U.S. patent application number 15/818322 was filed with the patent office on 2018-06-14 for methods for producing a paired tag from a nucleic acid sequence and methods of use thereof.
The applicant listed for this patent is Applied Biosystems, LLC. Invention is credited to Joel Malek, Kevin McKernan, Douglas SMITH.
Application Number | 20180163253 15/818322 |
Document ID | / |
Family ID | 34549481 |
Filed Date | 2018-06-14 |
United States Patent
Application |
20180163253 |
Kind Code |
A1 |
SMITH; Douglas ; et
al. |
June 14, 2018 |
METHODS FOR PRODUCING A PAIRED TAG FROM A NUCLEIC ACID SEQUENCE AND
METHODS OF USE THEREOF
Abstract
Methods for producing a paired tag from a nucleic acid sequence
are provided in which the paired tag comprises the 5' end tag and
3' end tag of the nucleic acid sequence. In one embodiment, the
nucleic acid sequence comprises two restriction endonuclease
recognition sites specific for a restriction endonuclease that
cleaves the nucleic acid sequence distally to the restriction
endonuclease recognition sites. In another embodiment, the nucleic
acid sequence further comprises restriction endonuclease
recognition sites specific for a rare cutting restriction
endonuclease. Methods of using paired tags are also provided. In
one embodiment, paired tags are used to characterize a nucleic acid
sequence. In a particular embodiment, the nucleic acid sequence is
a genome. In one embodiment, the characterization of a nucleic acid
sequence is karyotyping. Alternatively, in another embodiment, the
characterization of a nucleic acid sequence is mapping of the
sequence. In a further embodiment, a method is provided for
identifying nucleic acid sequences that encode at least two
interacting proteins.
Inventors: |
SMITH; Douglas; (Gloucester,
MA) ; Malek; Joel; (Beverly, MA) ; McKernan;
Kevin; (Woburn, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Applied Biosystems, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
34549481 |
Appl. No.: |
15/818322 |
Filed: |
November 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15075742 |
Mar 21, 2016 |
9822395 |
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15818322 |
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12468818 |
May 19, 2009 |
9309560 |
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15075742 |
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10978224 |
Oct 29, 2004 |
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12468818 |
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60516080 |
Oct 31, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/34 20130101;
C12Q 1/6806 20130101; C12Q 1/6816 20130101; C12Q 2521/313 20130101;
C12Q 2525/131 20130101; C12Q 1/6806 20130101; C12Q 1/6816 20130101;
C12Q 2521/501 20130101; C12Q 2525/191 20130101; C12Q 2521/313
20130101 |
International
Class: |
C12Q 1/6806 20060101
C12Q001/6806; C12Q 1/6816 20060101 C12Q001/6816; C12P 19/34
20060101 C12P019/34 |
Claims
1-3. (canceled)
4. A method for producing a paired tag from a first nucleic acid
sequence fragment, without cloning, comprising the steps of: a)
joining the 5' and 3' ends of a first nucleic acid sequence
fragment to at least one adapter; b) cleaving the adapter(s),
thereby producing a second nucleic acid sequence fragment with
compatible ends; c) circularizing the second nucleic acid sequence
fragment such that a 5' end of the first nucleic acid sequence
fragment is joined to a 3' end of the first nucleic acid sequence
fragment via a linker derived from the adapter(s), thereby
producing a circular nucleic acid molecule; d) cleaving the
circular nucleic acid molecule, thereby producing a paired tag
wherein a 5' end tag of the first nucleic acid sequence fragment is
joined to a 3' end tag of the first nucleic acid sequence fragment
via the linker.
5. The method of claim 4, wherein: a) the adapter comprises at
least two restriction endonuclease recognition sites specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site;
b) the adapter comprises a restriction endonuclease recognition
site specific for a rare cutting restriction endonuclease; c)
cleaving the adapter joined to the nucleic acid sequence fragment
with a rare cutting restriction endonuclease to produce the
compatible ends; and d) cleaving the circular nucleic acid molecule
with a restriction endonuclease that cleaves distally to the
restriction endonuclease recognition site.
6. The method of claim 5, wherein the restriction endonuclease
recognition sites specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site are immediately adjacent
to the 5' and 3' ends of the nucleic acid sequence fragment.
7-18. (canceled)
19. A method for characterizing a nucleic acid sequence, without
cloning, comprising the steps of: a) fragmenting a nucleic acid
sequence thereby producing a plurality of first nucleic acid
sequence fragments having a 5' end and a 3' end; b) joining the 5'
and 3' ends of each first nucleic acid sequence fragment to a first
linker such that the first linker is located between the 5' end and
the 3' end of each first nucleic acid sequence fragment in a
circular nucleic acid molecule; c) cleaving the circular nucleic
acid molecules, thereby producing a plurality of second nucleic
acid sequence fragments wherein a subset of the fragments comprise
a paired tag derived from each first nucleic acid sequence fragment
joined via the first linker; d) joining the 5' and 3' ends of each
second nucleic acid sequence fragment via a second linker such that
the second linker is located between the ends of each second
nucleic acid sequence fragment to form a plurality of second
circular nucleic acid molecules; e) amplifying the second circular
nucleic acid molecules two oligonucleotides complementary to
sequences present in the second linker, thereby producing a
plurality of amplified nucleic acid fragments; and f)
characterizing the 5' and 3' end tags of the plurality of amplified
nucleic acid fragments.
20-24. (canceled)
25. The method of claim 19, wherein the second nucleic acid
fragments are purified away from other nucleic acid fragments prior
to PCR amplification.
26. The method of claim 25, wherein the second nucleic acid
fragment is purified from other nucleic acid fragments by affinity
capture using a reagent specific for the first linker prior to
amplification.
27. The method of claim 26, wherein the first linker comprises a
biotin moiety to enable affinity capture.
28. The method of claim 26, wherein the first linker comprises a
sequence capable of forming a triple helix to enable affinity
capture.
29. The method of claim 26 in which the first linker comprises a
recognition site for a DNA binding protein to enable affinity
capture.
30. The method of claim 27, wherein the nucleic acid sequence is a
genome.
31. The method of claim 30, wherein the method karyotypes the
genome.
32. The method of claim 30, wherein the method sequences the
genome.
33-38. (canceled)
39. A composition comprising a circular nucleic acid molecule,
wherein sequence elements are arranged in the following circular
order: ##STR00002## wherein the 5' end tag and the 3' end tag
comprise a paired tag derived from a single contiguous nucleic acid
sequence fragment.
40. The composition of claim 39, wherein linker 1 comprises at
least two restriction endonuclease recognition sites specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and are oriented in such a way that one of the sites directs
cleavage within the 5' end tag or at the junction of the 5' end tag
and linker 2, and the other site directs cleavage within the 3' end
tag or at the junction of the 3' end tag and linker 2.
41. The composition of claim 39, wherein linker 2 comprises: a) at
least two restriction endonuclease recognition sites specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and are oriented in such a way that one of the sites directs
cleavage within the 5' end tag or at the junction of the 5' end tag
and linker 2 and, and the other site directs cleavage within the 3'
end tag or at the junction of the 3' end tag and linker 2; and b)
at least one recognition site for a rare-cutting restriction
endonuclease located between the two restriction endonuclease
recognition sites specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site.
42. The composition of claim 39, wherein linker 2 is
palindromic.
43. The composition of claim 39, wherein linker 2 comprises a
recognition site for a rare-cutting restriction endonuclease.
44. The composition of claim 39, wherein the composition is
amplified isothermally using oligonucleotide primers complementary
to sequences in either linker 1 or linker 2, or in both linker 1
and linker 2.
45. The composition of claim 39, wherein the composition is
amplified isothermally using oligonucleotide primers complementary
to sequences in either linker 1 or linker 2, and oriented in
opposite directions such that a linear nucleic acid fragment is
produced that preserves the orientation of the 5' end tag and the
3' end tag with respect to each other.
46-69. (canceled)
Description
RELATED APPLICATION
[0001] This application is a Divisional of U.S. application Ser.
No. 15/075,742 filed Mar. 21, 2016, which is a Continuation of U.S.
application Ser. No. 12/468,818 filed May 19, 2009 (now U.S. Pat.
No. 9,309,560), which is a Divisional of U.S. application Ser. No.
10/978,224 filed Oct. 29, 2004 (now abandoned), which application
claims the benefit under 35 U.S.C. .sctn. 119(e) of U.S.
Application No. 60/516,080 filed Oct. 31, 2003. The entire contents
of the aforementioned applications are incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] Whole genome shotgun sequencing, assembly and finishing is
typically the strategy of choice for microbial and fungal genome
sequencing. The cost-advantages and simplicity of the whole genome
approach relative to a BAC based or hybrid sequencing strategy
argue strongly for its continued development and application in
future sequencing projects. However, a major problem with the
BAC-based approaches is the high cost and operational burden
associated with the production of 15,000-25,000 individual BAC
subclone libraries, the 15-20% waste associated with re-sequencing
the vector, as well as the unavoidable E. coli contamination, the
need to deal with transposon and bacteriophage insertions, and the
20-50% waste in redundant sequencing of BAC overlaps. Although
these costs can be reduced by sequencing the BACs at low coverage
(using a hybrid BAC/WGS strategy, for example) or by using a
pooling strategy, they cannot be eliminated. The need to generate a
physical map by using restriction digest fingerprinting or by
complex pooling and sequence based mapping strategies adds
additional cost and operational overhead.
[0003] Thus, a need exists for more cost-efficient sequencing
methods and for better methods of generating a reliable
sequence-derived scaffold that can support the accurate selection
of clones to finish any desired region of the genome with reduced
operational burden, increased efficiency, elimination of problems
associated with transposon and bacteriophage insertions and
reduction in wasted time, effort and expense spent in redundant
sequencing. Additionally, a need exists to provide a reliable and
efficient method that facilitates whole genome assembly and/or
karyotyping of a genome, and which enables the reliable and
efficient detection of sequence inversion in a genome.
[0004] Current bacterial and yeast two-hybrid screening methods are
useful to discover the identity of two interacting proteins.
However, these methods suffer from the need for large numbers of
transformations to be performed: one for each bait to be analyzed
against one or more prey molecule. Although some methods have been
developed to permit pools of ten to one hundred baits to be
screened in parallel, these methods require additional handling
steps to deconvolute the identities of the individual baits.
Therefore, these methods only incrementally increase the efficiency
of conventional two-hybrid systems.
[0005] Thus, a need exists for a method to increase the efficiency
of two-hybrid systems for the identification of two interacting
proteins.
SUMMARY OF THE INVENTION
[0006] The present invention provides a method for producing a
paired tag from a nucleic acid sequence (also referred to herein as
a nucleotide sequence), wherein at least one restriction
endonuclease recognition site is present at the 5' and 3' end of
the nucleic acid sequence and upon cleavage results in production a
paired tag. In one embodiment the present invention provides a
method for producing a paired tag from a nucleic acid sequence,
wherein two restriction endonuclease recognition sites specific for
a restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
are present at the 5' and 3' ends of the nucleic acid sequence,
thereby producing a 5' tag and a 3' tag (a paired tag) from the
nucleic acid sequence upon cleavage by the restriction
endonuclease.
[0007] In another embodiment the present invention provides a
method for producing a paired tag from a nucleic acid sequence,
wherein the nucleic acid sequence comprises one restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence on both sides
of the recognition site.
[0008] In addition, the present invention provides a method for
producing a paired tag from a nucleic acid sequence, wherein the
nucleic acid sequence comprises two restriction endonuclease
recognition sites specific for a restriction endonuclease that
cleaves the nucleic acid sequence between the two recognition
sites.
[0009] The present invention also provides a method for producing a
paired tag from a nucleic acid sequence, wherein the nucleic acid
sequence comprises at least one linker, the at least one linker is
joined to the 5' and 3' ends of the nucleic acid sequence, wherein
producing a fragment on either or both sides of the linker produces
a paired tag from the nucleic acid sequence.
[0010] The present invention also provides a method for producing a
paired tag from a nucleic acid sequence, wherein the nucleic acid
sequence comprises at least one recombination site, wherein a
recombination event at this recombination site in the nucleic acid
sequence produces a paired tag from the nucleic acid sequence.
[0011] Furthermore, the present invention provides methods of using
a paired tag to characterize a sequence and identify nucleic acid
sequences that encode at least two interacting proteins.
[0012] In one embodiment, the invention is directed to a method for
producing a paired tag from a nucleic acid sequence, wherein the
nucleic acid sequence comprises two restriction endonuclease
recognition sites specific for a restriction endonuclease that
cleaves the nucleic acid sequence distally to the restriction
endonuclease recognition site, comprising the steps of cleaving the
restriction endonuclease recognition sites using the restriction
endonuclease that is specific for the restriction endonuclease
recognition sites, thereby producing a 5' end tag and a 3' end tag
from the nucleic acid sequence and joining the 5' end tag with the
3' end tag from the nucleic acid sequence, thereby producing a
paired tag from the nucleic acid sequence. In a particular
embodiment, the nucleic acid sequence further comprises at least
two restriction endonuclease recognition sites specific for a rare
cutting restriction endonuclease.
[0013] In another embodiment of the invention, a method is provided
for characterizing a nucleic acid sequence comprising the steps of
a) fragmenting a nucleic acid sequence thereby producing a
plurality of nucleic acid sequence fragments having a 5' end and a
3' end, b) introducing into the 5' end and into the 3' end of each
nucleic acid sequence fragment, (i) a restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, and (ii) a restriction
endonuclease recognition site specific for a rare cutting
restriction endonuclease, thereby producing modified nucleic acid
sequence fragments, c) cleaving the restriction endonuclease
recognition site specific for a rare cutting restriction
endonuclease in each modified nucleic acid sequence fragment with
the rare cutting restriction endonuclease, thereby producing a
plurality of nucleic acid sequence fragments having compatible
ends, d) maintaining the fragments having compatible ends under
conditions in which the compatible ends intramolecularly ligate,
thereby producing a plurality of circularized nucleic acid
sequences comprising two restriction endonuclease recognition sites
specific for restriction endonucleases that cleave the nucleic acid
sequence fragment distally to the restriction endonuclease
recognition sites, e) cleaving the circularized nucleic acid
sequences at the restriction endonuclease recognition sites with
the restriction endonucleases specific for the restriction
endonuclease recognition sites, thereby producing a plurality of
paired tags comprising a 5' end tag and a 3' end tag of the nucleic
acid sequence fragment, and f) characterizing the paired tags,
thereby characterizing the nucleic acid sequence. In one
embodiment, the nucleic acid sequence is a genome. In a further
embodiment, the method karyotypes the genome.
[0014] The invention also provides in one embodiment, a method for
characterizing a nucleic acid sequence comprising the steps of a)
fragmenting a nucleic acid sequence thereby producing a plurality
of nucleic acid sequence fragments having a 5' end and a 3' end, b)
introducing into the 5' end and into the 3' end of each nucleic
acid sequence fragment a restriction endonuclease recognition site
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site, thereby producing a plurality of modified nucleic
acid sequence fragments, c) cloning the modified nucleic acid
sequence fragments, thereby producing cloned nucleic acid sequence
fragments, d) cleaving the restriction endonuclease recognition
sites at the 5' end and the 3' end of the cloned nucleic acid
sequence fragments using the restriction endonuclease that is
specific for the restriction endonuclease recognition sites,
thereby producing a 5' end tag and a 3' end tag of each cloned
nucleic acid sequence fragment, e) joining the 5' end tag to the 3'
end tag of each cloned nucleic acid sequence fragment thereby
producing a plurality of paired tags; and f) characterizing the
paired tags, thereby characterizing the nucleic acid sequence.
[0015] In another aspect of the invention, provided herein is a
method for characterizing a nucleic acid sequence comprising the
steps of a) fragmenting a nucleic acid sequence thereby producing a
plurality of nucleic acid sequence fragments having a 5' end and a
3' end, b) cloning each nucleic acid sequence fragment, wherein a
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
is introduced into the 5' end and into the 3' end of each cloned
nucleic acid sequence fragment, thereby producing a plurality of
cloned nucleic acid sequence fragments, c) cleaving the restriction
endonuclease recognition sites at the 5' end and the 3' end of each
cloned nucleic acid sequence fragment using the restriction
endonuclease that is specific for the restriction endonuclease
recognition sites, thereby producing a 5' end tag and a 3' end tag
of the cloned nucleic acid sequence fragments, d) joining the 5'
end tag to the 3' end tag of each cloned nucleic acid sequence
fragment thereby producing a plurality of paired tags, and e)
characterizing the paired tags, thereby characterizing the nucleic
acid sequence.
