U.S. patent application number 13/544054 was filed with the patent office on 2013-01-17 for methods and transposon nucleic acids for generating a dna library.
This patent application is currently assigned to Finnzymes Oy. The applicant listed for this patent is Heli Haakana, Ian Kavanagh, Laura-Leena Kiiskinen. Invention is credited to Heli Haakana, Ian Kavanagh, Laura-Leena Kiiskinen.
Application Number | 20130017978 13/544054 |
Document ID | / |
Family ID | 47519245 |
Filed Date | 2013-01-17 |
United States Patent
Application |
20130017978 |
Kind Code |
A1 |
Kavanagh; Ian ; et
al. |
January 17, 2013 |
METHODS AND TRANSPOSON NUCLEIC ACIDS FOR GENERATING A DNA
LIBRARY
Abstract
A method for the generation of DNA fragmentation library based
on a transposition reaction in the presence of a transposon end
with an engineered cleaveage site providing facilitated downstream
handling of the produced DNA fragments, e.g., in the generation of
sequencing templates. Transposon nucleic acids comprising a
transposon end sequence and an engineered cleaveage site located in
the sequence, e.g., in Mu transposon end sequence, are
disclosed.
Inventors: |
Kavanagh; Ian; (Luzern,
CH) ; Kiiskinen; Laura-Leena; (Espoo, FI) ;
Haakana; Heli; (Espoo, FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kavanagh; Ian
Kiiskinen; Laura-Leena
Haakana; Heli |
Luzern
Espoo
Espoo |
|
CH
FI
FI |
|
|
Assignee: |
Finnzymes Oy
Vantaa
FI
|
Family ID: |
47519245 |
Appl. No.: |
13/544054 |
Filed: |
July 9, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61506371 |
Jul 11, 2011 |
|
|
|
Current U.S.
Class: |
506/26 ; 435/194;
536/23.1 |
Current CPC
Class: |
C40B 40/08 20130101;
C12N 15/1093 20130101; C12Q 1/6806 20130101 |
Class at
Publication: |
506/26 ;
536/23.1; 435/194 |
International
Class: |
C40B 50/06 20060101
C40B050/06; C12N 9/12 20060101 C12N009/12; C07H 21/04 20060101
C07H021/04 |
Claims
1. An in vitro method for generating a DNA library, the method
comprising the steps of: a) incubating a transposon complex
comprising a transposon having a transposon end, and a transposase,
with a target DNA of interest under conditions for carrying out a
transposition reaction, wherein the transposon end comprises a
transposon end sequence which is recognizable by the transposase,
and wherein the transposon end sequence comprises at least one
modified position, wherein the modified position introduce(s) a
cleavage site into the transposon end sequence, and wherein the
transposition reaction results in fragmentation of the target DNA
and incorporation of the transposon end into the 5' ends of the
fragmented target DNA; and b) incubating the fragmented target DNA
with an enzyme cleaving at the cleavage site so that the transposon
ends incorporated to the fragmented target DNA are cleaved at the
cleavage site.
2. The method of claim 1 further comprising c) amplifying the
cleaved, fragmented target DNA in an amplification reaction using a
first and second oligonucletide primer complementary to the part of
the transposon end retained in the 5' ends of the cleaved,
fragmented target DNA, wherein the first and second primer
optionally comprise 5' adaptor tails.
3. The method of claim 1 further comprising the step of contacting
the fragments of target DNA obtained from step a) or b) comprising
the transposon end at the 5' ends of the fragmented target DNA with
DNA polymerase having 5'-3' exonuclease or strand displacement
activity so that fully double-stranded DNA molecules are produced
from the fragments of target DNA.
4. The method of claim 2 further comprising the step of
denaturating the fully double-stranded DNA molecules to produce
single stranded DNA for use in the amplification reaction of step
c).
5. The method of claim 1, wherein the transposon end is a Mu
transposon end.
6. The method of claim 1, wherein the transposase is MuA
transposase.
7. The method of claim 1, wherein the enzyme is an N-glycosylase,
an endonuclease, or a restriction enzyme.
8. The method of claim 7, wherein the N-glycosylase is
uracil-N-glycosylase.
9. The method of claim 7, wherein the restriction enzyme is a
methylation specific restriction enzyme.
10. The method of claim 7, wherein the endonuclease is RNase H.
11. The method of claim 1, wherein the 5' adaptor tail of the first
and/or the second oligonucleotide primers comprise a tag selected
from the group consisting of an amplification tag, a sequencing
tag, a detection tag, and combinations thereof.
12. The method of claim 11, wherein the adaptor tail of the first
and the second oligonucleotide primers comprise a sequencing tag,
and the method further comprises denaturating the amplification
products obtained from step c) to produce single stranded DNA,
annealing the single stranded DNA to a solid support coated with an
oligonucleotide complementary to the sequencing tag and performing
a DNA sequencing reaction using the single stranded DNA which is
immobilized to a solid support as a template.
13. The method of claim 1, wherein the cleavage site in the
transposon end sequence is located 15-25 base pairs 5' direction
from the 3' joining end of the transposon end.
14. The method of claim 1, wherein the cleavage site in the
transposon end sequence is located within 25 base pairs 5'
direction from the 3' joining end.
15. The method of claim 1, wherein the cleavage site in the
transposon end sequence is located beyond 25 base pairs 5'
direction from the 3' joining end.
16. A modified transposon nucleic acid comprising a transposon end
sequence and a joining end, and an engineered cleaveage site
located in the transposon end sequence.
17. The modified transposon nucleic acid of claim 16 wherein the
engineered cleavage site is located 15-25 base pairs 5' direction
from the 3' joining end of the transposon end.
18. The modified transposon nucleic acid of claim 16, wherein the
transposon end sequence is Mu transposon end sequence.
19. The modified transposon nucleic acid of claim 16, wherein the
cleavage site is an uracil nucleic acid base, a plurality of
ribonucleic acid bases, or a methylated nucleic acid base
introduced into the transposon end sequence.
20. The modified transposon nucleic acid of claim 16, wherein the
cleavage site is a restriction enzyme site.
21. The modified transposon nucleic acid of claim 16, further
comprising a transposase and optionally DNA primers complementary
to a region of the modified transposon.
22. A method comprising use of the transposon nucleic acid
according to claim 16 for generating a DNA library or DNA
sequencing templates.
23. A kit comprising a transposase, a transposon, wherein the
transposase binds the transposon, and wherein the transposon is
modified to include a cleavage site. a first and second primer
complimentary to a region of the transposon, at least one
additional component selected from the group consisting of a
buffer, a polymerization enzyme, an N-glycosylase, an endonuclease,
and a restriction enzyme, and instructions for forming a DNA
library from a target DNA.
Description
[0001] This application claims priority to co-pending U.S.
application Ser. No. 61/506,371 filed Jul. 11, 2011, which is
expressly incorporated by reference herein in its entirety.
[0002] The present invention relates to the fields of DNA library
preparation and high throughput multiplex DNA sequencing. The
invention is directed to a method for the generation of DNA
fragmentation library based on a transposition reaction in the
presence of a transposon end with an engineered cleaveage site
providing facilitated downstream handling of the produced DNA
fragments, e.g., in the generation of sequencing templates. The
invention is further directed to transposon nucleic acids
consisting of a transposon end sequence and an engineered cleaveage
site in the sequence. In one embodiment, this transposon end
sequence is a Mu transposon end.
[0003] The term "DNA sequencing" generally refers to methodologies
aiming to determine the primary sequence information in a given
nucleic acid molecule. Traditionally, Maxam-Gilbert and Sanger
sequencing methodologies have been applied successfully for several
decades, as well as a pyrosequencing method. However, these
methodologies have been difficult to multiplex, as they require a
wealth of labor and equipment time, and the cost of sequencing is
excessive for entire genomes. These methodologies required each
nucleic acid target molecule to be individually processed, the
steps including, e.g., subcloning and transformation into E. coli
bacteria, extraction, purification, amplification and sequencing
reaction preparation and analysis.
[0004] Recently, several platforms have challenged these
conventional methods. So called "next-generation" technologies or
"massive parallel sequencing" platforms allow millions of nucleic
acid molecules to be sequenced simultaneously. The methods rely on
sequencing-by-synthesis approach, while certain other platforms are
based on sequencing-by-ligation technology. Although very
efficient, all of these new technologies rely on multiplication of
the sequencing templates. Thus, for each application, a pool of
sequencing templates need to be produced.
