U.S. patent application number 16/386746 was filed with the patent office on 2019-10-03 for transposon nucleic acids comprising a calibration sequence for dna sequencing.
The applicant listed for this patent is THERMO FISHER SCIENTIFIC BALTICS UAB. Invention is credited to Heli HAAKANA, Ian KAVANAGH, Laura-Leena KIISKINEN.
Application Number | 20190300937 16/386746 |
Document ID | / |
Family ID | 47556174 |
Filed Date | 2019-10-03 |
United States Patent
Application |
20190300937 |
Kind Code |
A1 |
KAVANAGH; Ian ; et
al. |
October 3, 2019 |
TRANSPOSON NUCLEIC ACIDS COMPRISING A CALIBRATION SEQUENCE FOR DNA
SEQUENCING
Abstract
Transposon nucleic acids comprising a transposon end sequence
and a calibration sequence for DNA sequencing in the transposon end
sequence. In one embodiment, the transposon end sequence is a Mu
transposon end. A method for the generation of DNA fragmentation
library based on a transposition reaction in the presence of a
transposon end with the calibration sequence providing facilitated
downstream handling of the produced DNA fragments, e.g., in the
generation of sequencing templates.
Inventors: |
KAVANAGH; Ian; (Luzern,
CH) ; KIISKINEN; Laura-Leena; (Espoo, FI) ;
HAAKANA; Heli; (Espoo, FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THERMO FISHER SCIENTIFIC BALTICS UAB |
Vilnius |
|
LT |
|
|
Family ID: |
47556174 |
Appl. No.: |
16/386746 |
Filed: |
April 17, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15791740 |
Oct 24, 2017 |
10308978 |
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16386746 |
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14836248 |
Aug 26, 2015 |
9834811 |
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15791740 |
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13553395 |
Jul 19, 2012 |
9145623 |
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14836248 |
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61509691 |
Jul 20, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C40B 50/06 20130101;
C12N 15/1093 20130101; C12Q 1/6806 20130101; C12Q 2525/131
20130101; C12Q 1/6806 20130101; C40B 40/08 20130101; C12Q 2525/155
20130101 |
International
Class: |
C12Q 1/6806 20060101
C12Q001/6806; C40B 50/06 20060101 C40B050/06; C12N 15/10 20060101
C12N015/10; C40B 40/08 20060101 C40B040/08 |
Claims
1. A nucleic acid comprising a modified transposon end sequence,
wherein (a) the modified transposon end sequence comprises (i) a
first strand having the nucleotide sequence of SEQ ID NO: 1 that is
modified to contain a calibration sequence that is four nucleotides
in length and (ii) a second strand that is complementary to the
first strand, and (b) the calibration sequence contains four
different nucleotide bases in any order and is located in the 3'
end region of sequence of SEQ ID NO: 1 3' of position 17 of SEQ ID
NO: 1.
2. The nucleic acid of claim 1, wherein the calibration sequence is
chosen from among the sequences of 5'-TCAG-3', 5'-GTCA-3' and
5'-TGCA-3'.
3. The nucleic acid of claim 1, wherein the modified transposon end
sequence comprises the nucleotide sequence of SEQ ID NO: 3, SEQ ID
NO: 4 or SEQ ID NO: 6.
4. The nucleic acid of claim 1, wherein the modified transposon end
sequence comprises a cleavage site.
5. An in vitro transpososome assembly reaction mixture comprising a
transposase, a nucleic acid comprising a modified transposon end
sequence, and reaction mixture components suitable for assembly of
transposoomes, wherein (a) the modified transposon end sequence
comprises (i) a first strand having the nucleotide sequence of SEQ
ID NO: 1 that is modified to contain a calibration sequence that is
four nucleotides in length and (ii) a second strand that is
complementary to the first strand, (b) the calibration sequence
contains four different nucleotide bases in any order and is
located in the 3' end region of sequence of SEQ ID NO: 1 3' of
position 17 of SEQ ID NO: 1 and (c) the modified transposon end
sequence and the transposase are capable of forming a
transpososome.
