNTPs are obtained in a fashion analogous to that
used for the determination of the relative ratios of solutions
containing two modified dNTPs as described in Examples 3 and 4 and
exemplified in FIG. 7. However, to determine the relative ratios of
modified to non-modified dNTPs in a dNTP mixture comprising
modified dNTPs for three of the four dNTPs, a three dimensional
titration rather than two dimensional titration would be
performed.
[0094] In another embodiment, the dNTP mixture comprises four
modified dNTPs, dNTP.sub.1, dNTP.sub.2, dNTP.sub.3 and dNTP.sub.4
wherein each modified dNTP is a fraction of the total amount of the
particular deoxynucleotide triphosphate base in the dNTP mixture.
Each dNTP.sub.1, dNTP.sub.2, dNTP.sub.3 and dNTP.sub.4 is a
modified dATP, dCTP, dGTP or dTTP, provided that dNTP.sub.1,
dNTP.sub.2, dNTP.sub.3 and dNTP.sub.4 are not the same modified
dNTP. The amount of the four nucleotide triphosphates in the dNTP
mixture comprising four modified dNTPs is determined by the ratio
(R.sub.4) of the product of the concentration of modified
dNTP.sub.1 ([modified dNTP.sub.1]), the concentration of modified
dNTP.sub.2 ([modified dNTP.sub.2]), the concentration of modified
dNTP.sub.3 ([modified dNTP.sub.3]) and the concentration of
modified dNTP.sub.4 ([modified dNTP.sub.4]) relative to the product
of the concentration of non-modified dNTP.sub.1 ([dNTP.sub.1]), the
concentration of non-modified dNTP.sub.2 ([dNTP.sub.2]), the
concentration of non-modified dNTP.sub.3 ([dNTP.sub.3]) and the
concentration of non-modified dNTP.sub.4 ([dNTP.sub.4]), which is
shown as follows:
R.sub.4=([modified dNTP.sub.1].times.[modified
dNTP.sub.2].times.[modified dNTP.sub.3].times.[modified
dNTP.sub.4])/( [dNTP.sub.1].times.[dNTP.sub.2-
].times.[dNTP.sub.3].times.[dNTP.sub.4])
[0095] In order to determine the concentration of each modified
dNTP.sub.1, dNTP.sub.2, dNTP.sub.3, and dNTP.sub.4 in the dNTP
mixture, an acceptable ratio of each modified and non-modified
dNTP.sub.1, dNTP.sub.2, dNTP.sub.3, and dNTP.sub.4 is individually
determined by titration. An acceptable ratio of each modified to
non-modified dNTP is obtained when a compromise between PCR
amplification toxicity is minimized and exonuclease protection is
maximized. In a preferred embodiment, an acceptable ratio is
obtained when PCR amplification yield and quality is identical to
reactions performed in the absence of any modified dNTP in the dNTP
mixture while exonuclease protection is essentially quantitative,
i.e., exonuclease digestion results in a mean overhang length of
less than 20 base pairs. However, sacrifices in amplification yield
may need to be tolerated so that adequate exonuclease protection
can be obtained. The type of experiment is exemplified in the
s-dNTP and b-dNTP titrations shown in FIGS. 6 and 9. After the
preferred ratio of each individual modified and non-modified
dNTP.sub.1, dNTP.sub.2, dNTP.sub.3, and dNTP.sub.4 is determined,
the total composition of modified and non-modified dNTP.sub.1,
dNTP.sub.2, dNTP.sub.3, and dNTP.sub.4 in the dNTP mixture is
determined by titration. The method for a dNTP mixture comprising
four modified dNTPs are obtained using methods analogous to that
utilized for the determination of the relative ratios of solutions
containing two or three modified nucleotides as described in
Examples 3 and 4 and exemplified in FIG. 7. A four dimensional
titration rather than a two or three dimensional titration would be
performed to determine the relative ratios of modified to
non-modified dNTPs in a dNTP mixture comprising modified dNTPs for
four dNTPs. See "Quality Improvement through Planned
Experimentation 2nd Edition", Ronald Moen, Thomas Nolan, Lloyd
Provost, McGraw Hill, N.Y., 1998, incorporated herein by
reference.
[0096] An amplicon is produced from the extension of the primers
when dNTPs, both modified and unmodified dNTPs, in the dNTP mixture
are enzymatically incorporated into the nucleotide sequence of the
amplicon. Thus, the newly synthesized amplicon contains both
modified and unmodified dNTPs. The incorporation of the modified
dNTPs impart resistance against enzymatic degradation by an
exonuclease at the site of incorporation of the modified dNTPs
thereby protecting the nucleotides of the amplicon from enzymatic
degradation by an exonuclease.
[0097] The extension of the first and second primers using the dNTP
mixture result in newly synthesized amplicons containing both
modified and unmodified dNTPs which have a first terminus
complimentary to a first ligation site sequence of the first
adaptor sequence and a second terminus complimentary to a second
ligation site sequence of a second adaptor sequence. Preferably,
the amplicon's termini are amplified sequences which correspond to
the 3' or 5' termini of the first and second primer sequences used
in PCR. In a preferred embodiment, the first ligation site sequence
of the first adaptor sequence and the second ligation site sequence
of the second adaptor sequence are the ends of a cloning vector.
Preferably, the termini of the amplicon are complimentary to the
first and second ligation site sequences of the cloning vector, and
more preferably, the 5' termini of the amplicons contain sequences
complimentary to the first and second ligation site sequences of
the cloning vector. In an even more preferred embodiment, the 5'
termini of the amplicons contain restriction enzyme recognition
sequences. Preferably, the termini of the amplicon contain
restriction enzyme recognition sequences corresponding to
restriction enzymes which leave a 5' overhang restriction site. In
a preferred embodiment, the termini of the amplicon are not
identical to each other to enable directional ligation into the
cloning vector.
[0098] In order to prepare amplicons which can be inserted in one
direction relative to the first adaptor sequence, the dNTP mixture
comprising modified dNTPs for at least one, and preferably, two of
the four dNTPs comprising dATP , dGTP, dCTP and dTTP is utilized to
extend first and second primers which contain a first and second
sequence, respectively, which is complimentary to a first and
second ligation site sequence of the adaptor sequence,
respectively. In a preferred embodiment, the first ligation site
sequence of the first adaptor sequence and the second ligation site
sequence of the second adaptor sequence are the ends of a cloning
vector and the amplicons are prepared for directional cloning into
the cloning vector.
[0099] The extension of the first and second primers using the dNTP
mixture comprising modified dNTPs for at least one, and preferably,
two of the four dNTPs result in newly synthesized amplicons
containing both modified and unmodified dNTPs. The amplicons have a
first terminus complimentary to a first ligation site sequence of
the first adaptor sequence and a second terminus complimentary to a
second ligation site sequence of the second adaptor sequence
wherein the termini of the amplicon are not identical to each
other. Preferably, the amplicon's termini are amplified sequences
which correspond to the 3' or 5' termini of the first and second
primer sequences used in PCR. In a preferred embodiment, the first
and second ligation site sequences of the first and second adaptor
sequences, respectively, are the ends of a cloning vector.
Preferably, the termini of the amplicon are complimentary to the
first and second ligation site sequences of the cloning vector, and
more preferably, the 5' termini of the amplicons contain sequences
complimentary to the first and second ligation site sequences of
the cloning vector wherein the first and second ligation site
sequences of the cloning vector are not identical. In an even more
preferred embodiment, the 5' termini of the amplicons contain
restriction enzyme recognition sequences. Preferably, the termini
of the amplicon contain restriction enzyme recognition sequences
corresponding to restriction enzymes which leave a 5' overhang
restriction site.