[0005] Tenkanen et al. (U.S. Pat. No. 6,593,113) was the first to
disclose an in vitro transposition reaction for DNA library
preparation comprising an in vitro transposition reaction and a PCR
amplification reaction to select sequencing templates. In the
method, the transposition reaction results in fragmentation of the
target DNA and the subsequent amplification reaction is carried out
in the presence of a fixed primer complementary to the known
sequence of the target DNA and a selective primer having a
complementary sequence to the end of a transposon DNA.
[0006] Grunenwald et al. (U.S. 20100120098) disclose methods for
using a transposase and a transposon end for generating extensive
fragmentation and 5'-tagging of double-stranded target DNA in
vitro. The method is based on the use of a DNA polymerase for
generating 5'- and 3'-tagged single-stranded DNA fragments after
fragmentation without performing a PCR amplification reaction. The
authors disclose tagged transposon ends, but the actual transposon
end sequence of the used transposons corresponds to native Tn5
transposon sequence. The tag domain combined with the native
transposon end can comprise a sequence or structure of a cleavage
site, in which case the method comprises a step of incubating the
tagged DNA fragments obtained from fragmentation step with a
cleavage enzyme. Grunenwald et al describes having the cleavage
site in a tag sequence that is attached to the 5'-end of the
transposon sequence, not in the transposon sequence itself.
[0007] In U.S. Pat. No. 7,172,882 (Savilahti et al.), a transposon
containing at least partly within its transposon ends a
modification with translation stop codons in three reading frames
is disclosed. The modified transposon was used for producing
deletion derivatives of polypeptides. Further, Laurent et al. (J.
Virology, vol. 74, No. 6, 2000, pp. 2760-2769) disclose that a Notl
restriction site can be engineered close to the transposon end and
in this way new restriction sites can be introduced into target DNA
through transposition.
[0008] What is still needed in the art are methods which facilitate
the downstream handling of the fragmented DNA obtained from the
transposition step, since the transposition products having
complementary transposon end sequences at both ends form
intramolecular loop structures when denatured to single stranded
DNA, shown schematically in FIG. 1. This is particularly a problem,
when the fragmented DNA is subjected to PCR amplification.
[0009] In one embodiment, the present invention provides an in
vitro method for generating a DNA library shown schematically in
FIG. 2 where the DNA sequences of the fragments from the
transposition reaction are, e.g.,
TABLE-US-00001 SEQ ID NO: 2 .......Insert from Target DNA.......gap
SEQ ID NO: 1 SEQ ID NO: 3
gap.................................................. SEQ ID NO:
12
[0010] and showing the product after gap-filling by a DNA
polymerase. The method comprises the steps of:
[0011] a) initiating a transposition reaction in the presence of a
transposon end, transposase enzyme, and in the presence of target
DNA, wherein the transposon end comprises a transposon end sequence
which is recognizable by a transposase, the transposon end sequence
comprising a modified position or modified positions, wherein the
modified position or positions introduce(s) a cleavage site into
the transposon end sequence, and wherein the transposition reaction
results in fragmentation of the target DNA and incorporation of the
transposon end into the 5' ends of the fragmented target DNA;
and
[0012] b) incubating the fragmented target DNA with an enzyme
specific to the cleavage site so that the transposon ends
incorporated to the fragmented target DNA are cleaved at the
cleavage site.
[0013] In one embodiment, the method further comprises c)
performing an amplification reaction using a first and second
oligonucletide primer complementary to the part of the transposon
end retained in the 5'' ends of the fragmented target DNA, wherein
the first and second primer may comprise 5' adaptor tails.
[0014] In one embodiment, a modified transposon nucleic acid
consisting of transposon end sequence and an engineered cleaveage
site located within the transposon end sequence is provided. In one
embodiment, the cleavage site is within 25 base pairs 5' direction
from the 3' joining end. In one embodiment, the cleavage site is
within not within 25 base pairs 5' direction from the 3' joining
end. In one embodiment, a modified transposon nucleic acid
consisting of transposon end sequence and an engineered cleaveage
site located 15-25 base pairs 5' direction from the 3' joining end
of the transposon end is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The patent or application file contains at least one drawing
executed in color. A Petition under 37 C.F.R. .sctn.1.84 requesting
acceptance of the color drawing is being filed separately. Copies
of this patent or patent application publication with color
drawing(s) will be provided by the Office upon request and payment
of the necessary fee.
[0016] FIG. 1 shows fragmented transposition products forming
intramolecular loop structures when denatured to single stranded
DNA.
[0017] FIG. 2 shows a transposition reaction on target DNA.
[0018] FIG. 3 shows four primer adaptor addition PCR where
amplicons that have different adaptor structures (A and B) at each
end will not be complementary, allowing the shorter primers to
anneal with greater efficiency and enriching this sequence during
amplification.
[0019] FIGS. 4A-D show denaturing PAGE gel analysis of lambda DNA
fragmentation using uracil containing transposon-transposase
complex.
[0020] FIG. 5A-E show transposon ends truncation using uracyl DNA
glycosylase (UDG) and EndoIV treatment.
[0021] FIG. 6A-D show denaturing PAGE gel analysis of lambda DNA
fragmentation using m5C containing transposon-transposase
complex.
[0022] FIG. 7A-D show denaturing PAGE gel analysis of lambda DNA
fragmentation using RNA/DNA hybrid regions containing
transposon-transposase complex.
[0023] The term "transposon", as used herein, refers to a nucleic
acid segment, which is recognized by a transposase or an integrase
enzyme and which is an essential component of a functional nucleic
acid-protein complex (i.e. a transpososome) capable of
transposition. In one embodiment, a minimal nucleic acid-protein
complex capable of transposition in a Mu transposition system
comprises four MuA transposase protein molecules and a pair of Mu
end sequences that are able to interact with MuA.
[0024] The term "transposase" as used herein refers to an enzyme,
which is a component of a functional nucleic acid-protein complex
capable of transposition and which is mediating transposition. The
term "transposase" also refers to integrases from retrotransposons
or of retroviral origin.
[0025] The expression "transposition reaction" used herein refers
to a reaction wherein a transposon inserts into a target nucleic
acid. Primary components in a transposition reaction are a
transposon and a transposase or an integrase enzyme. The method and
materials of the present invention are exemplified by employing in
vitro Mu transposition (Haapa et al. 1999; Savilahti et al. 1995).
Other transposition systems can be used as well, e.g., Tyl (Devine
and Boeke, 1994; International Patent Application WO 95/23875); Tn7
(Craig 1996); Tn 10 and IS 10 (Kleckner et al. 1996); Mariner
transposase (Lampe et al. 1996); Tcl (Vos et al. 1996, 10(6),
755-61); Tn5 (Park et al. 1992); P element (Kaufman and Rio 1992);
and Tn3 (Ichikawa and Ohtsubo, 1990), bacterial insertion sequences
(Ohtsubo and Sekine 1996), retroviruses (Varmus and Brown 1989),
and retrotransposon of yeast (Boeke, 1989).
[0026] The term "transposon end sequence" as used herein refers to
the nucleotide sequences at the distal ends of a transposon. The
transposon end sequences are responsible for identifying the
transposon for transposition; they are the DNA sequences the
transpose enzyme requires in order to form transpososome complex
and to perform transposition reaction. For MuA transposase, this
sequence is 50 bp long (SEQ ID NO. 1) and is described by
Goldhaber-Gordon et al., J Biol Chem. 277 (2002) 7703-7712, which
is hereby incorporated by reference in its entirety. A transposable
DNA of the present invention may comprise only one transposon end
sequence. The transposon end sequence in the transposable DNA
sequence is thus not linked to another transposon end sequence by
nucleotide sequence, i.e. the transposable DNA contains only one
transposase binding sequence. Thus, the transposable DNA comprises
a "transposon end" (see, e.g. Savilahti et al., 1995).
[0027] The term "transposase binding sequence" or "transposase
binding site" as used herein refers to the nucleotide sequences
that is always within the transposon end sequence whereto a
transposase specifically binds when mediating transposition. The
transposase binding sequence may however comprise more than one
site for the binding of transposase subunits.
[0028] The term "transposon joining strand" or "joining end" as
used herein means the end of that strand of the double-stranded
transposon DNA, which is joined by the transposase to the target
DNA at the insertion site.