6. The in vitro transpososome assembly reaction mixture of claim 5,
wherein the calibration sequence is chosen from among the sequences
of 5'-TCAG-3', 5'-GTCA-3' and 5'-TGCA-3'.
7. The in vitro transpososome assembly reaction mixture of claim 5,
wherein the modified transposon end sequence comprises the
nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or
SEQ ID NO: 6.
8. The in vitro transpososome assembly reaction mixture of claim 5,
wherein the modified transposon end sequence comprises the
nucleotide sequence of SEQ ID NO: 5.
9. A kit comprising a nucleic acid comprising a modified transposon
end sequence of claim 1, a transposase and a buffer for performing
a transposition reaction.
10. The kit of claim 9, further comprising a DNA polymerase.
11. A method for in vitro assembly of one or more transpososome
complexes, comprising contacting a transposase and a nucleic acid
comprising a modified transposon end sequence under a condition for
forming a transpososome complex, thereby forming one or more
transpososome complexes, wherein (a) the modified transposon end
sequence comprises (i) a first strand having the nucleotide
sequence of SEQ ID NO: 1 that is modified to contain a calibration
sequence that is four nucleotides in length and (ii) a second
strand that is complementary to the first strand, and (b) the
calibration sequence contains four different nucleotide bases in
any order and is located in the 3' end region of sequence of SEQ ID
NO: 1 3' of position 17 of SEQ ID NO: 1.
12. The method of claim 11, wherein the calibration sequence is
chosen from among the sequences of 5'-TCAG-3', 5'-GTCA-3' and
5'-TGCA-3'.
13. The method of claim 11, wherein the modified transposon end
sequence comprises the nucleotide sequence of SEQ ID NO: 3, SEQ ID
NO: 4 or SEQ ID NO: 6.
14. The method of claim 11, wherein the modified transposon end
sequence comprises a cleavage site.
15. The method of claim 13, further comprising contacting the one
or more transpososome complexes with a plurality of target nucleic
acids.
16. The method of claim 15, further comprising incubating the one
or more transpososome complexes and plurality of target nucleic
acids under a condition for performing a transposition reaction,
wherein the transposition reaction results in fragmentation of the
plurality of target nucleic acids and incorporation of a modified
transposon end sequence into the ends of the fragmented target
nucleic acids, thereby generating a plurality of target nucleic
acid fragments attached at both ends to a modified transposon end
sequence.
17. The method of claim 16, further comprising contacting a DNA
polymerase having 5'-3' exonuclease or strand displacement activity
with the plurality of target nucleic acid fragments to generate
fully double-stranded nucleic acid molecules.
18. The method of claim 17, further comprising denaturating the
fully double-stranded nucleic acid molecules to produce
single-stranded nucleic acids, and amplifying the single-stranded
nucleic acids.
19. The method of claim 18, further comprising sequencing the
single-stranded nucleic acids.
Description
[0001] This application is a continuation application of U.S.
patent application Ser. No. 15/791,740, filed Oct. 24, 2017, which
is a divisional application of U.S. patent application Ser. No.
14/836,248, filed Aug. 26, 2015, now issued as U.S. Pat. No.
9,834,811, which is a continuation application of U.S. patent
application Ser. No. 13/553,395, filed Jul. 19, 2012, now issued as
U.S. Pat. No. 9,145,623, which claims priority to U.S. Provisional
Application Ser. No. 61/509,691, filed Jul. 20, 2011, each of which
is expressly incorporated by reference herein in its entirety.
[0002] The invention relates to the field of high throughput
multiplex DNA sequencing. The invention is directed to transposon
nucleic acids comprising a transposon end sequence and a
calibration sequence for DNA sequencing in the transposon end
sequence. In one embodiment, this transposon end sequence is a Mu
transposon end. The invention is also directed to a method for
generation of a DNA fragmentation library based on a transposition
reaction in the presence of a transposon end with the calibration
sequence, providing facilitated downstream handling of the produced
DNA fragments, e.g., in the generation of sequencing templates.
BACKGROUND
[0003] "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] 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 needs to be produced. A major advancement for
template generation was the use of in vitro transposition
technology. The earliest in vitro transposition-assisted sequencing
template generation methodology (Tenkanen U.S. Pat. No. 6,593,113)
discloses a method in which 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.