[0100] The amplicons produced from this process have a variety of
uses. For example, RNA transcripts are synthesized from the
amplicons by in vitro DNA transcription. Transcription of RNA is
performed with the appropriate RNA polymerase, preferably, T3, T7,
or SP6, depending on the RNA polymerase promoter sites present
upstream of the DNA to be transcribed. Because these polymerases
are extremely promoter-specific, i.e., there is almost no
transcriptional cross-talk, virtually homogenous RNA can be
obtained using plasmid DNA (circular or linearized) or
enzymatically synthesized DNA (linear) as the template in a
transcription reaction. Melton et al. (Nucleic Acid Research (1984)
v. 12, p. 7035) describes a general procedure for RNA synthesis in
vitro. In short, DNA is mixed with an RNA polymerase plus the four
ribonucleoside triphosphates used in RNA synthesis. During an
incubation at 37.degree. C., large amounts of the desired RNA are
then generated by in vitro transcription. RNA transcripts may be
radiolabeled with .sup.32P-, .sup.33P-, .sup.35S-, or
.sup.3H-labeled ribonucleotides, depending upon the specific
application.
[0101] RNA transcripts may be used to generate probes for
hybridization to Northern or Southern blots, plaque and colony
lifts, tissue sections and chromosome spreads. RNA transcripts are
also useful for Si nuclease mapping, generation of antisense RNAs
to block translation, and mRNA synthesis for translation in
vitro.
[0102] Additionally, the amplicons may be used to synthesize
protein expression in vitro. The in vitro synthesis of proteins in
cell-free extracts is an important tool for molecular biologists
and has a variety of applications, including the rapid
identification of gene products, localization of mutations through
synthesis of truncated gene products, protein folding studies, and
incorporation of modified or unnatural amino acids for functional
studies. The use of in vitro translation systems can have
advantages over in vivo gene expression when the over-expressed
product is toxic to the host cell, when the product is insoluble or
forms inclusion bodies, or when the protein undergoes rapid
proteolytic degradation by intracellular proteases.
[0103] The most frequently used cell-free translation systems
consist of extracts from rabbit reticulocytes, wheat germ and
Escherichia coli. All are prepared as crude extracts containing all
the macromolecular components (70S or 80S ribosomes, tRNAs,
aminoacyl-tRNA synthetases, initiation, elongation and termination
factors, etc.) required for translation of exogenous RNA. Pelham et
al. (Eur. J. Biochem. (1976) v. 67, p. 247) describe a general
procedure for mRNA translation in vitro. In short, the lysates
containing the cellular components necessary for protein synthesis
are mixed with the subject mRNA and incubated at 30.degree. C. for
90 minutes followed by an analysis of the translation reaction for
expected products (i.e., SDS-PAGE analysis).
[0104] The amplicons containing modified dNTPs and unmodified dNTPs
are preferably purified and treated with an exonuclease.
Preferably, the amplicons were synthesized using a dNTP mixture
comprising modified dNTPs for at least two of the four nucleotide
triphosphates comprising dATP, dGTP, dCTP and dTTP and analogs
thereof. Even more preferably, the amplicons were synthesized using
a dNTP mixture comprising modified dATP and dGTP, and still more
preferably, alpha thiophosphorano dATPs and dGTPs or alpha
boranophosphorano dATPs and dGTPs, and most preferably, alpha
boranophosphorano dATPs and dGTPs. Accordingly, in a preferred
embodiment, the amplicons comprise alpha phosphate modified dATPs
and dGTPs.
[0105] Because the modified dNTPs impart resistance to enzymatic
degradation by blocking the action of an exonuclease that cleaves
normal phosphodiester bonds, the exonuclease will only digest
unmodified dNTPs which have been incorporated into the nucleotide
sequence of the amplicon. Preferably, the enzymatic degradation by
an exonuclease at the sites of incorporation of the modified dNTPs
is approximately 1.1 to 10 times slower than exonuclease
degradation of unmodified phosphodiester bond hydrolysis, more
preferably, the enzymatic degradation by an exonuclease at the
sites of incorporation of the modified dNTPs is approximately 10 to
1000 times slower than exonuclease degradation of unmodified
phosphodiester bond hydrolysis, even more preferably the enzymatic
degradation by an exonuclease at the sites of incorporation of the
modified dNTPs is approximately 1000 to 10,000 times slower than
exonuclease degradation of unmodified phosphodiester bond
hydrolysis, and most preferably, the enzymatic degradation by an
exonuclease at the sites of incorporation of the modified dNTPs is
approximately 10,000 to 100,000 slower than exonuclease degradation
of unmodified phosphodiester bond hydrolysis. In a preferred
embodiment, the enzymatic degradation by an exonuclease at the
sites of incorporation of the modified dNTPs is completely blocked
at each modified phosphodiester bond of the amplicon. The amplicons
are protected from enzymatic degradation by the exonuclease at the
sites of incorporation of the modified dNTPs and preferably, the
termini of the digested amplicons terminate at the sites of
incorporation of the modified dNTPs. The exonuclease digests the
termini of the amplicons thereby creating a single stranded
overhang sequence at each terminus of the amplicon.
[0106] Preferably, the single stranded overhang sequence at the
first terminus of the amplicon is complimentary to a first ligation
site sequence of the first adaptor sequence, and the single
stranded overhang sequence at the second terminus of the amplicon
is complimentary to the second ligation site sequence of the second
adaptor sequence. The sequences of the first and second ligation
site sequences of the first adaptor sequence and the second adaptor
sequence are not identical thereby enabling for the ligation of the
digested amplicon in one direction relative to the first and second
adaptor sequences. Preferably, the first and second ligation site
sequences of the first and second adaptor sequences, respectively,
are the ends of a doubly digested cloning vector and the digested
amplicon is preferably inserted into the cloning vector in one
direction relative to the vector, i.e., the digested amplicon is
directionally cloned into the vector.
[0107] The amplicon is treated with an exonuclease, wherein the
exonuclease can be a 5' to 3' exonuclease or a 3' to 5'
exonuclease. In a preferred embodiment, the amplicons are treated
with a 3' to 5' exonuclease, and more preferably, the amplicons are
treated with exonuclease III. Treating the amplicons with
exonuclease III digests the nucleotides of the 3' termini and
exposes single stranded overhang sequences at each 5' terminus of
the amplicons. Preferably, the single stranded overhang sequence at
the first 5' terminus of the digested amplicon is similar, and
preferably identical to the first primer's terminus which is
complimentary to a first ligation site sequence of the first
adaptor sequence; and the single stranded overhang sequence at the
second 5' terminus of the digested amplicon is similar, and
preferably identical to the second primer's terminus which is
complimentary to a second ligation site sequence of the second
adaptor sequence.
[0108] In a preferred embodiment, the single stranded overhang
sequences at the first and second 5' termini of the digested
amplicon are complimentary to the first and second ligation site
sequences in the first and second adaptor sequence, respectively.
Preferably, the single-stranded overhang sequence at the first and
second 5' terminus is approximately between one and ten nucleotides
in length, more preferably two to seven nucleotides in length,
still more preferably two to five nucleotides in length, and most
preferably four nucleotides in length. In a preferred embodiment,
the single overhang sequence at the first and second 5' terminus of
the digested amplicon has similar, and more preferably, the same
number of nucleotides as the first and second ligation site
sequence of the first and second adaptor sequences, respectively,
and the single overhang sequence at the first and second 5'
terminus of the digested amplicon is complimentary to the first and
second ligation site sequence of the first and second adaptor
sequences, respectively.