[0016] In one embodiment of the invention, provided herein is a
method for producing a paired tag from a first nucleic acid
sequence fragment, without cloning, comprising the steps of joining
the 5' and 3' ends of a nucleic acid sequence fragment via a linker
such that the linker is located between the 5' end and the 3' end
of the first nucleic acid sequence fragment in a circular nucleic
acid molecule, b) cleaving the circular nucleic acid molecule,
thereby producing a paired tag wherein a 5' end tag of the first
nucleic acid sequence fragment is joined to a 3' end tag of the
first nucleic acid sequence fragment via the linker. In one
embodiment, the linker comprises at least two restriction
endonuclease recognition sites specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, and the
circular nucleic acid molecule is cleaved with a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site to
thereby producing the paired tag. In a further embodiment, the
restriction endonuclease recognition sites are immediately adjacent
to the ends of the first nucleic acid sequence fragment.
[0017] In a further embodiment of the invention, provided herein is
a method for producing a paired tag from a first nucleic acid
sequence fragment, without cloning, comprising the steps of a)
joining the 5' and 3' ends of a first nucleic acid sequence
fragment to at least one adapter, b) cleaving the adapter(s),
thereby producing a second nucleic acid sequence fragment with
compatible ends, c) circularizing the second nucleic acid sequence
fragment such that a 5' end of the first nucleic acid sequence
fragment is joined to a 3' end of the first nucleic acid sequence
fragment via a linker derived from the adapter(s), thereby
producing a circular nucleic acid molecule, and cleaving the
circular nucleic acid molecule, thereby producing a paired tag
wherein a 5' end tag of the first nucleic acid sequence fragment is
joined to a 3' end tag of the first nucleic acid sequence fragment
via the linker. In a particular embodiment, the adapter comprises
at least two restriction endonuclease recognition sites specific
for a restriction endonuclease that cleaves the nucleic acid
sequence fragment distally to the restriction endonuclease
recognition site, the adapter comprises a restriction endonuclease
recognition site specific for a rare cutting restriction
endonuclease, cleaving the adapter joined to the nucleic acid
sequence fragment with a rare cutting restriction endonuclease to
produce the compatible ends, and cleaving the circular nucleic acid
molecule with a restriction endonuclease that cleaves distally to
the restriction endonuclease recognition site. In a further
embodiment, the restriction endonuclease recognition sites specific
for a restriction endonuclease that cleaves the nucleic acid
sequence fragment distally to the restriction endonuclease
recognition site are immediately adjacent to the 5' and 3' ends of
the nucleic acid sequence fragment.
[0018] In another embodiment, provided herein is a method for
producing a paired tag from a first nucleic acid sequence fragment,
without cloning, comprising the steps of a) joining the 5' and 3'
ends of a first nucleic acid sequence fragment via a first linker
such that the first linker is located between the 5' end and the 3'
end of the first nucleic acid sequence fragment in a first circular
nucleic acid molecule, b) cleaving the first circular nucleic acid
molecule, thereby producing a second nucleic acid sequence fragment
wherein a 5' end tag of the first nucleic acid sequence fragment is
joined to a 3' end tag of the first nucleic acid sequence fragment
via the first linker, c) joining a second linker to the 5' and 3'
ends of the second nucleic acid sequence fragment, and d)
amplifying the second nucleic acid fragment using an
oligonucleotide complementary to a sequence present in the second
linker.
[0019] In a further embodiment of the invention, provided herein is
a method for producing a paired tag from a first nucleic acid
sequence fragment, without cloning, comprising the steps of a)
joining the 5' and 3' ends of a first nucleic acid sequence
fragment via a first linker such that the first linker is located
between the 5' end and the 3' end of the first nucleic acid
sequence fragment in a first circular nucleic acid molecule, b)
cleaving the first circular nucleic acid molecule, thereby
producing a second nucleic acid sequence fragment wherein a 5' end
tag of the first nucleic acid sequence fragment is joined to a 3'
end tag of the first nucleic acid sequence fragment via the first
linker, c) joining the 5' and 3' ends of a second nucleic acid
sequence fragment via a second linker such that the second linker
is located between the 5' end and the 3' end of the first nucleic
acid sequence fragment in a second circular nucleic acid molecule,
and d) amplifying a nucleic acid sequence fragment from the second
circular nucleic acid molecule using two oligonucleotides
complementary to sequences present in the second linker. In one
embodiment, the second linker comprises a recognition site for a
rare-cutting restriction endonuclease, and the second circular
nucleic acid molecule is cleaved using a rare-cutting restriction
endonuclease that recognizes the site in the second linker. In
another embodiment, the first linker comprises at least two
restriction endonuclease recognition sites specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and the first circular nucleic acid molecule is cleaved with a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site.
In a particular embodiment, the restriction endonuclease
recognition sites specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site are immediately adjacent
to the ends of the first nucleic acid sequence fragment.
Furthermore, in one embodiment, the second nucleic acid fragment is
purified prior to amplification, for example by affinity capture
using a reagent specific for the first linker prior to
amplification. In one example, the first linker comprises a biotin
moiety, a sequence capable of forming a triple helix, or a
recognition site for a DNA binding protein.
[0020] In another embodiment of the invention, provided herein is a
method for characterizing a nucleic acid sequence comprising the
steps of a) fragmenting a nucleic acid sequence to produce a
plurality of nucleic acid sequence fragments having a 5' end and a
3' end, b) joining one or more first adapters to the 5' and 3'
ends, wherein the one or more first adapters are compatible for
promoting intramolecular ligation, thereby producing a plurality of
modified nucleic acid sequence fragments, intramolecularly ligating
the modified nucleic acid sequence fragments thereby producing a
plurality of circularized nucleic acid sequences, wherein the one
or more first adapters forms a linker between the 5' end and the 3'
end of the circularized nucleic acid sequence fragment, c)
fragmenting the circularized nucleic acid sequences by random
shearing or by other means thereby producing linear fragments
comprising the linker and a paired tag comprising the 5' end and a
3' end of the nucleic acid sequence fragment, d) joining a second
adapter to each of the 5' and 3' ends of the linear fragments to
produce a plurality of linear fragments having the second adapter
at each 5' and 3' end, e) amplifying the linear fragments using a
primer that is complementary to the second adapter thereby
amplifying the paired tags, and f) characterizing the paired tags,
thereby characterizing the nucleic acid sequence.
[0021] In another aspect of the invention, provided herein is a
method for characterizing a nucleic acid sequence comprising the
steps of a) fragmenting a nucleic acid sequence to produce a
plurality of nucleic acid sequence fragments each having a 5' end
and a 3' end, b) joining one or more first adapters to the 5' and
3' ends, wherein the one or more first adapters are compatible for
promoting intramolecular ligation, thereby producing a first
plurality of modified nucleic acid sequence fragments,
intramolecularly ligating the modified nucleic acid sequence
fragments thereby producing a first plurality of circularized
nucleic acid sequences, wherein the one or more first adapters
forms a first linker between the 5' end and the 3' end of the
circularized nucleic acid sequence fragment, c) fragmenting the
circularized nucleic acid fragments by random shearing or by other
means thereby producing linear fragments comprising the first
linker and a paired tag comprising the 5' end and a 3' end of the
nucleic acid sequence fragment, d) joining a second adapter to each
of the 5' and 3' ends of the fragments to produce a second
plurality of modified fragments having the second adapter at each
5' and 3' end, wherein the second adapter at the 5' and 3' ends are
compatible for promoting intramolecular ligation, intramolecularly
ligating the second plurality of modified nucleic acid sequence
fragments thereby producing a second plurality of circularized
nucleic acid sequences, wherein the second adapter at each 5' and
3' end form a second linker located between the 5' and 3' ends of
the second plurality of circularized nucleic acid sequences, and
wherein the second linker comprises two priming sites, f)
amplifying the second plurality of circularized nucleic acid
sequences using two different primers that are complementary to the
priming sites in the second linker, thereby amplifying the paired
tags, and g) characterizing the paired tags, thereby characterizing
the nucleic acid sequence.
[0022] In an alternative aspect of the invention, provided herein
is a method for characterizing a nucleic acid sequence comprising
the steps of a) fragmenting a nucleic acid sequence to produce a
plurality of nucleic acid sequence fragments having a 5' end and a
3' end, b) joining one or more first adapters to the 5' and 3'
ends, wherein the one or more first adapters are compatible for
promoting intramolecular ligation, thereby producing a first
plurality of modified nucleic acid sequence fragments,
intramolecularly ligating the first plurality of modified nucleic
acid sequence fragments thereby producing a first plurality of
circularized nucleic acid sequences, wherein the one or more first
adapters forms a first linker between the 5' end and the 3' end of
the circularized nucleic acid sequence fragment, c) fragmenting the
first plurality of circularized nucleic acid sequences by random
shearing or by other means, thereby producing first linear
fragments comprising the first linker and a paired tag comprising
the 5' end and a 3' end of the nucleic acid sequence fragment, d)
joining one or more second adapters to the each of the 5' and 3'
ends of the first linear fragments, wherein the one or more second
adapters are compatible for promoting intramolecular ligation,
thereby producing a second plurality of modified nucleic acid
sequence fragments, and intramolecularly ligating the second
plurality of modified nucleic acid sequence fragments to produce a
second plurality of circularized nucleic acid sequences, wherein
the one or more second adapters form a second linker between the 5'
end and the 3' end of the second circularized nucleic acid sequence
fragments, and wherein the second linker comprises two priming
sites separated by a rare-cutting restriction endonuclease cleavage
site, f) cleaving the second plurality of circularized nucleic acid
sequences using a rare-cutting restriction endonuclease that
recognizes the cleavage site in the second linker, thereby
producing a second plurality of linear fragments g) amplifying the
second plurality of linear fragments using two different primers
that are complementary to the priming sites in the second linker,
and h) characterizing the paired tags, thereby characterizing the
nucleic acid sequence.
[0023] In another aspect of the invention, provided herein is a
method for characterizing a nucleic acid sequence comprising the
steps of a) fragmenting a nucleic acid sequence to produce a
plurality of nucleic acid sequence fragments having a 5' end and a
3' end, b) joining one or more first adapters to the 5' and 3'
ends, wherein the one or more first adapters are compatible for
promoting intramolecular ligation, thereby producing a first
plurality of modified nucleic acid sequence fragments,
intramolecularly ligating the first plurality of modified nucleic
acid sequence fragments thereby producing a first plurality of
circularized nucleic acid sequences, wherein the one or more first
adapters forms a first linker between the 5' end and the 3' end of
the circularized nucleic acid sequence fragment, and wherein the
first linker comprises a sequence or chemical moiety that enables
isolation or separation (e.g., by affinity capture), c) fragmenting
the first plurality of circularized nucleic acid sequences by
random shearing or by other means, thereby producing linear
fragments comprising the first linker and a paired tag comprising
the 5' end and the 3' end of the nucleic acid sequence fragment, d)
purifying the linear fragments by affinity capture, e) joining one
or more second adapters to the each of the 5' and 3' ends of the
first linear fragments, wherein the one or more second adapters are
compatible for promoting intramolecular ligation, thereby producing
a second plurality of modified nucleic acid sequence fragments, and
intramolecularly ligating the second plurality of modified nucleic
acid sequence fragments to produce a second plurality of
circularized nucleic acid sequences, wherein the one or more second
adapters form a second linker between the 5' end and the 3' end of
the second circularized nucleic acid sequence fragments, and
wherein the second linker comprises two priming sites, f)
amplifying the nucleic acid sequence fragments using two different
primers that are complementary to the priming sites in the second
linker, and g) characterizing the paired tags, thereby
characterizing the nucleic acid sequence.
[0024] In one embodiment, provided herein is a composition
comprising nucleic acid sequence elements arranged in the following
order: linker 1-5' end tag-linker 2-3' end tag-linker 3 wherein the
5' end tag and the 3' end tag comprise a paired tag derived from a
single contiguous nucleic acid sequence fragment. In a particular
embodiment, linker 2 comprises at least two restriction
endonuclease recognition sites specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, and is
oriented in such a way that one of the sites directs cleavage
within the 5' end tag or at the junction of linker 1 and the 5' end
tag, and the other site directs cleavage within the 3' end tag or
at the junction of linker 3 and the 3' end tag. In another
embodiment, the linker 2 comprises at least two restriction
endonuclease recognition sites specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, and is
oriented in such a way that one of the sites directs cleavage
within the 5' end tag or at the junction of linker 1 and the 5' end
tag, and the other site directs cleavage within the 3' end tag or
at the junction of linker 3 and the 3' end tag, and at least one
recognition site for a rare-cutting restriction endonuclease
located between the two restriction endonuclease recognition sites
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site. In one example, linker 1 and linker 3 are the
same sequence in reverse orientation. In a further embodiment,
oligonucleotides complementary to sequences present in linker 1 and
linker 3 are used for amplification. In another example, linker 1
and linker 3 are derived by cleavage of a circular nucleic acid
molecule with a rare-cutting restriction endonuclease comprising a
recognition site between linker 1 and linker 3.
[0025] In a further embodiment of the invention, provided herein is
a composition comprising a circular nucleic acid molecule, wherein
sequence elements are arranged in the following circular order:
##STR00001##
wherein the 5' end tag and the 3' end tag comprise a paired tag
derived from a single contiguous nucleic acid sequence fragment. In
one embodiment, linker 1 comprises at least two restriction
endonuclease recognition sites specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, and are
oriented in such a way that one of the sites directs cleavage
within the 5' end tag or at the junction of the 5' end tag and
linker 2, and the other site directs cleavage within the 3' end tag
or at the junction of the 3' end tag and linker 2. I another
embodiment, linker 2 comprises at least two restriction
endonuclease recognition sites specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, and are
oriented in such a way that one of the sites directs cleavage
within the 5' end tag or at the junction of the 5' end tag and
linker 2 and, and the other site directs cleavage within the 3' end
tag or at the junction of the 3' end tag and linker 2, and at least
one recognition site for a rare-cutting restriction endonuclease
located between the two restriction endonuclease recognition sites
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site. In one embodiment, linker 2 is palindromic. In
another embodiment, linker 2 comprises a recognition site for a
rare-cutting restriction endonuclease. In a further embodiment,
oligonucleotide primers complementary to sequences in either linker
1 or linker 2, or in both linker 1 and linker 2 are used for
isothermic amplification, for example, oligonucleotide primers
complementary to sequences in either linker 1 or linker 2, and
oriented in opposite directions such that a linear nucleic acid
fragment is produced that preserves the orientation of the 5' end
tag and the 3' end tag with respect to each other are used.
[0026] In a further aspect of the invention, provided herein is a
method for characterizing a nucleic acid sequence comprising the
steps of a) fragmenting a nucleic acid sequence to produce a
plurality of nucleic acid sequence fragments having a 5' end and a
3' end, b) joining one or more first adapters to the 5' and 3'
ends, wherein the one or more first adapters are compatible for
promoting intramolecular ligation, thereby producing a first
plurality of modified nucleic acid sequence fragments,
intramolecularly ligating the first plurality of modified nucleic
acid sequence fragments thereby producing a first plurality of
circularized nucleic acid sequences, wherein the one or more first
adapters forms a first linker between the 5' end and the 3' end of
the circularized nucleic acid sequence fragment, and wherein the
first linker comprises a sequence or chemical moiety that enables
affinity capture, c) fragmenting the first plurality of
circularized nucleic acid sequences by random shearing or by other
means, thereby producing first linear fragments comprising the
first linker and a paired tag comprising the 5' end and a 3' end of
the nucleic acid sequence fragment, d) purifying the linear
fragments by affinity capture, e) joining one or more second
adapters to the each of the 5' and 3' ends of the first linear
fragments, wherein the one or more second adapters are compatible
for promoting intramolecular ligation, thereby producing a second
plurality of modified nucleic acid sequence fragments, and
intramolecularly ligating the second plurality of modified nucleic
acid sequence fragments to produce a second plurality of
circularized nucleic acid sequences, wherein the one or more second
adapters form a second linker between the 5' end and the 3' end of
the second circularized nucleic acid sequence fragments, and
wherein the second linker comprises two priming sites separated by
a rare-cutting restriction endonuclease cleavage site, f) cleaving
the second plurality of circularized nucleic acid sequences using a
rare-cutting restriction endonuclease that recognizes the cleavage
site in the second adapter, thereby producing a second plurality of
linear fragments, g) amplifying the second plurality of linear
fragments using two different primers that are complementary to the
priming sites in the second linker, h) characterizing the paired
tags, thereby characterizing the nucleic acid sequence.