[0029] The term "adaptor" or "adaptor tail" as used herein refers
to a non-target nucleic acid component, generally DNA, that
provides a means of addressing a nucleic acid fragment to which it
is joined. For example, in embodiments, an adaptor comprises a
nucleotide sequence that permits identification, recognition,
and/or molecular or biochemical manipulation of the DNA to which
the adaptor is attached (e.g., by providing a site for annealing an
oligonucleotide, such as a primer for extension by a DNA
polymerase, or an oligonucleotide for capture or for a ligation
reaction).
[0030] Transposon complexes form between a transposase enzyme and a
fragment of double stranded DNA that contains a specific binding
sequence for the enzyme, termed "transposon end". The sequence of
the transposon binding site can be modified with other bases, at
certain positions, without affecting the ability for transposon
complex to form a stable structure that can efficiently transpose
into target DNA. By manipulating the sequence of the transposon
end, the method provided properties to the fragmented target DNA
that can be utilized in downstream applications, particularly when
using the method for library preparation before sequencing. The
following are examples of how the disclosed method provided
simplified and more specific DNA fragmentation libraries:
[0031] 1. Inclusion of uracil in the transposon end sequence, which
can be used to cleave the resulting fragment of DNA in a downstream
step. This is useful for removing parts of the transposon end
sequence from the fragmented DNA, which improves downstream
amplification (e.g., by reducing intramolecular loop structures, as
a result of less complementary sequence) or reduces the amount of
transposon end sequence that would be read during sequencing (e.g.,
single molecule sequencing). The enzyme uracil glycosylase can be
used to remove the uracil from the DNA fragment specifically, since
uracil is a common nucleic acid base in RNA, but is not usually
present in DNA. The resulting abasic sites formed in DNA by uracil
glycosylase can be subsequently cleaved by heat, alkali-treatment,
or apurinic/apyrimidinic (AP) endonucleases that cleave
specifically at abasic sites, such as endonuclease IV.
[0032] 2. Inclusion of a restriction enzyme, including a
methylation specific restriction enzyme (inserting methylated base
into transposon end sequence) site into transposon end, as a way of
providing a method for reducing the transposon end sequence in
downstream steps by subsequent cleavage using the appropriate
restriction enzyme.
[0033] 3. Inclusion of ribonucleotides into transposon end, to form
either double-stranded RNA or RNA-DNA double-stranded hybrids in
the transposon end. Double-stranded RNA can be specifically
degraded by exoribonucleases recognizing double-stranded RNA, and
RNA/DNA hybrids can be degraded by using a combination of
ribonuclease that specifically degrades the RNA strand in RNA-DNA
hybrids (such as ribonuclease H) and a DNA exonuclease specific for
single-stranded DNA (such as exonuclease I).
[0034] Modified transposon end sequences comprising a uracil base,
an additional restriction site, or ribonucleotides can be produced,
e.g., by regular oligonucleotide synthesis.
[0035] In one embodiment, the invention provides a method for
generating a DNA library by:
[0036] a) initiating a transposition reaction in the presence of a
transposon end and in the presence of target DNA and a transposase,
wherein the transposon end comprises a transposon end sequence
which is recognizable by the transposase, the transposon end
sequence comprising a modified position or modified positions,
wherein the modified position or positions introduce(s) a cleavage
site into the transposon end sequence, and wherein the
transposition reaction results in fragmentation of the target DNA
and incorporation of the transposon end into the 5' ends of the
fragmented target DNA; and
[0037] b) incubating the fragmented target DNA with an enzyme
specific to the cleavage site so that the transposon ends
incorporated to the fragmented target DNA are cleaved at the
cleavage site.
[0038] In one embodiment, the method may further comprise step c)
performing an amplification reaction using a first and second
oligonucletide primer complementary to the part of the transposon
end retained in the 5' ends of the fragmented target DNA, wherein
the first and second primer may comprise 5' adaptor tails.
[0039] In one embodiment, the method further comprises the step of
contacting the fragments of target DNA obtained from step a) or b)
comprising the transposon end at the 5' ends of the fragmented
target DNA with DNA polymerase having 5'-3' exonuclease or strand
displacement activity so that fully double-stranded DNA molecules
are produced from the fragments of target DNA. This step is used to
fill the gaps generated in the transposition products in the
transposition reaction. The length of the gap is characteristic to
a certain transposition enzyme, e.g., for MuA the gap length is 5
nucleotides.
[0040] To prepare the transposition products for downstream steps,
such as PCR reaction, the method may comprise the further step of
denaturating the fully double-stranded DNA molecules to produce
single stranded DNA for use in the amplification reaction of step
c).
[0041] In one embodiment, the transposition system used in the
inventive method is based on MuA transposase enzyme. For the
method, one can assemble in vitro stable but catalytically inactive
Mu transposition complexes in conditions devoid of Mg.sup.2+ as
disclosed in Savilahti et al., 1995 and Savilahti and Mizuuchi
1996. In principle, any standard physiological buffer not
containing Mg.sup.2+ is suitable for the assembly of the inactive
Mu transposition complexes. In one embodiment, the in vitro
transpososome assembly reaction may contain 150 mM Tris-HCl pH 6.0,
50% (v/v) glycerol, 0.025% (w/v) Triton X-100, 150 mM NaCl, 0.1 mM
EDTA, 55 nM transposon DNA fragment, and 245 nM MuA. The reaction
volume may range from about 20 .mu.l to about 80 .mu.l. The
reaction is incubated at about 30.degree. C. for about 0.5 hours to
about 4 hours. In one embodiment, the assembly reaction is
incubated for 2 hours at about 30.degree. C. Mg.sup.2+ is added for
activation.
[0042] The enzyme used in step b) of the above method may be an
N-glycosylase, an endonuclease, or a restriction enzyme, such as
uracil-N-glycosylase or a methylation specific restriction enzyme,
respectively.
[0043] In one embodiment, the 5' adaptor tail of the first and/or
the second PCR primer(s) used in step c) of the method comprise one
or more of the following groups: an amplification tag, a sequencing
tag, and/or a detection tag.
[0044] The amplification tag is a nucleic acid sequence providing
specific sequence complementary to the oligonucleotide primer to be
used in the subsequent rounds of amplification. For example, the
sequence may be used for the purpose of facilitating amplification
of the nucleic acid obtained from step c).
[0045] The sequencing tag provides a nucleic acid sequence
permitting the use of the amplified DNA fragments obtained from
step c) as templates for next-generation sequencing. For example,
the sequencing tag may provide annealing sites for sequencing by
hybridization on a solid phase. The sequencing tag may be Roche
454A and 454B sequencing tags, Applied Biosystems' SOLID.TM.
sequencing tags, ILLUMINA.TM. SOLEXA.TM. sequencing tags, the
Pacific Biosciences' SMRT.TM. sequencing tags, Pollonator Polony
sequencing tags, and the Complete Genomics sequencing tags.
[0046] The detection tag comprises a sequence or a detectable
chemical or biochemical moiety for the purpose of facilitating
detection of the nucleic acid obtained from step c). Examples of
detection tags are fluorescent and chemiluminescent dyes such as
green fluorescent protein; and enzymes that are detectable in the
presence of a substrate, e.g., an alkaline phosphatase using an
appropriate substrate such as nitro-blue tetrazolium chloride (NBT)
and 5-bromo-4-chloro-3'-indolyphosphate p-toluidine (BCIP), or a
peroxidase with a suitable substrate. The detection tag may contain
a sequence whose purpose is to identify a source of a sample DNA.
By using different detection tags, e.g., barcodes, sequences from
multiple samples can be sequenced in the same instrument run and
identified by the sequence of the detection tag. Examples include
Illumina's index sequences in TruSeq DNA Sample Prep Kits, and
molecular barcodes in Life Technologies' SOLiD.TM. DNA Barcoding
Kits.
[0047] In one embodiment, the fragmentation products obtained from
step a) are subjected to two consecutive amplification steps,
wherein the first and the second PCR primer in step c), comprising
a first amplification step, comprise a tag that may be used by a
third and fourth PCR primer in a subsequent or second amplification
step. For instance, in step c) the tag is an amplification tag, and
in a subsequent amplification step, the tag in the third and fourth
PCR primer is a sequencing tag. It is also contemplated that the
first and second primer comprise different tags. In another
embodiment, the third and fourth PCR primers do not comprise an
adaptor tail.