[0109] The single overhang sequence at the first 5' terminus of the
digested amplicon is directionally ligated to the first adaptor
sequence to form an amplicon-adaptor complex. Preferably, the
adaptor sequence is a linear synthetic or enzymatically prepared
linear nucleic acid. In a preferred embodiment, the single overhang
sequence at the first and second 5' terminus of the digested
amplicon is directionally ligated to the first and second ligation
site sequence of the first and second adaptor sequences,
respectively, thereby producing a digested amplicon flanked by a
first and second adaptor sequence. In a preferred embodiment, the
digested amplicon flanked by the first and second adaptor sequence
is subjected to a second round of PCR amplification using primers
which are specific for the first and second adaptors.
[0110] In a preferred embodiment, RNA is synthesized from the
amplicon flanked by the first and second adaptor sequences by in
vitro transcription. See Melton et al., Nucleic Acid Res., 1984,
12: 7035. The synthesized RNA may be used to generate probes for
hybridization to Northern or Southern blots, plaque and colony
lifts, tissue sections and chromosome spreads. RNA transcripts are
also useful for S1 nuclease mapping, generation of antisense RNAs
to block translation, and mRNA synthesis for translation in vitro.
In another preferred embodiment, the amplicon flanked by the first
and second adaptor sequences is used to synthesize protein by in
vitro translation. See Pelham et al., Eur. J. Biochem., 1976, 67:
247.
[0111] In one preferred embodiment, the first and second ligation
site sequences of the first and second adaptor sequences are the
ends of a cloning vehicle such as a plasmid, vector, or bacterial
artificial chromosome. These cloning vehicles may be used for
subcloning and/or gene expression. Aside from being used to
propagate the digested amplicon, the adaptor sequence may be
designed to impart one or more desired properties that add
functionality to the adaptor sequence.
[0112] Preferably, the adaptor sequence comprises a nucleotide
sequence encoding for expression systems which will permit affinity
purification. In a preferred embodiment, the adaptor sequence may
comprise at least one epitope tag which may be c-myc,
polyhistidine, polyarginine, glutathione-S-transferase (GST) tag,
HA epitope, V5, Xpress.TM., and FLAG.RTM.. See Evan et al., Mol
Cell Biol. 5:3610-3616 (1985). The use of a polyarginine tag allows
for the polypeptide to be purified on a cation exchange resin. See
Sassenfeld, H. M. and Brewer, S. J. BioTechnology, 2:76 (1984);
U.S. Pat. No. 4,532,207. Preferably, the amplicon-adaptor complex
includes a nucleotide sequence encoding for
glutathione-S-transferase. The resulting polypeptide may be
selectively recovered on glutathione-agarose. See Smith, D. B. and
Johnson, K. S. Gene 67:31 (1988). In another preferred embodiment,
the adaptor sequence comprises a nucleotide sequence encoding
IgG-Sepharose can be used to affinity purify fusion proteins
containing staphylococcal protein A. See Uhlen, M. et al. Gene
23:369 (1983). In yet another preferred embodiment, the adaptor
sequence comprises a nucleotide sequence encoding the
maltose-binding protein domain from the malE gene of E. coli which
allows the affinity purification of the resulting polypeptide on
amylose resins.
[0113] In a preferred embodiment, the adaptor sequence is designed
to contain sequences encoding for a metal chelating sequence
composed of multiple or alternating histidine residues which would
allow the adaptor sequence to bind to a metal ion immobilized on a
resin or other matrix. Preferably, a metal chelating sequence may
comprise at least one histidine residue, at least one glycine
residue or a combination of alternating or multiple histidine
residues, which may be used in affinity purification techniques
using a Ni.sup.2+ binding metal resin. See e.g., U.S. Pat. Nos.
4,569,794, 5,310,663, 5,284,933 and 5,594,115 which are
incorporated herein by reference. Once the polypeptide is bound to
the metal resin, the adaptor sequence can be released by
protonation of its associated metal ion-binding ligand.
Dissociation is achieved by lowering the pH of the surrounding
buffer medium, a common method known in the art for eluting bound
proteins.
[0114] In another preferred embodiment, the adaptor sequence
comprises nucleic acid sequences encoding for an epitope tag.
Epitope tagging utilizes antibodies against guest peptides to study
protein localization at the cellular level and subcellular levels.
See Kolodziej, P. A. and Young, R. A., Methods Enzymol.,
194:508-519 (1991). Preferably, the digested amplicon is ligated to
the first adaptor sequence comprising a nucleotide sequence
encoding the epitope tag to form an amplicon-adaptor complex. The
amplicon-adaptor complex is introduced into a cell by a method such
as transformation. When the amplicon-adaptor complex is expressed
the result is a chimeric protein containing the epitope as a guest
peptide. If the epitope is exposed on the surface of the protein,
it is available for recognition by the epitope-specific antibody,
allowing the investigator to observe the protein within the cell
using immunofluorescence or other immunolocalization techniques.
Preferably, the amplicon-adaptor complex comprising such epitope
tags are used for purifying proteins utilizing affinity
purification techniques.
[0115] Preferably, the nucleotide sequence encoding the epitope tag
is located at the terminus of the adaptor sequence. After the
digested amplicon is directionally ligated to the adaptor sequence
to form an amplicon-adaptor complex, the amplicon-adaptor complex
is expressed. Preferably, the nucleotide sequence encoding the
epitope tag is located at the terminus of the amplicon-adaptor
complex, and more preferably, the nucleotide sequence encoding the
epitope tag is located at the amino-terminus of the
amplicon-adaptor complex. Generally, when the epitope is fused to
the amino or carboxy-terminus of the expressed protein, the epitope
is more accessible to the antibody for detection and less likely to
cause severe structural or functional perturbations.
[0116] In a preferred embodiment, the adaptor sequence comprises a
nucleotide sequence encoding for the FLAG.RTM. octapeptide
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 10). Preferably, the
FLAG.RTM. epitope is fused to the amino or carboxy-terminus of the
expressed amplicon-adaptor complex protein, and more preferably,
the FLAG.RTM. epitope is fused to the amino terminus of the
expressed amplicon-adaptor complex protein. The placement of
FLAG.RTM. octapeptide at the amino-terminus allows for the
amplicon-adaptor complex to be affinity purified on an
immuno-affinity resin containing an antibody specific for the
octapeptide. See Hopp, T. P., et al. Biotechnology, 6:1204 (1988);
Prickett, K. S., et al., BioTechniques, 7:580 (1989); and U.S. Pat.
No. 4,851,341. The original FLAG.RTM. sequence is recognized by two
antibodies, M1, M2, and a FLAG.RTM. sequence with an initiator
methionine attached is recognized by a third antibody, M5. The last
five amino acids of the FLAG.RTM. sequence is a recognition site
for the protease enterokinase, thus, allowing for removal of the
FLAG.RTM. epitope. More preferably, the adaptor sequence comprises
a nucleotide sequence encoding for multiple antigenic epitopes, and
even more preferably, the adaptor sequence comprises a nucleotide
sequence encoding for multiple FLAG.RTM. epitopes. See U.S. patent
application Ser. No. 09/415,000, filed Oct. 8, 1999, incorporated
herein.
[0117] The adaptor sequence may also comprise other elements such
as promoters, repressors, or enhancers, e.g., BNL, CMV, T7, T3,
SP6, Gal4, Tet On/Off. Preferably, the adaptor sequence comprises a
reporting element, e.g., fluorescing proteins (e.g. GFP etc.), beta
galactosidase (.beta.-gal), luciferase, and/or an antibiotic
resistant gene, (e.g. amp, neo, agr, kan, pur, hyg and etc.). The
usage of a reporting gene allows for clone selection after the
amplicon-adaptor complex is expressed. Other nucleotide sequences
which may be included in the adapter nucleic acid are sequences
that direct recombination or enable ligase independent cloning. The
adaptor sequence may also be designed to include sequences critical
to developing nucleic acid based diagnostics, increased specificity
diagnostics, SNP analysis or gene expression analysis.