[0027] In a further aspect of the invention, a method for
identifying nucleic acid sequences that encode at least two
interacting proteins is provided, comprising the steps of a)
combining (i) a first vector comprising (1) a nucleic acid sequence
that encodes a first protein that interacts with a second protein,
and (2) a first restriction endonuclease recognition site specific
for a restriction endonuclease that cleaves the nucleic acid
sequence fragment distally to the restriction endonuclease
recognition site, and (ii) a second vector comprising (1) a nucleic
acid sequence that encodes the second protein; and (2) a second
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
thereby producing a combination, b) optionally maintaining the
combination under conditions in which the first protein and the
second protein are expressed and interact, c) joining the first
vector with the second vector, thereby forming a contiguous nucleic
acid sequence that comprises (i) the nucleic acid sequence that
encodes a first protein that interacts with a second protein, (ii)
the first restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(iii) the nucleic acid sequence that encodes the second protein,
and (iv) the second restriction endonuclease recognition site
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site, d) cleaving the first restriction endonuclease
recognition site and the second restriction endonuclease
recognition site in the contiguous nucleic acid sequence with
restriction endonucleases that cleave the contiguous nucleic acid
sequence distally to the restriction endonuclease recognition
sites, thereby producing a 5' end tag and a 3' end tag of the
contiguous nucleic acid sequence, e) joining the 5' end tag to the
3' end tag, thereby producing a paired tag, and f) sequencing the
paired tag, thereby identifying nucleic acid sequences that encode
at least two interacting proteins.
[0028] In an additional embodiment of the invention, provided is a
method for identifying nucleic acid sequences that encode at least
two interacting proteins comprising the steps of a) combining (i) a
first vector comprising (1) a nucleic acid sequence that encodes a
first protein that interacts with a second protein, and (2) a first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and (ii) a second vector comprising (1) a nucleic acid sequence
that encodes the second protein, and (2) a second restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, thereby
producing a combination, b) optionally maintaining the combination
under conditions in which the first protein and the second protein
are expressed and interact, c) joining the first vector with the
second vector, thereby forming a contiguous nucleic acid sequence
that comprises (i) the nucleic acid sequence that encodes a first
protein that interacts with a second protein, (ii) the first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(iii) the nucleic acid sequence that encodes the second protein,
and (iv) the second restriction endonuclease recognition site
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site, d) sequencing the contiguous nucleic acid
sequence, thereby identifying nucleic acid sequences that encode at
least two interacting proteins.
[0029] In another embodiment of the invention, provided is a method
for identifying nucleic acid sequences that encode at least two
interacting proteins comprising the steps of a) combining, (i) a
first vector comprising (1) a nucleic acid sequence that encodes a
first protein that interacts with a second protein, and (2) a first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and (ii) second vector comprising (1) a nucleic acid sequence that
encodes the second protein; and (2) a second restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, thereby
producing a combination, b) optionally maintaining the combination
under conditions in which the first protein and the second protein
are expressed and interact, c) joining the first vector with the
second vector, thereby forming a contiguous nucleic acid sequence
that comprises (i) the nucleic acid sequence that encodes a first
protein that interacts with a second protein, (ii) the first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(iii) the nucleic acid sequence that encodes the second protein,
and (iv) the second restriction endonuclease recognition site
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site, d) cleaving the first restriction endonuclease
recognition site and the second restriction endonuclease
recognition site in the contiguous nucleic acid sequence with
restriction endonucleases that cleave the contiguous nucleic acid
sequence distally to the restriction endonuclease recognition
sites, thereby producing a paired tag comprising a 5' end tag and a
3' end tag of the contiguous nucleic acid sequence, e) sequencing
the paired tag, thereby identifying nucleic acid sequences that
encode at least two interacting proteins.
[0030] In a further embodiment of the invention, provided is a
method for identifying a plurality of nucleic acid sequences that
encode at least two interacting proteins comprising the steps of a)
combining (i) a plurality of first vectors each comprising (1) a
nucleic acid sequence that encodes a first protein that interact
with a second protein; and (2) a first restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, and (ii) a plurality of
second vectors each comprising (1) a nucleic acid sequence that
encodes the second protein; and (1) a second restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, thereby
producing a combination comprising a plurality of first vectors and
a plurality of second vectors, b) optionally maintaining the
combination under conditions in which the plurality of first
vectors encoding a first protein and the plurality of second
vectors encoding a second protein are expressed and the first
protein and second protein interact, c) joining the first vectors
with the second vectors, wherein the first vectors and second
vectors encode interacting proteins, thereby forming a plurality of
contiguous nucleic acid sequences that each comprise (i) the
nucleic acid sequence that encodes a first protein that interacts
with a second protein, (ii) the first restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, (iii) the nucleic acid
sequence that encodes the second protein, and (iv) the second
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
d) cleaving the first restriction endonuclease recognition site and
the second restriction endonuclease recognition site in each
contiguous nucleic acid sequence with restriction endonucleases
that cleave the contiguous nucleic acid sequence distally to the
restriction endonuclease recognition sites, thereby producing a
plurality of paired tags comprising a 5' end tag and a 3' end tag
of the contiguous nucleic acid sequence, e) joining the paired
tags; and f) sequencing the joined paired tags, thereby identifying
a plurality of nucleic acid sequences that encode at least two
interacting proteins.
[0031] In a further embodiment of the invention, a method for
identifying nucleic acid sequences that encode at least two
interacting proteins is provided, comprising the steps of a)
combining (i) a first vector comprising (1) a nucleic acid sequence
that encodes a first protein that interacts with a second protein,
(2) a first restriction endonuclease recognition site specific for
a restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site;
and (3) a second restriction endonuclease recognition site, and
(ii) a second vector comprising (1) a nucleic acid sequence that
encodes the second protein, (2) a third restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, and (3) a fourth
restriction endonuclease recognition site, thereby producing a
combination, b) optionally maintaining the combination under
conditions in which the first protein and the second protein are
expressed and interact, c) joining the first vector with the second
vector, thereby forming a contiguous nucleic acid sequence that
comprises (i) the nucleic acid sequence that encodes a first
protein that interacts with a second protein, (ii) the first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(iii) the second restriction endonuclease recognition site, (iv)
the nucleic acid sequence that encodes the second protein, (v) the
third restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and (vi) the fourth restriction endonuclease recognition site, d)
cleaving the second restriction endonuclease recognition site and
the fourth restriction endonuclease recognition site in the
contiguous nucleic acid sequence with restriction endonucleases
thereby producing compatible ends in the contiguous nucleic acid
sequence, e) maintaining the contiguous nucleic acid sequence under
conditions in which the compatible ends in the contiguous nucleic
acid sequence intramolecularly ligate, thereby producing a
circularized nucleic acid sequence, and f) sequencing the
circularized nucleic acid sequence, thereby identifying nucleic
acid sequences that encode at least two interacting proteins. In a
particular embodiment, the cleaving of the second restriction
endonuclease recognition site and the cleaving of the fourth
restriction endonuclease recognition site in the contiguous nucleic
acid sequence releases a fragment containing a recombined
site-specific recombinase recognition site between the nucleic acid
sequence that encodes a sequence from the first protein and a
sequence from the second protein that interact with each other.
[0032] In another aspect of the invention, provided is a method for
identifying nucleic acid sequences that encode at least two
interacting proteins comprising the steps of a) combining (i) a
first vector comprising (1) a nucleic acid sequence that encodes a
first protein that interacts with a second protein, (2) a first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and (3) a second restriction endonuclease recognition site, and
(ii) a second vector comprising, (1) a nucleic acid sequence that
encodes the second protein, (2) a third restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, and (3) a fourth
restriction endonuclease recognition site, thereby producing a
combination, b) optionally maintaining the combination under
conditions in which the first protein and the second protein are
expressed and interact, c) joining the first vector with the second
vector, thereby forming a contiguous nucleic acid sequence that
comprises (i) the nucleic acid sequence that encodes a first
protein that interacts with a second protein, (ii) the first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(iii) the second restriction endonuclease recognition site; (iv)
the nucleic acid sequence that encodes the second protein, and (v)
the third restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(vi) a fourth restriction endonuclease recognition site, d)
cleaving the second restriction endonuclease recognition site and
the fourth restriction endonuclease recognition site in the
contiguous nucleic acid sequence with restriction endonucleases,
thereby producing compatible ends in the contiguous nucleic acid
sequence, e) maintaining the contiguous nucleic acid sequence under
conditions in which the compatible ends in the contiguous nucleic
acid sequence intramolecularly ligate, thereby producing a
circularized nucleic acid sequence, f) cleaving the first
restriction endonuclease recognition site and the third restriction
endonuclease recognition site in the circularized nucleic acid
sequence with restriction endonucleases that cleave the
circularized nucleic acid sequence distally to the restriction
endonuclease recognition sites, thereby producing a paired tag
comprising a 5' end tag and a 3' end tag of the circularized
nucleic acid sequence, and g) sequencing the paired tag, thereby
identifying nucleic acid sequences that encode at least two
interacting proteins.
[0033] In a further embodiment of the invention, provided is a
method for identifying a plurality of nucleic acid sequences that
encode at least two interacting proteins comprising the steps of a)
combining (i) a plurality of first vectors each comprising (1) a
nucleic acid sequence that encodes a first protein that interacts
with a second protein, (2) a first restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, and (3) a second
restriction endonuclease recognition site, and (ii) a plurality of
second vector each comprising (1) a nucleic acid sequence that
encodes the second protein, (2) a third restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, and (3) a fourth
restriction endonuclease recognition, thereby producing a
combination comprising a plurality of first vectors and a plurality
of second vectors, b) maintaining the combination under conditions
in which the plurality of first vectors encoding a first protein
and the plurality of second vectors encoding a second protein are
expressed and the first protein and second protein interact c)
selecting the combinations of a first vector that encodes a first
protein and a second vector that encodes a second protein, wherein
the first protein interacts with the second protein, d) joining the
first vectors with the second vectors, wherein the first vectors
and second vectors encode interacting proteins, thereby forming a
plurality of contiguous nucleic acid sequences that each comprise
(i) the nucleic acid sequence that encodes a first protein that
interacts with a second protein, (ii) the first restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, (iii)
the second restriction endonuclease recognition site adjacent to
the first restriction endonuclease site, (iv) the nucleic acid
sequence that encodes the second protein, (v) the third restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, and (vi)
the fourth restriction endonuclease recognition site, d) cleaving
the second restriction endonuclease recognition site and the fourth
restriction endonuclease recognition site in the contiguous nucleic
acid sequence with restriction endonucleases that leave compatible
ends, e) maintaining the contiguous nucleic acid sequence under
conditions in which the compatible ends intramolecularly ligate,
thereby producing a circularized nucleic acid sequence that encodes
a sequence from the first protein and a sequence from the second
protein that interact with each other, f) cleaving the first
restriction endonuclease recognition site and the third restriction
endonuclease recognition site in each circularized nucleic acid
sequence with restriction endonucleases that cleave each
circularized nucleic acid sequence distally to the restriction
endonuclease recognition sites, thereby producing a plurality of
paired tags comprising a 5' end tag and a 3' end tag of the
circularized nucleic acid sequence, g) joining the paired tags, and
h) sequencing the joined paired tags, thereby identifying a
plurality of nucleic acid sequences that encode at least two
interacting proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The patent or application file contains at least one drawing
executed in color. 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.
[0035] FIG. 1 is a graph depicting the effect of clone size and
distribution.
[0036] FIG. 2 is a graph depicting the number of segmental
duplications identified in the rat genome versus the percent
identity of the cognate pairs.
[0037] FIG. 3 is a schematic of sequence assembly (derived from
Holt et al., Science 298:129-149 (2002)).
[0038] FIG. 4 is a schematic of paired tags in a concatemer.
[0039] FIG. 5 is an outline of the paired tag, two-hybrid
method.
[0040] FIG. 6A-6D are schematics outlining an example of a paired
tag protocol. FIG. 6A is a schematic outlining the vector, and the
steps of excising a stuffer fragment and preparation of inserts.
FIG. 6B is a schematic outlining the steps of cloning of the
inserts, optional exonuclease treatment, restriction endonuclease
digestion with MmeI, and ligation to a degenerate linker. FIG. 6C
is a schematic outlining the steps of PCR amplification, excising
of paired tags, concatenation of paired tags and cloning of paired
tags into a suitable vector, such as a sequencing vector. FIG. 6D
is a list of particular nucleic acid sequences of the
invention.
[0041] FIG. 7 is a schematic of one embodiment of paired tag
formation.
[0042] FIG. 8 is a schematic of one embodiment of paired tag
formation.
[0043] FIG. 9 is a schematic of one embodiment for generating
paired genome sequence tags.
[0044] FIG. 10 is a schematic of one embodiment for generating
paired genome sequence tags.
[0045] FIG. 11 is a schematic of an affinity tag with two unique
primer sequences.
[0046] FIG. 12 is a schematic of one embodiment for generating
paired genome sequence tags.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention provides a method for producing a
paired tag from a nucleic acid sequence (also referred to herein as
a nucleotide sequence). In one embodiment, the nucleic acid
sequence (e.g. DNA, RNA) comprises at least two restriction
endonuclease recognition sites specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site. In
another embodiment, the nucleic acid sequence comprises at least
one restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves both upstream and downstream
from the recognition site. The restriction endonuclease recognition
sites present in the nucleic acid sequence can be the same or
different. In addition, the restriction endonuclease recognition
sites present in the nucleic acid sequence can be cleaved by the
same or different restriction endonucleases.
[0048] The present invention also provides a method for producing a
paired tag from a nucleic acid sequence, wherein the nucleic acid
sequence comprises at least one recombination site, wherein a
recombination event at this recombination site in the nucleic acid
sequence produces a paired tag from the nucleic acid sequence.
[0049] As used herein, a "paired tag" is produced from a nucleic
acid sequence and comprises a 5' end of a nucleic acid sequence
(e.g., a contiguous nucleic acid sequence) paired or joined with
the 3' end of the same nucleic acid sequence, wherein a portion of
the internal sequence of the contiguous nucleic acid sequence is
removed. In one embodiment, a paired tag can be represented as:
[0050] 5' - - - - - - - - .box-solid. - - - - - - 3' wherein "5 - -
- - - " represents a 5' end tag, " - - - - - - 3" represents a 3'
end tag, and ".box-solid." represents a linker (or adapter) that
links the 5' end tag to the 3' end tag. Alternatively, a paired tag
can be represented as: [0051] .box-solid. - - - - - - - - 5' 3' - -
- - - - .box-solid. wherein " - - - - - - 5" represents a 5' end
tag, " - - - - - - 3" represents a 3' end tag, and ".box-solid."
represents an adapter (or linker). As will be recognized by the
person of skill in the art, the orientation of the 5' end tag and
3' end tag can be reversed. As discussed below, the linker or
adapter can comprise: at least one endonuclease recognition site,
(e.g., for a restriction endonuclease enzyme such as a rare cutting
enzyme, an enzyme that cleaves distally to its recognition
sequence); an overhang that is compatible with joining to a
complementary overhang from a restriction endonuclease digestion
product; an attachment capture moiety, such as biotin; primer sites
(for use in, e.g., amplification, RNA polymerase reactions); Kozak
sequence, promoter sequence, (e.g. T7 or SP6); and/or an
identifying moiety, such as a fluorescent label.
[0052] A paired tag is distinguished from a ditag since a ditag is
a randomized pairing of two tags usually from more than one nucleic
acid sequence, for example, a 5' end of sequence A and the 3' end
of sequence B. In contrast, a paired tag as described herein, is
not a randomized pairing of two tags, but the pairing of two tags
that are produced from a single contiguous nucleic acid
sequence.