[0048] In one embodiment, a modified transposon nucleic acid
consisting of transposon end sequence and an engineered cleaveage
site located 15-25 base pairs 5' direction from the 3' joining end
of the transposon end is also provided. The transposon end sequence
may be a Mu transposon end sequence
5'-TGAAGCGGCGCACGAAAAACGCGAAAGCGTTTCACGATAAATGCGAAAAC-3'; SEQ ID
NO.: 1). Shown in double-stranded form, native 50 bp MuA transposon
end sequence is:
TABLE-US-00002 SEQ ID NO.: 2
5'-GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCT TCA-3' SEQ ID
NO.: 3 3'-CAAAAGCGTAAATAGCACTTTGCGAAAGCGCAAAAAGCACGAGGCGA
AGT-5'
[0049] In one embodiment, SEQ ID NO. 1 is modified to include a
cleavage site.
[0050] In embodiments, the cleavage site is a uracil nucleic acid
base, a plurality of ribonucleic acid bases, or methylated nucleic
acid base introduced into the transposon end sequence. The cleavage
site can also be a restriction enzyme site.
EXAMPLE 1
Lambda DNA Fragmentation with Uracil Containing
Transposon-Transposase Complex and Subsequent Transposon Ends
Truncation Using UDG and Heat Treatment
[0051] The ability to remove transposon ends using uracyl DNA
glycosilase (UDG) was directly shown using transposon containing
uracyl base, lambda DNA as a fragmentation target, and UDG
treatment.
[0052] All enzymes and reagents were from Thermo Fisher Scientific
unless indicated otherwise. All oligonucleotides were synthesized
at Microsynth.
[0053] Oligonucleotide Ck4_UDG12ntMU (SEQ ID NO: 4) was 5'-labeled
using T4 PNK and [.gamma.-.sup.33P]-ATP; T4 PNK from reaction
mixture was removed by phenol-chloroform extraction, unincorporated
[.gamma.-.sup.33P]-ATP (Perkin Elmer) was removed by size exclusion
chromatography (Zeba.TM. Spin Desalting Column (7K MWCO)).
Transposon (final concentration 30 .mu.M) was prepared by annealing
of 17 pmol labeled and 583 pmol unlabeled Ck4_UDG12ntMU
5'-GTTTTCGCATTTATCGTGAAACGCTTTCGCGUTTTTCGTGCGTCAGTTCA-3' [0054]
(SEQ. ID NO.: 4) and 600 pmol unlabeled NCk4_UDG12ntMU [0055]
5'-TGCTGAACTGACGCACGAAAAACGCGAAAGCGTUTCACGATAAATGCGAAAAC-3' (SEQ ID
NO.: 5) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl.
Annealing program: 95.degree. C. for 5 min, 95-25.degree. C. 70
cycles for 40 seconds (1.degree. C./per cycle), 10.degree. C.
(Eppendorf Mastercycler epgradientS).
[0056] MuA--Transposon Complex (Transposon Mix) was formed in 120
mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.05% Triton X-100, 1 mM EDTA
and 10% glycerol (final conc. of transposon was 9.3 .mu.M and for
MuA Transposase 1.65 g/l). After 1 h incubation at 30.degree. C.
glycerol, NaCl and EDTA were added to final 47.2%, 200 mM and 2 mM
concentrations respectively. The solution was thoroughly mixed with
a tip. Transposon Mix was stored at -70.degree. C. for at least 16
hours.
[0057] Lambda DNA was fragmented in 12 separate tubes. In each tube
fragmentation of 100 ng of lambda DNA (dam-, dcm-) (12 reactions)
was carried out in 36 mM Tris-HCl (pH 8.0), 137 mM NaCl, 0.05%
Triton X-100, 10 mM MgCl.sub.2, 4.6% DMSO and 6.8% glycerol.
Immediately after adding the Transposon Mix (1.5 .mu.l to final
reaction volume 30 .mu.l), vortexing and a short spin-down, the
tube was incubated at 30.degree. C. for 5 minutes. The reaction was
stopped by adding 3 .mu.l of 4.4% SDS. After brief vortexing, the
tube was kept at room temperature.
[0058] Fragmented DNA was purified by Agencourt AMPure XP PCR
Purification system. The beads were taken to room temperature for
at least 30 minutes prior to starting the purification protocol and
thoroughly mixed before pipetting. Fragmented DNA was transferred
into a 1.5 ml tube (2 reaction mixes were coupled, so each of 6
tubes contained 66 .mu.l of fragmented DNA). Then 99 .mu.l of room
temperature Agencourt AMPure XP beads were added to the reaction
and mixed carefully by pipetting up and down ten times. The same
procedure was repeated with all six tubes of fragmented DNA.
Samples were incubated for 5 minutes at room temperature. After a
short spin, the tubes were placed in a magnetic rack until the
solutions were cleared. The supernatant was aspirated carefully
without disturbing the beads and discarded. The tubes were kept in
the rack and 800 .mu.l of freshly-prepared 70% ethanol was added.
After 30 seconds incubation all the supernatant was removed. The
ethanol wash step was repeated. The beads were air-dried on the
magnet by opening the tube caps for two minutes, allowing all
traces of ethanol to evaporate. The tubes were removed from the
magnetic rack, and the beads were suspended in 50 .mu.l of
nuclease-free water by pipetting up and down ten times. The tubes
were placed in the magnetic rack until the solution became clear
and 45-50 .mu.l of the supernatants (containing the purified
fragmented DNA) from each of six tubes without disturbing the
pellet were collected into a new sterile tube (total volume 287
.mu.l). After evaluation of the radioactivity level (cpm) on DE-81
filter paper, sample of purified fragmented DNA was
dried/evaporated in "Eppendorf concentrator 5301" to the final
volume of 27 .mu.l. The sample was divided into two parts: one for
control, and one for treatment with Uracil DNA Glycosylase.
[0059] Fragmented DNA (.about.0.9 pmol) was treated with Uracil-DNA
Glycosylase (UDG) in 20 mM Tris-HCl (10.times.pH 8.2 at 25.degree.
C.), 1 mM EDTA, 10 mM NaCl, 0.1 u/.mu.l UDG at 50.degree. C. for 10
min (total volume 25 .mu.l). The abasic sites formed in DNA by UDG
were subsequently cleaved by heat treatment (95.degree. C. for 10
min). The reaction mixture was desalted (Zeba.TM. Spin Desalting
Column (7K MWCO)), completely dried in "Eppendorf concentrator
5301" and dissolved in 1.times.Loading Dye (47.5% formamide,
0.0125% SDS, 0.0125% bromophenol blue, 0.0125% xylene cyanol FF,
0.0125% ethidium bromide, 0.25 mM EDTA).
[0060] Radioactively labeled samples of transposon (20000 cpm),
fragmented DNA (70000 cpm) and UDG treated fragmented DNA (70000
cpm) were analyzed on the 10% denaturing polyacrylamide/urea gel
using 89 mM Tris, 89 mM boric acid, 2 mM EDTA (10.times.pH 8.3) as
the running buffer. Electrophoresis was performed for 1.25 h at 24
V/cm at 50.degree. C. (Biorad, DCode Universal Mutation Detection
System). Radiolabeled bands were detected using Typhoon Trio imager
(GE Healthcare).
[0061] FIG. 4 shows denaturing PAGE gel analysis of lambda DNA
fragmentation using uracil containing transposon-transposase
complex. FIG. 4A L--GeneRuler.TM. 50 bp DNA Ladder (was labeled
using T4 DNA kinase and [.gamma.-.sup.33P]-ATP), L1--GeneRuler.TM.
Ultra Low Range DNA Ladder (was labeled using T4 DNA kinase and
[.gamma.-.sup.33P]-ATP), 1--Transposon (contains labeled
Ck4_UDG12nt_MU (SEQ ID NO: 4)) (20000 cpm), 2--Fragmented Lambda
DNA (dam-, dcm-) (70000 cpm), 3--Fragmented Lambda DNA (dam-, dcm-)
after treatment with UDG (70000 cpm). FIG. 4B is transposon
(contains 5' labeled Ck4_UDG12nt_MU (SEQ ID NO: 4)), radioactively
labeled oligonucleotide has grey background, and uracil has black
background. FIG. 4C is fragmented Lambda DNA (contains 5' labeled
Ck4_UDG12nt_MU), radioactively labeled counterpart of DNA has grey
background and uracil has black background. FIG. 4D shows
transposon ends removal by UDG and heat treatment, radioactively
labeled counterpart of DNA has grey background.
