U.S. patent application number 10/024677 was filed with the patent office on 2003-10-16 for method for assembly of multiple dna fragments.
Invention is credited to Sharon, Gil.
Application Number | 20030194786 10/024677 |
Document ID | / |
Family ID | 11069867 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030194786 |
Kind Code |
A1 |
Sharon, Gil |
October 16, 2003 |
Method for assembly of multiple DNA fragments
Abstract
A method for assembling two or more DNA fragments with high
efficiency, comprises: a) providing, for each DNA fragment, at
least one protruding terminus, or "overhang", capable of hydrogen
bonding to a complementary sequence on at least one strand of a
second DNA fragment, said overhang having at least 15 bases; and b)
mixing two or more said DNA fragments under conditions suitable to
promote joining thereof.
Inventors: |
Sharon, Gil; (Mevasseret
Zion, IL) |
Correspondence
Address: |
LERNER, DAVID, LITTENBERG,
KRUMHOLZ & MENTLIK
600 SOUTH AVENUE WEST
WESTFIELD
NJ
07090
US
|
Family ID: |
11069867 |
Appl. No.: |
10/024677 |
Filed: |
December 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10024677 |
Dec 18, 2001 |
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09383143 |
Aug 25, 1999 |
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6372429 |
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09383143 |
Aug 25, 1999 |
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PCT/IL98/00096 |
Feb 26, 1998 |
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Current U.S.
Class: |
435/91.2 ;
536/23.2 |
Current CPC
Class: |
C12N 15/66 20130101;
C12Q 1/6811 20130101; C12N 15/10 20130101 |
Class at
Publication: |
435/91.2 ;
536/23.2 |
International
Class: |
C12P 019/34; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 1997 |
IL |
120,339 |
Claims
1. A method for assembling two or more DNA fragments with high
efficiency, comprising: a) providing, for each DNA fragment, at
least one protruding terminus, or "overhang", capable of hydrogen
bonding to a complementary sequence on at least one strand of a
second DNA fragment, said overhang and said complementary sequence
having at least 15 bases; and b) mixing two or more said DNA
fragments under conditions suitable to promote joining thereof.
2. A method according to claim 1, wherein the number of fragments
joined is three or more.
3. A method according to claim 1 or 2, wherein the overhang has at
least 20 bases.
4. A method according to claim 1 or 2, wherein the number of bases
in the overhang is between about 20 and about 30.
5. A method according to claim 2, wherein the molar ratio of each
DNA pair is between about 1:1 and 1:50
6. A method according to any one of claims 1 to 5, essentially as
described and with particular reference to the examples.
7. A DNA construct, whenever prepared by the method of any one of
claims 1 to 5.
8. A DNA fragment comprising an overhang of at least 15 nucleotides
or an end portion suitable to be converted into such an
overhang.
9. A DNA fragment as claimed in claim 8, for use in the method of
claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of International
Application No. PCT/IL98/00096, filed on Feb. 26, 1998, the
disclosure of which is incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of genetic
engineering. More particularly, the invention relates to a novel
method for assembling two or more DNA fragments with high
efficiency and yield.
BACKGROUND OF THE INVENTION
[0003] Building DNA constructs is the basis of genetic engineering.
Manipulating recombinant DNA and its incorporation into vectors is
common knowledge in the art. For example, fragments obtained by PCR
techniques, may be incorporated into plasmids for cloning purposes.
Generating specific DNA constructs by joining together different
DNA segments, each encoding specific properties and/or possessing
specific functions, is also known in the art. The state of the art
has been published in a very large number of books, articles,
patent applications, patents and the like, and is usually readily
available and known to all of skill in the art. For example, a
comprehensive account of DNA cloning procedures is provided in the
three volume text by Sambrook et al. (1989) entitled "Molecular
Cloning--a Laboratory Manual" 2nd edition, Cold Spring Harbor
Laboratory Press. This extensive account of the prior art
techniques for the combination of DNA fragments, cloning and
expression thereof, is included herein by reference, in its
entirety.
[0004] According to the present state of the art, the DNA fragments
comprising the plasmid to be constructed are cleaved by restriction
enzymes from larger entities. Next, they are connected utilizing
the enzyme Ligase, which forms phosphodiester bonds between DNA
fragments. Restriction enzymes cleave DNA leaving blunt or
staggered ends (protruding short single stranded termini) also
called overhangs. The efficiency of the ligation of blunt ended
fragments is very low. Using staggered ended fragments elevates the
efficiency of the ligation process, because the two fragments are
being held together by the hydrogen-bonds between the complementary
overhangs. Because the overhangs produced by most of the
restriction enzymes are very short (2-4 bases), the connections
between the complementary overhangs are weak and not very specific.
Thus, when constructing a plasmid from restriction enzyme enzyme
produced fragments, the yield is very low and there are many
illegitimate ligations, leading to undesired products. It is
therefore necessary to amplify the product usually by transfecting
cells of choice such as bacterial cells, and then to identify and
isolate colonies containing the DNA of choice from the ones
containing undesired products. Only then can another DNA fragment
be added by repetition of the whole procedure. As a result of this
inefficient processes the building of complex DNA constructs is a
laborious, time consuming and expensive process, in which each step
necessitates the successful completion of the former. The
construction of complex molecules may take several weeks to several
months. Sometimes the completion of such constructs is not achieved
at all. Another major drawback of the above mentioned method is
that the availability of restriction site does not always coincide
the construction demands. One way to overcome the above mentioned
problems is to use short artificial DNA molecules called "linkers".
This however further complicates the construction process, reduces
the yield, increases the percentage of wrong constructs and
sometimes add undesired foreign sequences.
[0005] Several methods suggest connecting DNA fragments by
producing longer complementary overhangs (8-14 nucleotides). These
connections are more stable than the ones produced by restriction
enzymes. In fact, they are stable enough to render the ligation
step unnecessary and the bacterial cells may be transformed right
after the fragments are connected. The phosphodiester bonds are
generated later, by the endogenous bacterial ligation
machinery.
[0006] One prior method uses single strand extensions that are
created by adding nucleotides at the 3' end of a DNA strand in a
template-independent fashion (Roychoudhury, R. Gene Amplif. Anal.
2:41-83, 1981). The enzyme used in this method, Terminal
Transferase, incorporates nucleotides at the end of a
double-stranded DNA fragment, thus creating a single-stranded tail.
Since the enzyme uses the nucleotides randomly, the only way to
ensure that the single-stranded tail will be complementary to a
corresponding overhang created on a second DNA molecule, is to
provide for each extension only one of the four nucleotides. The
overhangs created with this method must therefore be homopolymeric,
so that only four types of overhangs can be used, corresponding to
the residues dA, dC, dG or dT. Since the overhangs created on both
termini of a DNA fragment must be identical, cloning with this
method is directionless and can only involve two fragments that are
connected to each other at both ends, forming a circular molecule.
Furthermore, the length of the overhangs cannot be specifically
controlled. Finally, the method necessarily introduces an unwanted
stretch of nucleotides into the final construct, the length of
which cannot be determined exactly, making the method unsuitable
for the purpose of cloning into expression vectors where the
reading frame must be preserved.
[0007] According to another method (the commercial product
"PCR-Direct.TM.", manufactured by CLONTECH, Inc., USA) the
overhangs are generated utilizing the exonuclease activity of a DNA
polymerase.
[0008] U.S. Pat. No. 5,137,814 describes another method in which
the overhang is generated by providing at least one dU residue
instead of dT, close to the terminus of the fragment. The position
of the dU residues determines the length of the overhang. This
method involves an a-purination of the Uracil bases. The
a-purinated residues no longer have hydrogen-bond connections with
their complementary bases on the opposite strand. Moreover, they
destabilize the hydrogen bonds of their neighboring bases as well.
The resulting 3' protruding termini may connect to a complementary
single strand sequence. A commercial product using this method is
the CloneAmp.RTM. pAMP1 System (manufactured by Life Technologies,
Inc., USA). In the aforesaid method a 12-base overhang is used.
[0009] The use of overhangs longer than 4 nucleotides for the
purpose of fragment cloning is also described in several other
publications. Rashtchian et al. (Anal. Biochem. 206, p. 91-97,
1992), describe 12 nucleotide overhangs generated by Uracil DNA
Glycosylase (UDG) to achieve high-efficiency cloning of single
inserts into a vector. Kuijper et al. (Gene 112, p. 147-155, 1992)
and Aslanidis et al. (PCR Methods Appl. 4:172-177, 1994), describe
a cloning method wherein T4 polymerase is used together with a
predetermined dNTP to generate overhangs of a certain length in PCR
products. This method requires a specific sequence to be present in
the PCR primer. Hsiao et al. (NAR 21, p. 5528-5529, 1993) and Yang
et al. (NAR 21, 1889-1893, 1993) disclose generation of overhangs
by the exonucleolytic activity of Exonuclease III (Exo III) or of
T4 polymerase. Overhangs of 12 (Aslanidis), 8 (Yang) and 10-14
(Hsiao) nucleotides are disclosed.
[0010] The probability of producing a joint molecule composed from
three molecules is the probability of joining of the first two
molecules, multiplied by the probability of joining of the second
and third molecules. If the joining of any two molecule is a rare
event, due to the relatively short overhang, the joining of three
or more molecules becomes practically useless for cloning purposes,
because of the resulting low efficiency.
[0011] It is therefore clear that the present state of the art is
not satisfactory and there is a need for an improved method by
which several DNA fragments can be effectively joined together in a
directional, predetermined way, in one single-step. Such a method
will remove the limitations imposed on genetic engineering by the
aforementioned methods, and will thus revolutionize the way in
which genetic engineering is performed.
SUMMARY OF THE INVENTION
[0012] In one aspect, the invention is directed to a method for
assembling two or more DNA fragments with high efficiency,
comprising:
[0013] a) providing, for each DNA fragment, at least one protruding
terminus, or "overhang", capable of hydrogen bonding to a
complementary sequence on at least one strand of a second DNA
fragment, said overhang having at least 15 bases; and
[0014] b) mixing two or more said DNA fragments under conditions
suitable to promote joining thereof.
[0015] The method of the invention is based on the very surprising
finding that increasing the number of bases in complementary
overhang from 12 to at least 15, permits to reduce the ratio of the
reagents from about 1:100-300 to about 1:1. At the same time, this
permits to join a plurality of fragments (3 or more), because of
the high efficiency of the process.
[0016] According to one embodiment of the invention three or more
fragments are joined by the method of the invention, and the molar
ratio of each DNA pairs is about 1:1 to 1:50.
[0017] The present invention provides a DNA fragment comprising an
overhang of at least 15 nucleotides or an end portion suitable to
be converted into such an overhang. The invention further provides
said DNA fragment, for use in the above method of the
invention.
[0018] According to another preferred embodiment of the invention,
the number of bases in the overhang is between 20 and 30. While, as
stated above, the improvement of the process efficacy is very
dramatic when passing from 12 to 15 bases, there is still a further
steep improvement in the efficacy. when increasing the number of
bases to about 20. This increase reaches a plateau above 20 bases.
Taking into account that, when joining a number of fragments, the
overall efficiency of the process decreases, it is of course
desirable to employ the lowest number of bases in the overhang
which still provides for increased joining efficiency. As stated,
in most cases this optimal length will be in the neighborhood of 20
bases.