[0053] Paired tags facilitate the assembly (such as whole genome
assembly, or genome mapping) of a nucleic acid sequence, such as a
genomic DNA sequence, even if either tag (for example, the 5' tag)
is generated from a non-informative sequence (for example, a repeat
sequence) and the other tag in the pair (for example, the 3' tag)
is generated from an informative sequence based on the paired tag's
"signature". A paired tag's signature is derived from the size of
the original nucleic acid sequence from which the paired tag
represents the 5' end and Tend of the paired tag's nucleic acid
sequence. The random association of tags to form ditags does not
retain any signature as the two tags in the ditag generally do not
represent the 5' end and 3' end of any contiguous nucleic acid
sequence. In addition, a paired tag can identify the presence of an
inverted nucleic acid sequence in, for example, a genomic DNA
sample, because of the paired tag's signature. Randomly associated
tags that form ditags cannot detect the presence of an inverted
nucleic acid sequence because the ditag does not retain a
signature. For example, a database version of one genome places
tags in the order of: X-Y-Z-A in a contiguous sequence. Paired tags
from this sequence generates the following two paired tags: X-Y and
Z-A. In a comparison genome, for example, from a cancer cell, the
paired tags from the same contiguous sequence generates the
following two paired tags: X-Z and Y-A. These two paired tags
indicates the order of the tags in the contiguous sequence of the
cancer cell genome as: X-Z-Y-A. Thus, it is determined that the
fragment Y-Z is inverted. Ditags will not have sufficient
information to determine if a contiguous sequence has an inversion
due to the random association of any two tags together.
[0054] A contiguous nucleic acid sequence is a nucleic acid
sequence having a sequential sequence of nucleic acids, for
example, deoxyribonucleic acids, ribonucleic acids, derivatives or
analogs thereof, and combinations thereof, as will be understood by
one of skill in the art.
[0055] In one embodiment, a "contiguous nucleic acid sequence" is a
nucleic acid fragment. Such a nucleic acid fragment can be
obtained, for example, from sheared DNA, such as genomic DNA, from
enzyme-digested DNA, for example, restriction enzyme-digested or
non-specific endonuclease-digested DNA, or a combination thereof,
or from a modified genomic DNA fragment derived by treatment of
genomic DNA, for example, with one or more nucleic acid modifying
enzymes. In a further embodiment, the contiguous nucleic acid
sequence comprises at least two nucleic acid sequences that encode
at least two interacting proteins, wherein the at least two nucleic
acid sequences have been joined to form a contiguous nucleic acid
sequence.
[0056] A "5' end tag" (also referred to as a "5' tag") and a "3'
end tag" (also referred to as a "3' tag") of a contiguous nucleic
acid sequence can be short nucleic acid sequences, for example, the
5' end tag or 3' end tag can be from about 6 to about 80
nucleotides, from about 6 to about 600 nucleotides, from about 6 to
about 1200 nucleotides or longer, from about 10 to about 80
nucleotides, from about 10 to about 1200 nucleotides, from about 10
to about 1500 nucleotides or longer in length that are from the 5'
end and 3' end, respectively, of the contiguous nucleic acid
sequence. In one embodiment, the 5' end tag and/or the 3' end tag
are about 14 nucleotides, about 20 nucleotides or about 27
nucleotides. The 5' end tag and a 3' end tag are generally
sufficient in length to identify the contiguous nucleic acid
sequence from which they were produced. In one embodiment, the 5'
end tag and/or the 3' end tag are produced after cleavage of the
contiguous nucleic acid sequence with a restriction endonuclease
having a recognition site located at the 5' and/or 3' end of the
contiguous nucleic acid sequence. In a particular embodiment, the
restriction endonuclease cleaves the contiguous nucleic acid
sequence distally to (outside of) its restriction endonuclease
recognition site. The 5' end tag and/or 3' end tag can also be
produced after cleavage by other fragmentation means, such as
random shearing, treatment with non-specific endonucleases or other
fragmentation methods as will be understood by one skilled in the
art. In some embodiments, cleavage can occur in a linker or adapter
sequence, in other embodiments, cleavage can occur outside a linker
or adapter sequence, such as in a genomic DNA fragment.
[0057] Traditional classification of restriction endonucleases
(restriction enzymes) generally divide restriction endonucleases
according to their subunit composition, cleavage position,
sequence-specificity and co-factor requirements. For example,
restriction endonucleases can be classified as type I, type II,
type IIs, typeIIB, type III or type IV restriction endonucleases.
Restriction endonucleases are also characterized according to their
cleavage frequency for a particular sequence, such as a genomic
sequence. For example, a restriction endonuclease can be
characterized as a "rare-cutting restriction endonuclease" or a
"rare-cutting restriction enzyme" based on the rarity or
infrequency of its recognition site in a nucleic acid sequence,
e.g., a genomic sequence. Examples of rare-cutting restriction
endonucleases are well known to those of skill in the art, and
include, for example, AsiSI, Notl. Further examples of enzymes that
are rare-cutting include homing endonucleases. Homing endonucleases
are rare-cutting enzymes encoded by introns and inteins (Belfort M.
and Roberts R. J., Nucleic Acids Res. 25:3379-88 (1997)). Examples
of homing endonucleases include I-CeuI, I-SceI, PI-PspI and
PI-SceI.
[0058] In one embodiment, the restriction endonuclease for use in
the methods of the invention includes one or more restriction
endonucleases that cleave a nucleic acid distally to its
restriction endonuclease recognition site. As used herein,
"restriction endonucleases that cleave a nucleic acid distally to
its restriction endonuclease recognition site" refers to a
restriction endonuclease that recognizes a particular site within a
nucleic acid sequence and cleaves this nucleic acid sequence
outside the region of the recognition site (cleavage occurs at a
site which is distal or outside the site recognized by the
restriction endonuclease). In one embodiment, a restriction
endonuclease that cleaves a nucleic acid distally to its
restriction endonuclease recognition site cleaves on one side of
the restriction endonuclease recognition site (for example,
upstream or downstream of the recognition site). In another
embodiment, restriction endonuclease that cleaves a nucleic acid
distally to its restriction endonuclease recognition site cleaves
on both sides of the restriction endonuclease recognition site (for
example, upstream and downstream of the recognition site). In
another embodiment, the restriction endonuclease cleaves once
between two restriction endonuclease recognition sites. Examples of
such restriction endonucleases are well known in the art, and
include restriction endonucleases classified as Type I (e.g., CfrA
I, Eco377 I, Hind I, KpnA, IngoAV, StySK I), Type lus (e.g., MmeI,
Fok I, Bsg I, Bpm I, Mbo II, and Alw I), Type IIB (e.g. AlfI, AloI,
BaeI, BcgI, BplI, BsaXI, BsIFI, Bsp24I, CjeI, CjePI, CspCI, FalI,
HaeIV, Hin4I, PpiI, and PsrI), Type III (e.g., EcoP I, EcoP15I,
Hine I, Hinf III) and Type IV (e.g., Eco57 I, Bcg I [recently
re-classified as Type IIB], BseMII) restriction endonucleases.
[0059] The present invention provides in one embodiment, a method
for producing a paired tag from a nucleic acid sequence. In one
embodiment, the nucleic acid sequence comprises at least one
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
distally to the restriction endonuclease recognition site. In
another embodiment, the nucleic acid sequence comprises two
restriction endonuclease recognition sites specific for a
restriction endonuclease that cleaves the nucleic acid sequence
distally to the restriction endonuclease recognition site. In a
particular embodiment, the restriction endonuclease recognition
site specific for a restriction endonuclease that cleaves the
nucleic acid sequence distally to the recognition site is adjacent
to the nucleic acid sequence of interest. These restriction
endonuclease recognition sites are cleaved using the restriction
endonuclease that is specific for the restriction endonuclease
recognition sites. As a result of this cleavage, a 5' end tag and a
3' end tag is produced from the nucleic acid sequence. The 5' end
tag is joined with the 3' end tag of the nucleic acid sequence to
produce a paired tag from the nucleic acid sequence. The two
restriction endonuclease recognition sites specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
can be the same or different.
[0060] In another embodiment, the invention provides a method for
generating paired tags from the 5' and 3' ends of a set of genomic
DNA fragments, wherein the tags are generated by fragmentation
means other than by cleavage with restriction endonucleases that
cleave distally to their recognition sites, for example, by random
shearing or by other non-specific cleavage methods. This is
achieved, for example, by performing two rounds of intramolecular
ligation in which a linker is incorporated between the 5' and 3'
ends of the genomic DNA fragments, with a fragmentation step
performed between the first and second intramolecular ligation
steps. This method produces 5' and 3' end tags of the genomic DNA
fragment that can be long in length, thereby facilitating long
sequence read lengths.
[0061] As used herein, "joining" refers to methods such as
ligation, annealing or recombination used to adhere one component
to another. Recombination can be achieved by any methods known in
the art. For example, recombination can be a Cre/Lox recombination.
In one embodiment, the recombination is a between a lox71 site and
a lox66 site. In another embodiment, joining of a nucleic acid
sequence to another nucleic acid sequence is performed by
intermolecular ligation. For example, two nucleic acid sequences
can be joined to form one contiguous nucleic acid sequence. A
typical example of intermolecular ligation is cloning a nucleic
acid sequence into a vector. A vector is generally understood in
the art, and is understood to contain an origin of replication
("ori"). In another embodiment, intermolecular ligation can be
achieved using a non-vector vehicle (also referred to herein as
"without cloning"). Without cloning means that a joining of nucleic
acid sequence is not an accomplished using vector is not used to
clone into. For example, a DNA sequence or an oligonucleotide, such
as a linker or an adapter can be intermolecularly ligated to the
nucleic acid sequence of interest to facilitate joining of nucleic
acid sequences.
[0062] In one embodiment, intermolecular ligation is between two
nucleic acid sequences having sticky ends ("sticky ends ligation").
In an alternative embodiment, intermolecular ligation is between
two nucleic acid sequences having blunt ends ("blunt end
ligation"). In another example, intermolecular ligation is between
a number of paired tags, for example, to form a concatemer. In a
further embodiment, intermolecular ligation is between at least
three components, such as two paired tags and a linker, or two tags
and a linker. The linker functions to link two components together,
for example, the linking of one or more paired tags together. As
used herein, a "linker" is a nucleic acid fragment, such as DNA, as
is understood in the art. The linker can be double-stranded or
single-stranded. In general, the linker acts to link two components
together, such as linking the 5' end of a nucleic acid sequence to
the 3' end of a nucleic acid sequence. Furthermore, a linker can
comprise: at least one endonuclease recognition site, (e.g., for a
restriction endonuclease enzyme such as a rare cutting enzyme, an
enzyme that cleaves distally to its recognition sequence); an
overhang that is compatible with joining to a complementary
overhang from a restriction endonuclease digestion product; an
attachment moiety, such as biotin; primer sites, for use in, (e.g.,
amplification, RNA polymerase reactions; Kozak sequence, promoter
sequence), (e.g. T7 or SP6; and/or an identifying moiety, such as a
fluorescent label). A double-stranded linker can be produced by
annealing two oligonucleotides, for example, and typically ranges
between about 6 to about 1000 base pairs in length, but more
typically is between about 30 and about 100 base pairs in length.
In one embodiment, the linker provides a link between two
components having non-complementary ends. In another embodiment,
the linker is a degenerate linker. As used herein, a "degenerate
linker" is a linker having in at least one position, a plurality of
possible nucleotides, as will be understood by those of skill in
the art. In a particular embodiment, the linker has the sequence
NNATGATGNN (SEQ ID NO: 1), where N=any nucleotide. In another
embodiment, the linker comprises a restriction endonuclease
recognition site. In a particular embodiment, the linker comprises
a rare cutting restriction endonuclease recognition site. For
example, the linker having SEQ ID NO: 1 allows the 3' overhangs of
paired tags to come together when the paired tag produces a
sequence comprising "linker-5' TAG-3'TAG-linker". In another
example, the linker and the paired tag produces a sequence
comprising "5' TAG-linker-3' TAG". Other examples are those that
generate the arrangement "linker-5' TAG-linker-3' TAG-linker". As
will be recognized by one of skill in the art, any of the linker
moieties can be replaced by a vector.
[0063] In another embodiment, joining of two nucleic acid sequences
is performed by intramolecular ligation. Intramolecular ligation
includes, for example, the ligation of the 5' end of a nucleic acid
sequence fragment to the 3' end of the same nucleic acid sequence
fragment. In one embodiment, intramolecular ligation produces a
circular nucleic acid sequence. For example, in one embodiment,
joining of a 5' end tag to the 3' end tag of a contiguous sequence
is performed by intramolecular ligation. In a further embodiment,
joining of a 5' end tag to the 3' end tag of a contiguous sequence
is performed by intramolecular ligation, wherein the 5' end tag
nucleic acid sequence and the 3' end tag nucleic acid sequence
have, for example, sticky ends ("sticky ends ligation"). In another
embodiment, joining of a 5' t end tag to the 3' end tag comprised
in a contiguous sequence is performed by intramolecular ligation,
wherein the 5' end tag nucleic acid sequence and the 3' end tag
nucleic acid sequence have, for example, blunt ends ("blunt end
ligation").
[0064] In one embodiment, intramolecular ligation is performed by
ligation of a nucleic acid sequence fragment in the presence of a
linker such that one end of the linker is first ligated to one end
of the nucleic acid sequence fragment by intermolecular ligation,
then the other end of the linker is ligated to the other end of the
nucleic acid sequence fragment by intramolecular ligation. This
results in a circular molecule in which the linker is inserted
between the two ends of the nucleic acid sequence fragment.
Typically, the ends of the linker and the nucleic acid sequence
fragment are complementary to each other, but not to themselves to
avoid self-ligation of the inserts and linkers.
[0065] In the method of the invention, conditions that favor
intramolecular ligation over intermolecular ligation are used when
attempting to circularize DNA molecules in order to avoid chimeric
ligation (i.e., the ligation of 5' and 3' ends from two different
DNA molecules which results in the production of ditags).
Conditions that favor intramolecular ligation over intermolecular
ligation are known in the art. In one embodiment, intramolecular
ligation is favored over intermolecular ligation by performing
ligation at low DNA concentrations, and also in the presence of
crowding reagents like polyethylene glycol (PEG) at low salt
concentrations (Pfeiffer and Zimmerman, Nucl. Acids Res. (1983)
11(22): 7853-7871). Ligation at low DNA concentration can be
expensive and impractical since large reaction volumes are used at
high ligase concentration but dilute DNA concentration. The use of
PEG increases the reaction rate, therefore, long reaction times can
still result in intermolecular products. In addition, volume
exclusion does not eliminate diffusion of DNA molecules such that
given enough time, DNA molecules will diffuse within reach of one
another and ligate to one another. To overcome these problems,
water-in-oil emulsions can be used. Water-in-oil emulsions have
been described by Dressman et al. for single molecule PCR (Dressman
et al., PNAS (2003), 100(15): 8817-8822). By creating a
water-in-oil emulsion, billions of micro-reaction bubbles, for
example, 10 micrometers in diameter can be generated. Using a
dilute enough DNA concentration can ensure that only one or less
than one molecule of DNA exists in any given micro-reactor. Under
such conditions, long reaction times and additives (such as PEG,
MgCl.sub.2, DMSO) which increase the reaction rate of ligase
(Alexander et al., Nuc. Acids Res. (2003) 31(12): 3208-3216) can be
utilized without any risk of intermolecular ligation.
[0066] In one embodiment, emulsion ligation of a nucleic acid
sequence fragment is performed in the presence of a linker or
adapter, such that the linker or adapter is incorporated into the
resulting circular molecules between the 5' and 3' ends of the
nucleic acid sequence fragment. In another embodiment, emulsion
ligation of a nucleic acid sequence fragment is performed in the
presence of a substrate, for example, a magnetic bead coupled to a
linker or adaptor, such that the resulting circularized DNA becomes
immobilized (covalently or non-covalently) onto the substrate. In
each of these embodiments, the concentration of nucleic acid
sequence fragments, linkers, and beads can be modulated
independently to maximize intramolecular ligation or, if relevant,
immobilization of an individual nucleic acid sequence fragment onto
a single bead.