[0062] Synthetic oligonucleotide Ck4_UDG12ntMU (SEQ ID NO: 4)
containing uracyl base in the middle of the sequence was
radioactively labeled at its 5' end and annealed with another
uracyl containing oligonucleotide NCk4_UDG12ntMU (SEQ ID NO: 5) in
such a way that double stranded MuA transposon with uracyl bases at
both strands was generated (FIG. 4A, lane 1 and FIG. 4B). MuA
transposase and uracyl containing transposon complex was formed and
used for subsequent lambda DNA fragmentation (FIG. 4A, lane 2 and
FIG. 4C). Fragmented DNA with transposon sequences at the ends was
purified. Uracyl bases in the transposon sequence part of DNA
fragments were removed using UDG. Generated abase sites were
hydrolyzed by heat treatment (FIG. 4A lane 3 and FIG. 4D).
[0063] This experiment clearly indicates that unnecessary
transposon sequence present at both ends of randomly fragmented
target DNA were effectively removed by combined UDG and heat
treatment, meanwhile target genomic DNA without uracyl bases in it
remained intact. Resulting DNA ends could be designed to be
compatible with appropriate downstream applications, providing
additional flexibility in subsequent experiment design.
EXAMPLE 2
Lambda DNA Fragmentation with Uracil Containing
Transposon-Transposase Complex and Subsequent Transposon Ends
Truncation Using UDG and Endonuclease Treatment
[0064] FIG. 5A shows double stranded transposon containing uracil
bases (shown in black background) used to form
transposon-transposase complex. FIG. 5B shows fragmented Lambda DNA
after fragmentation with uracyl containing transposon-transposase
complex. FIG. 5C shows transposon ends removal by UDG and EndoIV
treatment. FIG. 5D shows Agilent 2100 Bioanalyzer (HS chip)
analysis of lambda DNA library before and after UDG/EndoIV
treatment--full picture. FIG. 5E shows Agilent 2100 Bioanalyzer (HS
chip) analysis of lambda DNA library before and after UDG/EndoIV
treatment--DNA library peaks are zoomed in.
[0065] All enzymes and reagents were from Thermo Fisher Scientific
unless indicated otherwise. All oligonucleotides were synthesized
at Microsynth. Transposon (final concentration 100 .mu.M) was
prepared by annealing Ck4_UDG12ntMU (SEQ ID NO: 4) and
NCk4_UDG12ntMU (SEQ ID NO: 5) in 10 mM Tris-HCl (pH 8.0), 1 mM
EDTA, 50 mM NaCl. The annealing program was: 95.degree. C. for 5
min, 95-25.degree. C. 70 cycles for 40 seconds (1.degree. C./per
cycle), 10.degree. C. (Eppendorf Mastercycler epgradientS).
[0066] MuA--Transposon Complex (Transposon Mix) was formed in 120
mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.05% Triton X-100, 1 mM EDTA
and 10% glycerol (final concentration of transposon was 8.0 .mu.M
and for MuA Transposase 1.65 g/l. After 1 h incubation at
30.degree. C. glycerol, NaCl and EDTA were added to final 47.2%,
200 mM and 2 mM concentrations respectively. The solution was
thoroughly mixed with a tip. Transposon Mix was stored at
-70.degree. C. for at least 16 hours.
[0067] Lambda DNA was fragmented in six separate tubes. In each
tube fragmentation of 100 ng of lambda DNA (dam-, dcm-) (6
reactions) was carried out in 36 mM Tris-HCl (pH 8.0), 137 mM NaCl,
0.05% Triton X-100, 10 mM MgCl.sub.2, 4.6% DMSO and 6.8% Glycerol.
Immediately after adding the Transposon Mix (1.5 .mu.l to final
reaction volume 30 .mu.l), vortexing, and a short spin-down, the
tube was incubated at 30.degree. C. for 5 minutes. The reaction was
stopped by adding 3 .mu.l of 4.4% SDS. After brief vortexing, the
tube was kept at room temperature.
[0068] Fragmented DNA was purified by Agencourt AMPure XP PCR
Purification system. The beads were taken to room temperature for
at least 30 minutes prior to starting the purification protocol and
thoroughly mixed before pipetting. Fragmented DNA was transferred
into a 2 ml tube (three reaction mixes were combined, so each of
two tubes contained 99 .mu.l of fragmented DNA). Then 148.5 .mu.l
of room temperature Agencourt AMPure XP beads were added to the
reaction and mixed carefully by pipetting up and down ten times.
The same procedure was repeated with the second tube of fragmented
DNA. Samples were incubated for five minutes at room temperature.
After a short spin, the tubes were placed in a magnetic rack until
the solutions were cleared. The supernatant was aspirated carefully
without disturbing the beads and discarded. The tubes were kept in
the rack and 1200 .mu.l of freshly-prepared 70% ethanol was added.
After 30 seconds incubation all the supernatant was removed. The
ethanol wash step was repeated. The beads were air-dried on the
magnet by opening the tube caps for 2-5 minutes, allowing all
traces of ethanol to evaporate. The tubes were removed from the
magnetic rack, and the beads were suspended in 37 .mu.l of
nuclease-free water by pipetting up and down ten times. The tubes
were placed in the magnetic rack until the solution became clear
and 35-40 .mu.l of the supernatants (containing the purified
fragmented DNA) from both tubes without disturbing the pellet were
collected into a new sterile tube (total volume 75 .mu.l).
[0069] Fragmented DNA (75 .mu.l was divided for 25 .mu.l into 3
wells) was loaded into E-Gel.RTM. SizeSelect 2% agarose gel
(Invitrogen/Life Technologies) and 200-250 bp fraction was
collected (75 .mu.l). Invitrogen 50 bp DNA Ladder (10 .mu.l of
40-fold dilution) was used as size marker.
[0070] Fragmented DNA (5 .mu.l) fraction of 200-250 bp was treated
with Uracil-DNA Glycosylase (UDG) and Endonuclease IV (E. coli)
(Endo IV) in 20 mM Tris-HCl (10.times.pH 8.2 at 25.degree. C.), 1
mM EDTA, 10 mM NaCl, 2.5 u UDG, 2 u Endo IV at 37.degree. C. for 30
min (total volume 30 .mu.l). The same reaction--UDG/-Endo IV
and--UDG/+Endo IV were made as controls. All samples after reaction
were purified using GeneJet PCR purification Kit (Thermo Fisher
Scientific), eluted with 40 .mu.l elution buffer and
dried/evaporated in "Eppendorf concentrator 5301" to a final volume
of 5 .mu.l. Purified DNA products (1 .mu.l out of 5 .mu.l
concentrated) were analyzed using an Agilent 2100 Bioanalyzer
(Agilent High Sensitivity DNA Kit; Agilent Biotechnologies).
[0071] The ability to remove transposon ends using uracyl DNA
glycosilase (UDG) was shown in direct experiment using transposon
containing uracyl base, lambda DNA as a fragmentation target, and
UDG/EndoIV treatment. Synthetic oligonucleotide Ck4_UDG12ntMU (SEQ
ID NO: 4) containing uracyl base in the middle of the sequence was
annealed with another uracyl containing oligonucleotide
NCk4_UDG12ntMU (SEQ ID NO: 5) in such a way that double stranded
MuA transposon with uracyl bases at both strands was generated
(FIG. 5A). MuA transposase and uracyl containing transposon complex
was formed and used for subsequent lambda DNA fragmentation (FIG.
5B). Fragmented DNA with transposon sequences at the ends was
purified. Subsequently DNA library was size-selected in agarose gel
to be in the range of 200-250 bp. Uracyl bases in the transposon
sequence part of DNA fragments were removed using UDG. Finally,
generated abase sites were hydrolyzed by EndoIV treatment (FIG.
5C), purified, and analyzed on Agilent Bioanalyzer High Sensitivity
chip. UDG and EndoIV treatment truncates uracyl containing
transposon ends resulting in DNA library shift to shorter fragment
range (FIG. 5D). This example clearly indicated that unnecessary
transposon sequence present at both ends of randomly fragmented
target DNA was effectively removed by combined UDG and Endo IV
treatment, meanwhile target genomic DNA without uracyl bases in it
remained intact. Resulting DNA ends could be designed to be
compatible with appropriate downstream applications providing
additional flexibility in subsequent experiment design.
EXAMPLE 3
[0072] Lambda DNA Fragmentation with m5C Containing
Transposon-Transposase Complex and Subsequent Transposon Ends
Truncation Using Methylation Sensitive Restriction Eendonuclease
Sgel Treatment
[0073] All enzymes and reagents were from Thermo Fisher Scientific
unless indicated otherwise. All oligonucleotides were synthesized
at Eurofins MWG Operon.