[0019] As will be appreciated by the skilled person, this is most
surprising because such a dramatic increase in binding ability of
fragments (from almost no binding when overhangs are 12 bases long
to almost 100% binding when the overhangs are 21 bases long) was
unexpectable. Although some improvement could have been expected by
increasing the length of the overhang, such a dramatic improvement
(two orders of magnitude) was entirely surprising. The art, in
fact, has continued to use stepwise cloning techniques, with all
the inherent disadvantages, and has not attempted in practice to
use overhangs longer than 12 bases.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1-6 illustrate plasmids produced in Example 3. In the
FIGS. 1-6, the dark, cross-hatched bar depicts the fragment
amplified from pBR322 which carries the Amp.sup.r gene and the
origin of replication. The empty bar depicts the fragment PCR
amplified from pBR322 which carries the Tet.sup.r gene. The hatched
bar depicts the PCR amplified fragment from pACYC184 which carries
the Cm.sup.r gene. The arrows mark primers, where the arrow head is
the 3' end of the primer, and the wavy tails mark the various
repetitive sequences at the 5' end of the primers. The primer's
number is given above or below the arrow. The black area denotes
repetitive sequences situated at the connection between two
fragments. FIG. 12 illustrates the plasmid produced in example 4.
The empty, cross-hatched and hatched bars have the above meanings.
The arrows mark the primers which are used for the seamless
junction of the fragments.
[0021] FIG. 1 is plasmid pGS101. There is an overlap of 12 bp
between every two fragments which are joined together. The plasmid
is assembled from three fragments.
[0022] FIG. 2 is plasmid pGS102. There is an overlap of 12 bp
between every two fragments which are joined together. The plasmid
is assembled from four fragments.
[0023] FIG. 3 is plasmid pGS103. There is an overlap of 12 bp
between every two fragments which are joined together. The plasmid
is assembled from four fragments.
[0024] FIG. 4 is plasmid pGS201. There is an overlap of 20-21 bp
between every two fragments which are joined together. The plasmid
is assembled from three fragments.
[0025] FIG. 5 is plasmid pGS202. There is an overlap of 20-21 bp
between every two fragments which are joined together. The plasmid
is assembled from four fragments.
[0026] FIG. 6 is plasmid pGS203. There is an overlap of 20-21 bp
between every two fragments which are joined together. The plasmid
is assembled from four fragments.
[0027] FIG. 7 shows the results obtained by an Agarose gel run, to
check fusion ability of identical fragments with different overhang
lengths.
[0028] FIG. 8 shows the colonies that grew on plates following
transformation with constructs illustrated in FIGS. 1-6. The
results of constructs made with overhangs of 12 bases are shown on
the left and those with overhangs of 20-21 bases are shown on the
right. FIGS. 8 A, B, C, D, E, F, and G correspond to plasmids
pGS101, pGS201, pGS102, pGS202, pGS103 and pGS203, respectively.
The plating was carried out on selective plates that would minimize
background noise as explained in the text.
[0029] FIG. 9 illustrates the constructs made in Example 3, along
with the number of transformants obtained;
[0030] FIG. 10 are the results of two gel runs of the products
obtained in Example 3;
[0031] FIG. 11 shows the results of gel runs of the products
obtained in Example 2;
[0032] FIG. 12 shows a construct made by joining 3 different
fragments using exonuclease III-created overhangs in the experiment
of example 4;
[0033] FIG. 13 shows a construct made by joining 8 different
fragments in the experiment of example 5.
DETAILED DESCRIPTION OF THE INVENTION
General Procedures
[0034] Overview
[0035] The present invention is based on the surprising discovery
that by increasing the length of complementary overhangs of two DNA
fragments from 12 bases, as in the prior art, to 15 bases or more,
the tendency of the fragments to be connected to each other rises,
steeply increasing, from close to 0% when 12 base overhangs are
used, to almost 100% when around 20 bases are employed. When long
complementary overhangs of 15 bases or more are used, a large
number of DNA fragments can be assembled in a single step in a
predetermined, directional manner. In addition, when connections
between just two fragments are made, the yield of cloned products
is much higher than that of the prior art, when operating according
to the invention.
[0036] A number of procedures can be used in order to create these
overhangs. In the examples detailed below, three different
procedures were used.
[0037] The First Procedure
[0038] The first procedure is used in examples 1-3. Examples 1 and
2 illustrate the difference in the connecting ability of fragments
with complementary overhangs of different lengths in-vitro by
subjecting the connected fragments to agarose gel analysis. In the
third example, successful cloning of a plasmid assembled from five
fragments using the procedure was demonstrated. The fragments were
connected to each other in-vitro, and then transformed into
bacterial cells.
[0039] The procedure is as follows:
[0040] The fragments which are to be attached to each other in a
directional fashion are preferably prepared by utilizing the
well-established Polymerase Chain Reaction (PCR). This provides
suitable amounts of the fragments and is especially preferred when
the fragments to be used are originally obtained or available only
in small amounts, for example from restriction-endonuclease treated
plasmid DNA, genomic DNA, or cDNA libraries in which the desired
fragments are present in small amounts. In this way, the original
fragments are greatly amplified and by virtue of the use of
pre-selected specific primers, the ends of the PCR-amplified
fragments will contain the desired pre-selected complementary
sequences.
[0041] The primers are designed such that they consist of two
regions: A 3' region that is complementary to the template and a 5'
region that contains a sequence complementary to a sequence at the
5' end of the fragment it is designed to join. In this 5' region,
several dT residues are substituted by dU residues. During PCR, the
thermostable DNA Polymerase does not distinguish between the dT and
dU residues and incorporates dA opposite both of these residues in
the newly synthesized strand..
[0042] Following PCR, the fragments are mixed together and the
enzyme Uracyl DNA Glycosylase (UDG) is added to the reaction
mixture. UDG specifically attacks the dU bases and a-purinates
them. The a-purinated residues no longer have hydrogen bonds with
their complementary bases. Furthermore, they destabilize the
hydrogen bonds of their neighboring bases as well. As a result, the
double stranded structure near the terminus falls apart. Two open
strands are created at the end of the segment: 1. A 5' single
strand with several a-purinated residues that can no longer form
hydrogen bonds with a complementary strand. 2. A 3' single strand
with no a-purinated residues that can form such bonds. Although
this single strand is not, strictly speaking, an overhang, it is
equivalent to it and behaves as such for all practical purposes.
The complementary regions of DNA generated in this procedure are
made of repetitive sequences. Therefore, it can be used to
illustrate that the length of the exposed ends is the key parameter
in determining the tendency of fragments to be connected to one
other: The difference between the constructs in the various
experiments is in the lengths of the repetitive complementary
regions and not in the sequence.
[0043] Two fragments that have such complementary single strands
can form hydrogen-bond connections with each other. If such
fragments are used to transform bacterial cells, their 5'
a-purinated single strands are degraded by cellular enzymes.
Furthermore, endogenous ligases convert the joint fragments into a
single DNA molecule. Details of the procedure are described in
Examples 1-3 below.
[0044] The Second Procedure
[0045] In Example 4, a second procedure was used. This procedure
uses the enzyme Exonuclease III (herein after ExoIII) to generate
the overhangs. Other enzymes having an exonuclease activity such as
T4 DNA polymerase or T7 DNA polymerase may be used as well. In
addition to the above mentioned enzymes which have 3'.fwdarw.5'
exonuclease activity, enzymes with 5'.fwdarw.3' exonuclease
activity may also be used, depending on the circumstances.
Procedures similar to this one are disclosed in the above Hsiao et
al. and Yang et al. The specific procedure is elaborated in a
copending application of the same applicant herein (filed on the
same day as this application, and identified as Attorney Docket
4190/96, Israeli Patent Application No. 120378). The procedure can
be applied for the joining of PCR fragments as in examples 1-3 as
well as for the joining of a mixture of DNA fragments that are
generated by restriction enzyme cleavage together with PCR
generated fragments. In example 4 the fragments were produced by
PCR to generate a plasmid assembled from three segments.
[0046] The procedure is as follows:
[0047] The terminal portions of the fragments that are to be joined
have to be complementary to each other. This can be achieved by
producing the PCR fragments in the following way: The primers of a
first DNA fragment that is to be PCR produced and is to be joined
to a second and to a third fragment should be synthesized having
two regions: 1. A 3' region that is complementary to the template.
2. A 5' region that is complementary to the terminal portion of the
fragment that is to be joined to the first fragment. The length of
the complementary regions should be 15 nucleotides (nt) or
more.
[0048] The various DNA fragments to be joined are mixed together
and an exonuclease enzyme is added to the mixture. In the case of
example 4, ExoIII was used. ExoIll is an exonuclease that catalyzes
the stepwise removal of mononucleotides from 3' hydroxyl termini of
double stranded DNA. It is added in access and the reaction is
incubated at 6.degree. C. to ensure slow digestion. The reaction is
allowed to proceed for a time sufficient to digest the ends of the
fragments, exposing 5' overhangs. These overhangs usually consist
of a region which is longer than the complementary portion.
[0049] Once the reaction is stopped, the temperature is raised to
75.degree. C. The reaction mixture is then cooled slowly. This
facilitates the joining of complementary overhangs while minimizing
illegitimate connections. Following the cooling, dNTPs and T7 DNA
Polymerase are added in order to fill any gaps that may have been
formed due to ExoIII overdigestion. At this stage, the mixture is
used to transform bacterial cells. Further elaboration of the
procedure is given in Example 4 below.
[0050] The Third Procedure
[0051] In example 5 a third procedure was used. This procedure is
similar, in many respects to the first one but has the advantage
that the junctions between the various fragments require no
addition of linker nucleotides. An elaborated description of this
procedure is disclosed in a copending application (filed on the
same day as this application, and identified as Attorney Docket
4149/96, Israeli Patent Application No. 120377). As will be
demonstrated below, a single dU residue near the termini of the
fragments, along with UDG and the reagent N,N
dimethylethylenediamine were employed in this procedure to generate
seamless connections between fragments. In example 5, the cloning
of a plasmid assembled from eight separate segments is
demonstrated. To date, this is the only disclosure of an assembly
of a plasmid from so many segments in a single step.
[0052] The procedure is as follows:
[0053] This procedure allows the formation of true 3' overhangs
using a single dU residue in each of the primers to be connected.
It is based on the ability to chemically create a nick at the 3'
end of an a-purinated dU residue.
[0054] The fragments are produced by PCR. As in the other examples,
the terminal portions of the fragments that are to be joined have
to be complementary to each other. As described in example 4, this
is achieved by producing the PCR fragments in the following way:
The primers of a DNA fragment that is to be PCR produced and is to
be connected to two other fragments are synthesized having two
regions: 1. A 3' region that is complementary to the template. 2. A
5' region that is complementary to the terminal portion of the
fragment that is to be joined to the said fragment. The length of
the complementary regions should be 15 nt or more. In each primer,
a dT residue is substituted by a dU residue at the junction between
the complementary and non complementary regions.
[0055] The various DNA fragments to be joined are mixed together
and UDG, as well as N,N dimethylethylenediamine are added to the
mixture. The enzyme a-purinates the dU base and the chemical
generates a nick at the 3' side of the a-purinated residue.
[0056] Following the creation of the above mentioned nicks, the
temperature is raised to 70.degree. C. in order to disconnect the
short single-strand stretch 5' of the a-purinated dU residues from
the rest of the fragment. Thus, 3' overhangs which constitute the
complementary regions are created. After removing the nicked
oligonucleotides, the reaction is cooled slowly to allow joining
between the complementary overhangs while minimizing illegitimate
connections. Following the cooling, the mixtures are used to
transform bacterial cells. Further elaboration of the procedure is
found in Example 5 below.
[0057] The examples to follow are provided for the purpose of
illustration only, and it is clear that the invention is not
limited to any particular method or procedure for creating the
aforesaid overhangs.