[0067] In another embodiment, emulsion ligation of a nucleic acid
sequence fragment is performed in the presence of a substrate, for
example, a magnetic bead coupled to a linker or adaptor, such that
the resulting circularized DNA becomes immobilized onto the
substrate. In each of these embodiments, the concentration of
nucleic acid sequence fragments, linkers, and beads can be
modulated independently to maximize intramolecular ligation or, if
relevant, immobilization of an individual nucleic acid sequence
fragment onto a single bead. As used herein, "immobilized" means
attached to a surface by covalent or non-covalent attachment means,
as understood in the art. As used herein, a "substrate" is a solid
or polymeric support such as a silicon or glass surface, a magnetic
bead, a semisolid bead, a gel, or a polymeric coating applied to
the another material, as is understood in the art.
[0068] Circularized nucleic acid molecules produced by
intramolecular ligation with an intervening linker may be purified
by a variety of methods known in the art, such as by gel
electrophoresis, by treatment with an exonuclease (e.g., Bal31 or
"plasmid-safe" DNase) to remove contaminating linear molecules.
Nucleic acid molecules incorporating a linker between the 5' and 3'
ends of the starting nucleic acid sequence fragment can be purified
by affinity capture using a number of methods known in the art,
such as the use of a DNA binding protein that binds to the linker
specifically, by triplex hybridization using a nucleic acid
sequence complementary to the linker, or by means of a biotin
moiety covalently attached to the linker. Affinity capture methods
typically involve the use of capture reagents attached to a
substrate such as a solid surface, magnetic bead, or semisolid bead
or resin.
[0069] A nucleic acid sequence, fragment, or paired tag having
compatible ends are understood by one of skill in the art to mean
that the ends are compatible with joining to another nucleic acid
sequence, fragment or paired tag as provided herein. Compatible
ends can be "sticky ends" having a 5' and/or 3' overhang, or
alternatively, compatible ends can be "blunt ends" having no 5'
and/or 3' overhang. Sticky ends permit sequence-dependent ligation,
whereas blunt ends permit sequence-independent ligation. Compatible
ends are produced by any known methods that are standard in the
art. For example, compatible ends of a nucleic acid sequence are
produced by restriction endonuclease digestion of the 5' and/or 3'
end. In another embodiment, compatible ends of a nucleic acid
sequence are produced by introducing (for example, by annealing,
ligating, or recombination) an adapter to the 5' end and/or 3' end
of the nucleic acid sequence, wherein the adapter comprises a
compatible end, or alternatively, the adapter comprises a
recognition site for a restriction endonuclease that produces a
compatible end on cleavage. As used herein, an "adapter" is, for
example, nucleic acid fragment, such as DNA (double stranded,
single stranded), as will be understood by one of skill in the art.
An adapter can be used to modify the 5' end and/or the 3' end of a
nucleic acid sequence. In one embodiment, the adapter can comprise:
at least one endonuclease recognition site, e.g., for a restriction
endonuclease enzyme such as a rare cutting enzyme, an enzyme that
cleaves distally to its recognition sequence; an overhang that is
compatible with joining to a complementary overhang from a
restriction endonuclease digestion product; an attachment moiety,
such as biotin; and/or an identifying moiety, such as a fluorescent
label. Such adapters can be produced, for example, by annealing a
pair of oligonucleotides having the appropriate nucleic acid
sequences. In another embodiment, compatible ends of a nucleic acid
sequence are produced by introducing (for example, by annealing,
ligating, or recombination) the nucleic acid sequence into a
vector. In one embodiment, the nucleic acid sequence is cloned into
a vector, wherein the vector comprises at least one recognition
site for a restriction endonuclease in proximity, for example,
adjacent to where the nucleic acid sequence is cloned into, and
wherein the recognition site produces a compatible end on
cleavage.
[0070] In some aspects of the invention as disclosed herein, the
use of an adapter or a linker can be used interchangeably, as will
be understood by the person of skill in the art.
[0071] As used herein, a "vector" is, for example, a plasmid, phage
or phagemid, as will be understood by one of skill in the art. A
vector as is understood by one of skill in the art to contain an
origin of replication ("ori") for DNA replication in a host
organism (for example, E. coli). As used herein, "cloning" is the
propagation of a nucleic acid sequence in a vector in a viable host
cell, such as E. coli, as will be understood by one of skill in the
art.
[0072] A variety of in vitro amplification methods are known in the
art, including the polymerase chain reaction, "PCR", and
amplification methods that can be performed under isothermal
conditions, collectively referred to as "isothermal amplification",
which include, for example, rolling circle amplification (RCA),
strand displacement amplification (SDA), multiple displacement
amplification (MDA), and methods involving the use of a DNA
dependent RNA polymerase (such as T7 RNA polymerase) and RNaseH.
Compositions comprising paired tags and introduced nucleic acid
sequences, such as linkers, adapters, or vectors can readily be
amplified in vitro by using such methods. Furthermore, it is well
understood in the art that nucleic acid sequences may be amplified
on substrates if one of the primers used in the amplification is
immobilized on the substrate.
[0073] In another embodiment of the invention, a method is provided
for producing a paired tag from a nucleic acid sequence, wherein
the nucleic acid sequence comprises two restriction endonuclease
recognition sites specific for a restriction endonuclease that
cleaves the nucleic acid sequence distally to the restriction
endonuclease recognition site, and at least two restriction
endonuclease recognition sites specific for a rare cutting
restriction endonuclease. In one embodiment, endonuclease
recognition sites specific for a rare cutting restriction
endonuclease occur minimally in a sequence (e.g. once, twice, etc.)
are specifically recognized by a single rare (unique) cutting
restriction endonuclease. In another embodiment, endonuclease
recognition sites specific for a rare cutting restriction
endonuclease are specifically recognized by multiple rare cutting
restriction endonuclease. Thus, in one embodiment of the invention,
a method is provided for producing a paired tag from a nucleic acid
sequence comprising cleaving the nucleic acid sequence using the
restriction endonuclease that cleaves the nucleic acid sequence
distally to the restriction endonuclease recognition sites, thereby
producing a 5' end tag and a 3' end tag from the nucleic acid
sequence. The 5' end tag is joined with the 3' end tag of the
nucleic acid sequence to produce a paired tag from the nucleic acid
sequence. The paired tag can be further cleaved with a rare cutting
restriction endonuclease, which produces a paired tag having
compatible ends.
[0074] In an alternative embodiment, the present invention is drawn
to a method for producing a paired tag from a nucleic acid sequence
that comprises at least one restriction endonuclease recognition
site specific for a restriction endonuclease that cleaves the
nucleic acid sequence distally to the restriction endonuclease
recognition site, and at least two restriction endonuclease
recognition sites specific for a rare cutting restriction
endonuclease. The nucleic acid sequence is cleaved with the rare
cutting restriction endonuclease restriction endonuclease to
produce nucleic acid sequence fragments that have compatible ends
and which nucleic acid sequence fragments comprise at least one
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves distally to the recognition
site. The fragments are intramolecularly ligated to produce a
circularized nucleic acid sequence. The circularized nucleic acid
sequences are cleaved with the restriction endonuclease that
cleaves the nucleic acid sequence distally to the restriction
endonuclease recognition site which results in the production of a
paired tag comprising a 5' end tag of the nucleic acid sequence and
a 3' end tag of the nucleic acid sequence.
[0075] A nucleic acid sequence that comprises two restriction
endonuclease recognition sites specific for a restriction
endonuclease that cleaves the nucleic acid sequence distally to the
restriction endonuclease recognition site, and/or at least one
restriction endonuclease recognition sites specific for a rare
cutting restriction endonuclease can be naturally-occurring, or
prepared using techniques that are standard in the art. Methods for
introducing into the 5' end and/or into the 3' end of a nucleic
acid sequence fragment a restriction endonuclease recognition site
are well known in the art. For example, in one embodiment, a method
for introducing into the 5' end and/or into the 3' end of a nucleic
acid sequence fragment a restriction endonuclease recognition site
comprises joining (e.g., ligating or annealing) to the 5' end
and/or to the 3' end a nucleic acid sequence that comprises the
restriction endonuclease recognition site or, alternatively, forms
the restriction endonuclease recognition site once joined to the 5'
end and/or to the 3' end of the nucleic acid sequence fragment. In
another embodiment, a method for introducing a restriction
endonuclease recognition site into the 5' end and/or into the 3'
end of a nucleic acid sequence fragment comprises cloning the
nucleic acid sequence fragment into a vector, wherein the 5' end
and/or 3' end of the nucleic acid sequence fragment are cloned
adjacent to one or more restriction endonuclease recognition sites
present in the vector. Alternatively, in another embodiment, a
method for introducing a restriction endonuclease recognition site
into the 5' end and/or into the 3' end of a nucleic acid sequence
fragment comprises cloning the nucleic acid sequence fragment into
a vector, wherein the 5' end and/or 3' end of the nucleic acid
sequence fragment once cloned into the vector produces one or more
restriction endonuclease recognition sites at the 5' end and/or 3'
end of the nucleic acid sequence fragment.
[0076] A nucleic acid sequence fragment can be prepared by a
variety of methods. These methods are generally referred to herein
as the "fragmenting" of a nucleic acid sequence. For example,
fragmenting of a nucleic acid sequence can be achieved by shearing
(e.g. by mechanical means such as nebulization, hydrodynamic
shearing through a small orifice, or sonication) the nucleic acid
sequence or digesting the nucleic acid sequence with an enzyme,
such as a restriction endonuclease or a non-specific endonuclease.
The nucleic acid sequence fragments obtained can be of any size
(e.g., molecular weight, length, etc.). In one embodiment, nucleic
acid sequence fragments of a specific size (e.g., approximately
greater than about 1 mb, about 200 kb, about 100 kb, about 80 kb,
about 40 kb, about 20 kb, about 10 kb, about 3 kb, about 1.5 kb,
about 1 kb, about 500 bases, about 200 bases and ranges thereof)
are isolated, for example, by gel electrophoresis purification and
extraction, by filtration methods, such as column filtration, or by
other size fractionation methods that are standard in the art.
[0077] The production of a paired tag from a nucleic acid sequence
has many applications. Provided in one embodiment of the invention
is a method for characterizing a nucleic acid sequence. In a
particular embodiment, characterizing a nucleic acid sequence
comprises sequencing the paired tags produced from the nucleic acid
sequence. Sequencing methods are standard in the art, and include,
for example, traditional sequencing using the Maxam and Gilbert
technique or the Sanger method, or by hybridization to an array or
microarray of known oligonucleotides on, for example, a chip.
Alternative approaches include sequencing by synthesis methods in
which primer-template complexes are immobilized, for example, to a
substrate such as a polymer, a magnetic bead, or a solid surface,
and are extended using a DNA polymerase or DNA ligase in the
presence of labeled substrates such that the addition products can
be characterized to determine the DNA sequence.
[0078] In one embodiment, the nucleic acid sequence to be
characterized is a genome. A genome is the genomic DNA of a cell or
organism. In one embodiment, the genome is of a prokaryote,
eukaryote, plant, virus, fungus, or an isolated cell thereof. In
another embodiment, the genome is a known (previously characterized
or sequenced) genome. In a further embodiment, the genome is an
unknown (not previously characterized or sequenced) genome. In one
embodiment, characterizing a genome comprises karyotyping the
genome. Karyotyping is the analysis of the genome of a cell or
organism. In another embodiment, characterizing the genome
comprises polymorphism discovery or genotyping to identify
differences between two or more nucleic acid sequences derived from
different sources.
[0079] In one embodiment of the invention, a method for
characterizing a nucleic acid sequence is provided which comprises
fragmenting a nucleic acid sequence to produce a plurality of
nucleic acid sequence fragments having a 5' end and a 3' end. A
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
and a restriction endonuclease recognition site specific for a rare
cutting restriction endonuclease are introduced into the 5' end and
into the 3' end (either upstream or downstream as appropriate) of
each nucleic acid sequence fragment to produce modified nucleic
acid sequence fragments. The modified nucleic acid sequence
fragments are cleaved with the rare cutting restriction
endonuclease specific for the previously introduced rare cutting
restriction endonuclease recognition site, which produces a
plurality of nucleic acid sequence fragments having compatible
ends. The fragments are intramolecularly ligated to produce a
plurality of circularized nucleic acid sequences comprising the two
restriction endonuclease recognition sites specific for restriction
endonucleases that cleave the nucleic acid fragment distally to the
restriction endonuclease recognition sites. The circularized
nucleic acid sequences are cleaved at the restriction endonuclease
recognition sites with the restriction endonucleases specific for
the restriction endonuclease recognition sites to produce a
plurality of paired tags comprising a 5' end tag and a 3' end tag
of the nucleic acid fragment. These the paired tags are
characterized, which thus characterizes the nucleic acid sequence
of interest.
[0080] In an alternative embodiment of the invention, a method for
characterizing a nucleic acid sequence comprises, fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. A restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid fragment distally to the
restriction endonuclease recognition site is introduced into the 5'
end and into the 3' end of each nucleic acid sequence fragment,
thus producing a plurality of modified nucleic acid sequence
fragments. The modified nucleic acid sequence fragments are, for
example, cloned into a vector. The cloned nucleic acid sequence
fragments are cleaved at the restriction endonuclease recognition
sites at the 5' end and the 3' end of the cloned nucleic acid
sequence fragments using the restriction endonuclease that is
specific for the restriction endonuclease recognition sites, which
produces a 5' end tag and a 3' end tag of each cloned nucleic acid
sequence fragment. The 5' end tag and a 3' end tag of each cloned
nucleic acid sequence fragment are joined to produce a plurality of
paired tags which can be characterized, thereby permitting the
characterizing of the nucleic acid sequence.
[0081] In another embodiment of the invention, a method for
characterizing a nucleic acid sequence comprises, fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. A restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid fragment distally to the
restriction endonuclease recognition site is introduced into the 5'
end and into the 3' end of each nucleic acid sequence fragment,
thus producing a plurality of modified nucleic acid sequence
fragments. The modified nucleic acid sequence fragments are
intramolecularly ligated to produce circularized modified nucleic
acid sequence fragments. Intramolecular ligation can be achieved,
for example, by emulsion ligation. The circularized modified
nucleic acid sequence fragments are cleaved at the restriction
endonuclease recognition sites introduced at the 5' end and the 3'
end of the modified nucleic acid sequence fragments using the
restriction endonuclease specific for the restriction endonuclease
recognition sites, which produces a 5' end tag and a 3' end tag of
each modified nucleic acid sequence fragment. The 5' end tag and a
3' end tag of each nucleic acid sequence fragment are joined (i.e.,
concatemerized) to produce a plurality of paired tags (i.e., a
concatemer) which can be characterized, thereby permitting the
characterizing of the nucleic acid sequence.
[0082] In a further embodiment of the invention, a method for
characterizing a nucleic acid sequence comprises, fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. Each nucleic acid
sequence fragment is cloned to introduce into the 5' end and into
the 3' end of each cloned nucleic acid sequence fragment, a
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site.
The cloned fragments are cleaved at the restriction endonuclease
recognition sites at the 5' end and the 3' end of each cloned
nucleic acid sequence fragment using the restriction endonuclease
that is specific for the restriction endonuclease recognition
sites, thereby producing a 5' end tag and a 3' end tag of the
cloned nucleic acid fragments. The 5' end tag and the 3' end tag of
each cloned nucleic acid are joined to produce a plurality of
paired tags. These paired tags can now be characterized, thereby
characterizing the nucleic acid sequence.
[0083] In another embodiment of the invention, a method for
characterizing a nucleic acid sequence comprises fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. One or more
linkers and/or adapters are joined to the ends, and an
intramolecular ligation step is performed to produce a plurality of
circularized nucleic acid sequences, wherein an intervening linker
is introduced between the 5' end and the 3' end a nucleic acid
sequence fragment. The circularized molecules are fragmented by
random shearing or by other means and an adapter is joining to the
ends of the fragments to produce a plurality of linear nucleic acid
molecules wherein an adapter is introduced at each end. The
resulting nucleic acid molecules containing the 5' and 3' ends of a
nucleic acid sequence fragment (paired tag) are amplified by PCR
using a universal primer complementary to the adapter sequences.