[0005] In vitro transposition methodology has also been applied to
"next generation" sequencing platforms. Grunenwald (U.S. Patent
Application 20100120098) disclose methods 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.
[0006] Many "next-generation" sequencing instruments require a
specific calibration sequence to be read first as a part of the
sequence to be analyzed (e.g. ion torrent PGM and Roche 454 Genome
Sequencer FLX System). This calibration sequence has known bases in
particular order and it calibrates the instrument so that it is
capable of differentiating the signal generated from different
bases during the DNA sequencing reaction. It is necessary that each
of the sequencing templates comprises this calibration
sequence.
[0007] Methods that facilitate the downstream handling of the
fragmented DNA obtained from the transposition step are needed.
SUMMARY
[0008] The invention is related to the modification of a transposon
end sequence so that it includes a calibration sequence for DNA
sequencing. When the transposon end sequence is inserted into the
target DNA in the fragmentation reaction, the calibration sequence
is simultaneously incorporated into the target sequence.
[0009] A modified transposon nucleic acid comprising a transposon
end sequence and an engineered calibration sequence for DNA
sequencing in the transposon end sequence, and a kit for DNA
sequencing containing the modified transposon nucleic acid.
[0010] An in vitro method for generating a DNA library by
incubating a transposon complex comprising a transposon nucleic
acid and a transposase with a target DNA of interest under
conditions for carrying out a transposition reaction, where the
transposon nucleic acid comprises a transposon end sequence
recognizable by the transposase, where the transposon end sequence
comprises a calibration sequence for DNA sequencing, and where the
transposition reaction results in fragmentation of the target DNA
and incorporation of the transposon end into the 5' end of the
fragmented target DNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] 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.
[0012] FIG. 1 is a gel showing a complex formation with MuA
transposase and varying transposon end sequences.
[0013] FIGS. 2A-C show human genomic DNA fragmented with different
amounts of transpososome complexes.
[0014] FIG. 3 is a gel showing a complex formation similar to FIG.
1, but with transposon end of SEQ ID NO: 5.
[0015] FIGS. 4A-B are similar to FIGS. 2A-C, but with transposon
end of SEQ ID NO: 5.
[0016] FIGS. 5A-B are similar to FIGS. 2A-C, but with transposon
end of SEQ ID NO: 6.
[0017] FIG. 6 shows a transposition reaction with Mu transposon end
sequences on target DNA.
[0018] FIG. 7 shows downstream sequencing reaction with target DNA
comprising an incorporated transposon end with calibration
sequence.
DETAILED DESCRIPTION
[0019] The terms "calibration sequence" or "key sequence" as used
herein generally refer to a nucleic acid sequence that can be used
to calibrate a DNA sequencing system. Thus, in embodiments, the
particular bases, the order of the bases, and the number of bases
that are present in a calibration sequence depends on the
requirements of a particular DNA sequencing system.
[0020] In one embodiment, the calibration sequence is a
four-nucleotide-long nucleic acid sequence of the four known bases
(A, T, C and G) in particular order incorporated into target DNA to
be sequenced. The calibration sequence calibrates the sequencing
instrument for each sample so that it is capable of differentiating
the signal generated from different bases during the DNA sequencing
reaction. For example, sequences TCAG, GTCA and TGCA can be used as
calibration sequences. However, the four bases could be presented
in the calibration sequence in any possible order. In embodiments,
the calibration sequence may be longer than four nucleotides, e.g.,
the calibration sequence may be five, six, seven, eight, nine, ten,
or more nucleotides long. The calibration sequence may also
comprise bases in addition to, or in place of, the four known bases
A, T, G, and C. For example, the calibration sequence may contain
derivatized and/or artificial nucleotide bases; such modified bases
are known to one skilled in the art.
[0021] The term "transposon", as used herein, refers to a nucleic
acid segment that is recognized by a transposase or an integrase
enzyme and 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
transposon end sequences that are able to interact with MuA (FIG.