EXAMPLE 1
[0058] In Vitro Hybridization of Fragments with Complementary
Overhangs of Different Lengths
[0059] Overview
[0060] In order to demonstrate the superiority of connection
between DNA fragments having long overhangs, the degree of
inter-connection of fragments with different lengths of overhangs
was tested. Five pairs of fragments, each containing different
length of the complementary region, were produced by PCR. Each pair
consisted of: 1). A fragment of about 1400 bp designated Tet, which
was PCR amplified from the Tet.sup.r gene (conferring resistance to
tetracyclin) of pBR 322. 2). A fragment of about 1700 bp designated
Amp, which was PCR amplified from the Ampr gene (conferring
resistance to ampicillin) of pBR322. One of the two primers that
was used to amplify the Tet fragment in each pair contained, in
addition to a region which was complementary to the template DNA, a
5' tail with repeats of three bases CUA. Likewise, one of the
primers used for Amp amplification contained a 5' tail with repeats
of UAG. Note, that CUA and UAG are complementary to one another. In
the first pair, the tail consisted of four repeats (12 bases) and
the tails of the second, third, fourth and fifth pairs consisted of
five, six, seven and ten repeats respectively (15, 18, 21 and 30
bases).
[0061] The Amp fragment and the Tet fragment of each pair were
mixed, and their 3' overhang exposed utilizing UDG. After an
annealing time period the mixtures were subjected to agarose gel
analysis (FIG. 7).
[0062] Primers and PCR Conditions
[0063] For each of the 5 reactions, two fragments were produced. An
Amp.sup.r fragment and a Tet.sup.r fragment. The only variable
between the five Amp.sup.r fragments (or between the five Tet
fragments) was the length, but not the sequence, of the
complementary region.
[0064] The hybridization rate between pairs of DNA fragments was
tested.
[0065] A) The Primers and Production of the Fragments
[0066] In each of the primers below, and in the following examples,
the * symbol is written below the dU residue in order to emphasize
it. All of the primer sequences given herein are written in 5' to
3' direction.
1 Primer SEQ. ID NO. 1 - Tet sense primer (also designated 31160) -
upstream of Tet (PBR322: 1-22) --irrelevant tail- 22 nt
AGCTCCTGA-TTCTCATGTTTGACAGCTTATC Tet antisense primers note: the 5
primers below differ from one another only by the number of CUA
repeats at their 5'end. Primer SEQ. ID NO. 2 (also designated 3781)
- downstream of Tet (pBR322: 1449-1425) 25nt ---12 nt (CUA)x4---
TGG CCA GGA CCC AAC GCT GCC CGA G * Primer SEQ. ID NO. 3 (also
designated 4082) - downstream of Tet (pBR322: 1449-1425) 25nt ---15
nt (CUA)x5--- TGG CCA GGA CCC AAC GCT GCC CGA G * Primer SEQ. ID
NO. 4 (also designated 4353) - downstream of Tet (pBR322:
1449-1425) 25nt ---18 nt (CUA)x6--- TGG CCA GGA CCC AAC GCT GCC CGA
G * Primer SEQ. ID NO. 5 (also designated 4635) - downstream of Tet
(pBR322: 1449-1425) 25nt ---21 nt (CUA)x7--- TGG CCA GGA CCC AAC
GCT GCC CGA G * Primer SEQ. ID NO. 6 (also designated 5535) -
downstream of Tet (pBR322: 1449-1425) 25nt ---30 nt (CUA)x10--- TGG
CCA GGA CCC AAC GCT GCC CGA G * (Note that primers SEQ. ID NO. 2-6
are homologously complementary to primers SEQ. ID NO. 8-12
respectively at their 5'ends) Primer SEQ. ID NO. 7-Amp sense primer
(also designated 4142) - downstream of Amp (pBR322 2460-2479)
-----------irrelevant tail-------- 20 nt:
ATTGGTGCCCTTAAACGCCTG-AACGCAGGAAAGAACATGTG note: the 5 primers
below differ from one another only by the number of UAG repeats at
their 5'end. Amp antisense primers Primer SEQ. ID NO. 8 (also
designated 36107) - upstream of Amp (pBR322: 4136-4159) 24nt ---12
nt (UAG)x4---AAG AGT ATG AGT ATT CAA CAT TTC * Primer SEQ. ID NO. 9
(also designated 3993) - upstream of Amp (pBR322: 4136-4159) 24nt
---15 nt (UAG)x5---AAG AGT ATG AGT ATT CAA CAT TTC * Primer SEQ. ID
NO. 10 (also designated 4270) - upstream of Amp (pBR322: 4136-4159)
24nt ---18 nt (UAG)x6---AAG AGT ATG AGT ATT CAA CAT TTC * Primer
SEQ. ID NO. 11 (also designated 4560) - upstream of Amp (pBR322:
4136-4159) 24nt --21 nt (UAG)x7---AAG AGT ATG AGT ATT CAA CAT TTC *
Primer SEQ. ID NO. 12 (also designated 5434) - upstream of Amp
(pBR322: 4136-4159) 24nt ---30 nt (UAG)x10---AAG AGT ATG AGT ATT
CAA CAT TTC *
[0067] Note that primers SEQ. ID NO. 8-12 are homologously
complementary to primers SEQ. ID NO. 2-6 respectively at their 5'
ends.
[0068] The template for the PCR reaction was pBR 322 DNA. The full
sequence and maps of the various regions of this plasmid is well
known and can be accessed from GenBank database under accession no.
J01749 (pBR322).
[0069] The various fragments were produced by PCR, as follows:
2 PCR of Tet fragments (the number at the right denotes the length
of the overhang) Tet 12 Tet 15 Tet 18 Tet 21 Tet 30 10 .mu.l SEQ
SEQ SEQ SEQ SEQ (0.1 .mu.g/.mu.l) ID 2 ID 3 ID 4 ID 5 ID 6 10 .mu.l
SEQ SEQ SEQ SEQ SEQ (0.1 .mu.g/.mu.l) ID 1 ID 1 ID 1 ID 1 ID 1
pBR322 DNA 40 ng 40 ng 40 ng 40 ng 40 ng 180 .mu.l PCR mix in each
tube PCR of Amp fragments (the number at the right denotes the
length of the overhang) Amp 12 Amp 15 Amp 18 Amp 21 Amp 30 10 .mu.l
SEQ SEQ SEQ SEQ SEQ (0.1 .mu.g/.mu.l) ID 8 ID 9 ID 10 ID 11 ID 12
10 .mu.l SEQ SEQ SEQ SEQ SEQ (0.1 .mu.g/.mu.l) ID 7 ID 7 ID 7 ID 7
ID 7 pBR322 DNA 40 ng 40 ng 40 ng 40 ng 40 ng 180 .mu.l PCR mix in
each tube PCR mix: dNTPs (2.5 mM each) 128 .mu.l Buff .times. 1O
160 .mu.l H.sub.2O 1087 .mu.l Taq DNA Polymerase 5 U/.mu.l .sup.
6.4 .mu.l.sup.
[0070] Temperature regime
[0071] 94.degree. C. 40 sec
[0072] 40.degree. C. 2 min
[0073] 72.degree. C. 4 min
[0074] 30 cycles
[0075] 72.degree. C. 5 min
[0076] 6.degree. C. infinitely
[0077] All the required fragments were obtained. The concentration
of each fragment was determined by measuring the density of the
band created by each DNA fragment on an agarose gel. The volumes
used for the following experiment were corrected accordingly.
[0078] B) Checking Fusion Ability
[0079] Each pair of fragments having the same length of repeats was
put in a different Eppendorf tube. The DNA was incubated at
37.degree. C. with UDG in order to create the overhangs and allow
hybridization (see details below). The products of the reaction
were subjected to electrophoresis on an agarose gel at 60V for 60
min, stained with ethidium bromide and photographed under U.V.
light (FIG. 7). Amp fragments are 1.7 kb in length, Tet fragments
are 1.4 kb, and fragments of Amp+Tet that have hybridized to each
other are 3.1 kb in length. The difference in the density between
the Amp band, the Tet band and the Amp+Tet band, reflect the
ability of the fragments to adhere to each other. Comparisons could
thus be made between the adherence abilities of fragment-pairs
having different overhang-lengths.
[0080] The Amp and Tet fragments (of each pair) were mixed together
as follows
[0081] (The number of the mix is equivalent to the number of the
complementary overhang).
3 Mix 12 15 18 21 30 Amp frag. 3 .mu.l 1.5 .mu.l 1 .mu.l 1 .mu.l 1
.mu.l Tet frag. 3 .mu.l 2 .mu.l 2.5 .mu.l 1.5 .mu.l 2 .mu.l
H.sub.2O 2 .mu.l 4.5 .mu.l 4.5 .mu.l 5.5 .mu.l 5 .mu.l UDG Buff.
.times. 10 1 .mu.l 1 .mu.l 1 .mu.l 1 .mu.l 1 .mu.l
[0082] UDG Buff.x10=200 mM Tris HCl pH 8.4, 500 mM KCl, 15 mM
MgCl.sub.2
[0083] The content of each of the tubes was divided into two. In
the first tube, 0.5 .mu.l UDG was added. In the second, 0.5 .mu.l
H.sub.2O was added as a blank control. The tubes were incubated at
37.degree. C. for 5 hours. Then, the content of the tubes was
transferred into an 0.8% TAE Agarose gel. The gel was run at 60V
for 60 min, stained with ethidium bromide and photographed under
U.V. light. Results are shown in FIG. 7.
4 OVERHANG 12 15 18 21 30 Tube No. 1 2 3 4 5 6 7 8 9 10 UDG
H.sub.2O UDG H.sub.2O UDG H.sub.2O UDG H.sub.2O UDG H.sub.2O Lane
on the gel: 1 2 3 4 5 6 7 8 9 10
[0084] The experiment shows: 1) Almost no fusion is observed when
the overhangs are 12 bp long. 2) In lane 7, there are more Tet
fragments then Amp fragments. Therefore, even though there are
almost no free Amp fragments left, some free Tet fragments still
exist. 3) Both in lanes 7 and 9, there are no free Amp fragments
left. There is an outstanding ability of overhangs of 21 and 30 bp
to adhere to one another.
[0085] In conclusion, the results of the experiment indicate
that:
[0086] 1) Almost no fusion is observed when the overhangs are 12 bp
long.
[0087] 2) There is a dramatic increase in adherence starting with
overhangs 15 bases long.
[0088] 3) There is an outstanding ability of almost 100% of
overhangs of 21 and 30 bases to adhere to one another.
EXAMPLE 2
Multi DNA Fragment Assembly (MDFA) In-vitro: Comparison Between
Fragments with Overhangs of 12 and 20-21 Bases
[0089] I. Overview:
[0090] The following experiment was carried out in order to show
the stronger affinity and higher stability of connection between
fragments with overhangs of 21 bases versus the connection of
fragments having overhangs of 12 bases. Two sets of fragments were
generated by PCR. In the first set, the length of the complementary
regions at each end of the fragments was 12 bp. In the second set,
the length was 20-21 bp. The complementary regions were repeats of
3 or 5 bases. Therefore, the sequence of the complementary regions
in analogous fragments of the two sets can be regarded as
identical, the only difference being the overall length of the
repeated region. Four different fragments of each set were
generated by PCR, mixed together and 3' complementary overhangs
were exposed utilizing UDG. After an annealing time period, they
were run on agarose gel in order to check the degree of the
connection between the fragments (FIG. 11).
[0091] II. Primers and PCR Conditions:
[0092] Four unique fragments of each set (one set with 12 bp
complementary sequences and the other set with 21 bp complementary
sequences) were amplified by PCR.