The paired tags are then characterized, thereby characterizing the
nucleic acid sequence.
[0084] In an alternative embodiment of the invention a method for
characterizing a nucleic acid sequence comprises fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. One or more
linkers and/or adapters are joined to the ends, and an
intramolecular ligation is performed to produce a plurality of
circularized nucleic acid sequences, wherein an intervening first
linker is introduced between the 5' end and the 3' end a nucleic
acid sequence fragment. The circularized molecules are fragmented
by random shearing or by other means, and adapters are joined to
the ends of the fragments. A second intramolecular ligation is
performed to produce a plurality of circularized nucleic acid
sequences, wherein an intervening second linker is introduced
between the ends of the fragments, wherein the second linker
comprises two priming sites. The molecules containing the 5' and 3'
ends of a nucleic acid sequence fragment (the paired tag) are
amplified by PCR using two different primers that are complementary
to the priming sites in the second linker. The paired tags are then
characterized, thereby characterizing the nucleic acid
sequence.
[0085] In another embodiment of the invention a method for
characterizing a nucleic acid sequence comprises fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. One or more
linkers and/or adapters are joined to the ends, and an
intramolecular ligation is performed to produce a plurality of
circularized nucleic acid sequences, wherein an intervening first
linker is introduced between the 5' end and the 3' end a nucleic
acid sequence fragment. The circularized molecules are fragmented
by random shearing or by other means, and adapters are joined to
the ends of the fragments. A second intramolecular ligation is
performed to produce a plurality of circularized nucleic acid
sequences, wherein an intervening second linker is introduced
between the ends of the fragments, wherein the second linker
comprises two priming sites separated by a rare-cutting restriction
endonuclease cleavage site. The molecules containing the 5' and 3'
ends of a nucleic acid sequence fragment (the paired tag) are
cleaved using a rare-cutting restriction endonuclease that
recognizes the cleavage site in the linker. Linearization of the
circular DNA molecules is not required, but improves the efficiency
of PCR amplification in the next step. The resulting linear
molecules are amplified by PCR using two different primers that are
complementary to the priming sites in the second linker. The paired
tags are then characterized, thereby characterizing the nucleic
acid sequence.
[0086] In an alternate embodiment of the invention a method for
characterizing a nucleic acid sequence comprises fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. One or more
linkers and/or adapters are joined to the ends, and an
intramolecular ligation is performed to produce a plurality of
circularized nucleic acid sequences, wherein an intervening first
linker containing a sequence or chemical moiety that enables
affinity capture is introduced between the Send and the 3' end a
nucleic acid sequence fragment. The circularized molecules are
fragmented by random shearing or by other means, and adapters are
joined to the ends of the fragments. The molecules containing the
5' and 3' ends of a nucleic acid sequence fragment are purified by
affinity capture using a reagent specific for the first linker. A
second intramolecular ligation is then performed to produce a
plurality of circularized nucleic acid sequences, wherein an
intervening second linker is introduced between the ends of the
fragments, wherein the second linker comprises two priming sites.
The molecules containing the 5' and 3' ends of a nucleic acid
sequence fragment (the paired tag) are amplified by PCR using two
different primers that are complementary to the priming sites in
the second linker. The paired tags are then characterized, thereby
characterizing the nucleic acid sequence.
[0087] A second purification step, for example, by affinity capture
using a reagent specific for a sequence in the first linker, may be
added prior to PCR amplification. Alternatively, the first
purification step could be omitted and a single purification step
could be performed prior to PCR amplification.
[0088] In another embodiment of the invention a method for
characterizing a nucleic acid sequence comprises fragmenting a
nucleic acid sequence to produce a plurality of nucleic acid
sequence fragments having a 5' end and a 3' end. One or more
linkers and/or adapters are joined to the ends, and an
intramolecular ligation is performed to produce a plurality of
circularized nucleic acid sequences, wherein an intervening first
linker containing a sequence or chemical moiety that enables
affinity capture is introduced between the 5' end and the 3' end a
nucleic acid sequence fragment. The circularized molecules are
fragmented by random shearing or by other means, and adapters are
joined to the ends of the fragments. The molecules containing the
5' and 3' ends of a nucleic acid sequence fragment are purified by
affinity capture using a reagent specific for the first linker. A
second intramolecular ligation is then performed to produce a
plurality of circularized nucleic acid sequences, wherein an
intervening second linker is introduced between the ends of the
fragments, wherein the second linker comprises two priming sites
separated by a rare-cutting restriction endonuclease cleavage site.
The molecules containing the 5' and 3' ends of a nucleic acid
sequence fragment (the paired tag) are cleaved using a rare-cutting
restriction endonuclease that recognizes the cleavage site in the
linker. Linearization of the circular DNA molecules is not
required, but improves the efficiency of PCR amplification in the
next step. The resulting linear molecules are amplified by PCR
using two different primers that are complementary to the priming
sites in the second linker. The paired tags are then characterized,
thereby characterizing the nucleic acid sequence. When two
intramolecular ligation steps are performed, both the 5' end and
the 3' end of a nucleic acid sequence fragment paired tag can be
sequenced from the same DNA strand by using separate primers that
are complementary to the sequences in the first and second linkers.
The use of two distinct PCR primers enables amplification on a
substrate (e.g., on the surface of a chip or a magnetic bead)
wherein only one of the two primers is attached to the substrate,
which results in all of the molecules being amplified in a unique
orientation with respect to the substrate.
[0089] In a further embodiment of the invention, a method is
provided for identifying nucleic acid sequences that encode at
least two interacting proteins. Typically, this method comprises
combining (1) a first vector that comprises a nucleic acid sequence
that encodes a first protein that interacts with a second protein
and a first restriction endonuclease recognition site specific for
a restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
and (2) a second vector that comprises a nucleic acid sequence that
encodes the second protein and a second restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site. Typically, this
combination of vectors is maintained under conditions in which the
first protein and the second protein are expressed and interact.
Once interacting first and second interacting proteins are
identified, the first and second vectors encoding same are selected
for identification. Identification of interacting proteins use
methods that are standard in the art (e.g., 2 or 3 hybrid systems
such as yeast, bacteria and mammalian). The vectors encoding the
two interacting proteins are joined to form a contiguous nucleic
acid sequence which comprises the nucleic acid sequence that
encodes the first protein, the first restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, the nucleic acid
sequence that encodes the second protein, and the second
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site.
This contiguous nucleic acid sequence is cleaved at the first and
second restriction endonuclease recognition sites with restriction
endonucleases that cleave the contiguous nucleic acid sequence
distally to the restriction endonuclease recognition sites to
produce a 5' end tag and a 3' end tag of the contiguous nucleic
acid sequence. The 5' end tag is joined to the 3' end tag to
produce a paired tag which can be sequenced, thereby identifying
the nucleic acid sequences that encode the at least two interacting
proteins. In one embodiment, the paired tag produced from one
combination of vectors that encode at least two interacting
proteins can be joined with one or more paired tags produced from
other combinations of vectors that encode at least two interacting
proteins, to produce a concatemer that can be sequenced. Sequencing
of such concatemers provides for high throughput analyses and
increased efficiency.
[0090] In another embodiment of the invention, a method for
identifying nucleic acid sequences that encode at least two
interacting proteins is provided. This method provides combining a
first vector which can comprise (1) a nucleic acid sequence that
encodes a first protein that interacts with a second protein and
(2) a first restriction endonuclease recognition site specific for
a restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
and a second vector which can comprise (1) a nucleic acid sequence
that encodes the second protein, and (2) a second restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, which
produces a combination. The combination is maintained under
conditions the first protein and the second protein are expressed
and interact. The first vector and second vector are joined to form
a contiguous nucleic acid sequence that comprises (i) the nucleic
acid sequence that encodes a first protein that interacts with a
second protein, (ii) the first restriction endonuclease recognition
site specific for a restriction endonuclease that cleaves the
nucleic acid sequence fragment distally to the restriction
endonuclease recognition site, (iii) the nucleic acid sequence that
encodes the second protein, and (iv) the second restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site. The
contiguous nucleic acid sequence is sequenced, thereby identifying
nucleic acid sequences that encode at least two interacting
proteins.
[0091] In an alternative embodiment of the invention, a method for
identifying nucleic acid sequences that encode at least two
interacting proteins is provided. The method combines a first
vector which can comprise (1) a nucleic acid sequence that encodes
a first protein that interacts with a second protein, and (2) a
first restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
and a second vector which can comprise (1) a nucleic acid sequence
that encodes the second protein and (2) a second restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, which
produces a combination. The combination is maintained under
conditions in which the first protein and the second protein are
expressed and interact. The first vector is joined with the second
vector, thereby forming a contiguous nucleic acid sequence that
comprises (i) the nucleic acid sequence that encodes a first
protein that interacts with a second protein, (ii) the first
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(iii) the nucleic acid sequence that encodes the second protein and
(iv) the second restriction endonuclease recognition site specific
for a restriction endonuclease that cleaves the nucleic acid
sequence fragment distally to the restriction endonuclease
recognition site. The first restriction endonuclease recognition
site and the second restriction endonuclease recognition site in
the contiguous nucleic acid sequence are cleaved with restriction
endonucleases that cleave the contiguous nucleic acid sequence
distally to the restriction endonuclease recognition sites, thereby
producing a paired tag comprising a 5' end tag and a 3' end tag of
the contiguous nucleic acid sequence. The paired tag is sequenced,
thereby identifying nucleic acid sequences that encode at least two
interacting proteins.
[0092] In a further embodiment of the invention, a method is
provided for identifying a plurality of nucleic acid sequences that
encode at least two interacting proteins. In a particular
embodiment, the method provides for screening multiple nucleic
acids, such as, for example, a library of nucleic acids, as will be
appreciated by one of skill in the art. The method comprises
combining a plurality of first vectors each can comprise (1) a
nucleic acid sequence that encodes a first protein that interact
with a second protein and (2) a first restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site and a plurality of second
vectors each comprising (1) a nucleic acid sequence that encodes
the second protein and (2) a second restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site, thereby producing a
combination comprising a plurality of first vectors and a plurality
of second vectors. The combination is maintained under conditions
in which the plurality of first vectors encoding a first protein
and the plurality of second vectors encoding a second protein are
expressed and the first protein and second protein interact. The
first vectors are joined with the second vectors, wherein the first
vectors and second vectors encode interacting proteins, thereby
forming a plurality of contiguous nucleic acid sequences. The
plurality of contiguous nucleic acid sequences each comprise (i)
the nucleic acid sequence that encodes a first protein that
interacts with a second protein, (ii) the first restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, (iii)
the nucleic acid sequence that encodes the second protein and (iv)
the second restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site.
In each contiguous nucleic acid sequence, the first restriction
endonuclease recognition site and the second restriction
endonuclease recognition site are cleaved with the restriction
endonucleases that cleave the contiguous nucleic acid sequence
distally to the restriction endonuclease recognition sites, thereby
producing a plurality of paired tags comprising a 5' end tag and a
3' end tag of the contiguous nucleic acid sequence. The paired tags
are joined and sequenced, thereby identifying a plurality of
nucleic acid sequences that encode at least two interacting
proteins.
[0093] Also provided in the invention is a method for identifying
nucleic acid sequences that encode at least two interacting
proteins comprising combining a first vector which can comprise (1)
a nucleic acid sequence that encodes a first protein that interacts
with a second protein and (2) a first restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site and (3) a second
restriction endonuclease recognition site, which in one embodiment,
can be adjacent to the first restriction endonuclease recognition
site and a second vector which can comprise (1) a nucleic acid
sequence that encodes the second protein (2) a third restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, and (3)
a fourth restriction endonuclease recognition site, which in one
embodiment, can be adjacent to the third restriction endonuclease
recognition site, thereby producing a combination. The combination
is maintained under conditions in which the first protein and the
second protein are expressed and interact. The first vector is
joined with the second vector, thereby forming a contiguous nucleic
acid sequence that comprises (i) the nucleic acid sequence that
encodes a first protein that interacts with a second protein, (ii)
the first restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
(iii) the second restriction endonuclease recognition site, (iv)
the nucleic acid sequence that encodes the second protein, (v) the
third restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and (vi) the fourth restriction endonuclease recognition site. The
second restriction endonuclease recognition site and the fourth
restriction endonuclease recognition site in the contiguous nucleic
acid sequence are cleaved with restriction endonucleases to produce
compatible ends in the contiguous nucleic acid sequence. The
nucleic acid sequence is maintained under conditions in which the
compatible ends in the contiguous nucleic acid sequence
intramolecularly ligate, thereby producing a circularized nucleic
acid sequence. The circularized nucleic acid sequence is sequenced,
thereby identifying nucleic acid sequences that encode at least two
interacting proteins.
[0094] In a further embodiment of the invention, a method for
identifying nucleic acid sequences that encode at least two
interacting proteins is provided. The method comprises combining a
first vector which can comprise (1) a nucleic acid sequence that
encodes a first protein that interacts with a second protein, (2) a
first restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site;
and (3) a second restriction endonuclease recognition site, which
in one embodiment, can be adjacent to the first restriction
endonuclease recognition site and a second vector which can
comprise (1) a nucleic acid sequence that encodes the second
protein (2) a third restriction endonuclease recognition site
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site and (3) a fourth restriction endonuclease
recognition site, which in one embodiment, can be adjacent to the
third restriction endonuclease recognition site, thereby producing
a combination. The combination is maintained under conditions in
which the first protein and the second protein are expressed and
interact. The first vector is joined with the second vector,
thereby forming a contiguous nucleic acid sequence that comprises
(i) the nucleic acid sequence that encodes a first protein that
interacts with a second protein, (ii) the first restriction
endonuclease recognition site specific for a restriction
endonuclease that cleaves the nucleic acid sequence fragment
distally to the restriction endonuclease recognition site, (iii)
the second restriction endonuclease recognition site, (iv) the
nucleic acid sequence that encodes the second protein, (v) the
third restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site
and (vi) a fourth restriction endonuclease recognition site. The
second restriction endonuclease recognition site and the fourth
restriction endonuclease recognition site in the contiguous nucleic
acid sequence are cleaved with restriction endonucleases, thereby
producing compatible ends in the contiguous nucleic acid sequence.
The contiguous nucleic acid sequence is maintained under conditions
in which the compatible ends in the contiguous nucleic acid
sequence intramolecularly ligate, thereby producing a circularized
nucleic acid sequence. The first restriction endonuclease
recognition site and the third restriction endonuclease recognition
site in the circularized nucleic acid sequence are cleaved with
restriction endonucleases that cleave the circularized nucleic acid
sequence distally to the restriction endonuclease recognition
sites, thereby producing a paired tag comprising a 5' end tag and a
3' end tag of the circularized nucleic acid sequence. The paired
tag is sequenced, thereby identifying nucleic acid sequences that
encode at least two interacting proteins.