[0118] In one preferred embodiment, the digested amplicon is
ligated to a cloning vehicle, and more preferably, a cloning
vector. Preferably, the first and second ligation site sequences of
the first and second adaptor sequences, respectively, are the ends
of a cloning vector thereby resulting in a cloning vector
comprising a ligation site flanked by a first and second ligation
site sequence to which the digested amplicon may be ligated. The
first and second ligation site sequences are complimentary to the
single stranded overhang sequences at the first and second terminus
of the digested amplicon, respectively, wherein the nucleotide
sequences of the first and second ligation site sequences are not
identical. The difference in the "sticky ends" or cohesive ends of
each digested amplicon's termini and the complementarity between
the first and second terminus of the digested amplicon and the
first and second ligation site sequences, respectively, allow for
the digested amplicons to be directionally inserted into the
cloning vector.
[0119] Preferably, the synthesis of amplicons using a dNTP mixture
comprising modified dNTPs followed by exonuclease digestion results
in a higher preponderance of directional clones. In a preferred
embodiment, a high percentage of the digested amplicons are
inserted directionally into the cloning vector. Preferably, 80% of
the digested amplicons are inserted directionally into the cloning
vector, more preferably, 90% of the digested amplicons are inserted
directionally into the cloning vector, and most preferably, 99% of
the digested amplicons are inserted directionally into the cloning
vector.
[0120] The resulting cloning vector is used to transform a host
microorganism. The transformants are isolated and analyzed for the
presence of the target nucleic acid. The transformants are then
multiplied in culture to cause replication of the vector. Various
procedures and materials for preparing recombinant vectors,
transforming host cells with the vectors, replicating the vector
and expressing polypeptide and proteins are discussed by Old and
Primrose, Principles of Gene Manipulation, (2d Ed. 1981).
[0121] The digested amplicon may be inserted into any of a variety
of conventional cloning vectors. Although plasmids are preferred,
the vector may be alternatively a bacteriophage or cosmid. If
cloning takes place in mammalian or plant cells, viruses can be
used as vectors. If a plasmid is employed, it may be obtained from
a natural source or artificially synthesized. The particular
plasmid chosen should be compatible with the particular cells
serving as the host, whether a bacteria such as Escherichia coli
(E. coli), yeast, or other unicellular microorganism. The plasmid
should have the proper origin of replication (replicon) for the
particular host cell chosen. Any variety of cell that is
transformable may serve as a host cell. Examples include but are
not limited to, E. coli XL1-blue, DH5-.alpha., HB101, JM101, JM103,
JM109, etc. Other bacterial hosts may include Bacillus or
Pseudomonas species and the like. By way of example, eukaryotic
host cells may include Saccharomyces species.
[0122] This invention also contemplates kits for directional
ligation of a nucleic acid to an adaptor sequence. This invention
further contemplates kits for cloning of a nucleic acid to an
adaptor sequence. Such kits may include, for example, the dNTP
mixture, instructions for using the dNTP mixture and other
components necessary for directional ligation or for cloning. The
kit may be in the form of a test kit, that is, in a packaged
collection or combination as appropriate for the needs of the user
and any analytical instrumentation involved. Minimally, the kit
will comprise the dNTP mixture comprising modified dNTPs which,
when incorporated into a polynucleotide, impart resistance against
enzymatic digestion by an exonuclease at the sites of incorporation
of the modified dNTPs. Preferably, the kit furthers contain an
exonuclease, preferably a 3' to 5' exonuclease, and more
preferably, exonuclease III. The kit can, of course, also include
appropriate packaging, containers, labeling, buffers, and controls
for directional ligation of a nucleic acid to an adaptor sequence
or for cloning a nucleic acid into a cloning vector.
[0123] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention. However, those of skill in
the art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention, therefore all
matter set forth or shown in the accompanying drawings is to be
interpreted as illustrative and not in a limiting sense.
EXAMPLES
Example 1
In vitro Incorporation of Thiodeoxypurines into DNA using
Polymerases
[0124] The universality with respect to thermostable polymerase was
checked using a variety of commercially available polymerases. The
experiment was performed with respect to product quality and
quantity for a normal (0.8 mM dNTP) vs. thiodeoxypurine containing
dNTP (sdNTP) mix. The experiment was carried out using buffers and
reagents with the exception of the nucleotide mix as described or
supplied by the enzyme supplier. Taq, REDTaq, AccuTaqLA, KlenTaqLA
were obtained from Sigma-Aldrich, St. Louis, Mo. Pfu Turbo was
obtained from Startagene. Vent and Deep Vent were obtained from New
England Biolabs. Pwo was obtained from Roche. UlTma was obtained
from Perkin Elmer. Amplification products were analyzed on a 0.2
agarose gel and visualized by ethidium bromide staining. No attempt
was made to optimize cycling parameters for any particular
enzyme.
[0125] FIG. 2 demonstrates that product quality was independent of
nucleotide mix as the products from a normal vs. osdNTP were
identically composed. Vent/Deep Vent lack of product was not
followed up since neither enzyme is licensed for PCR and each have
a reputation for being problematic. Notably from the gel, all
enzymes seem to have somewhat of a reduced yield using the sdNTP
mix formulation.
[0126] FIG. 3 summarizes the amplification and protection yields
from a .sup.32p dNTP containing PCR. Yields were measured by TCA
precipitation. The top panel shows overall yields of amplification
(incorporation) products for all enzymes normalized to Taq/dNTP.
Yields were generally within 2.times. of Taq. The middle panel
shows yields of sdNTP products normalized to dNTP. The inclusion of
thiodeoxypurines resulted in a reduction of amplicon yield for all
enzymes tested. The reduction is likely inconsequential for Taq,
REDTaq, AmpliTaqLA, KlenTaq and Ultma.
Example 2
Stability of Phosphothioate Bonds to Hydrolysis by Exonuclease
III
[0127] This experiment demonstrates that thiodeoxypurines
incorporated into the amplicons demonstrated resistance to
Exonuclease III hydrolysis. The lower panel of FIG. 3 demonstrates
that while all products were degraded from approximately 50% or
less (+exo/-exo) for the all deoxynucleotide amplifications, the
addition of ExoIII to the thiodeoxypurine containing reactions
resulted in protections ranging from approximately 80 to 250%.
[0128] Protection of nucleotides greater than 100% may occur if the
polymerase is active enough during ExoIII degradation to
incorporate additional label. This was demonstrated by performing
PCR in the absence of a labeled radionucleotide which was followed
immediately by addition of the radionucleotide as part of the
ExoIII addition (FIG. 4). Using the sdNTP mix, all enzymes
incorporated nucleotides significantly above background.
[0129] FIG. 5 demonstrates that ExoIII and polymerase were not
competitively adding/removing nucleotides to produce an abundance
of blunt ends. PCR was performed in the absence of labeled dNTP
followed by addition of ExoIII/.sup.32PdCTP. Taq DNA polymerase was
added to half of the samples and followed by incubation at
72.degree. C. to completely fill in the ExoIII generated 5'
overhangs. As shown, re-incorporation during ExoIII digestion
replaced an insignificant portion (approximately 5-15%) of the
nucleotides removed by ExoIII. These data demonstrated that it is
safe to use the sdNTP nucleotide mix with most thermostable DNA
polymerases.