[0074] Transposon 1 (final concentration 90 .mu.M) was prepared by
annealing Cut-key4 (Sgel-MU)
5-GTTTTCGCATTTATmCGTGAAACGCTTTCGCGTTTTTCGTGCGTCAGTTCA-3'(SEQ ID
NO.:6) and Non-cut-key4
5'-TGCTGAACTGACGCACGAAAAACGCGAAAGCGTTTCACGATAAATGCGAAAAC-3' (SEQ.
ID NO.: 7) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl (total
volume 25 .mu.l). The annealing program was: 95.degree. C. for 5
min, 95-25.degree. C. 70 cycles for 40 seconds (1.degree. C./per
cycle), 10.degree. C. (Eppendorf Mastercycler epgradientS).
Transposon 2 (final concentration 86 .mu.M) was prepared by
annealing Cut-key4 [0075]
5-GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGTCAGTTCA-3' (SEQ ID
NO.: 8) and Non-cut-key4 (Sgel-MU) [0076]
5'-TGCTGAACTGACGmCACGAAAAACGCGAAAGCGTTTCACGATAAATGCGAAAAC-3' (SEQ.
ID NO.: 9) using the same conditions for Transposon 1 (total volume
25 .mu.l).
[0077] MuA--Transposon Complex 1 (Transposon Mix 1 for sample 1)
was formed in 120 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.05% Triton
X-100, 1 mM EDTA and 10% glycerol (final conc. of transposon 1 was
8.0 .mu.M and 1.65 g/l of MuA transposase). After 1 h incubation at
30.degree. C. glycerol, NaCl and EDTA were added to final 47.2%,
200 mM and 2 mM concentrations. The solution was thoroughly mixed
with a tip. Transposon Mix 1 was stored at -70.degree. C. for at
least 16 hours. MuA--Transposon Complex 2 (Transposon Mix 2 for
sample 2) was formed using the same conditions as MuA - Transposon
Complex 1, except transposon 2 was used instead of transposon
1.
[0078] Lambda DNA was fragmented in three separate tubes with
Transposon Mix 1 (sample 1) and in three separate tubes with
Transposon Mix 2 (sample 2). In each tube fragmentation of 100 ng
of lambda DNA (dam-, dcm-) (3 reactions with Transposon Mix 1 and 3
reactions with Transposon Mix 2) was carried out in 36 mM Tris-HCl
(pH 8.0), 137 mM NaCl, 0.05% Triton X-100, 10 mM MgCl.sub.2, 4.6%
DMSO and 6.8% glycerol. Immediately after adding the Transposon Mix
1 or 2 (1.5 .mu.l to final reaction volume 30 .mu.l), vortexing,
and a short spin-down, the tube was incubated at 30.degree. C. for
5 minutes. The reaction was stopped by adding 3 .mu.l of 4.4% SDS.
After a brief vortexing, the tube was kept at room temperature.
[0079] Fragmented DNA was purified by Agencourt AMPure XP PCR
Purification system. The beads were taken to room temperature for
at least 30 minutes prior to starting the purification protocol and
thoroughly mixed before pipetting. Fragmented DNA was transferred
into a 1.5 ml tube. Then 49.5 .mu.l of room temperature Agencourt
AMPure XP beads were added to the reaction and mixed carefully by
pipetting up and down ten times. The same procedure was repeated
with all five remaining tubes of fragmented DNA. Samples were
incubated for five minutes at room temperature. After a short spin,
the tubes were placed in a magnetic rack until the solutions were
cleared. The supernatant was aspirated carefully without disturbing
the beads and discarded. The tubes were kept in the rack and 400
.mu.l of freshly-prepared 70% ethanol was added. After 30 seconds
incubation, all the supernatant was removed. The ethanol wash step
was repeated. The beads were air-dried on the magnet by opening the
tube caps for two minutes, allowing all traces of ethanol to
evaporate. The tubes were removed from the magnetic rack, and the
beads were suspended in 25 .mu.l of nuclease-free water by
pipetting up and down ten times. The tubes were placed in the
magnetic rack until the solution became clear and 20-25 .mu.l of
the supernatants (containing the purified fragmented DNA) from each
of three tubes (fragmentation with Transposon Mix 1 or Transposon
Mix 2) without disturbing the pellet were collected into a new
sterile tube (total volumes .about.70 .mu.l for sample 1 and for
sample 2). Samples 1 and 2 of purified fragmented DNA were
dried/evaporated in "Eppendorf concentrator 5301" to the final
volumes of 14.5 and 15.5 .mu.l
[0080] Transposon 1, Transposon 2 and Fragmented DNA was 5'-labeled
using T4 PNK and [.gamma.-.sup.33P]-ATP (Perkin Elmer);
unincorporated [.gamma.-.sup.33P]-ATP was removed by size exclusion
chromatography (Zeba.TM. Spin Desalting Column (7K MWCO)). The
level of radioactive labeling (cpm) was evaluated on DE-81 filter
paper. Sample 1 and sample 2 were divided into two parts: for
control and for treatment with Sgel.
[0081] Fragmented DNA (.about.6 ng) was treated with Sgel in 10 mM
Tris-HCl (pH 8.0 at 37.degree. C.), 5 mM MgCl.sub.2, 100 mM KCl,
0.02% Triton X-100, 0.1 mg/ml BSA and 50 or 500 u/.mu.g DNA Sgel
[dilution buffer for Sgel: 10 mM Tris-HCl (pH 7.4 at 25.degree.
C.), 100 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 50%
glycerol] at 37.degree. C. for 45 or 60 min (total volume 20
.mu.l). Sgel was subsequently inactivated by heat treatment
(65.degree. C. for 20 min). Reaction mixtures were desalted
(Zeba.TM. Spin Desalting Column (7K MWCO)), completely dried in
"Eppendorf concentrator 5301" and dissolved in 1X Loading Dye
(47.5% formamide, 0.0125% SDS, 0.0125% bromophenol blue, 0.0125%
xylene cyanol FF, 0.0125% ethidium bromide, 0.25 mM EDTA).
[0082] Radioactively labeled transposon (samples 1 and 2) (20000
cpm), fragmented Lambda DNA (samples 1 and 2) (70000 cpm) and Sgel
treated fragmented Lambda DNA (samples 1 and 2) (70000 cpm) were
analyzed on the 10% denaturing polyacrylamide/urea gel using 89 mM
Tris, 89 mM boric acid, 2 mM EDTA (10.times.pH 8.3) as the running
buffer. Electrophoresis was performed for one h at 24 V/cm at
50.degree. C. (Biorad, DCode Universal Mutation Detection System).
Radiolabeled bands were detected using Typhoon Trio imager (GE
Healthcare).
[0083] FIG. 6A shows denaturing PAGE gel analysis of lambda DNA
fragmentation using m5C containing transposon-transposase complex;
L--GeneRuler.TM. 50 bp DNA Ladder (was labeled using T4 DNA kinase
and [.gamma.-.sup.33P]-ATP), L1--GeneRuler.TM. Ultra Low Range DNA
Ladder (was labeled using T4 DNA kinase and
[.gamma.-.sup.33P]-ATP), 1--Transposon 1 (5' labeled, contains
Cut-key4 (Sgel-MU) (SEQ ID NO: 6) and Non-cut-key4 (SEQ ID NO: 7))
(20000 cpm), 2--Fragmented Lambda DNA (dam-, dcm-) 1 (contains
transposon 1) (70000 cpm), 3-4 Fragmented Lambda DNA (dam-, dcm-) 1
after treatment with 50 u Sgel/.mu.g DNA for 45 and 60 min
respectively (70000 cpm), 5-6 Fragmented Lambda DNA (dam-, dcm-) 1
after treatment with 500 u Sgel/.mu.g DNA for 45 min and 60 min
respectively (70000 cpm), 7--Transposon 2 (5' labeled, contains
Cut-key4 (Sgel-MU) (SEQ ID NO: 6) and Non-cut-key4 (SEQ ID NO: 7))
(20000 cpm), 8--Fragmented Lambda DNA (dam-, dcm-) 2 (contains
transposon 2) (70000 cpm), 9-10 Fragmented Lambda DNA (dam-, dcm-)
2 after treatment with 50 u Sgel/.mu.g DNA for 45 min and 60 min
respectively (70000 cpm), 11-12 Fragmented Lambda DNA (dam-, dcm-)
2 after treatment with 500 u Sgel/.mu.g DNA for 45 min and 60 min
respectively (70000 cpm).