6) where the DNA sequences of the fragments from the transposition
reaction are, e.g.,
TABLE-US-00001 SEQ ID NO: 1 Insert from Target DNA gap SEQ ID NO: 7
SEQ ID NO: 8 gap SEQ ID NO: 9
and showing the product after gap-filling by a DNA polymerase.
[0022] The term "transposase" as used herein refers to an enzyme
that 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.
[0023] A "transposition reaction" as used herein refers to a
reaction where 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 invention are exemplified by employing in vitro Mu
transposition (Haapa et al. 1999 and Savilahti et al. 1995). Other
transposition systems can be used, e.g., Tyl (Devine and Boeke,
1994, and 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).
[0024] A "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 to form a transpososome complex and to
perform a transposition reaction. For MuA transposase, this
sequence is 50 bp long (SEQ ID NO. 1) 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 a
nucleotide sequence, i.e., the transposable DNA contains only one
transposase binding sequence. Thus, the transposable DNA comprises
a "transposon end" (e.g., Savilahti et al., 1995).
[0025] A "transposase binding sequence" or "transposase binding
site" as used herein refers to the nucleotide sequences that are
always within the transposon end sequence where a transposase
specifically binds when mediating transposition. The transposase
binding sequence may comprise more than one site for binding
transposase subunits.
[0026] A "transposon joining strand" or "joining end" as used
herein means the end of that strand of the double-stranded
transposon DNA that is joined by the transposase to the target DNA
at the insertion site.
[0027] 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).
[0028] Many "next-generation" sequencing instruments (e.g. ion
torrent PGM and Roche 454 Genome Sequencer FLX System) require a
specific calibration sequence to be read first as a part of the
sequence to be analyzed. Because the in vitro transposition
technology is already used to fragment target DNA for sequencing,
the disclosed method provides transposon end sequences that include
the calibration sequence. In this way, the calibration sequence
would be incorporated to the target DNA during the fragmentation
step. To reduce unusable sequence reads, this calibration sequence
may be designed as close to the 3' inserted end of the transposon
end (i.e., the joining end) as possible.
[0029] The MuA transposase recognizes a certain transposon end
sequence of 50 bp (SEQ ID NO:1) but tolerates some variation at
certain positions (Goldhaber-Gordon et al., J Biol Chem. 277 (2002)
7703-7712). Various options for including a calibrator sequence
into the transposon end were designed.
[0030] In one embodiment, a modified transposon nucleic acid
comprising a transposon end sequence and an engineered calibration
sequence for DNA sequencing in the transposon end sequence is
provided. The transposon end sequence may be a Mu transposon end
sequence, and the Mu transposon end sequence may be any of SEQ ID
NOS: 3-6. In one embodiment, the Mu transposon end sequence is SEQ
ID NO: 5.
[0031] In one embodiment, a modified transposon nucleic acid
comprising a transposon end sequence and an engineered calibration
sequence for DNA sequencing in the transposon end sequence is
provided, where the transposon end sequence further contains an
engineered cleavage site. An engineered cleavage site in the
transposon end sequence can be 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). In embodiments, the
engineered cleavage site may be the incorporation of a uracil base
or a restriction site. Modified transposon end sequences comprising
an engineered calibration sequence for DNA sequencing and
optionally an uracil base or an additional restriction site can be
produced, e.g., by regular oligonucleotide synthesis.
[0032] In one embodiment, an in vitro method for generating a DNA
library is provided. The method incubates a transposon complex
comprising a transposon nucleic acid and a transposase with a
target DNA of interest under conditions for carrying out a
transposition reaction. Transposon nucleic acid comprises a
transposon end sequence that is recognizable by the transposase,
and where the transposon end sequence comprises a calibration
sequence for DNA sequencing. The transposition reaction results in
fragmentation of the target DNA, and incorporates the transposon
end into the 5' end of the fragmented target DNA.
[0033] In one embodiment, the method further comprises the step of
amplifying the fragmented target DNA in an amplification reaction
using a first and second oligonucleotide primer complementary to
the transposon end in the 5' ends of the fragmented target DNA. The
first and second primer optionally comprise 5' adaptor tails.
[0034] In one embodiment, the method further comprises the step of
contacting the fragments of target DNA 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.