[0093] The fragments were designated TetA, TetB, Amp, and CmB.
[0094] TetA was PCR amplified from pBR322 (bases 1-937).
[0095] TetB was PCR amplified from pBR322 (bases 1067-1449).
[0096] Amp was PCR amplified from pBR322 (bases 2460-4159).
[0097] CmB was PCR amplified from pACYC184 (bases 3768-4086).
[0098] The fragments were designed to connect with each other in
the following order: TetA-TetB-Amp-CmB. The primers were
synthesized with 3' ends that would enable making PCR fragments
from pBR322 and pACYC184 templates as in the former experiments.
Two sets of 8 primers were synthesized. In each set, three
junctions with complementary repeats were designed. A junction
between the TetA and the TetB fragments, a junction between the
TetB and the Amp fragments and a junction between the Amp and the
CmB fragments. Each of the repeats in a set was different and was
complementary-homologous only to one of the other repeats. The PCR
fragments that were produced could therefore be connected to one
another via the repeats, to form a linear fusion product.
Primers of the First Set: Primers Containing Repeats or
Near-repeats of 12 Nucleotides with dUs at Their 3' Ends
[0099]
5 Primer SEQ ID NO. 13 - Tet A sense (also designated 37101)
upstream of Tet (pBR322: 1-25) -------12 nt----- 25nt AU CAUAU
CAUAU TTC TCA TGT TTG ACA GCT TAT CAT C * 1 * * * * Primer SEQ ID
NO. 15 - Tet A antisense (also designated 37103) -------12 nt-----
25nt(pBR322: 913-937) UA CUAUA CUAUA CAT GCC GGC GAT AAT GGC CTG
CTT C * * * * * Primer SEQ ID NO. 16 - Tet B sense (also designated
37104) --------12 nt---- 25nt(pBR322: 1067-1091) UAUAG UAUAG UA GTA
GAT GAC GAC CAT CAG GGA CAG C * * * * * Primer SEQ ID NO. 2 Tet B
antisense downstream of Tet (pBR322: 1449-1425) ----12 nt CUAx4---
25 nt CUA CUA CUA CUA TGG CCA GGA CCC AAC GCT GCC CGA G * * * *
Primer SEQ ID NO. 8- Amp sense upstream of Amp (pBR 322: 4159-4136)
----12 nt UAGx4 - 24 nt UAG UAG UAG UAG AAG AGT ATG AGT ATT CAA CAT
TTC * * * * Primer SEQ ID NO. 18 - antisense Amp (also designated
36108) downstream of ORI ----12 nt AUGx4 - pBR322 (2460-2483) AUG
AUG AUG AUG AAC GCA GGA AAG AAC ATG TGA GCA * * * * Primer SEQ ID
NO. 17- Cm B sense (also designated 37106) -------12 nt 25nt
(pACYG1S4: 4086-4062) AU CUAAU CUAAU TAC GGT GAA AAC CTG GCG TAT
TTC C * * * * * Primer SEQ ID NO. 14 - Cm B antisense (also
designated 3866) -----12 nt CAUx4---- 26nt (pACYC184: 3768-3793)
CAU CAU CAU CAU GAG GCG TTT AAG GGC ACC AAT AAC TG * * * *
[0100] PCR Reactions
[0101] (The number on the right denotes the length of the
overhang)
6 reaction Tet A12 Tet B12 Amp12 Cm B12 20 ml primer 1 SEQ ID 13
SEQ ID 16 SEQ ID 18 SEQ ID 14 20 ml primer 2 SEQ ID 15 SEQ ID 2 SEQ
ID 8 SEQ ID 17 (Primer concentration: 0.1 .mu.g/.mu.l) expected 961
bp 406 bp 1724 bp 340 bp fragment size: mix 2 .mu.l dNTP's (2.5 mM
each) 112 .mu.l 2.5 .mu.l 10xBuff. 140 .mu.l 18 .mu.l H2O 1008
.mu.l 0.1 .mu.l Taq (5 u/.mu.l) 5.6 .mu.l put 360 .mu.l mix in each
tube. add primers add 1 .mu.l DNA pBR322 pBR322 pACYC184 pBR322
(DNA concentration: 40 ng/.mu.l)
[0102] Temperature regime
[0103] 94.degree. C. 40 sec
[0104] 40.degree. C. 2 min
[0105] 72.degree. C. 4 min
[0106] 30 cycles
[0107] 72.degree. C. 5 min
[0108] 6.degree. C. infinitely
Primers of the Second Set: Primers Containing Repeats of 21
Nucleotides with dUs at Their 3' Ends
[0109]
7 Primer SEQ. ID NO. 19 - TetA sense (also designated 4554) -
upstream of Tet (pBR322: 1-25) ------------20 ntGAUAUx4------- 25
nt CAUAU CAUAU CAUAU CAUAU TTC TCA TGT TTG ACA GCT TAT CAT C * * *
* * * * Primer SEQ. ID NO. 21 - TetA antisense (also designated
4558) - (pBR322: 913-937) ------------20 ntCUAUAx4------- 25 nt
CUAUA CUAUA CUAUA CUAUA CAT GCC GGC GAT AAT GGC CTG CTT C * * * * *
* * * Primer SEQ. ID NO. 22 - TetB sense (also designated 4559) -
(pBR322: 1067-1091) ------------20 ntUAUAGx4------- 25 nt UAUAG
UAUAG UAUAG UAUAG GTA GAT GAC GAC CAT CAG GGA CAG C * * * * * * * *
Primer SEQ. ID NO. 5 - TetB antisense downstream of Tet (pBR322:
1449-1425) ------------21 ntCUAx4----------- 25 nt CUA CUA CUA CUA
CUA CUA CUA TGG CCA GGA CCC AAC GCT GCC CGA G * * * * * * * Primer
SEQ. ID NO. 11 - Amp sense - pBR322 (4159-4136) -------21 nt
UAGx7---------- 24nt UAG UAG UAG UAG UAG UAG UAG AAG AGT ATG AGT
ATT CAA CAT TTC * * * * * * * Primer SEQ. ID NO. 24 Amp antisense
(also designated 4561) - pBR322 coding (2460-2483) -------20 nt
AUGx7---------- 24nt AUG AUG AUG AUG AUG AUG AUG AAC GCA GGA AAG
AAC ATG TGA GCA * * * * * * * Primer SEQ. ID NO.23- Cm B sense
(also designated 4556) - (pACYC184: 4086-4062) -------20 nt
CUAAUx4---------- 24nt CUAAU CUAAU CUAAU CUAAU TAC GGT GAA AAC CTG
GCC TAT TTC C * * * * * * * * Primer SEQ. ID NO. 20 - CmB antisense
(also designated 4743) - (pACYCI84: 3768-3793) -------21 nt
CAUx7---------- 26nt CAU CAU CAU CAU CAU CAU CAU CAG GCG TTT AAG
GGC ACC AAT AAC TG * * * * * * *
[0110] PCR Reactions
8 reaction Tet A21 Tet B21 Amp21 Cm B21 20 .mu.l primer 1 Seq. ID
19 Seq. ID 22 Seq. ID 11 Seq. ID 20 20 .mu.l primer 2 Seq. ID 21
Seq. ID 5 Seq. ID 24 Seq. ID 23 (primer concentration: 0.1
.mu.g/.mu.l) expected 977 bp 423 bp 1742 bp 357 bp fragment size:
mix x128 2 .mu.l dNTP's (2.5 mM each) 256 .mu.l 2.5 .mu.l 10xBuff.
320 .mu.l 18 .mu.l H2O 2304 .mu.l 0.1 .mu.l Taq 5 U/.mu.l 12.8
.mu.l (dNTP concentration: 2.5 mM each) put 360 ml mix in each
tube. add primers add 1 .mu.l DNA pBR322 pBR322 pACYC184 pBR322
(DNA concentration: 40 ng/.mu..lambda.)
[0111] Temperature regime
[0112] 94.degree. C. 40 sec
[0113] 40.degree. C. 2 min
[0114] 72.degree. C. 4 min
[0115] 30 cycles
[0116] 72.degree. C. 5 min
[0117] 6.degree. C. infinitely
[0118] III. Fusion of the Fragments
[0119] After the amplification, the fragments were mixed and 3'
complementary overhangs were exposed utilizing UDG. Fusions of
either 2 fragments, 3 fragments or 4 fragments of the two sets
(i.e., of fragments that were created with primers of the two
above-described sets) were performed. After the fusion the
reactions were run on a 0.8% agarose gel in 1.times. TAE stained
with ethidium bromide and photographed under U.V. light (FIG.
11).
[0120] In order to demonstrate the size of the various fragments,
they were run in separate lanes (TetA, TetB, Amp, and CmB, in lanes
1, 4, 7, and 10 respectively). Only fragments of the first set were
run, since those of the second set have identical lengths. The
various mixtures that included the fused fragments were also
run.
[0121] Reactions and lanes no. on the gel:
9 1.8 .mu.l 1. Tet A12 1.8 .mu.l 1.8 .mu.l 13.6 .mu.l 2 .mu.l 0.8
.mu.l 2. Tet A21 Tet B21 H.sub.2O UDG Buff .times. 10 UDG 1.8 .mu.l
1.8 .mu.l 13.6 .mu.l 2 .mu.l 0.8 .mu.l 3. Tet A12 Tet B12 H.sub.2O
UDG Buff .times. 10 UDG 1.8 .mu.l 4. Tet B12 1.8 .mu.l 1.8 .mu.l
1.8 .mu.l 11.8 .mu.l 2 .mu.l 0.8 .mu.l 5. Tet A21 Tet B21 Amp21
H.sub.2O UDGBuff .times. 1O UDG 1.8 .mu.l 1.8 .mu.l 1.8 .mu.l 11.8
.mu.l 2 .mu.l 0.8 .mu.l 6. Tet A12 Tet B12 Amp12 H.sub.2O UDGBuff
.times. 1O UDG 1.8 .mu.l 7. Amp12 1.8 .mu.l 1.8 .mu.l 1.8 .mu.l 1.8
.mu.l 10 .mu.l 2 .mu.l 0.8 .mu.l 8. Tet A21 Tet B21 Amp21 CmB21
H.sub.2O UDG Buff .times. 10 UDG 1.8 .mu.l 1.8 .mu.l 1.8 .mu.l 1.8
.mu.l 10 .mu.l 2 .mu.l 0.8 .mu.l 9. Tet A12 Tet B12 Amp12 CmB12
H.sub.2O UDG Buff .times. 10 UDG 1.8 .mu.l 10. CmB12
[0122] 11. Molecular Weight Markers
[0123] the concentration of all the DNA fragments was 0.09
pmol/.mu.l.
[0124] UDG Buff.x10=200 mM Tris HCT pH 8.4, 500 mM KCl, 15 mM
MgCl.sub.2
[0125] The reactions were incubated at 37.degree. C. for 5 hours
and then run on a gel. The results are shown in FIG. 11. The order
of the lanes is shown at the top, and the sizes of the molecular
weight markers are indicated at the right side of the
photograph.
[0126] From the above results, the following conclusions can be
drawn:
[0127] 1. Detectable fusion products are observed when the
overhangs are 21 bp long, but not when they are 12 bp long.
[0128] 2. Since the fusion efficiency of 21 bp overhangs is high
but less then 100%, products of partial fusions are also observed.
In lanes 2, 5 and 8, the band which appears highest on the gel, has
the length of the required end product.
[0129] 3. The results demonstrate that the amount of the required
product decreases when the number of fragments required to generate
it, increases.