[0095] Further provided in the invention is a method for
identifying a plurality of nucleic acid sequences that encode at
least two interacting proteins. The method comprises combining a
plurality of first vectors each can comprise (1) a nucleic acid
sequence that encodes a first protein that interacts with a second
protein, (2) a first restriction endonuclease recognition site
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site, and (3) a second restriction endonuclease
recognition site, which in one embodiment, can be adjacent to the
first restriction endonuclease recognition site and a plurality of
second vector each comprising (1) a nucleic acid sequence that
encodes the second protein, (2) a third restriction endonuclease
recognition site specific for a restriction endonuclease that
cleaves the nucleic acid sequence fragment distally to the
restriction endonuclease recognition site and (3) a fourth
restriction endonuclease recognition site, which in one embodiment,
can be adjacent to the third restriction endonuclease recognition
site, thereby producing a combination comprising a plurality of
first vectors and a plurality of second vector. The combination is
maintained under conditions in which the plurality of first vectors
encoding a first protein and the plurality of second vectors
encoding a second protein are expressed and the first protein and
second protein interact. The first vectors are joined the second
vectors, wherein the first vectors and second vectors encode
interacting proteins, thereby forming a plurality of contiguous
nucleic acid sequences that each can comprise (i) the nucleic acid
sequence that encodes a first protein that interacts with a second
protein, (ii) the first restriction endonuclease recognition site
specific for a restriction endonuclease that cleaves the nucleic
acid sequence fragment distally to the restriction endonuclease
recognition site, (iii) the second restriction endonuclease
recognition site, which in one embodiment, can be adjacent to the
first restriction endonuclease recognition site, (iv) the nucleic
acid sequence that encodes the second protein, (v) the third
restriction endonuclease recognition site specific for a
restriction endonuclease that cleaves the nucleic acid sequence
fragment distally to the restriction endonuclease recognition site,
and (vi) the fourth restriction endonuclease recognition site,
which in one embodiment, can be adjacent to the third restriction
endonuclease recognition site. The second restriction endonuclease
recognition site and the fourth restriction endonuclease
recognition site in the contiguous nucleic acid sequence are
cleaved with restriction endonucleases that produce compatible
ends. The contiguous nucleic acid sequence with compatible ends is
maintained under conditions in which the sequence that encodes a
sequence from the first protein and a sequence from the second
protein that interact with each other. The first restriction
endonuclease recognition site and the third restriction
endonuclease recognition site in each circularized nucleic acid
sequence is cleaved with restriction endonucleases that cleave each
contiguous nucleic acid sequence distally to the restriction
endonuclease recognition sites, thereby producing a plurality of
paired tags comprising a 5' end tag and a 3' end tag of the
circularized nucleic acid sequence. The paired tags are joined and
sequenced, thereby identifying a plurality of nucleic acid
sequences that encode at least two interacting proteins.
[0096] In one embodiment of the invention, joining is a
site-specific intermolecular recombination. In another embodiment,
joining is a site-specific intermolecular recombination at mutant
site specific recombinase recognition sites, wherein recombination
produces recombined sites having a decreased ability to
subsequently recombine. In a further embodiment of the invention,
the joining is site-specific intermolecular recombination between a
lox71 site and a lox66 site. In a still further embodiment,
cleaving the second restriction endonuclease recognition site and
cleaving the fourth restriction endonuclease recognition site in
the contiguous nucleic acid sequence releases a fragment containing
a recombined site-specific recombinase recognition site between the
nucleic acid sequence that encodes a sequence from the first
protein and a sequence from the second protein that interact with
each other.
Example 1 Whole Genome Assembly
[0097] The whole genome shotgun sequencing, assembly and finishing
paradigm is generally the strategy of choice for microbial and
fungal genome sequencing. Contrary to the expectations of some,
recent advances in the development and application of whole genome
assembly (WGA) software, such as Arachne, the Celera Assembler,
Phusion, or Jazz, have also demonstrated that it is straightforward
to produce a high quality assembly of plant and mammalian genomes
using such tools (Mural, Adams et al. 2002; Jaffe, Butler et al.
2003). The cost-advantages and simplicity of the whole genome
approach relative to a BAC based or hybrid sequencing strategy
argue strongly for its continued development and application in
future sequencing projects. One problem with the BAC based
approaches is the high cost and operational burden associated with
the production of 15,000-25,000 individual BAC subclone libraries,
the 15-20% waste associated with re-sequencing the vector as well
as the unavoidable E. coli contamination, the need to deal with
transposon and bacteriophage insertions, and the 20-50% waste in
redundant sequencing of BAC overlaps. Although these costs can be
reduced by sequencing the BACs at low coverage (using a hybrid
BAC/WGS strategy, for example) or by using a pooling strategy, they
cannot be eliminated. The need to generate a physical map by using
restriction digest fingerprinting or by complex pooling and
sequence based mapping strategies adds additional cost and
operational overhead.
[0098] For genomic sequencing and assembly, the preferred library
that is constructed has a narrow fragment size distribution (Jaffe
et al.). Using restriction enzyme digestion can lead to a reduced
representation library because of the difficulty in size selection
of a restriction-digested genome (Altshuler et al.). For a
particular size range of genomic fragments to be achieved,
restriction enzyme recognition sites are necessary to be spaced
appropriately in order to achieve the desired results. In contrast,
physical shearing permits the whole genome to be fragmented into a
particular size range regardless of where restriction enzyme
recognition sites are found in the genome. Additionally, using in
vivo library construction requires the library to be cloned into
vectors and transformed into the preferred host E. coli. This
results in a cloning bias which typically under-represents AT-rich
regions of the genome (Mead et al.). In a preferred method, the
production of a genomic library would not be dependent on the use
of vectors and E. coli, thereby minimizing cloning biases. For
example, performing emulsion ligations in combination with Emulsion
PCR avoids the necessity of E. coli and permits DNA amplification
on a solid support for future sequencing.
[0099] A critical question regarding the WGS approach is whether it
is possible to generate enough long range scaffolding information
to avoid misassemblies of genomic regions involving complex
repeats, significant levels of allelic polymorphism, or recent
segmental duplications. One aim is to provide a reliable
sequence-derived genome map that can support the accurate selection
of clones to finish any desired region of the genome. As pointed
out by Jaffe et al., (supra), the C57BL6/J mouse genome has certain
characteristics that may have expedited the whole genome assembly
process. These include the inbred status and the apparently low
number of recent segmental duplications compared to the human
genome. In addition, a significant number of large insert reads
were generated (about 4-fold clone coverage from 10 kb inserts,
9-fold from 40 kb inserts, and 14 fold from 200 kb inserts),
although this was a strategic decision that could be applied to any
genome. As stated above, the recent improvements in the ability to
produce paired end sequences (paired tags) from large insert clones
make it possible to significantly increase the number of such
sequences compared to that used in the mouse project. A more
detailed discussion of issues relating to segmental duplications
and sequence polymorphism is presented below.
[0100] Clearly, independent mapping data (genetic or RH markers, or
BAC overlap data, for example) is helpful for placing supercontigs
in relation to each other, especially where BAC end data are
missing or ambiguous. However, it is possible to generate such
information by other means (see below), or to collect the necessary
data to disambiguate such regions through targeted post-assembly
mapping efforts (for example, using sequence data from unmapped
supercontigs to identify overlapping clones directly or to augment
other existing mapping resources). The method provided herein
increases the number of Fosmid ends and other long range links such
that they occur at a spacing of at least one every 500 bp in the
assembly, more than four times the density used in the mouse
project, and four times the number required to achieve statistical
closure of supercontigs.
[0101] Mathematical modeling studies (Ru-Fang Yeh, 1999) indicate
that genome closure, as expressed in terms of supercontig length,
can be significantly accelerated by using a high level of clone
coverage and a broad range of insert sizes in large whole genome
shotgun projects. This is illustrated in FIG. 1 (computed from the
Yeh model), in which the sigmoidal curves indicate the theoretical
length of supercontigs that would be produced using our proposed
mix of insert sizes (green curve) versus a single insert size (blue
curve), for a non-repetitive 3 Gb genome and a 100% paired end
rate.
[0102] Supercontig closure can be significantly accelerated by
using high clone coverage with a mix of insert sizes.
[0103] As shown in FIG. 1, there is a significant increase in the
expected contig length (from .about.98 kb to .about.567 kb) and
decrease in contig number (from .about.30,000 to .about.5,300) as
the sequence coverage goes up from about 7 to about 9 fold coverage
(approximately equivalent to an increase in Phred Q20 coverage from
about 6 to about 8 fold).
[0104] Experiments can determine the effect of insert size and
modification of distance to improve the assembly. By focusing on
accurate sizing of libraries from which reads are derived, the
assembly based on more careful sizing of inserts is refined. For
the special case of comparative sequencing applications within a
species it is possible to define the distance constraints very
precisely based on comparison of the paired end data to a reference
sequence. All WGS reads can be pre-processed this way and entered
into the assembly program.
[0105] Given the high level of shotgun coverage and the large
amount of long range linking information that is obtained, genomic
assemblies are generated with an average contig length of
approximately 125 kb, and with supercontigs spanning a large
proportion of the euchromatic chromosome arms. Gap in the large
insert scaffold can be bridged through the generation and mapping
of paired end GST sequences. Thus, completion of the project having
very few clone gaps remaining will be achieved.
Segmental Duplications
[0106] FIG. 2 graphs the information on the numbers of segmental
duplications identified in the rat genome by Evan Eichler and
colleagues versus the % identity of the cognate pairs (referred to
here as duplicons). The number of duplications in the rat genome is
similar to that in the human genome, about 3.7% vs about 4%,
respectively. It is clear from the graph that there is a trend
towards stronger similarity as the size of the duplicated region
goes up. This phenomenon has been described previously, and
presumably reflects degradation of segmental duplications over
evolutionary timescales. Notably, more than 99.9% of the
duplications are less than 70 kb in size, and these are generally
less than 98% identical.
[0107] For duplications up to 70 kb in size, the large number of
Fosmid sequence links generated in this assembly method (occurring
every 500 bp or so; less than the average read length), will allow
the Arachne software to connect nearly all of the sequence reads
falling within the duplicated region to the unique sequences
flanking it on either side (the Fosmid links to either side will
still meet at the center). This, in turn, will allow clear
segregation of the four chromosomal haplotypes within each pair of
duplicons, considering especially the high expected accuracy of the
component sequences (.about.4 fold Q20 coverage).
[0108] Larger duplications that collapsed in the assembly can be
detected as extended regions with high coverage
(.about.16.times.Q20), and are readily deconvoluted using the
Fosmid and BAC-end information to link out to the unique sequences
on each side. To correctly segregate and precisely map the two
nearly identical sequences for a particular duplication of special
interest, the representative BACs from each duplicon can be used to
generate finished sequences.
Polymorphism
[0109] Sequences with polymorphism rates that fall significantly
below the mismatch threshold settings of the assembly software (as
implied above; typically about 1% mismatch for bases of Phred 20
quality and about 0.2% mismatch for bases of Phred 30 quality) will
be well tolerated and correctly assembled by the software, in most
cases. In the resulting assembly, these regions will be
characterized by high-quality discrepancies in contigs where the
assembly is otherwise strongly supported by mate pair information,
allowing SNPs to be detected and characterized.
[0110] In the case of Anopheles, the Celera assembler was tuned to
accept a higher mismatch frequency than usual to allow most regions
with a high-density of SNPs to be assembled correctly (Holt,
Subramanian et al. 2002). However, some regions remained where both
haplotypes were assembled into separate contigs in the same
supercontig causing "pseudo tandem duplication", or where one
haplotype was left out of the supercontig as an entirely separate
element (see FIG. 3). An understanding of the origin of these
effects and their consequences in terms of mate pair conflicts
between the associated contigs, however, allows detection and
correction of such sequences. For example, a contig that represents
a correctly assembled region with low heterozygosity would have
mate pair bridges to two separate contigs representing the two
different haplotypes of an adjoining region with high
heterozygosity. These mate pair conflicts will force the assembler
to either shift one of the polymorphic contigs out of position or
to exclude it from the supercontig. Algorithmic improvements can be
made to the assembly software to deal with such situations
automatically. Other problems due to genomic inversions, or large
palindromic sequences, can be detected by a different pattern of
characteristic mate pair conflicts.
[0111] Regular assemblies of the reads will help monitor the
quality of libraries on an ongoing basis. Assemblies using Arachne
2.0.1 can be monitored for number and N50 length of contigs, number
and N50 length of supercontigs, average fold coverage, for the
occurrence of contigs with local coverage in a sliding window that
deviates significantly from the expected levels, and for mate pair
conflicts as described above. If there are previously sequenced
clones from the genome, comparisons to these regions can be
conducted using Arachne to evaluate the assembly quality. Arachne's
output assists in detecting deviations of observed K-mer
frequencies versus expected, for example, which can be
monitored.
New Sequenced Based Mapping Tools
[0112] It will be useful to develop sequence-based mapping tools
that can compliment the WGS and BAC end sequencing data without
adding significant incremental cost or operational overhead.
[0113] A high density of BAC- and Fosmid-end sequence pairs can
obviate the need for independent mapping data in most regions of
the genome by providing a highly redundant network of long-range
clone links. However, it is possible that certain regions that are
incompatible with these cloning systems would not be represented.
Wherever such sequences occur in the genome, a persistent break in
the large insert scaffold will be encountered. A linking strategy
to jump over such sequences, such as that applied by Celera to
produce "virtual 50 kb clones" (Venter, Adams et al. 2001) can be
used to overcome these instances. However, by replacing the
standard restriction based jumping library strategy with a novel
long SAGE-like approach (Velculescu, Zhang et al. 1995), large
numbers of long range links (from about 20 to about 100 kb) can be
created using short sequence tags (from about 20 to about 25 bases)
with approximately 20 times the efficiency of standard paired end
sequencing. By using such a strategy, a genomic tag spacing of 500
bp can be generated with only about 300,000 sequencing reads.
Unlike BAC- or Fosmid-end sequencing, the tags can be cloned in
high copy number vectors that are compatible with our current high
copy prepping methods. In addition, short genomic sequence
stretches, punctuated by linker sequences, may allow efficient
generation of tag pairs for AT-rich sequences that are often
difficult to clone in large insert E. coli based vector systems.
Thus, the method provides long range linking information where it
is otherwise very difficult to generate.
[0114] This method efficiently produces paired tag pairs from
randomly sheared or restriction digested fragment ends with any
desired length up to about 100 kb, ensuring that only contiguous
paired end tags are generated (see, for example, FIG. 4). This
method is also referred to herein as Paired Genomic Sequence
Tagging, or Paired GST. Paired GST libraries are distinctly
different from conventional Digital karyotyping ("DK") libraries in
that the paired tags are generated from the ends of individual
genomic fragments.
[0115] In one method, several different restriction enzymes with
hexanucleotide recognition sites that generate 4 base overhangs are
used (for example, to avoid sequence bias) to partially digest the
target DNA, creating a collection of fragments averaging 40 kb in
size. The genomic fragments are treated with a single-strand
specific endo- or exonuclease to remove the single stranded
overhangs, destroying the restriction sites and leaving one base of
the original hexanucleotide recognition site at each end of the
fragments. This method generates sequence tags of approximately 25
base if a MmeI site is juxtaposed to the ends of the fragments
because the reach of MmeI is 20 bases and 5 bases can be inferred
from the destroyed restriction sites. The use of restriction
enzymes can be avoided (e.g., the starting DNA can be fragmented by
shearing) at the cost of 5 bases in the tag length. The method
utilizes in vitro paired tag generation via intramolecular ligation
in dilute solution and does not necessarily require any passage of
large fragments through E. coli, thereby avoiding any cloning bias.
The resulting tag pairs will bridge any persistent gaps in the high
coverage plasmid and Fosmid/BAC clone scaffold.
[0116] Although tags of about 25 bases will generally be unique in
a 3 Gb genome (frequency of 1 in 10.sup.15), tags with one or both
ends falling in a repeated sequence may not be used to reliably
force a join between two contigs or supercontigs. However, a
genomic spacing of approximately 50 kb between tag pair elements
(insert length) and an average spacing of approximately 500 bases
between tags in the genome would result in an average of
approximately 50 redundant links across any given point in the
genome. Even if half of these tag pairs must be excluded for
assembly purposes due to repeats, the remaining linking information
across any given point would equate to approximately 1,250 bases of
unique sequence information (approximately 25 paired tags of
approximately 50 bases each), and would be mutually confirming,
resulting in an astronomically small probability of joining error.
Tag pairs with only one repeated end can be used later to confirm
joins that were made during assembly using the unique
information.
Example 2 Paired Genome Sequence Tag Method
[0117] Typically, a collection of genomic DNA fragments is produced
by fragmentation using one or more restriction endonucleases, or by
shearing, with subsequent repair of the ends to produce blunt-ended
fragments. Fragments of a specific size range are purified by gel
electrophoresis, followed by extraction from a gel slice by
electroelution, extraction with chaotropic salts and glass beads,
or enzymatic dissolution of the gel followed by phenol extraction
and ethanol precipitation. The fragments are ligated to an adapter
containing a type Ils restriction enzyme site juxtaposed to a blunt
end and the resulting circularized products are subsequently
digested with the enzyme to create short tags specific to the ends
of the DNA fragment. These tags, which remain connected to each
other through the process via the adapter are ligated to form
concatemers of paired tags. The concatemers are sequenced and the
paired tags extracted from the sequence. The paired information can
be used to improve shotgun genome assemblies or detect regions of a
genome that have been rearranged/translocated, amplified and/or
deleted.