[0035] To prepare the transposition products for downstream steps,
such as polymerase chain reaction (PCR), 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.
[0036] In one embodiment, the transposition system used in the
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 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 four hours. In one embodiment, the assembly reaction is
incubated for two hours at about 30.degree. C. Mg.sup.2+ is added
for activation.
[0037] In case the transposon end sequence comprises an engineered
cleavage site, the method can comprise a further step of 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. The cleaving enzyme
may be an N-glycosylase or a restriction enzyme, such as
uracil-N-glycosylase or a methylation specific restriction enzyme,
respectively.
[0038] In one embodiment, the 5' adaptor tail of the first and/or
second PCR primer(s) used in the method comprise one or more of the
following groups: an amplification tag, a sequencing tag, and/or a
detection tag.
[0039] The amplification tag is a nucleic acid sequence providing
specific sequence complementary to an oligonucleotide primer to be
used in the subsequent rounds of amplification. For example, the
sequence may be used for facilitating amplification of the nucleic
acid obtained. Examples of detection tags are fluorescent and
chemiluminescent dyes, a green fluorescent protein, and enzymes
that are detectable in the presence of a substrate, e.g., an
alkaline phosphatase with NBT plus BCIP, or a peroxidase with a
suitable substrate. By using different detection tags, i.e.
barcodes, sequences from multiple samples can be sequenced in the
same instrument run and identified by the sequence of the detection
tag. Examples are Illumina's index sequences in TruSeq DNA Sample
Prep Kits, or Molecular barcodes in Life Technologies' SOLiD.TM.
DNA Barcoding Kits.
[0040] 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. Such 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.
[0041] The detection tag comprises a sequence or a detectable
chemical or biochemical moiety for facilitating detection of the
nucleic acid obtained from the amplification step.
[0042] In one embodiment, a kit for use in DNA sequencing is
provided. The kit comprises at least a tranposon nucleic acid
comprising transposon end sequence and an engineered calibration
sequence for DNA sequencing in the transposon end sequence. In one
embodiment, the tranposon nucleic acid is a Mu transposon end
sequence. In one embodiment, the Mu transposon end sequence is
selected from SEQ ID NOS: 3-6. In one embodiment, the Mu transposon
end sequence is SEQ ID NO:5. In one embodiment, the tranposon
nucleic acid further comprises an engineered cleavage site. The kit
may also comprise additional components, e.g., buffers for
performing transposition reaction, buffers for DNA sequencing,
control DNA, transposase enzyme, and DNA polymerase. The kit can be
packaged in a suitable container with instructions for using the
kit.
[0043] 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. The present invention is further
described in the following examples, which are not intended to
limit the scope of the invention.
Example 1
Native Sequence of the Inserted Strand of MuA Recognition
Transposon End
[0044] In various embodiments, a calibration sequence is four bases
long and contains each of the four bases (A, T, G, C) sequentially
in a row but in any order. The native MuA recognition sequence only
contains these at underlined positions which are far from the
3'-end.
TABLE-US-00002 (SEQ ID NO: 1)
GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCTTCA
If these sequences were used as calibrators, a minimum of 34 bases
of the inserted transposon sequence would have to be read in every
target fragment before reaching the sequence of interest, wasting
sequencing reagents and instrument run time. The accuracy of
sequence reading also gradually decreases after each cycle, so the
best possible accuracy would always be "wasted" on reading the
transposon end sequence. In addition, the bases in the "native
calibrator sequences" are not necessarily in the preferred order,
as defined by the instruments' requirements. That is why the MuA
recognition sequence needs to be modified near the 3'-end in order
to be used in library generation for instruments that require a
calibration sequence. Other transposons, such as the native Tn5
recognition transposon sequence, do not contain the four bases in a
row anywhere in its sequence.