[0130] This example clearly demonstrated the advantage of using
fragments with long overhangs. No end products were observed when
using fragments with 12 nt overhangs. When using fragments with
20-21 nt overhangs, the fusion of 2-3 and 4 fragments is clearly
demonstrated.
EXAMPLE 3
Multi DNA Fragment Assembly (MDFA) In-vivo: Comparison Between
Fragments with Overhangs of 12 and 20-21 Bases
[0131] The following experiment illustrates the advantage of
connecting fragments having 20-21 base overhangs as compared to
fragments having 12 base overhangs, in vivo. Fragments having
either 12 base overhangs or 20-21 base overhangs were produced by
PCR and connected, forming circular plasmids. These plasmids were
transformed into bacterial cells and the colonies were
examined.
[0132] Two sets of plasmids were constructed, as depicted in FIGS.
1-6. In the first set, fragments containing 12 base overhangs were
used (FIGS. 1-3). In the second, fragments containing 20-21 base
overhangs were used (FIGS. 4-6).
[0133] Each set contained three plasmids:
[0134] 1. A plasmid constructed out of the three basic
fragments.
[0135] an Amp.sup.r fragment (hereinafter Amp) which contained the
Ampicillin resistance gene.
[0136] a Tef.sup.r fragment (hereinafter Tet) which contained the
Tetracycline resistance gene.
[0137] a Cm.sup.r fragment (hereinafter Cm) which contained the
Chloramphenicol resistance gene.
[0138] 2. A plasmid constructed out of four fragments; the said Amp
and Cm fragments and two fragments designated TetA and TetB, which
are two separate regions of the Tet gene.
[0139] 3. A plasmid constructed out of four fragments; the said Amp
and Tet fragments and two fragments designated CmA and CmB, which
are two separate regions of the Cm gene.
[0140] The plasmids of the first set are equivalent to the plasmids
of the second set except for the length of the overhangs produced.
The overhangs of the fragments were designed and produced by adding
short repeats of either 3 or 5 bases. Each of the repeats in a set,
was different and was complementary to one of the other repeats.
The PCR fragments that were produced could therefore be connected
to one another via the repeats to form a full circular plasmid (see
FIGS. 1-6). Analogous primers of the two sets were nearly
identical, the only difference being in the length of the repeat
sequence. In the first set, the overall lengths of the repeats was
12 bp. In the second, the overall length of the repeats was 20-21
bp. In all cases, the dT residues in the repeats have been changed
to dU residues. The 5' termini of fragments containing the repeats
could therefore be dissociated from the complementary strand by
a-purinating the dU residues, exposing a 3' overhang of the
required size.
[0141] After amplifying the fragments comprising each plasmid they
were mixed and their termini exposed utilizing UDG. After an
annealing time period each mixture was used to transform bacterial
cells, as detailed below.
[0142] A) Primers and PCR Conditions
[0143] Primers were synthesized with 3' ends that would enable
generating PCR fragments from pBR322 and pACYC184 templates. The
full sequence and maps of the various regions of these plasmids are
well known and can be accessed from GenBank database under
accession nos. J01749 (pbR322), and X06403 (pACYC184).
[0144] Two sets of primers were synthesized:
First Set of Primers: Primers Containing Repeats or Near-repeats of
12 Nucleotides with dUs at Their 3' Ends
[0145]
10 Seq. ID NO. 13 - Tet A sense upstream of Tet (pBR322: 1-25)
--------12 nt------- 25 nt AU CAUAU CAUAU TTC TCA TGT TTG ACA GCT
TAT CAT C * * * * * Seq. ID NO. 15 - Tet A antisense downstream of
Tet (pBR322: 9 13-937) --------12 nt------- 25 nt UA CUAUA CUAUA
CAT GCC GGC GAT AAT GGC CTG CTT C * * * * * Seq. ID NO. 16 - Tet B
sense (pBR322: 1067-1091) --------12 nt------- 25 nt UAUAG UAUAG UA
GTA GAT GAC GAC CAT CAG GGA CAG C * * * * * Seq. ID NO. 2 - Tet B
antisense downstream of Tet (pBR322: 1449-1425) --------12 nt
CUAx4---- 25 nt CUA CUA CUA CUA TGG CCA GGA CCC AAC GCT GCC CGA G *
* * * Seq. ID NO. 18 - Amp sense - upstream of Amp (pBR322
2460-2483) --------12 bpAUGx4------- 24 nt AUG AUG AUG AUG AAC GCA
GGA AAG AAC ATG TGA GCA * * * * Seq. ID NO. 8 - Amp antisense -
downstream of Amp (pBR322: 4159-4136) --------12 bpUAGx4------- 24
nt UAG UAG UAG UAG AAG AGT ATG AGT ATT CAA CAT TTC * * * * Seq. ID
NO. 17- Cm B sense - (pACYC184: 4086-4062) --------12 bp------- 25
nt AU CUAAU CUAAU TAC GGT GAA AAC CTG GCC TAT TTC C * * * * * Seq.
ID NO. 14 - Cm B antisense downstream of Cm (pACYC184: 3768-3793)
-----12 ntCAUx4----- 26 nt CAU CAU CAU CAU CAG GCG TTT AAG GGC ACC
AAT AAC TG * * * * Seq. ID NO. 25 - Cm A sense (also designated
37102) - upstream of Cm (pACYC184: 240-216) --------12 nt------- 25
nt AUAUG AUAUG AU TCA GGA GCT AAG GAA GCT AAA ATG G * * * * * Seq.
ID NO. 26 - Cm A antisense (also designated 37105) - (pACYC184:
22-46) --------12 bnt------- 25 nt AUUAG AUUAG AU CAG GCG GGC AAG
AAT GTG AAT AAA G ** ** *
[0146] PCR Reactions
11 reaction Tet A12 Tet B12 Cm B12 Cm A12 20 .mu.l primer 1 SEQ. ID
13 SEQ. ID 16 SEQ. ID 14 SEQ. ID 25 20 .mu.l primer 2 SEQ. ID 15
SEQ. ID 2 SEQ. ID 17 SEQ. ID 26 (primer concentration: 0.1
.mu.g/.mu.l) expected 961 bp 406 bp 340 bp 241 bp fragment size:
reaction Tet12 Cm12 Amp12 20 .mu.l primer 1 SEQ. ID 13 SEQ. ID 14
SEQ. ID 18 20 .mu.l primer 2 SEQ. ID 2 SEQ. ID 25 SEQ. ID 8 (primer
concentration: 0.1 .mu.g/.mu.l) expected 1473 bp 739 bp 1724 bp
fragment size mix x128 2 .mu.l dNTP's (2.5 mM each) 256 .mu.l 2.5
.mu.l 10xBuff. 320 .mu.l 18 .mu.l H2O 2304 .mu.l 0.1 .mu.l Taq (5
U/.mu.l) 12.8 .mu.l put 360 .mu.l mix in each tube. add primers add
1 .mu.l DNA (40 ng/.mu.l): Tet A12 Tet B12 Cm B12 Cm A12 pBR322
pBR322 pACYC184 pACYC84 Tet12 Cm12 Amp12 pBR322 pACYC184 pBR322
[0147] Temperature regime
[0148] 94.degree. C. 40 sec
[0149] 40 .degree. C. 2 min
[0150] 72 .degree. C. 4 min
[0151] 30 cycles
[0152] 72.degree. C. 5 min
[0153] 6 .degree. C. infinitely
Second Set of Primers: Primers Containing Repeats of 21 Nucleotides
with dUs at Their 3' Ends
[0154]
12 SEQ. ID NO. 19- TetA sense - upstream of Tet (pBR322: 1-25)
---------20 nt CAUAUx4-------- 25 nt CAUAU CAUAU CAUAU CAUAU TTC
TCA TGT TTG ACA GCT TAT CAT C * * * * * * * * SEQ. ID NO. 5 TetB
antisense - downstream of Tet (pBR322: 1449-1425) ---------21 nt
CUAx7-------- 25 nt CUA CUA CUA CUA CUA CUA CUA TGG CCA GGA CCC AAC
GCT GCC CGA G * * * * * * * SEQ. ID NO. 21 - TetA antisense
(pBR322: 913-937) ---------20 nt CUAUAx4-------- 25 nt CUAUA CUAUA
CUAUA CUAUA CAT GCC GGC GAT AAT GGC CTG CTT C * * * * * * * * SEQ.
ID NO. 22 - TetB sense - (pBR322: 1067-1091) ---------20 nt
UAUAGx4-------- 25 nt UAUAG UAUAG UAUAG UAUAG GTA GAT GAC GAC CAT
CAG GGA CAG C * * * * * * * * SEQ. ID NO. 20- CmB antisense
(pACYC184: 3768-3793) ---------20 nt CAUx7-------- 26 nt CAU CAU
CAU CAU CAU CAU CAU CAG GCG TTT AAG GGC ACC AAT AAC TG * * * * * *
* SEQ. ID NO.27- CmA sense (also designated 4555) - (pACYC184:
240-216) ---------20 nt AUAUGx4-------- 25 nt AUAUG AUAUG AUAUG
AUAUG TCA GGA GCT AAG GAA GCT AAA ATG G * * * * * * * * SEQ. ID
NO.28- Cm A antisense (also designated 4557) - (pACYC184: 22-46)
---------20 nt AUUAGx4------- 25 nt AUUAG AUUAG AUUAG AUUAG CAG GCG
GGC AAG AAT GTG AAT AAA G ** ** ** ** SEQ. ID NO.23- Cm B sense
(pACYC184: 4086-4062) ---------20 nt CUAAUx4----- 25 nt CUAAU CUAAU
CUAAU CUAAU TAC GGT GAA AAC CTG GCC TAT TTC C * * * * * * * * SEQ.
ID NO. 11 - Amp antisense pBR322 (4159-4136) ---------20 nt
UAGx7-------- 24 nt UAG UAG UAG UAG UAG UAG UAG AAG AGT ATG AGT ATT
CAA CAT TTC * * * * * * * SEQ. ID NO. -24 Amp sense pBR322
(2460-2483) ---------21 nt AUGx7-------- 24 nt AUG AUG AUG AUG AUG
AUG AUG AAC GCA GGA AAG AAC ATG TGA GCA * * * * * * *
[0155] PCR Reactions
13 reaction Tet A21 Tet B21 Cm B21 Cm A21 20 .mu.l primer 1 SEQ. ID
19 SEQ. ID 22 SEQ. ID 20 SEQ. ID 28 20 .mu.l primer 2 SEQ. ID 21
SEQ. ID 5 SEQ. ID 23 SEQ. ID 27 (primer concentration: 0.1
.mu.g/.mu.l) expected 977 bp 423 bp 357 bp 257 bp fragment size:
reaction Tet 21 Cm21 Amp21 20 .mu.l primer 1 SEQ. ID 19 SEQ. ID 20
SEQ. ID 11 20 .mu.l primer 2 SEQ. ID 5 SEQ. ID 27 SEQ. ID 24
(primer concentration: 0.1 .mu.g/.mu.l) expected 1489 bp 756 bp
1742 bp fragment size: mix x128 2 .mu.l dNTP's (2.5 mM each) 256
.mu.l 2.5 .mu.l 10xBuff. 320 .mu.l 18 .mu.l H2O 2304 .mu.l 0.1
.mu.l Taq (5 u/.mu.l) 12.8 .mu.l
[0156] put 360 .mu.l mix in each tube. add primers
[0157] add 1 .mu.l DNA (40 ng/.mu.l)
14 Tet A21 Tet B21 Cm B21 Cm A21 pBR322 pBR322 pACYC184 pACYC84 Tet
21 Cm21 Amp21 pBR322 pACYC184 pBR322 Temperature regime 94.degree.
C. 40 sec 40.degree. C. 2 min 72.degree. C. 4 min 30 cycles
72.degree. C. 5 min 6.degree. C. infinitely
[0158] The required plasmids are illustrated in FIGS. 1 through 6.