Example 3
Optimization of Incorporation of Thiodeoxynucleotides and
Exonuclease Digestion
[0130] The shortcoming of using a single nucleotide for controlling
exoIII digestion is sequence specific over and under-digestion. For
over-digestion it is conceivable that target sequences could be
amplified that are under-represented by the exonuclease controlling
nucleotide (e.g. low abundance dG sequences have a low occurrence
of sdG occupation). In this event, over-digestion of the amplicon
would result in a population of clonable duplex DNAs and single
stranded-ligation competing sequences. Such a situation would
undoubtedly result in diminished cloning efficiencies relative to
single strand deficient duplex amplicons. This is most likely going
to be problematic for relatively short and/or high AT (low GC)
amplicons. Under-digestion results from incomplete exposure of the
cohesive 5' ends upon exoIII digestion leading to reduced cloning
efficiency from sub-optimal insert: plasmid stoicheometry and from
competitive inhibitory duplexes. Using one modified nucleotide
(sdGTP) produced amplicons whose sdG content was 30%. There was a
30% chance that every G position would be occupied by a thioG.
Exonuclease III then has a 30% chance of stopping at each G.
Palindromic restriction sites can contain between one and two dGs
opposite dC of a 5' overhang. Table 1 contains the expected
proportions of A, B and C after exoIII digestion of amplicons
designed to be cloned between dG containing and non-dG containing
restriction sites (one end probabilities) and between two dG
contining sites (both ends). The main lesson from this exercise is
that the concentration of competitive ends (column C) approaches
and exceeds the concentration of desired amplicon (column A). With
the exception of the both end-two dG containing restriction
examples (Table 1 column B bold), amplicons B are of little
concern.
1TABLE 1 Reduction of ligation efficiencies due to 30% dGTPaS
occupation. Fraction of digested amplicon.sup.a One end.sup.b Both
ends.sup.c Recognition A B C A B C sequence.sup.d Affected
enzymes.sup.e 0.7 0 0.3 0.49 0.09 0.42 {circumflex over ( )}CDDD
Bsp11I, AflIII, NcoI, StyI, DsaI, BspHI, SpeI, AvrII, StyI, NheI,
XbaI {circumflex over ( )}DCDD XhoI, AvaI, BsoBI, SmlI {circumflex
over ( )}DDCD HindIII, SfcI, ApaLI {circumflex over ( )}DDDC DpnII,
MboI, Sau3AI, BglII, BstYI, BamHI, BclI, BsiWI, Acc65I, BanI, BsrGI
N{circumflex over ( )}CGN MaeII, AclI, BsaHI, MspI, HpaII, HinP1I,
NarI, TaqI, ClaI, BspDI, XhoI, AvaI, BsoBI, SmlI, AccI, BstBI
N{circumflex over ( )}GCN None 0.49 0 0.51 0.24 0.26 0.50
{circumflex over ( )}CCGG AgeI, BsrFI, BsaWI, XmaI, AvaI, BsoBI,
NgoMIV, BsrFI, BspEI, BsaWI {circumflex over ( )}CGCG MluI, AflIII,
DsaI, BssHII {circumflex over ( )}GCGC KasI, BanI {circumflex over
( )}GGCC EagI, EaeI, Bsp120I
[0131] based upon 30% dGTPaS occupation.
[0132] Amplicon to be cloned into plasmid digested with non-dG
overhang (e.g. EcoRI) and dG containing overhang (e.g. affected
enzyme column).
[0133] Amplicon to be cloned into plasmid digested with two dG
containing overhang restrioction enzymes (e.g. one or two enzymes
from affected enzyme column).
[0134] D=not C, {circumflex over ( )}=cut position
[0135] Palindromic Tetra- and hexa-nucleotide recognition sequences
table (New England Biolabs)
[0136] Having established that phosphorothioate bonds can provide
protection from the hydrolytic action of exonuclease III, this
experiment was undertaken to understand the possibility of
formulating a PCR invisible nucleotide mixture that would produce
uniformly gapped ligation products independent of sequence would
have nuclease resistant phosphodiesters statistically represented
at all positions. With phosphorothioates, this could be
accomplished by including all four sdNTPs. The final total
nucleotide concentration used in this formulation was 0.8 mM, a
concentration that is commonly recommended in many PCR protocols.
At this concentration, the free Mg.sup.++ concentration should not
change relative to a "normal" or typical PCR. It should be noted
that the 0.8 mM concentration is only required if, as here, it is
desired that the reaction formulation of the reaction buffer is to
be ignored. It is possible that other nucleotide formulations could
be investigated, however, it would be prudent to ascertain their
effect on free magnesium concentration and adjust accordingly as
PCR performance is often dependent on magnesium concentration.
[0137] Sequence independent gap size or probability would occur
when all nucleotides are replaced by odNTPS at identical frequency.
Such formulation would meet the following criteria: 2 0.8 mM = [
dATP ] + [ dCTP ] + [ dGTP ] + [ dTTP ] + [ sdATP ] + [ sdCTP ] + [
sdGTP ] + [ sdTTP ] and
k.sub.As[sdATP]=k.sub.Cs[sdCTP=k.sub.Gs[sdGTP]=k.sub.Ts[sdTTP]
[0138] wherein k.sub.Ns=incorporation rate constant.
[0139] The possibility of formulating the above solution was to
titrate PCRs (lambda 500 mer) with each thionucleotide. PCR was
performed using lambda control primers (Perkin Elmer) and lambda
DNA (Sigma) as template. All reagents (1.times. PCR buffer, 0.05
u/.mu.l Taq DNA polymerase) with the exception of the nucleotide
composition were from Sigma and used without modification. Cycling
conditions were 94.degree. C./30 sec., 68.degree. C./30 sec.,
72.degree. C./60 sec. for 30 cycles.
[0140] PCR was performed using an invariant .sup.32PDN'TP tracer
and 0.2 mM=[dNTP]+[sdNTP] (N and N' are not identical).
Thionucleotide incorporation was ascertained by resistance to
ExoIII digestion. As shown in the top panel of FIG. 6, PCR is
tolerant of thionucleotides in the order G>A>T.apprxeq.C. The
lower panel of FIG. 6 shows that the amplicons were preferentially
protected from exoIII digestion in the order G>A>T>C. PCR
yield data indicated that the concentration of any of the dNTPs
should remain above 0.1 mM in order to minimally impact PCR yield.
This allowed for 0.4 mM to be available for sdNTP.
[0141] Supposing that each thionucleotide was equally distributed
(i.e. [sdNTP]=0.1 mM each), then the concentrations of sdNTP would
be sub-optimal for protection. Instead, thiopurines were chosen for
optimization since they are best utilized by Taq during
amplification. Although this relaxed the requirement for sequence
independent ExoIII digestion termination, it still expanded the
repertoire of sequences relative to the single thionucleotide
example. The formulation criteria then became:
0.8 mM=[dATP]+[dCTP]+[dGTP]+[dTTP]+[sdATP]+[sdGTP]
and
k.sub.As[sdATP]=k.sub.Gs[sdGTP].
[0142] From the above thionucleotide titration yield data, the
concentration of each dNTP was greater than or equal to 0.1 mM. PCR
yields for a sdATP/sdGTP titration at [dNTP]=0.125 mM each
(N=A,C,G,T) were from ca. 85-92% (0.3 mM sdATP to 0.3 mM sdGTP) and
PCR yields were 59-72% at [dNTP]=0.1 mM each. 100% was defined at
[dNTP]=0.2 mM each for these experiments. From this data, an
acceptable formulation would be:
[dNTP]=0.125 mM each and 0.3 mM=[sdATP]+[sdGTP].