[0045] When the transposon end sequence and MuA transposase were
incubated together in a suitable buffer, they formed transpososome
complexes (FIG. 1). The higher molecular weight DNA band of about
300 bp represents transposon end DNA bound to transpososome
complexes (mobility in the gel electrophoresis is retarded due to
protein binding) and the smaller DNA band (50 bp) is the free
transposon DNA, i.e. not complexed by MuA. M: Molecular weight
marker. wt: Native MuA transposon end +MuA transposase. 1:
Transposon end of SEQ ID NO:4. 2: Transposon end of SEQ ID NO:2. 3:
Transposon end of SEQ ID NO:3. -MuA: Control reaction with native
MuA transposon end DNA sequence (SEQ ID NO: 1) without MuA
transposase.
[0046] When these complexes (formed using wild-type MuA transposon
end DNA and MuA transposase) were incubated with target DNA, the
transposon sequences were inserted into DNA and the target DNA was
fragmented, as shown in FIGS. 2A-C. FIG. 2A: 0.05 g/L MuA in the
fragmentation reaction. FIG. 2B: 0.15 g/l MuA. FIG. 2C: 0.2 g/l
MuA, gDNA fragmented with MuA. Control contains MuA complexes but
no gDNA.
TABLE-US-00003 TABLE 1 Composition of the complex formation
reaction in FIG. 1. The following components of the complex
formation reaction were incubated for one hour at 30.degree. C. MuA
transposase 0.44 g/l Transposon DNA 3.0 .mu.M Tris-HCl pH 8 120.42
mM HEPES pH 7.6 2.6 mM EDTA 1.05 mM DTT 0.10 mM NaCl 102.1 mM KCl
52 mM Triton X-100 0.05 % glycerol 12.08 % DMSO 10 %
Different amounts of the complex were incubated with human genomic
DNA at 30.degree. C. for one hour (Table 2), and each reaction was
replicated eight times. The replicates were combined after the
fragmentation, DNA was purified with QIAGEN MinElute PCR
Purification Kit, and analyzed with Agilent 2100 Bioanalyzer
instrument (FIG. 2).
TABLE-US-00004 TABLE 2 Example of final composition of the
fragmentation reaction. For the fragmentation reaction, the MuA
transposon and transposon DNA concentrations were varied in
different experiments, whereas the concentration of other
components was kept constant. MuA transposon 0.05 g/l Transposon
DNA 0.341 .mu.M gDNA 100 ng Tris-HCl pH 8 40 mM EDTA 0.33 mM NaCl
100 mM MgCl.sub.2 10 mM Triton X-100 0.05 % glycerol 10 % DMSO 3.3
%
Example 2
Changing of the Mu 3'-End to Include the Current Ion Torrent Key
Sequence (TCAG)
TABLE-US-00005 [0047] (SEQ ID NO: 2)
GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCTCAG
This transposon end sequence was tested. MuA transposase did not
form complexes with this sequence and thus there was no
transposition activity (see FIG. 1).
Example 3
Converting the Fourth T of Mu End Sequence to G (Counting from the
3'-End) to Yield GTCA Key
TABLE-US-00006 [0048] (SEQ ID NO: 3)
GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCGTCA
This was tested for transposition activity and MuA transposase
formed complexes with this sequence and was also active (FIG.
1).
Example 4
Converting the Third T of Mu End Sequence to G to Yield TGCA
Key
TABLE-US-00007 [0049] (SEQ ID NO: 4)
GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGCCGCTGCA
This was tested for transposition activity and MuA transposase
formed complexes with this sequence and was also active (FIG.
1).
Example 5
Modification of the 5th, 6th, and 8th Bases of Mu End Sequence into
G, a, and T, Respectively, to Yield the Current Ion Torrent Key
TABLE-US-00008 [0050] (SEQ ID NO: 5)
GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTGCGTCAGTTCA
This transposon end sequence worked well with MuA transposase in
complex formation, as shown in FIG. 3, which shows complex
formation similar to FIG. 1, but with transposon end of SEQ ID NO:
5, and in fragmentation, as shown in FIG. 4, which is similar to
FIG. 2, but with transposon end of SEQ ID NO: 5, i.e. showing human
genomic DNA fragmented with different amounts of transpososome
complexes. FIG. 4A: 0.05 g/L MuA in the fragmentation reaction.
FIG. 4B: 0.15 g/l MuA, with gDNA fragmented with MuA and control
that contains MuA complexes but no gDNA.