FIGS. 1-3 depict plasmids constructed from fragments containing 12
nucleotide (nt) overhangs. FIGS. 4-6 depicts plasmids constructed
from fragments containing 21 nt overhangs.
[0159] B. Connecting the Fragments
[0160] The PCR fragments were mixed and their 3' overhangs exposed
by utilizing UDG. The various reactions are given below. Two
identical independent experiments were conducted. The DNA fragments
for each experiment were prepared independently as well.
[0161] The concentration of the DNA fragments was 0.05
pmol/.mu.l.
[0162] UDG Buff.x10=200 M Tris HCl pH 8.4, 500 mM KCl, 15 mM
MgCl.sub.2.
[0163] Reactions mixtures:
15 1 .mu.l 1 .mu.l 1 .mu.l 1 .mu.l 7 .mu.l 1.2 .mu.l 0.8 .mu.l 1.
Amp12 Tet12 Cm12 H.sub.2O H.sub.2O UDG Buff. UDG .times. 10 2.
Amp12 Tet12 CmA12 CmB12 H.sub.2O UDG Buff. UDG .times. 10 3. Amp12
TetA12 TetB12 Cm12 H.sub.2O UDG Buff. UDG .times. 10 4. Amp21 Tet21
Cm21 H.sub.2O H.sub.2O UDG Buff. UDG .times. 10 5. Amp21 Tet21
CmA21 CmB21 H.sub.2O UDG Buff. UDG .times. 10 6. Amp21 TetA21
TetB21 Cm21 H.sub.2O UDG Buff. UDG .times. 10
[0164] The reactions were incubated at 37.degree. C. for 5 hours. 1
.mu.l of each reaction was used in the electroporation of 20 .mu.l
GibcoBRL ElectroMax DH10B cells. After the transformation the cells
of reactions 1 and 4 were plated on LB plates containing
Ampicillin, Tetracycline and Chloramphenicol, for selection (FIGS.
8A and 8B respectively). Likewise, the cells of reactions 2 and 5
were plated on LB plates containing Ampicillin and Tetracycline
(FIGS. 8C and 8D respectively). Cells of reactions 3 and 6 were
plated on LB plates containing Ampicillin and Chloramphenicol
(FIGS. 8E and 8F respectively). The fusion fragments CmA-CmB and
TetA-TetB contain only the upstream and downstream fragments of
Cm.sup.r and Tet.sup.r. In addition they are interconnected by
short linker repeats. They cannot therefore confer resistance to
chloramphenicol or tetracycline.
[0165] An illustration of the constructs along with the number of
transformants obtained, is given in FIG. 9. In the figure, colonies
marked with a * were checked by PCR as detailed below and proven to
contain plasmids with rearrangements rather than the required
constructs.
[0166] Further Examination of the Constructs by PCR
[0167] The 6 transformants of reactions 2 as well as 16
representatives from reaction 5 were PCR tested in order to check
whether they contain the desired constructs: Since these
transfornants were resistant to both Ampicillin and Tetracycline it
was assumed that these fragments had fused correctly to each other.
It was desired to know whether the Cm A and Cm B were present as
well. Therefore the existence of the Cm fragments was tested by
checking the various transformants by PCR with primers SEQ. ID NO.
14 and SEQ. ID NO. 25. Since in this constructs, Cm A (which
encompasses part of the 5' region of Cm) is 257 bp long and Cm B
(which encompasses part of the 3' region of Cm) is 357 bp long, the
overall size of the expected PCR product should be 614 bp long,
some 142 bp shorter than the size of a complete Cm fragment (756 bp
long).
[0168] In FIG. 10(a), the six left lanes are of the transformants
obtained from reaction 2, the next five lanes are from reaction 5.
The right lane is a full Cm-fragment control.
[0169] In FIG. 10(b), the right lane is a full Cm-fragment control.
The rest of the lanes are of more transformants from reaction
5.
[0170] The results indicate that none of the transformants in
reaction 2 contain the desired constructs. In reaction 5, 9 out of
16 transformants contain the desired product.
[0171] Transformants from reactions 4, 5 and 6 were further tested
by PCR to check if they contain the desired fragments in the
desired order, as detailed below: reaction:
16 A B C D Tet Cm A Tet A Cm B Amp Amp Tet B primer 1 SEQ. ID 13
SEQ. ID 26 SEQ. ID 17 SEQ. ID 16 primer 2 SEQ. ID 2 SEQ. ID 15 SEQ.
ID 8 SEQ. ID 18 expected fragment size: 1473 bp 1180 bp 2043 bp
1207 bp
[0172] The tests carried out according to the above, verified that
the transformants from reactions 5 and 6 (which were plasmids
constructed from fragments having 21 nt overhangs) contained the
desired fragments in the correct order. In reaction 5, 9 out of 16
transformants contained the desired product. In reaction 6, 2 out
of 2 transformants contained the desired product.
[0173] This experiment clearly demonstrates the advantage of
constructing plasmids from fragments with long overhangs (around 21
nt). Almost no colonies were obtained in the 3 experiments (1-3)
that used fragments with overhangs of 12 nt. The only 6 colonies
that were observed did not contain the correct construct. In
contrast, in the experiments that used fragments with overhangs of
21 nt, colonies containing the correct product were readily
obtained.
EXAMPLE 4
Assembly of Three DNA Fragments Using ExoIII-created Overhangs
[0174] Exonuclease III (ExoIII) of E. coli was used in order to
create complementary overhangs of 20 bases or longer. ExoIII
digests one strand of blunt-ended DNA or DNA containing 5'
overhangs, in a 3' to 5' direction, thus creating a 5' overhang or
enlarging an existing 5' overhang. By regulating the temperature
and time of the reaction one can control the extent of the
digestion, hence the length of the overhang.
[0175] The formation of a construct made of three fragments is
illustrated. Using the same method, plasmids made out of four and
five segments were also constructed (not shown).
[0176] The plasmid illustrated in FIG. 12 was assembled by the
joining of three independently produced DNA fragments. These three
DNA fragments are:
[0177] a) a DNA fragment of 1739 base pairs (bp) containing the
Amp.sup.r gene and the ColE1-ori region;
[0178] b) a DNA fragment of 1466 bp containing the Tet.sup.r gene;
and
[0179] c) a DNA fragment of 745 bp containing the Cm.sup.r
gene.
[0180] These fragments are illustrated schematically in FIG. 12
showing their relative positioning one to the other, namely, that
the Cm.sup.r fragment was to be connected at its one end to one end
of the Tet.sup.r fragment and at its other end to one end of the
Amp.sup.r+ColE1-ori fragment, and likewise, the other ends of the
Tet.sup.r and Amp.sup.r+ColE1-ori fragments were to be connected to
each other to provide for a circular DNA molecule, being the
desired plasmid, having the above predetermined order of the three
fragments.
[0181] To produce the above three DNA fragments, the aforementioned
PCR procedure was carried out using the following primers and
template DNA:
[0182] a) The 1739 bp Amp.sup.r+ColE1-ori DNA fragment was
synthesized by the PCR procedure using primers SEQ. ID NO. 29 (also
designated 4142) and SEQ. ID NO. 30 (also designated 3884) and
pBR322 as the template DNA. The concentrations of the primers and
template DNA, as well as the other PCR conditions, are as indicated
above. The relative direction of the primers with respect to the
synthesis of the Amp.sup.r+ColE1-ori fragment as it is positioned
in the completed plasmid product is as depicted schematically in
FIG. 12. Thus, primer SEQ. ID NO. 29 was synthesized to have a
predetermined sequence so as to provide for the desired junction
region between the Amp.sup.r+ColE1-ori fragment and the Cm.sup.r
fragment, and primer SEQ. ID NO. 30 was synthesized to have a
predetermined sequence so as to provide for the desired junction
between the Amp.sup.r+ColE1-ORI fragment and the Tet.sup.r
fragment.
[0183] b) The 1466 bp Tet.sup.r DNA fragment was synthesized by the
PCR procedure using primers SEQ. ID NO. 31 (also designated 31160)
and SEQ. ID NO. 32 (also designated 30397) and pBR322 as the
template DNA. The concentrations of the primers and template DNA,
as well as the other PCR conditions, are as indicated above. The
relative direction of the primers with respect to the synthesis of
the Tef fragment as it is positioned in the completed plasmid
product is as depicted schematically in FIG. 12.
[0184] c) The 745 bp Cm.sup.r DNA fragment was synthesized by the
PCR procedure using primers SEQ. ID NO. 33 (also designated 3595)
and SEQ. ID NO. 34 (also designated 4143) and pACYC 184 as the
template DNA. The relative direction of the primers with respect to
the synthesis of the Cm.sup.r fragment as it is positioned in the
completed plasmid product is as depicted schematically in FIG.
12.
[0185] The sequences of the above primers are as follows:
[0186] The arrows inside the sequences mark the junction
points.
[0187] primers SEQ. ID NO. 29 and SEQ. ID NO. 30
[0188] for the Amp.sup.r+ColE1-ori segment:
17 part of Cm.sup.r region sequence part of Amp.sup.r region
sequence primer SEQ. ID NO. 29: ATTGGTGCCCTTAAACGCCTG.dwnarw.AACG-
CAGGAAAGAACATGTG (also designated 4142) part of Tet.sup.r region
sequence part of Amp.sup.r region sequence primer SEQ. ID NO. 30:
AGCGTTGGGTCCTGG.dwnarw.CCAAAGAGTATGAGTATTCAACA (also designated
3884) primers SEQ. ID NO. 31 and SEQ. ID NO. 32 for the Tet.sup.r
segment: part of Cm.sup.r region sequence part of Tet.sup.r region
sequence primer SEQ. ID NO.31:
AGCTCCTGA.dwnarw.TTCTCATGTTTGACAGCTTATC (also designated 31160)
part of Amp.sup.r +ColE1-ori region sequence part of Tet.sup.r
region sequence primer SEQ. ID NO. 32:
ATACTCTT.dwnarw.TGGCCAGGACCCAACGCTGCCC (also designated 30397)
primers SEQ. ID NO. 33 and SEQ. ID NO. 34 for the Cm.sup.r segment:
part of Tet.sup.r region sequence part of Cm.sup.r region sequence
primer SEQ. ID NO. 33: AAACATGAGAA.dwnarw.TCAGGAGCTAAGGAAGCTAAAATG
(also designated 3595) The Amp region The CM region primer SEQ. ID
NO. 34: ATGTTCTTTCCTGCGTT.dwnarw.CAGGCGTTTAAGGGCACCAATAAC (also
designated 4143)
[0189] In view of the fact that the original primer sequences were
derived only from the Tet.sup.r, Cm.sup.r and Amp.sup.r+ColE1-ori
region genes, with no introduction of any additional ("linker") DNA
sequences whatsoever, the above junctions between said genes are
"seamless".
[0190] The PCR was carried out according to the description in the
former examples. Each PCR fragment, separately, was then subjected
to agarose-gel purification using a commercial kit, namely,
Bio-Rad's Prep-A-Gene.TM. purification kit and following the
manufacturer's instructions.
[0191] Once purified, each PCR fragment was then quantitated by
determining the DNA concentration of each fragment by standard
procedures, using Pharmacia's Gene-Quant.TM. RNA/DNA calculator and
following the manufacturer's instructions.