[0143] The optimal ratio of sdATP to sdGTP (i.e. such that
k.sub.As[sdATP]=k.sub.Gs[sdGTP]) was measured as shown in FIG. 7.
Although these conditions were not those used in the final
formulation, the quantity measured is the ratio of
thiodeoxypurines. Inspection of the figure revealed that
k.sub.As[sdATP]=k.sub.Gs[sdGTP] at 0.3 mM sdATP/0.2 mM sdGTP or
[sdATP]/[sdGTP]=3/2. The formulation was finalized by titrating
thiodeoxypurine (sdATP/sdGTP=3/2) vs. deoxypurine (dATP/dGTP=1).
FIG. 8 (top panel) demonstrates that PCR product yield begins to
suffer at concentrations of thiodeoxypurine above 0.3 mM. The final
formulation was: [dNTP]=0.125 mM each, 0.18 mM [sdATP] and 0.12 mM
[sdGTP]. The lower panel of FIG. 8 reports the occupation of purine
sites by dPuMP as a function of dPuTP concentration. The above
formulation produces amplicons that are 97% dPuMP/3% sdPuMP (i.e.
dAMP occupation was 97% at [dPuTP]=0.25 mM). The expected 5'
overhang length for a random sequence duplex is calculated as
(0.5+0.5*0.97).sup.n (i.e. 50% chance dPy plus 50% chance of 97%
chance dPu at nucleotide position n). From this, it is apparent
that all but very short amplicons should remain duplex after ExoIII
digestion (data not shown).
[0144] In random sequence DNA, this nucleotide solution will
produce amplicons that are 50% blocked to exoIII digestion by base
43. The probability of encountering a dG deficient sequence
decrease as 0.75.sup.n compared to a dPu deficient sequence at
0.5.sup.n i.e., a dG deficient sequence is 1.5.sup.n more likely
than is a dPu deficient sequence. Table 2 demonstrates how quickly
this difference manifests itself as a function of base position.
The advantage of sdPu incorporation vs. sdG is given by:
(P.sub.ntgP.sub.N)/(P.sub.notPuP'.sub.N).sup.n
[0145] where P.sub.notG is the probabilty of not G (i.e. is A, C or
T), P.sub.N is probability of dNMP (A+C+G+T), P.sub.notPu is
probability of not A or G (i.e. C+T), P'.sub.oN is probability of
dNMP (A+C+G+T) or:
(0.75(0.75+0.25P.sub.oG))/(0.5 (0.5+0.5P.sub.oPu)).sup.n
[0146] where P.sub.oG and P.sub.oPu are the probabilities of an
dGMP (70%) and dPuMP (97%) at base position n (column 3, Table 2).
These data demonstrate that though specific base occupation has
been reduced using sdPuTP, the effect is superior protection for a
larger cross section of sequences.
2TABLE 2 Relative probabilities of encountering dG vs dPu deficient
sequences as a function base position dG vs. dPu deficiency sdPu
vs. sdG advantage Base probabilities (0.75(0.75 + 0.25 .times.
0.7))/ Position (0.75/0.5).sup.n (0.5(0.5 + 0.5 .times.
0.97)).sup.n 1 1.5 1.4 5 7.59 5.6 10 57.7 31 15 438 170 20 3,325
960 25 25,251 5,400 30 191,751 30,000 35 1,456,109 160,000 40
11,057,332 930,000 45 83,966,617 5,200,000 50 637,621,500
29,000,000 55 >human genome 1.6 .times. 10.sup.8
[0147] Table 3 contains expected proportions of "ligatable" ends
from sdPuTP containing PCR products.
3TABLE 3 Reduction of ligation efficiencies due to 6% sdPuTP
occupation Fraction of digested amplicon.sup.a One end.sup.b Both
ends.sup.c A B C A B C Recognition sequence.sup.d N/A N/A N/A 0.88
0.003 0.11 {circumflex over ( )}PuPuPyPy, {circumflex over (
)}PuPyPuPy, {circumflex over ( )}PyPuPyPu, {circumflex over (
)}PyPyPuPu N/A N/A N/A 0.94 0.0009 0.058 N{circumflex over (
)}PyPuN, N{circumflex over ( )}PuPyN
[0148] Calculated using 3% sdPuTP occupation.
[0149] All four base palindromes contain purines making it
impossible to have an unaffected end.
[0150] Amplicon to be cloned into plasmid digested with one or two
four base overhang producing restriction enzymes.
[0151] Pu=dA or dG, Py=dC or dT, {circumflex over ( )}=cut
position
[0152] A comparison of Tables 1 and 3 reveals that inclusion of an
additional thionucleotide reduces the relative abundance of
competitive ligation inhibitors to inconsequential levels. However,
a nucleotide mixture containing a set of three or four modified
nucleotides could be developed that would produce low enough
occupancies at any position within the amplicon thereby retaining a
minimization of competitive ligation inhibitors.
[0153] Although there was a loss in product yield using the osdNTP
mix, the mix did not alter product quality relative to PCR using
non-modified dNTP mixtures (data not shown).
Example 4
Optimization of Incorporation of bdNTPs and Exonuclease
Digestion
[0154] The methods used to develop a phosphoroborano containing
deoxynucleotide mix that rendered amplicons protected from exoIII
overdigestion was analogous with the thiophosphate work described
in Example 3. Specifically, 1) titration of all four
phosphoroboranes in a quantitative PCR to establish nucleotide
biases, 2) optimization of favored phosphoroborane ratios (i.e.
bdATP, bdGTP), 3) titration of optimized phosphoroborane mixture
with dNTPs. PCR was performed using lambda control primers (Perkin
Elmer) and lambda DNA (Sigma) as template. All reagents (1.times.
PCR buffer, 0.05 u/.mu.l Taq DNA polymerase) with the exception of
the nucleotide composition were from Sigma and used without
modification. Cycling conditions were 94.degree. C./30 sec.,
68.degree. C./30 sec., 72OC/60 sec for 30 cycles.
[0155] FIG. 9 contains titration results for the four
phosphoroboranes (bdNTP, N=A, C, G, T). These data show that PCR
yields are negatively impacted by phosphoroboranes in the order
G<A<C<T and that ExoIII protection increases in the order
G>A>C.apprxeq.T. These results indicate that bdPyTPs were
incorporated inefficiently and the insensitivity of PCR yield to
bdPyTP concentration may be largely a result of dPyTP dilution,
i.e., that bdPyTPs are not particularly inhibitory. As with the
thionucleotides, it is evident that Taq polymerase prefers bdPuTPs
and that in keeping with a 0.8 mM total nucleotide triphosphate
concentration, there would again be little room to include all
bdNTPs in a reaction mix. A reasonable compromise again would be to
formulate a mix that contains dNTP+bdPuTP at a total of 0.8 mM. As
with the thiophosphates, this is not a requirement if one is
willing to investigate the affect other nucleotide formulations
have on free magnesium concentration and hence PCR performance.
FIGS. 10 and 11 contain bdATP/bdGTP and dPuTP/bdPuTP optimization
results respectively. PCR was performed using lambda control
primers (Perkin Elmer) and lambda DNA (Sigma) as template. All
reagents (1.times. PCR buffer, 0.05 u/.mu.l Taq DNA polymerase)
with the exception of the nucleotide composition were from Sigma
and used without modification. Cycling conditions were 94.degree.