[0084] FIG. 6B shows transposon 1 (5' labeled, contains Cut-key4
(Sgel-MU) (SEQ ID NO: 6) and Non-cut-key4 (SEQ ID NO: 7));
Transposon 2 (5' labeled, contains Cut-key4 (SEQ ID NO: 8) and
Non-cut-key4 (Sgel-MU) (SEQ ID NO: 9)); methylated C shown with
black background. FIG. 6C shows fragmented Lambda DNA 1 (5'
labeled, contains Cut-key4 (Sgel-MU) (SEQ ID NO: 6) and
Non-cut-key4 (SEQ ID NO: 7)); Fragmented Lambda DNA 2 (5' labeled,
contains Cut-key4 (SEQ ID NO: 8) and Non-cut-key4 (Sgel-MU) (SEQ ID
NO: 9)); recognition and cleavage sequence of Sgel are denoted by
solid line rectangle and dashed lines respectively; radioactively
labeled part of fragmented DNA has grey background. FIG. 6D shows
transposon ends removal by Sgel; recognition and cleavage sequence
of Sgel are denoted by solid line rectangle and dashed lines
respectively; radioactively labeled counterpart of cleaved DNA has
grey background.
[0085] The ability to remove transposon ends using Sgel was shown
in a direct experiment using transposon containing m5C (FIG. 6B),
lambda DNA as a fragmentation target and methylation sensitive
restriction endonuclease Sgel treatment. Synthetic oligonucleotide
Cut-key4 (Sgel-MU) containing m5C (SEQ ID NO: 6) was annealed with
complementary oligonucleotide Non-cut-key4 (SEQ ID NO: 7) in such a
way that double stranded MuA transposon 1 (for sample 1) with m5C
at one strand was generated (FIG. 6A, lanes 1 and 7, and FIG. 6B).
Alternatively synthetic oligonucleotide Non-cut-key4 (Sgel-MU)
containing m5C (SEQ ID NO: 9) was annealed with complementary
oligonucleotide Cut-key4 (SEQ ID NO: 8) in such a way that double
stranded MuA transposon 2 (for sample 2) with m5C at one strand was
generated (FIG. 6A, lanes 1 and 7, and FIG. 6B). MuA transposase
and m5C containing transposon 1 or 2 complex was formed and used
for subsequent lambda DNA fragmentation (FIG. 6A, lanes 2 and 8,
and FIG. 6C). Fragmented DNA with transposon 1 or 2 sequences at
the ends was purified and 5'-labeled using T4 PNK and
[.gamma.-.sup.33P]-ATP. DNA fragments containing m5C in the
transposon 1 or 2 sequence part were recognized and cleaved by
methylation sensitive restriction endonuclease Sgel. As a result
radioactive label was removed from fragmented DNA library (DNA
bands start to disappear) and either 22, 27 nucleotides long
fragments of transposon 1 (sample 1) or 23, 26 nucleotides long
fragments of transposon 2 (sample 2) origin were visualized (FIG.
6A, lanes 3-6 and 9-12, and FIG. 6D). This example clearly
indicated that unnecessary transposon sequence present at both ends
of randomly fragmented target DNA could be effectively removed by
Sgel, meanwhile target genomic DNA without m5C in it remained
intact. Resulting DNA ends could be designed to be compatible with
appropriate downstream applications providing additional
flexibility in subsequent experiment design.
EXAMPLE 4
Lambda DNA Fragmentation with Transposon (RNA/DNA
Hybrid)-Transposase Complex and Subsequent Transposon Ends
Truncation Using RNase H
[0086] All enzymes and reagents were from Thermo Fisher Scientific
unless indicated otherwise. Hybrid RNA/DNA oligonucleotides were
synthesized at Thermo Scientific Dharmacon. [0087] Transposon
(final concentration 30 .mu.M) was prepared by annealing
CK_RNR/DNR.sub.--2
5'-GTTTTCGCATTTATCGTGAAACGCTTTCrGrCrGrTTTTTCGTGCGTCAGTTCA-3' (SEQ
ID NO.: 10) and NCK_RNR/DNR.sub.--2 [0088]
5'-TGCTGAACTGACGCACGAAAAACGCGAAAGCGrUrUrUrCACGATAAATGCGAAAAC-3'
[0089] (SEQ ID NO.: 11) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 50
mM NaCl (total volume 20 .mu.l). Annealing program: 95.degree. C.
for 5 min, 95-25.degree. C. 70 cycles for 40 seconds (1.degree.
C./per cycle), 10.degree. C. (Eppendorf Mastercycler
epgradientS).
[0090] MuA--Transposon Complex (Transposon Mix) was formed in 120
mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.05% Triton X-100, 1 mM EDTA
and 10% glycerol (final concentration of transposon was 9.3 .mu.M
and for MuA Transposase 1.65 g/l. After one hour incubation at
30.degree. C., glycerol, NaCl, and EDTA were added to final 47.2%,
200 mM and 2 mM concentrations respectively. The solution was
thoroughly mixed with a tip. Transposon Mix was stored at
-70.degree. C. for at least 16 hours.
[0091] Lambda DNA was fragmented in three separate tubes with
Transposon Mix. In each tube fragmentation of 100 ng of lambda DNA
(dam-, dcm-) (3 reactions) was carried out in 36 mM Tris-HCl (pH
8.0), 137 mM NaCl, 0.05% Triton X-100, 10 mM MgCl.sub.2, 4.6% DMSO
and 6.8% glycerol. Immediately after adding the Transposon Mix (1.5
.mu.l to final reaction volume 30 .mu.l), vortexing, and a short
spin-down, the tube was incubated at 30.degree. C. for five
minutes. The reaction was stopped by adding 3 .mu.l of 4.4% SDS.
After brief vortexing, the tube was kept at room temperature.
[0092] Fragmented DNA was purified by Agencourt AMPure XP PCR
Purification system. The beads were taken to room temperature for
at least 30 minutes prior to starting the purification protocol and
thoroughly mixed before pipetting. Fragmented DNA was transferred
into a 1.5 ml tube. Then, 49.5 .mu.l of room temperature Agencourt
AMPure XP was added to the reaction and mixed carefully by
pipetting up and down ten times. The same procedure was repeated
with the two remaining tubes of fragmented DNA. Samples were
incubated for five minutes at room temperature. After a short spin,
the tubes were placed in a magnetic rack until the solutions were
cleared. The supernatant was aspirated carefully without disturbing
the beads and discarded. The tubes were kept in the rack and 400
.mu.l of freshly-prepared 70% ethanol was added. After 30 seconds
incubation all the supernatant was removed. The ethanol wash step
was repeated. The beads were air-dried on the magnet by opening the
tube caps for two minutes, allowing all traces of ethanol to
evaporate. The tubes were removed from the magnetic rack, and the
beads were suspended in 25 .mu.l of nuclease-free water by
pipetting up and down ten times. The tubes were placed in the
magnetic rack until the solution became clear and 20-25 .mu.l of
the supernatants (contains the purified fragmented DNA) from each
of three tubes without disturbing the pellet were collected into a
new sterile tube (total volume about 70 .mu.l).
[0093] Transposon and fragmented DNA were 5'-labeled using T4 PNK
and [.gamma.-.sup.33P]-ATP(Perkin Elmer); T4 PNK from reaction
mixture was removed by phenol-chloroform extraction, unincorporated
[.gamma.-.sup.33P]-ATP was removed by size exclusion chromatography
(Zeba.TM. Spin Desalting Column (7K MWCO)). The level of
radioactive labeling (cpm) was evaluated on DE-81 filter paper.
Fragmented DNA was concentrated in "Eppendorf concentrator 5301"
and divided into three parts: for control without any additional
treatment, for control "-RNase H", and for treatment with RNase
H.
[0094] Fragmented DNA (about 14% from all concentrated fragmented
DNA volume) was treated with RNase H in 20 mM Tris-HCl (10.times.pH
7.8), 40 mM KCl, 8 mM MgCl2, 1 mM DTT and 2.5 u RNase H at
37.degree. C. for 60 min (total volume 20 .mu.l). The same reaction
"-RNase H" was made as a negative control. Reaction mixtures were
desalted (Zeba.TM. Spin Desalting Column (7K MWCO)), completely
dried in "Eppendorf concentrator 5301" and dissolved in
1.times.Loading Dye (47.5% formamide, 0.0125% SDS, 0.0125%
bromophenol blue, 0.0125% xylene cyanol FF, 0.0125% ethidium
bromide, 0.25 mM EDTA).