[0192] The PCR fragments were then subjected to Exo III digestion
and subsequent joining procedure. The various PCR fragments were
mixed together (0.15 pmol DNA for each fragment), in a cooled
(6.degree. C.) reaction mixture of 12 .mu.l containing: 1.2 .mu.l
10.times. TA buffer (330 mM Tris-acetate, pH 7.8; 660 mM potassium
acetate, 100 mM magnesium acetate and 5 mM DTT); 0.8 .mu.l Exo III
(200 U/.mu.l purchased from Epicentre Technologies); and sterile
double distilled H.sub.2O to make up the final volume of 12 .mu.l.
In practice, the PCR fragments were mixed into a precooled, namely,
6.degree. C., TA buffer solution made up to 11.2 .mu.l with the
H.sub.2O, to which was then added the 0.8 .mu.l Exo III. Adding the
Exo III last provides for better control over the Exo III reaction,
which reaction is controlled by the time of incubation of the PCR
fragments with the Exo III. Following the Exo III addition, the
reaction mixture was then incubated at 6.degree. C. for 40 mins.
(the time necessary to achieve more than 20 nucleotide degradation
of each DNA strand in the 3'-5' direction under the above
conditions of temperature=6.degree. C. and concentration of Exo
III).
[0193] The Exo III reaction was then stopped by performing a phenol
extraction. This is done by adding to the above reaction mixture an
equal volume=12 .mu.l of a 1:1 (v/v) phenol/chloroform mixture
which causes denaturation of the Exo III. The aqueous phase was
then separated from the above phenol/chloroform mixture, this
aqueous phase containing the PCR fragments. The separated aqueous
phase was then subjected to three washes with chloroform to yield a
final aqueous phase having an essentially purified mixture of the
Exo III-digested PCR fragments.
[0194] In order to prevent both the evaporation of the buffer and
the drying of the fragments, 40 .mu.l of mineral oil was added to
the vessel containing the ExoIII-digested PCR fragments. The
mixture was then heated to 75.degree. C., at which temperature it
was further incubated for one hour. After this incubation, the
mixture is slowly cooled, under conditions providing only a
2.degree. C. decrease in temperature per hour, until it reached
37.degree. C. (the heating and cooling to provide for specific
complementary interactions between complementary overhangs on the
PCR fragments and to prevent non-specific interactions). This
heating and cooling represents the first stage of the specific
joining between the fragments, the joining by way of hydrogen
bonding between complementary overhangs.
[0195] Once the above mixture has reached 37.degree. C., it was
then subjected to the final stage of the joining, including the
filling in reaction, as follows:
[0196] To the cooled (at 37.degree. C.) mixture of now essentially
joined (by hydrogen bonding of complementary overhangs) PCR
fragments there was added 10 .mu.l of the `Synthesis Mixture`,
which contains:
[0197] 1 .mu.l of 20 mM ATP
[0198] 4 .mu.l of 2.5 mM dNTPs (=dATP, dTTP, dCTP and dGTP in equal
amounts, concentration of each=2.5 mM)
[0199] 1 .mu.l of 10.times. TA buffer (see above for
constituents)
[0200] 1 .mu.l of T7 DNA polymerase (5 U/.mu.l, purchased from
USB)
[0201] 1 .mu.l of T4 DNA ligase (10 U/.mu.l, purchased from
Epicentre Technologies)
[0202] 2 .mu.l of sterile double-distilled H.sub.2O
[0203] Total volume: 10 .mu.l
[0204] The above synthesis mixture had the T7 DNA polymerase and
dNTPs to facilitate the filling in of gaps in the junction regions
between the joined fragments, as well as the ATP and the T4 ligase
to covalently join the DNA strands together once the filling in of
gaps has been completed.
[0205] Following the addition of the synthesis mixture, the
resulting reaction mixture was then incubated for two hours at
37.degree. C. After this incubation, the reaction mixture was then
ethanol precipitated under standard conditions to finally yield a
pellet of precipitated DNA which was essentially the completed DNA
construct composed of the joined PCR fragments. This DNA pellet was
resuspended in 5 .mu.l sterile double-distilled H.sub.2O and is
ready for further analysis or use.
[0206] Following the preparation of the desired construct described
above from the three PCR fragments, this construct was analyzed for
its biological activity, namely, whether or not it could confer
resistance to all three antibiotics when introduced into bacterial
cells. Thus, electroporation of electrocompetent DH10B E. coli
cells was performed using a 2 .mu.l aliquot of a 5 .mu.l final
product containing the DNA construct. After electroporation, the
cells were first plated on agar plates containing ampicillin. The
results revealed more than 1000 colonies on these plates,
indicating that more than 1000 originally transformed cells
received a DNA construct having at least an active Amp.sup.r gene.
Of these Amp.sup.r colonies, 40 were chosen at random, as a test
sample, and were plated on both tetracycline- and
chloramphenicol-containing agar plates. All 40 of these test
colonies grew on these plates as well, indicating that they were
also Tet.sup.r and Cm.sup.r Hence, it is concluded that at least
these 40 colonies received an intact construct in which all of the
Tet.sup.r, Cm.sup.r and Amp.sup.r genes were intact and fully
expressible. Some of the colonies were tested further by PCR, as
indicated above. The PCR bands that appeared were of the expected
sizes.
[0207] In the above example, the ligation and fill-in steps have
been used for the sake of completeness and to illustrate possible
alternative procedures. However, as will be easily understood by
the skilled person, said ligation and fill-in steps are not
necessary, and the procedure exemplified above can be carried out
without using such steps. Examples of such procedures without said
steps have not been given, for the sake of brevity.
EXAMPLE 5
Assembly of 8 DNA Fragments into a Circularized Plasmid Using
UDG-created Overhangs
[0208] The plasmid to be constructed was designed to consist of
eight fragments, each to be prepared separately by PCR
amplification and then joined in a specific directional fashion to
provide a circularized plasmid as the end-product. For the purposes
of exemplifying the method of the present invention, it was chosen
to combine eight PCR fragments which together encompass four
regions, as detailed below, thus carrying out a specific
directional connection of eight independent PCR fragments to form a
single active plasmid construct, and this by an essentially
one-step procedure in accordance with the present invention, a
result never obtained or attempted in the prior art.
[0209] This example is similar to Example 1, as the overhangs are
created by the use of dU in the primer and the enzyme UDG to expose
the overhang. However, in the present example, only one dU residue
per primer is used. Therefore, the procedure of exposing the
overhang comprises an additional reagent, N,N
Dimethyl-ethylenediamine. The use of this reagent is explained in
farther detail in the section of general procedures above and in
the above mentioned copending patent application, identified as
Attorney Docket 4149/96, Israeli Patent Application No. 120337. In
addition, the present example uses primers that contain, at their
5' end, the natural gene sequence of the fragment to which they are
to be joined. The junction is therefore seamless: no unnecessary
residues are added at the junctions. In contrast, Example 1 uses
primers that contain at their 5' end an irrelevant sequence whose
only purpose is to serve as an overhang, since several dU residues
must be used in the 5' part of the primer sequence. This irrelevant
sequence is then introduced into the resulting fragment between the
natural gene sequences of the fragments that are joined. Thus, the
use of only one dU nucleotide, as in the present example, enables
the seamless joining of fragments.
[0210] In FIG. 13, there is shown schematically the plasmid that
was designed and produced by the method of the present invention.
This plasmid carries four independent antibiotic resistance genes,
for resistance to Ampicillin (Amp.sup.r gene, or hereinafter Amp);
Tetracycline (Tet.sup.r gene, or hereinafter Tet); Chloramphenicol
(Cm.sup.r gene, or hereinafter Cm) and Kanamycin (Kn.sup.r gene, or
hereinafter Kn) The plasmid also carries the ColE1 origin of
replication (ColE1-ori), which in this specific instance is
situated next to the Amp.sup.r gene. Hence, such a plasmid is
capable of being replicated in a host cell and will endow the host
cell with resistance to all four types of antibiotic.
[0211] Accordingly, it is possible to readily select for those host
cells transformed by this plasmid by growing the transform cells in
the presence of one of the antibiotics and then to screen for the
resistance to the other antibiotics. In order to verify that
mis-connections did not occur, additional verification tests may be
carried out. One such test carried out included testing transformed
colonies by restriction enzyme analysis. Plasmid DNA was prepared
from a number of transformed colonies and checked by restriction
enzymes.
[0212] For this eight fragments construction, the following was
carried out:
[0213] (i) Preparation of the Specific Primers for the PCR
Amplification.
[0214] As shown in FIG. 13, and as outlined above, it was desired
to construct a circularized plasmid having four regions:
[0215] (a) a Tet.sup.r region.
[0216] (b) an Amp.sup.r+ColE1-ori region.
[0217] (c) a Cm.sup.r region.
[0218] (d) a Kn.sup.r region.
[0219] The order of the connection is depicted in FIG. 13.
[0220] To achieve this task, eight separate fragments were
designed:
18 Name of fragment Size(bp) plasmid Location 1 574 pBR322
3603-4159 2 1171 pBR322 2460-3624 3 481 PACYC18 4021-240 4 293
PACYC18 3768-4043 5 862 pBR322 1-848 6 631 pBR322 827-1449 7 569
PACYC17 1809-2368 8 652 PACYC17 2349-2991
[0221] Written are the names of the fragments, their sizes, the
plasmid from which they were PCR amplified, the exact location on
the original plasmid of the site to be amplified and the numbers of
the primers that were used to amplify the fragments. The full
sequence and maps of the various regions of these plasmids are well
known and can be accessed from GenBank database under accession
nos. J01749 (pBR322), X06403 (pACYCI84) and X06402 (pACYC177).
[0222] The AmpB fragment includes the 5' part of the Amp fragment
and the ColE1-ori sequence. The AmpA fragment includes the 3' part
of the Amp fragment. The CmA fragment includes the 5' part of the
Cm fragment and the CmB fragment includes the 3' part of the Cm
fragment. The TetB fragment includes the 5' part of the Tet
fragment and the TetA fragment includes the 3' part of the Tet
fragment. The KnA fragment includes the 5' part of the Kn fragment
and the KnB fragment includes the 3' part of the Kn fragment.
[0223] Each primer consists of two regions: a 3' region
complementary to the DNA to be amplified, and a 5' region
complementary to the fragment it should be connected to. Using
standard automated procedures to produce polynucleotide oligomers
(Applied Biosystems, U.S.A.), the following primers were
synthesized:
[0224] Primers for the Amplification of the AmpA Fragments
[0225] primer SEQ. ID NO. 35, also designated 241365: internal
Amp.sup.r region
19 primer SEQ. ID NO. 35, also designated 241365: internal
Amp.sup.r region ATTGCTGCAGGCATCGTGGTGUCA * primer SEQ. ID NO. 36,
also designated. 3885: Cm.sup.r region Amp.sup.r region
AGCGTTGGGTCCTGGCCA - AAGAGTAUGAGTATTCAA * Primers for the
amp4fication of the AmpB fragments primer SEQ. ID NO. 37, also
designated 27342: Cm.sup.r region Amp.sup.r region ACGCCTG -
AACGCAGGAAAGAACAUGTG * primer SEQ. ID NO. 38, also designated
241366: internal Amp.sup.r region ACACCACGATGCCTGCAGCAAUGG *
Primers for the amplification of the CmA fragments primer SEQ. ID
NO. 39, also designated 40122: Kn.sup.r region Cm.sup.r region AGG
CCT GGT ATG AGT C - TCA GGA GCU AAG GAA GCT AAA ATG * primer SEQ.