C./30 sec., 68.degree. C./30 sec., 72.degree. C./60 sec for 30
cycles. The optimal bdATP/bdGTP ratio is 2.5 and verified by the
overlapping data in the lower panel of FIG. 11. The useful (high
protection levels) and un-impeding (little or no effect on PCR
yield) concentrations of bdPuTP range from approximately 0.1 to 0.3
mM bdPuTP. The mean 5' overhangs at these concentrations are 81 and
4 bases respectively. From these data it is clear that a nucleotide
mix could be formulated using bdNTPs.
Example 5
Directional Cloning of a PCR Product into a Vector
[0156] The present example is provided to demonstrate the
exonuclease recession technique for providing directional cloning
of a PCR product into a vector. The clonability of amplicons
prepared using the above described nucleotide formulations and
ExoIII digestion were compared with amplicons cloned using
restriction enzyme generation of the cohesive ends. Until this
point, amplification had been indiscriminately performed using Taq
or REDTaq DNA polymerases. Accutaq LA and REDTaq were used for the
below experiments. For integrity, Exoclone vs. restriction enzyme
cloning was compared. Thus, the lambda 500mer was amplified using
primer set BamHI 1 (SEQ ID NO: 1) and XbaI 1 (SEQ ID NO: 2) for
Exoclone, and a primer set designed for digestion with BamHI/XbaI
(BamHI 2: cut BamHI 1 lam 5' GCACG GGATCC GAT GAG TTC GTG TCC GTA
CAA CTG (SEQ ID NO: 3), XbaI 2: cut XbaI 1 lam 5' GCACG TCTAGA GGT
TAT CGA AAT CAG CCA CAG CGC (SEQ ID NO: 4), recognition site
underlined). After PCR the amplicons were respectively digested
with ExoIII, BamHI/XbaI, ligated with BamHI/XbaI cut pUC19 and
transformed into competent E. coli DH5a (Life Technologies). PCR
was performed using above described primers and lambda DNA (Sigma)
as template. All reagents (1.times. PCR buffer, 0.05 u/.mu.l Taq
DNA polymerase) with the exception of the nucleotide composition
were from Sigma and used without modification. Cycling conditions
were 94.degree. C./30 sec., 68.degree. C./30 sec., 72.degree. C./60
sec for 30 cycles. Restriction digestion was performed using
successive digestions by the restriction enzymes using a silica
bind and elute DNA purification method (Qiagen PCR purification
kit) between and after digestions. Ligation was at 16.degree. C.
for two hours using reagents from a ligation kit (Sigma).
[0157] The plasmids were isolated and subjected to PCR across the
inserts. Amplification across the insert (primer set EcopUC (SEQ ID
NO: 5)/HindpUC (SEQ ID NO: 6), FIG. 12) yielded amplicons that were
larger than from pUC19 and the same as an amplification product
from a previously prepared construct containing an identical insert
(FIG. 13A lanes 1-4 vs. 5 and 6 respectively). FIG. 13B
demonstrates that all of the clones were directional. That is,
primer pairs EcopUC (SEQ ID NO: 5)/Lambda R (SEQ ID NO: 8) and
HindpUC (SEQ ID NO: 6)/Lambda L (SEQ ID NO: 7) produced no PCR
products while primer pairs EcopUC (SEQ ID NO: 5)/Lambda L (SEQ ID
NO: 7) and HindpUC (SEQ ID NO: 6)/Lambda R (SEQ ID NO: 8) produced
PCR products. For additional evidence, one of the RE clones (pBX)
was cut using BamHI, XbaI and BamHI/XbaI (FIG. 14). The double
digest yielded the expected "insert" restriction fragment (FIG. 14
lane 4). The singly cut plasmid had a higher electrophoretic
mobility than did singly cut pUC19 (compare FIG. 14 lanes 1, 3 vs.
4, 5). Removal of the insert by double digestion resulted in a
fragment that comigrated with cut pUC19 (lane 4 vs. 5-7).
Example 6
Phosphoborano Modified Nucleotides vs. Phosphorothioate Modified
Nucleotides
[0158] This experiment was conducted in order to determine whether
any difference in cloning exists between the use of a mixture
containing phosphoborano modified nucleotides and a mixture
containing phosphorothioate modified nucleotides. FIG. 15
demonstrates the expected overhang length as a function of modified
nucleotide concentration. Clearly, phosphoborano nucleotides can be
used at higher occupation rates with out impacting PCR
performance.
[0159] Applicants then determined whether there was some advantage
to ligating DNAs with shorter rather than longer 5' overhangs. For
this PCR reactions (template=lambda DNA, primer set=BamHI 1 (SEQ ID
NO: 1)/XbaI 1 (SEQ ID NO: 2)) at two bdPuTP and one sdPuTP (FIG.
15) concentration were ligated after exoIII digestion to BamHI/XbaI
cut pUC19. PCR was performed using above described primers and
lambda DNA (Sigma) as template. All reagents (1.times. PCR buffer,
0.05 u/.mu.l Taq DNA polymerase) with the exception of the
nucleotide composition were from Sigma and used without
modification. Cycling conditions were 94.degree. C./30 sec.,
68.degree. C./30 sec., 72.degree. C./60 sec for 30 cycles. Ligation
reactions were serially diluted 10.times. to obtain relative
ligation efficiencies from the PCRs. FIG. 16 shows resulting PCR
products. Ligation was at 16.degree. C. for two hours using
reagents from a ligation kit (Sigma). Lanes marked bx were parallel
PCR performed with an authentic clone. All ligation reactions
produced a relatively high molecular weight PCR product while only
EcopUC/lambda L and HindpUC/lambda R produced products that
co-migrated with the authentic clone amplification products. The
high molecular weight products are likely amplification of
dimerized insert. Relative ligation efficiencies were estimated by
quantifying the bands from FIG. 16 using a BioRad gel doc image
analysis system.
Example 7
Preponderance or Lack Thereof of Mutations at Single Strand
Overhangs
[0160] Transformations using plasmids that potentially contain
relatively long tracts of single strand sequence is non-typical. In
all likelihood, cellular polymerases would repair the single
stranded "lesion" making the inserted DNA entirely duplex. It is
however not inconceivable that cellular repair enzymes could react
unexpectedly and introduce unwanted mutations in the "lesion"
region.
[0161] To investigate the cellular response fidelity, bacterial
Alkaline Phosphatase (SEQ ID NO: 8) was PCR amplified from a gene
bearing plasmid and cloned in E. coli (Nova Blue). Amplification
was performed using the thionucleotide mix described in Example 3.
96 clones were sequenced using fluorescent cycle sequencing kit
(Applied Biosystems) and analyzed for mutations as a function of
mutation position vs. duplex probability. The results are shown in
Table 4.
4TABLE 4 Base position and fraction duplex at mutation sites Base
Position Fraction Duplex Number of mutations 23 0.579477 1 24
0.579477 1 29 0.604708 1 34 0.65072 1 55 0.82316 1 137 0.987638 2
158 0.993342 1 198 0.997814 1 207 0.998068 1 209 0.998184 4 214
0.998492 2 215 0.998582 1 234 0.999188 1 241 0.999404 1 248
0.999505 1 250 0.999535 2 260 0.999679 2 262 0.999698 1 297
0.999865 1 302 0.999895 1 304 0.999895 2 309 0.999912 2
[0162] The fraction (probability) duplex at each position was
calculated for each position according to the probability of a base
position containing 97% dATP , 100% dCTP, 97% dGTP and 100% dTTP.
FIG. 17 ("duplex" curve, left axis) shows the fraction duplex and
the frequency of mutation as a function of sequence position. It is
clear that no correlation exists between mutational frequency and
degree of single stranded overhang. As shown in the Table 4, there
were no mutations found in regions of highest single stranded
probability (i.e. duplex probabilities <50%) and the
preponderance of mutations occurred at sites that have virtually no
probability of being single stranded (i.e. duplex probability is
approximately 1).