Example 6
Modification of the 10th, and 11th Bases of Mu End Sequence into a
and C, Respectively, to Yield
TABLE-US-00009 [0051] (SEQ ID NO: 6)
GTTTTCGCATTTATCGTGAAACGCTTTCGCGTTTTTCGTCAGCCGCTTCA
This transposon end sequence worked well with MuA transposase in
complex formation and in fragmentation, as shown in FIG. 5, which
is similar to FIG. 2, but with transposon end of SEQ ID NO: 6, i.e.
showing human genomic DNA fragmented with different amounts of
transpososome complexes. FIG. 5A: 0.05 g/L MuA in the fragmentation
reaction. FIG. 5B: 0.15 g/l MuA, with gDNA fragmented with MuA and
control that contains MuA complexes but no gDNA.
REFERENCES
[0052] Boeke J. D. 1989. Transposable elements in Saccharomyces
cerevisiae in Mobile DNA. [0053] Craig N. L. 1996. Transposon Tn7.
Curr. Top. Microbiol. Immunol. 204: 27-48. [0054] Devine, S. E. and
Boeke, J. D., Nucleic Acids Research, 1994, 22(18): 3765-3772.
[0055] Goldhaber-Gordon et al., J Biol Chem. 277 (2002) 7703-7712
[0056] Haapa, S. et al., Nucleic Acids Research, vol. 27, No. 13,
1999, pp. 2777-2784 [0057] Ichikawa H. and Ohtsubo E., J. Biol.
Chem., 1990, 265(31): 18829-32. [0058] Kaufman P. and Rio D. C.
1992. Cell, 69(1): 27-39. [0059] Kleckner N., Chalmers R. M., Kwon
D., Sakai J. and Bolland S. TnlO and IS10 Transposition and
chromosome rearrangements: mechanism and regulation in vivo and in
vitro. Curr. Top. Microbiol. Immunol., 1996, 204: 49-82. [0060]
Lampe D. J., Churchill M. E. A. and Robertson H. M., EMBO J., 1996,
15(19): 5470-5479. [0061] Ohtsubo E. & Sekine Y. Bacterial
insertion sequences. Curr. Top. Microbiol. Immunol., 1996,
204:1-26. [0062] Park B. T., Jeong M. H. and Kim B. H., Taehan
Misaengmul Hakhoechi, 1992, 27(4): 381-9. [0063] 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. [0064] 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. [0065] 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. [0066] Vos J. C., Baere I. And Plasterk R. H. A., Genes
Dev., 1996, 10(6): 755-61.
[0067] 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 Jul.
17, 2012 1:49 P.M. and file size of 1.98 KB.
Sequence CWU 1
1
9150DNAArtificial SequenceBacteriophage Mu 1gttttcgcat ttatcgtgaa
acgctttcgc gtttttcgtg cgccgcttca 50250DNAArtificial
SequenceModified Mu end sequence 2gttttcgcat ttatcgtgaa acgctttcgc
gtttttcgtg cgccgctcag 50350DNAArtificial SequenceModified Mu end
sequence 3gttttcgcat ttatcgtgaa acgctttcgc gtttttcgtg cgccgcgtca
50450DNAArtificial SequenceModified Mu end sequence 4gttttcgcat
ttatcgtgaa acgctttcgc gtttttcgtg cgccgctgca 50550DNAArtificial
SequenceModified Mu end sequence 5gttttcgcat ttatcgtgaa acgctttcgc
gtttttcgtg cgtcagttca 50650DNAArtificial SequenceModified Mu end
sequence 6gttttcgcat ttatcgtgaa acgctttcgc gtttttcgtc agccgcttca
50750DNAArtificial Sequencetransposon 7tgaagcggcg cacgaaaaac
gcgaaagcgt ttcacgataa atgcgaaaac 50850DNAArtificial
Sequencetransposon 8caaaagcgta aatagcactt tgcgaaagcg caaaaagcac
gaggcgaagt 50950DNAArtificial Sequencetransposon 9acttcgccgc
gtgctttttg cgctttcgca aagtgctatt tacgcttttg 50
* * * * *