ID NO. 40, also designated 27343: internal Cm.sup.r region
ATTGGCTGAGACGAAAAACATAUTCTC * Primers for the amplification of the
CmB fragment primer SEQ. ID NO. 41, also designated 25596: internal
Cm.sup.r region ATATGTTTTTCGTCTCAGCCAAUCC * primer SEQ. ID NO. 42,
also designated 4144: Amp.sup.r region Cm.sup.r region
ATGTTCTTTCCTGCGTT - CAGGCGUTTAAGGGCACCAATAAC * Primers for the
amplification of the TetA fraginent: primer SEQ. ID NO. 43, also
designated 27341: internal Tet .sup.r region
ATACCGCAAGCGACAGGCCGAUCATCG * primer SEQ. ID NO. 44, also
designated 36176: Kn antisense Tet sense ACGTGGCTTTGTTG -
TTCTCATGUTTGACAGCTTATC * Primers for the amplification of the TetB
fraginent: primer SEQ. ID NO. 45, also designated 30402: Amp.sup.r
region Tet.sup.r region ATACTCTT - TGGCCAGGACCCAACGCUGCCC * primer
SEQ. ID NO. 46, also designated 25595: internal Tet .sup.r region
ATCGGCCTGTCGCTTGCGGTAUTCG * Primers for the amplification of the
KnA fragment: primer SEQ. ID NO. 47, also designated 31254: Tet
region Kn region ACATGAGAA - CAACAAAGCCACGUTGTGTCTC * primer SEQ.
ID NO. 48, also designated 25953: internal Kn region
AGACGAAATACGCGATCGCUGTTAA * Primers for the ampilfication of the
KnB fragment: primer SEQ. ID NO. 49, also designated 25952:
internal Kn region AGCGATCGCGTATTTCGTCUCGCTC * primer SEQ. ID NO.
50, also designated 31253: Cm region Kn region AGCTCCTGA -
GACTCATACCAGGCCUGAATCG *
[0226] (ii) Preparation of the PCR Fragments
[0227] PCR reactions were carried out as in the other examples
above. Following PCR synthesis of the individual fragments, each
fragment was purified by standard agarose-gel purification
techniques using the commercially available Bio-Rad
"Prep-A-Gene.TM." DNA purification kit and adhering to the
manufacturer's instructions. Following purification, the
concentration of the purified fragment DNA was determined by
standard procedures using the Pharmacia "Gene-Quant.TM. RNA/DNA
Calculator" and adhering to the manufacturer's instructions.
[0228] (iii) Connection of the PCR-produced Fragments
[0229] The eight PCR fragments as produced and purified according
to the above-mentioned procedure, were connected to each other in a
one-step reaction mixture in a single reaction vessel. This was
achieved by mixing the fragments together in a 25 .mu.l reaction
mixture that included: 0.15 pmol of each fragment, 2.5 .mu.l buffer
(200 mM Tris-HCl pH 8.4, 500 mM KCl, 15 mM MgCl.sub.2), 2.5 .mu.l
of 1M N,N-Dimethyl-ethylenediamine and 6.25 units of UDG
(GibcoBRL). The mixture was incubated at 37.degree. C. for 4 hours
and then transferred to 70.degree. C. for 5 minutes to facilitate
dissociation of the short nicked single-stranded DNA from the 5'
ends of the fragments. After dissociation the short nicked
single-stranded DNA were removed using "QlAquick PCR purification
kit" (QIAGEN) adhering to the manufacturer's instructions. Before
adding the first buffer of the kit, 200 .mu.l of hot (70.degree.
C.) buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl.sub.2)
was added in order to minimize the reannealing of the short nicked
single-stranded DNA. The DNA was eluted in 30 .mu.l of
double-distilled water (DDW). 27 .mu.l of the DNA was incubated
with 3 .mu.l of buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM
MgCl2) in a water-bath, at 70.degree. C. The bath was shut down and
the temperature was slowly decreased to 37.degree. C. This allows
for the joining between the complementary overhangs, while
minimizing illegitimate connection.
[0230] (iv) Analysis of the Efficiency of Joining of the
Fragments
[0231] A 1 .mu.l sample of the above DNA, containing the newly
constructed plasmid, made from joining the 8 separate fragments,
was used to transform E. coli DH10B cells by electroporation. In
the transformation procedure 20 .mu.l of electrocompetent
"ElectroMax" cells (purchased from Gibco BRL) were mixed with the
above DNA sample and subjected to electroporation in a commercially
available apparatus (BioRad "E. coli Pulser Apparatus" set at 1.8
kV and operated according to the manufacturer's instructions).
[0232] Following electroporation (transformation) the cells were
plated on LB Agar plate containing 100 mM Ampicillin (to select for
transformants having Ampicillin resistance by virtue of having
being transformed with a DNA carrying the Amp.sup.r gene). On the
day after, three colonies were picked and checked for resistance to
Chloramphenicol, Tetracycline and Kanamycin by plating on Agar
plates containing the appropriate antibiotics.
[0233] The results showed that the three colonies were resistant to
all the four antibiotics. The colonies were further checked by
restriction enzyme analysis and proved to be correct (data not
shown).
[0234] The above result is very significant since it shows that it
is possible to join correctly 8 separate DNA fragments in a
specific directional manner, in a single reaction mixture by an
essentially one-step procedure.
[0235] All the above description of preferred embodiments and
examples have been provided for the purpose of illustration, and
are not meant to limit the invention. Many modifications can be
made in the methods, materials and conditions, and many different
products and results can be obtained, using different numbers of
overhangs and different numbers of fragments to be joined, all
without exceeding the scope of the invention.
Sequence CWU 1
1
50 1 31 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 agctcctgat tctcatgttt gacagcttat c 31 2 37 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 2
cuacuacuac uatggccagg acccaacgct gcccgag 37 3 40 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 3
cuacuacuac uacuatggcc aggacccaac gctgcccgag 40 4 43 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 4
cuacuacuac uacuacuatg gccaggaccc aacgctgccc gag 43 5 46 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
5 cuacuacuac uacuacuacu atggccagga cccaacgctg cccgag 46 6 55 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
6 cuacuacuac uacuacuacu acuacuacua tggccaggac ccaacgctgc ccgag 55 7
41 DNA Artificial Sequence Description of Artificial Sequence
Primer 7 attggtgccc ttaaacgcct gaacgcagga aagaacatgt g 41 8 36 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
8 uaguaguagu agaagagtat gagtattcaa catttc 36 9 39 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 9
uaguaguagu aguagaagag tatgagtatt caacatttc 39 10 42 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 10
uaguaguagu aguaguagaa gagtatgagt attcaacatt tc 42 11 45 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
11 uaguaguagu aguaguagua gaagagtatg agtattcaac atttc 45 12 54 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
12 uaguaguagu aguaguagua guaguaguag aagagtatga gtattcaaca tttc 54
13 37 DNA Artificial Sequence Description of Combined DNA/RNA
Molecule Primer 13 aucauaucau auttctcatg tttgacagct tatcatc 37 14
38 DNA Artificial Sequence Description of Combined DNA/RNA Molecule
Primer 14 caucaucauc aucaggcgtt taagggcacc aataactg 38 15 37 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
15 uacuauacua uacatgccgg cgataatggc ctgcttc 37 16 37 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 16
uauaguauag uagtagatga cgaccatcag ggacagc 37 17 37 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 17
aucuaaucua autacggtga aaacctggcc tatttcc 37 18 36 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 18
augaugauga ugaacgcagg aaagaacatg tgagca 36 19 45 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 19
cauaucauau cauaucauau ttctcatgtt tgacagctta tcatc 45 20 47 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
20 caucaucauc aucaucauca ucaggcgttt aagggcacca ataactg 47 21 45 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
21 cuauacuaua cuauacuaua catgccggcg ataatggcct gcttc 45 22 45 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
22 uauaguauag uauaguauag gtagatgacg accatcaggg acagc 45 23 45 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
23 augaugauga ugaugaugau gaacgcagga aagaacatgt gagca 45 24 45 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
24 augaugauga ugaugaugau gaacgcagga aagaacatgt gagca 45 25 37 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
25 auaugauaug autcaggagc taaggaagct aaaatgg 37 26 37 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 26
auuagauuag aucaggcggg caagaatgtg aataaag 37 27 45 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 27
auaugauaug auaugauaug tcaggagcta aggaagctaa aatgg 45 28 45 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
28 auuagauuag auuagauuag caggcgggca agaatgtgaa taaag 45 29 41 DNA
Artificial Sequence Description of Artificial Sequence Primer 29
attggtgccc ttaaacgcct gaacgcagga aagaacatgt g 41 30 38 DNA
Artificial Sequence Description of Artificial Sequence Primer 30
agcgttgggt cctggccaaa gagtatgagt attcaaca 38 31 31 DNA Artificial
Sequence Description of Artificial Sequence Primer 31 agctcctgat
tctcatgttt gacagcttat c 31 32 30 DNA Artificial Sequence
Description of Artificial Sequence Primer 32 atactctttg gccaggaccc
aacgctgccc 30 33 35 DNA Artificial Sequence Description of
Artificial Sequence Primer 33 aaacatgaga atcaggagct aaggaagcta
aaatg 35 34 41 DNA Artificial Sequence Description of Artificial
Sequence Primer 34 atgttctttc ctgcgttcag gcgtttaagg gcaccaataa c 41
35 24 DNA Artificial Sequence Description of Combined DNA/RNA
Molecule Primer 35 attgctgcag gcatcgtggt guca 24 36 36 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
36 agcgttgggt cctggccaaa gagtaugagt attcaa 36 37 27 DNA Artificial
Sequence Description of Combined DNA/RNA Molecule Primer 37
acgcctgaac gcaggaaaga acaugtg 27 38 24 DNA Artificial Sequence
Description of Combined DNA/RNA Molecule Primer 38 acaccacgat
gcctgcagca augg 24 39 40 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Primer 39 aggcctggta tgagtctcag
gagcuaagga agctaaaatg 40 40 27 DNA Artificial Sequence Description
of Combined DNA/RNA Molecule Primer 40 attggctgag acgaaaaaca
tautctc 27 41 25 DNA Artificial Sequence Description of Combined
DNA/RNA Molecule Primer 41 atatgttttt cgtctcagcc aaucc 25 42 41 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
42 atgttctttc ctgcgttcag gcguttaagg gcaccaataa c 41 43 27 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
43 ataccgcaag cgacaggccg aucatcg 27 44 36 DNA Artificial Sequence
Description of Combined DNA/RNA Molecule Primer 44 acgtggcttt
gttgttctca tguttgacag cttatc 36 45 30 DNA Artificial Sequence
Description of Combined DNA/RNA Molecule Primer 45 atactctttg
gccaggaccc aacgcugccc 30 46 25 DNA Artificial Sequence Description
of Combined DNA/RNA Molecule Primer 46 atcggcctgt cgcttgcggt autcg
25 47 31 DNA Artificial Sequence Description of Combined DNA/RNA
Molecule Primer 47 acatgagaac aacaaagcca cgutgtgtct c 31 48 25 DNA
Artificial Sequence Description of Combined DNA/RNA Molecule Primer
48 agacgaaata cgcgatcgcu gttaa 25 49 25 DNA Artificial Sequence
Description of Combined DNA/RNA Molecule Primer 49 agcgatcgcg
tatttcgtcu cgctc 25 50 31 DNA Artificial Sequence Description of
Combined DNA/RNA Molecule Primer 50 agctcctgag actcatacca
ggccugaatc g 31
* * * * *