[0163] From this data, it is clear that cloning DNA containing
significant lengths of single stranded overhang introduces no
increased opportunity for sequence mutation.
Example 8
Generation of an Expression Library
[0164] The fact that exposure of amplicon cohesive bases is
amplicon sequence independent lends this method to generation of
expression libraries. 96 gene targets from E. coli were cloned
using the methods described herein. 89 successful PCR reactions
generated 70 clones displaying 95% or greater sequence homology
with the target gene sequence. This corresponds to an approximately
80% success rate neglecting PCR failure, i.e., PCR failure can be
due to many factors (primer design, cycling conditions, solution
formulation and etc.) outside of the scope of the current
invention. Such a success rate suggests that reliably generating
expression libraries using the current processes disclosed herein
is feasible.
Example 9
Self Ligation
[0165] As demonstrated in FIG. 18, the exonuclease digested
amplicons are self-ligatable.
[0166] A 500 base pair fragment was amplified from lambda 500 mer
DNA using a dNTP mixture containing s dATP and s dGTP, and primers
specific for BamHI (lane B), EcoRI (lane E), HindIII (lane H), SalI
(lane S), XbaI (lane X). The amplicon was digested with ExoIII and
followed by ligation at 16.degree. C. for 1 hour. Lane U contains
an amplified fragment which was not cut with exonuclease III. As
shown, all digested amplicons produced ligation products
demonstrating the premise of self-ligation.
[0167] Self ligation would be helpful if one were to attempt to use
this methodology in a gene shuffling or similar
combinatorial/molecular evolution experiment. That is, one could
take a variety of sequences, do nick translation or random priming
using the dNTP mixture containing modified dNTPs, digest with
exonucleaseIII then ligate to an adaptor. It is anticipated that
some of the sequences express an enzyme/protein with an altered
(improved) property.
Example 10
Ligation of Amplicons to Specifically Designed Adaptor
Sequences
[0168] FIG. 19 outlines the design of adapters designed to add
attributes for in vitro translation experiment. Specifically, a 5'
adapter nucleic acid was designed to contain the T7 promoter and a
FLAG.RTM. octapeptide coding sequence. The stop codon was
engineered into the 3' adapter. Since there is a sense strandedness
to the design, i.e., ligation of an amplicon to the adapters orient
with the 5' adapter upstream of a genes sense strand and the stop
codon 3' of the gene's sense strand, ligation to a gene followed by
amplification of the ligation product using adapter based
amplification primers would be most efficient if the ligation
proceeded directionally. Cohesive ends one and two were engineered
into the 5' and 3' adapters respectively for this purpose. Six
clones of these constructs were sequenced to corroborate that the
constructs were assembled as expected. In each case the ligation
orientation was shown to be as expected and without error.
[0169] FIG. 20 shows amplification products before (lanes 1, 2) and
after (lanes 3, 4) ligation of the above adapter sequence to the
digested amplicons (prepared by s-dATP/s-dGTP containing
amplification mixture followed by ExoIII digestion) of genes p53
and IkB, respectively. Amplification of the ligation mixture using
adapter specific primers clearly yielded amplicons that are longer
than those obtained by amplifying with gene specific primers.
[0170] It is to be understood that the present invention has been
described in detail by way of illustration and example in order to
acquaint others skilled in the art with the invention, its
principles, and its practical application. Further, the specific
embodiments of the present invention as set forth are not intended
as being exhaustive or limiting of the invention, and that many
alternatives, modifications, and variations will be apparent to
those skilled in the art in light of the foregoing examples and
detailed description. Accordingly, this invention is intended to
embrace all such alternatives, modifications, and variations that
fall within the spirit and scope of the following claims. While
some of the examples and descriptions above include some
conclusions about the way the invention may function, the inventors
do not intend to be bound by those conclusions and functions, but
puts them forth only as possible explanations.
Sequence CWU 1
1
14 1 28 DNA Lambda phage misc_feature (1)..(28) Primer for
Polymerase Chain Reaction (PCR) 1 gatcgatgag ttcgtgtccg tacaactg 28
2 28 DNA Lambda phage misc_feature (1)..(28) Primer for Polymerase
Chain Reaction (PCR) 2 ctagggttat cgaaatcagc cacagcgc 28 3 35 DNA
Lambda phage misc_feature (1)..(35) Primer for Polymerase Chain
Reaction (PCR) 3 gcacgggatc cgatgagttc gtgtccgtac aactg 35 4 35 DNA
Lambda phage misc_feature (1)..(35) Primer for Polymerase Chain
Reaction (PCR) 4 gcacgtctag aggttatcga aatcagccac agcgc 35 5 23 DNA
Lambda phage misc_feature (1)..(23) Primer for Polymerase Chain
Reaction (PCR) 5 cagtcacgac gttgtaaaac gac 23 6 23 DNA Lambda phage
misc_feature (1)..(23) Primer for Polymerase Chain Reaction (PCR) 6
cacaggaaac agctatgacc atg 23 7 23 DNA Lambda phage misc_feature
(1)..(23) Primer for Polymerase Chain Reaction (PCR) 7 ggcaatcagt
tcatctttcg tca 23 8 22 DNA Lambda phage misc_feature (1)..(22)
Primer for Polymerase Chain Reaction (PCR) 8 gtgtggcagc cgaaatgaca
ga 22 9 558 DNA Escherichia coli 9 agcttctcga gatgcctgtt ctggaaaacc
gggctgctca gggcgatatt actgcacccg 60 gcggtgctcg ccgtttaacg
ggtgatcaga ctgccgctct gcgtgattct cttagcgata 120 aacctgcaaa
aaatattatt ttgctgattg gcgatgggat gggggactcg gaaattactg 180
ccgcacgtaa ttatgccgaa ggtgcgggcg gcttttttaa aggtatagat gccttaccgc
240 ttaccgggca atacactcac tatgcgctga ataaaaaaac cggcaaaccg
gactacgtca 300 ccgactcggc tgcatcagca accgcctggt caaccggtgt
caaaacctat aacggcgcgc 360 tgggcgtcga tattcacgaa aaagatcacc
caacgattct ggaaatggca aaagccgcag 420 gtctggcgac cggtaacgtt
tctaccgcag agttgcagga tgccacgccc gctgcgctgg 480 tggcacatgt
gacctcgcgc aaatgctacg gtccgagcgc gaccagtgaa aaatgtccgg 540
gtaacgctct ggaaaaag 558 10 8 PRT Artificial Sequence TAG USED TO
PURIFY RECOMINANT PROTEIN 10 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 11
60 DNA Artificial Sequence forward primer containing T7 promotoer
and FLAG octapeptide designed to incorporate these sequences into
amplification products 11 ggatgctaat acgactcact atagggagaa
gggccaccat ggactacaaa gacgatgacg 60 12 60 DNA Artificial Sequence
complimentary sequence to SEQ ID 11 containing cohesive end to
enhance directional ligation to a gene for amplification 12
cctacgatta tgctgagtga tatccctctt cccggtggta cctgatgttt ctgctactgc
60 13 50 DNA Artificial Sequence complimentary sequence to SEQ ID
14 containing a cohesive end to enhance directional ligation to a
gene for amplification 13 gatcttgaaa actaaccata cgtcatgtgc
ccaccagcct tgtcctaata 50 14 46 DNA Artificial Sequence reverse
primer containing a stop codon designed to incorporate these
sequences into PCR amplification products 14 aacttttgat tggtatgcag
tacacgggtg gtcggaacag gattat 46
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