[0095] Radioactively labeled samples of transposon (20000 cpm),
fragmented Lambda DNA and fragmented Lambda DNA (70000 cpm)
.+-.RNase H treatment were heated at 70.degree. C. for five min,
chilled on ice for three min, and analyzed on 10% denaturing
polyacrylamide/urea gel using 89 mM Tris, 89 mM boric acid, 2 mM
EDTA (10.times.pH 8.3) as the running buffer. Electrophoresis was
performed for one h at 24 V/cm at 50.degree. C. (Biorad, DCode
Universal Mutation Detection System). Radiolabeled bands were
detected using Typhoon Trio imager (GE Healthcare).
[0096] FIG. 7A shows denaturing PAGE gel analysis of lambda DNA
fragmentation using RNA/DNA hybrid regions containing
transposon-transposase complex; L--GeneRuler.TM. 50 bp DNA Ladder
(was labeled using T4 DNA kinase and [.gamma.-.sup.33P]-ATP),
L1--GeneRuler.TM. Ultra Low Range DNA Ladder (was labeled using T4
DNA kinase and [.gamma.-.sup.33P]-ATP), 1--Transposon (5' labeled,
contains CK_RNR/DNR.sub.--2 (SEQ ID NO: 10) and NCK_RNR/DNR.sub.--2
(SEQ ID NO: 11)) (20000 cpm), 2--Fragmented Lambda DNA (dam-, dcm-)
(70000 cpm), 3--Fragmented Lambda DNA (dam-, dcm-) after incubation
in the buffer without RNase H (70000 cpm), 4--Fragmented Lambda DNA
(dam-, dcm-) after treatment with RNase H (70000 cpm).
[0097] FIG. 7B shows transposon containing RNA/DNA hybrid (5'
labeled, contains CK_RNR/DNR.sub.--2 (SEQ ID NO: 10) and
NCK_RNR/DNR.sub.--2 (SEQ ID NO: 11)). FIG. 7C shows fragmented
Lambda DNA (5' labeled, contains CK_RNR/DNR.sub.--2 (SEQ ID NO: 10)
and NCK_RNR/DNR.sub.--2 (SEQ ID NO: 11)); radioactively labeled
counterpart of DNA has grey background. FIG. 7D shows transposon
ends removal by RNase H; radioactively labeled counterpart of DNA
has grey background.
[0098] The ability to remove transposon ends using RNase H was
shown using transposon (containing two 4 bp length RNA/DNA hybrid
regions), lambda DNA as a fragmentation target, and RNase H
treatment. Synthetic oligonucleotides CK_RNR/DNR.sub.--2 (SEQ ID
NO.: 10) and NCK_RNR/DNR.sub.--2 (SEQ ID NO.: 11) containing 4 bp
length RNR insert in the middle of their sequences were annealed in
such a way that double stranded MuA transposon with two separated 4
bp length RNA/DNA hybrid regions were generated (FIG. 7A lane 1,
and FIG. 7B). MuA transposase and two separated 4 bp length RNA/DNA
hybrid regions containing transposon complex was formed and used
for subsequent lambda DNA fragmentation (FIG. 7A lanes 2 and FIG.
7C). Fragmented DNA with transposon sequences at the ends was
purified and 5'-labeled using T4 PNK and [.gamma.-.sup.33P]-ATP.
Fragmented DNA library was incubated in a buffer without RNase H
(FIG. 7A lane 3) and with RNase H (FIG. 7A lane 4, and FIG. 7D). As
a result of RNase H treatment the sequence of transposon at the
region of RNA/DNA hybrid was hydrolyzed at the expected positions.
This example clearly indicated that unnecessary transposon sequence
present at both ends of randomly fragmented target DNA could be
effectively removed by RNase H treatment, meanwhile target genomic
DNA without RNR/DNA hybrid region in it will remain intact.
Resulting DNA ends could be designed to be compatible with
appropriate downstream applications providing additional
flexibility in subsequent experiment design.
[0099] The publications and other materials used herein to
illuminate the background of the invention, and in particular, to
provide additional details with respect to its practice, are
incorporated herein by reference in their entirety. The disclosure
and examples are not intended to limit the scope of the
invention.
REFERENCES
[0100] Boeke J. D. 1989. Transposable elements in Saccharomyces
cerevisiae in Mobile DNA. [0101] Craig N. L. 1996. Transposon Tn7.
Curr. Top. Microbiol. Immunol. 204: 27-48. [0102] Devine, S.E. and
Boeke, J.D., Nucleic Acids Research, 1994, 22(18): 3765-3772.
[0103] Haapa, S. et al., Nucleic Acids Research, vol. 27, No. 13,
1999, pp. 2777-2784 [0104] Ichikawa H. and Ohtsubo E., J. Biol.
Chem., 1990, 265(31): 18829-32. [0105] Kaufman P. and Rio D. C.
1992. Cell, 69(1): 27-39. [0106] Kleckner N., Chalmers R. M., Kwon
D., Sakai J. and Bolland S. TnIO and IS10 [0107] Transposition and
chromosome rearrangements: mechanism and regulation in vivo and in
vitro. Curr. Top. Microbiol. Immunol., 1996, 204: 49-82. [0108]
Lampe D. J., Churchill M. E. A. and Robertson H. M., EMBO J.,1996,
15(19): 5470-5479. [0109] Ohtsubo E. & Sekine Y. Bacterial
insertion sequences. Curr. Top. Microbiol. Immunol., 1996,
204:1-26. [0110] Park B. T., Jeong M. H. and Kim B. H., Taehan
Misaengmul Hakhoechi, 1992, 27(4): 381-9. [0111] Savilahti, H. and
K. Mizuuchi. 1996. Mu transpositional recombination: donor DNA
cleavage and strand transfer in trans by the Mu transposase. Cell
85:271-280. [0112] Savilahti, H., P. A. Rice, and K. Mizuuchi.
1995. The phage Mu transpososome core: DNA requirements for
assembly and function. EMBO J. 14:4893-4903. [0113] Varmus H and
Brown. P. A. 1989. Retroviruses, in Mobile DNA. Berg D. E. and Howe
M. eds. American society for microbiology, Washington D. C.
pp.53-108. [0114] Vos J. C., Baere I. And Plasterk R. H. A., Genes
Dev., 1996,10(6): 755-61.
[0115] Applicants incorporate by reference the material contained
in the accompanying computer readable Sequence Listing identified
as Sequence Listing_ST25.txt, having a file creation date of July
6, 2012 10:43 A.M. and file size of 2.71 KB.
Sequence CWU 1
1
12150DNAArtificial Sequencetransposon 1tgaagcggcg cacgaaaaac
gcgaaagcgt ttcacgataa atgcgaaaac 50250DNAArtificial
Sequencetransposon 2gttttcgcat ttatcgtgaa acgctttcgc gtttttcgtg
cgccgcttca 50350DNAArtificial Sequencetransposon 3caaaagcgta
aatagcactt tgcgaaagcg caaaaagcac gaggcgaagt 50450DNAArtificial
Sequencetransposon 4gttttcgcat ttatcgtgaa acgctttcgc guttttcgtg
cgtcagttca 50553DNAArtificial Sequencetransposon 5tgctgaactg
acgcacgaaa aacgcgaaag cgtutcacga taaatgcgaa aac 53650DNAArtificial
Sequencetransposon 6gttttcgcat ttatcgtgaa acgctttcgc gtttttcgtg
cgtcagttca 50753DNAArtificial Sequencetransposon 7tgctgaactg
acgcacgaaa aacgcgaaag cgtttcacga taaatgcgaa aac 53850DNAArtificial
Sequencetransposon 8gttttcgcat ttatcgtgaa acgctttcgc gtttttcgtg
cgtcagttca 50953DNAArtificial Sequencetransposon 9tgctgaactg
acgcacgaaa aacgcgaaag cgtttcacga taaatgcgaa aac 531050DNAArtificial
Sequencetransposon 10gttttcgcat ttatcgtgaa acgctttcgc gtttttcgtg
cgtcagttca 501153DNAArtificial Sequencetransposon 11tgctgaactg
acgcacgaaa aacgcgaaag cguuucacga taaatgcgaa aac 531250DNAArtificial
Sequencetransposon 12acttcgccgc gtgctttttg cgctttcgca aagtgctatt
tacgcttttg 50
* * * * *