[0118] In one method, adapters containing a Typel Is site followed
by a rare-cutting restriction site are ligated on to the DNA in a
manner that brings the Typells site immediately next to the end of
the prepared DNA. The adapters are created in such a way as to stop
long concatemers of adapters from forming during the ligation
process. Excess unligated linkers are removed using gel
purification, solid phase reversible immobilization (SPRI),
filtration, or selective precipitation. The adapted DNA is digested
with the rare-cutting restriction enzyme generating compatible
sites at the end of each adapted DNA strand. The DNA is ligated at
a molarity encouraging high intramolecular ligation rather than
intermolecular ligation, or in an emulsion. This creates circles of
DNA with a fragment of unknown DNA and a joined linker containing
two divergent Typells restriction sites. These circles are digested
with the Typells enzyme creating a structure of: a tag, the joined
linker, another tag. These tags come from the 5' and 3' ends of the
prepared DNA fragment. These tags are purified from a gel and can,
optionally, be concatemerized to form DNA strands containing
numerous paired tags. In an alternative method, the tags are
purified and can be sequence in parallel on a large scale. The
concatemerized tags can either be cloned directly, or amplified
using the Polymerase Chain Reaction (PCR) amplification with Phi29
or similar polymerases prior to cloning. Tags and their paring
information are decoded using DNA sequencing.
[0119] In a second method, a linker containing a portion of a
Typells restriction followed by a non-compatible restriction site
such as BstXI site, is ligated onto either end of the prepared DNA
fragments. Excess linker is removed using a purification method
such as gel purification, solid phase immobilization, solid phase
reversible immobilization (SPRI), filtration, or selective
precipitation. The DNA fragments with linkers attached are ligated
to an adapter/vector which comprises a compatible form of the BstXI
or similar site, the remainder of the Typells site with internal
divergent primer sites at a molarity that encourages
circularization of the DNA on the vector/adapter. The circles are
selected on a gel and digested with the Typells restriction site
creating linear fragments with structure: a tag, the
adapter/vector, followed by another tag. These tags come from the
5' and 3' ends of the prepared DNA fragment and are blunted using
appropriate enzymes prior to the subsequent ligation. The structure
is ligated at a low molarity to encourage high intramolecular
ligation rather than intermolecular ligation, or alternatively, in
an emulsion under conditions that favor ligation of a single
adapter/vector to a single prepared genomic fragment. The circles
that are formed contain primer sites outside of the ligated tag
pair. These primer sites are used to amplify the paired tags plus
flanking sites. The amplification product is digested with the rare
cut site and the tag pair purified, or the flanking sites removed
using streptavidin purification if the primers were labeled with
biotin. The purified tag pairs are concatemerized and cloned. Tag
pair information is obtained by sequencing of the concatemers.
Example 3 Paired Tag Two-Hybrid
[0120] This method provides a linked pair of bait and prey
molecules that derive from cells that screen positive for an
interaction. The bait-prey pairs can be sequenced individually, for
example, from plasmids, PCR or rolling circle amplification (RCA)
products, or from short sequence paired tags. The paired tags can
be catenated into longer molecules and sequenced using conventional
unidirectional or paired end sequencing methods to generate
approximately a 6-10 tag pair sequences from each sequencing read.
The linking of bait and prey tags allows thousands of baits to be
screened against thousands of preys (e.g., complete libraries of
both) simultaneously in a single transformation and screening
experiment. The catenation of tag pairs allows approximately 10 or
more pairs to be characterized for the cost of one hi-copy plasmid
sequencing read (depending on the read length). By using a paired
end sequencing approach, approximately 12-20 tag pairs can be
generated from each plasmid template isolated.
[0121] The principle of the method illustrated below for the
bacterial two-hybrid system (Dove and Hochschild, Genes and
Development 12:745-754, 1998) in FIG. 5. Clone libraries containing
the bait and prey ORFs are typically generated in the pBT and pTRG
plasmids according to established methods (for example, the ORFs
can be prescreened by sequencing to select a genomic subset
representing only the particular ORFs, sub-ORFs or domains of
interest). The pBT and pTRG plasmids can be modified to contain a
loxP or other phage attachment site at the cloning sites where ORFs
are inserted. The cloning site modification may also contain, for
example, a typells restriction endonuclease cleavage site (such as
MmeI) in such an orientation that restriction digestion will result
in the generation of a short tag sequence adjacent to the loxP or
other phage attachment site. Another modification includes a
rare-cutting restriction endonuclease site to allow excision of the
loxP site from the recombined product (described below). Cells
containing individual bait plasmids are transformed with prey
plasmids, or alternatively the prey molecules are introduced into
cells by conjugation or by phage infection. The reciprocal
experiment can also be performed (for example, transforming cells
containing prey plasmids with a bait library). The cells into which
the constructs are transferred can additionally contain an
inducible Cre recombinase or other phage integrase gene in addition
to the appropriate genetic components for the two-hybrid screening.
The presence of interacting bait and prey pairs are detected, for
example, by selection on agar plates containing ampicillin or
carbenicillin using techniques that are standard in the art.
Expression of the recombinase is induced in the selected cells to
allow recombination between the two plasmids. If two different
mutant loxP sites are used (e.g., lox71 and lox66) on the bait and
prey constructs, respectively, then a unidirectional recombination
event can be induced upon Cre expression (see Albert, et al., Plant
J. 7:649-59, 1995, and Zhang and Lutz, Nucleic Acids Research
30:e90, 2002). This will increase the efficiency of generation of
the recombined product, pJOlN, and will ensure that the bait-prey
pairs do not undergo any further recombination after initial
formation (because the lox site between them will be a double
mutant). While this is advantageous, it is not absolutely required
for the successful practice of the method.
[0122] After recombination, the pJOlN plasmid can be isolated by
standard methods, or the bait-prey pair can be amplified, for
example, by PCR using primers flanking the 3' ends of the bait and
prey genes. The bait and prey ORFs can be identified, for example,
by complete sequencing, or by primer-directed sequencing outward
from the loxP site (or form another sequence introduced into the
starting plasmids flanking the loxP sites, e.g., an M13 sequencing
primer binding site).
[0123] Alternatively, plasmid DNA can be prepared from a large pool
of selected colonies and digested with MmeI (or another
distally-cleaving restriction endonuclease, such as a type-lus
enzyme) to release approximately 85 bp fragments containing the
loxp site flanked by bait and prey tags. Alternatively, the pooled
plasmid molecules can be cut at a pair of rare-cutting endonuclease
sites (e.g., AsiSI) flanking the mutant loxP site between the bait
and prey ORFs and the plasmids can be recircularized to eliminate
that loxP site prior to MmeI digestion. This will remove the
approximately 34 bp lox site, resulting in approximately 56 bp
fragments after MmeI digestion, which can be purified, for example,
by gel electrophoresis. A linker with appropriate overhanging ends
(e.g., two base 3' overhangs of random sequence to match the MmeI
ends) is ligated to the purified fragments (this preserves the full
tag length of 20 bp). Digestion of the ligated linkers at a
rare-cutting restriction endonuclease recognition site contained
within them (e.g., AscI) will result in compatible termini on all
of the fragments allowing them to be ligated together (for example,
after a suitable purification step to remove excess linkers and
linker fragments) to form a concatemer. After catenation of the
fragments, the DNA can be fragmented, for example, sheared or
subjected to shearing, or to partial digestion with AscI to
generate fragments of approximately 1.5-2 kb in size. These
fragments are typically cloned into high copy number vectors, such
as pSMART or pUC for sequencing. Sequencing of the tag pairs is
accomplished by standard methods.
[0124] The method herein described is illustrated in conjunction
with the bacterial two-hybrid system. However, it can also be used
in conjunction with yeast two-hybrid screening using the
appropriate vectors. The Cre/loxP recombination system (or other
site-specific recombination system can be used as will be
understood by one of skill in the art) can be used with inducible
expression of the heterologous Cre recombinase in the appropriate
yeast host. In one method, to improve the recovery of large amounts
of plasmid from yeast cells, the plasmids can be amplified by
passage through E. coli, or by rolling circle amplification.
Alternatively, the region including the bait-prey tags can be
amplified by PCR using primers that hybridize outside of the cloned
ORF sequences (e.g., in the fused genes, promoter regions or
multiple cloning sites).
[0125] The tagging method generates paired tags from bait and prey
molecules that originally existed on different plasmids that were
introduced into the target cells at different times. Paired tag
formation can also be accomplished by cloning bait and prey
molecules into a modified two-hybrid vector in which the two ORFs
are appropriately arranged in a single vector with relation to the
distally-cleaving restriction enzyme, such as MmeI or other
type-lus restriction sites, so that the intervening regions are
excised enzymatically. This permits the bait-prey paired tags to be
generated without using recombination.
Example 4 Method for Generating Paired Genome Sequence Tags
[0126] Detecting genome amplifications, deletions and
translocations has been shown to pinpoint genes of interest in
various disease states including cancer and mental retardation.
Paired-end sequences have been shown capable of detecting these
regions of change in previously sequenced genomes in addition to
improving the assembly of novel shotgun sequences. In this method
of "Digital Karyotyping," translocations, amplifications and
deletions can be detected in a previously sequenced genome using
"Paired Genome Sequence Tags" (PGSTs). These tags can be produced,
for example, using a Type Ils restriction enzyme similar to those
generated in Digital Karyotyping and SAGE. The tags in PGST,
however, are generated from both ends of DNA fragments and remain
associated through concatemerization and sequencing steps. This
produces paired tags a defined distance from each other in the
genome. Greater than about 15 tag pairs can be obtained from a
single sequence. The tags can be used to detect regions of
amplification and deletion in previously sequenced genomes. The
additional information obtained through pairing also allows the
detection of translocations. This method also applies to generating
paired tag information for scaffolding genome shotgun assemblies.
In this method, paired-end sequence information is produced in an
extremely efficient manner that is free from cloning biases and is
a valuable method in genome shotgun sequencing.
Creation of a PGST Linker
[0127] An oligonucleotide and its complement containing two BstXI
restriction recognition sites separated by an RsaI recognition site
were synthesized. The oligonucleotide has the sequence:
TABLE-US-00001 (SEQ ID NO: 2)
AGCTTCCATGGATTTGGAGTACACCAAATCCATGGT
In this example, the BstXI sites were designed to have the same
recognition site sequence and which were not complementary to each
other so that cleavage with BstXI would not generate a 5' and 3'
end which could self-ligate. Furthermore, the oligonucleotide and
its complement were designed to contain one nucleotide base of a
Type lus restriction enzyme recognition site, in this example,
MmeI, as well as a portion of an additional restriction enzyme
recognition site, in this case BamHI, which was used later for
concatenation of the paired genome sequence tags (also referred to
herein as paired tags).
[0128] The oligonucleotide and its complement were annealed to
produce a double stranded oligonucleotide which was inserted into a
vector, such as pUC 18 or pUC 19, between an M13R primer site/SP6
promoter and an T7 promoter/M13F primer site. The vector was
digested with the restriction enzyme PvuII, which has two
recognition sites that flank the M13R primer site/SP6 promoter and
T7 promoter/M13F primer sites, to release a fragment having the
order: [0129] PvuII-M13R/Sp6-BstXI-RsaI-BstXI-T7/M13F-PvuII-. This
fragment was purified using agarose gel electrophoresis and the
fragment circularized on itself by intramolecular ligation. The
circularized fragment was then digested with the restriction enzyme
RsaI to create a linear linker having the order: [0130]
-BstXI-Sp6/M13R-PvuII-M13F/T7-BstXI-. This linker was inserted by
blunt end cloning into an E. coli-based vector (such as the pAGEN
vector) which did not contain any of the primer sites contained in
the above linker. The linker construct was transformed into an E.
coli strain for maintenance, propagation and amplification of the
linker, using standard protocols as will be understood by one of
skill in the art.
Preparation of the Linker for Use in the System
[0131] A plasmid DNA preparation from the strain containing the
linker construct was digested with the restriction enzyme BstXI,
thereby releasing the linker and creating identical,
non-complementary, 3' overhangs on either side of the linker.
Preparation of DNA of Interest for Ligation to the Linker
[0132] The nucleic acid sequence or DNA of interest, in this
example, the E. coli genome, was sheared using a mechanical method
(either nebulization, hydrodynamic shearing through a small
orifice, or sonication). The randomly sheared DNA was end repaired
using nucleotides (dNTPs) with T4 DNA Polymerase which has 5'-3'
polymerase activity as well as 3'-5' exonuclease activity which
fills in 3' overhangs and cuts back 5' overhangs, respectively. In
this example, the T4 DNA Polymerase with the free nucleotides
(dNTPs) filled in the BstXI 3' overhangs. This end-repaired
randomly sheared DNA was separated by size using agarose gel
electrophoresis. Fragments of the desired size, in this example,
2-3 kilobases (kb), were cut from the gel and purified using
standard gel purification columns. The purified, size selected DNA
fragments were ligated to specific DNA adaptors in vast molar
excess to ensure that the DNA fragments neither circularized nor
ligated to the end of another DNA fragment. The DNA adaptors were
complementary to the digested BstXI sites of the linker prepared
earlier. The adapter has the following top and bottom strands:
TABLE-US-00002 5' CCGACA 3' (SEQ ID NO: 3) (top strand) |||||| 3'
CCTAGGCTGT 5' (SEQ ID NO: 4) (bottom strand)
The blunt end of the adapter is ligated to the fragment of interest
while the overhang is complementary to the overhang left after
digestion of the DNA linker with BstXI. Excess adapter was removed
by agarose gel purification of the 2-3 kb adapted DNA
fragments.
Ligation of the Adapted DNA Fragments to the Linker
[0133] The adapted DNA fragments were ligated to the DNA linker
(prepared above) using the following formulas to determine the
molar ratio of adapted DNA fragments to DNA linker to ensure the
greatest number of circular fragments comprising one adapted DNA
fragment and one DNA linker generated by intramolecular
interactions during ligation (Current Protocols in Human Genetics
(1994) "Construction of Chromosome Jumping and Linking Libraries in
E. coli" by Drumm, Eds. Nicholas C. Dracoploi et al.):
J=K/S.sup.1/2 N=J/(I+J) Where, K is the constant 63.4 micrograms
per milliliter (.mu.g/mL), S is the size of the DNA fragment
ligated to the linker in units of Kilobases (kb), N is the fraction
of intramolecular interactions (e.g., 0.95=95% intramolecular
interactions), and I is the concentration of DNA in micrograms per
milliliter (.mu.g/mL) to obtain the desired fraction of
intramolecular interactions.
[0134] Ligation of the DNA linker to the adapted DNA fragments
creates a full MmeI site and a full BamHI site on either end of the
adapted DNA fragment due to the design of the linker and the
adapter. The circular DNA was purified from the other products of
the ligation reaction by size separation on an agarose gel. The
circular DNA was digested with MmeI releasing all of the DNA
fragment except a 20/18 bp tag on either side of the DNA linker
(i.e., a paired tag on the linker). The 20 bp is the number of
nucleotides remaining from the restriction endonuclease recognition
site in the 5 prime to 3 prime direction of the DNA upon digestion
with the restriction enzyme. Likewise, the 18 bp refers to the
number of nucleotides remaining from the enzyme recognition site on
the 3 prime to 5 prime strand upon digestion with the restriction
enzyme.
[0135] The DNA linker plus the paired tags from the DNA fragment of
interest were separated from other digestion fragments by size
selection using agarose gel electrophoresis using standard
protocols. The DNA linker plus the paired tags were blunt ended to
remove the 3' overhang using T4 DNA Polymerase to leave 18 bp of
each of tag in the paired tag. The DNA linker plus blunt-ended
paired tag was subsequently purified using phenol/chloroform
extraction. Each DNA linker plus the blunt-ended paired tags were
self-ligated to form a circular DNA using the above formula to
ensure intramolecular interactions leading to formation of single
circles such that the paired tags were now adjacent to each other.
This circularization also caused the primer sites (M13R/Sp6 and
T7/M13F) on the DNA linker flanking the paired tags to become
convergent (i.e., while the DNA linker is linear, the PCR primer
sites are directed away from each other (divergent), but upon
circularization, the primer sites are directed toward each other
thereby allowing amplification of the intervening DNA (the paired
tags).
[0136] Using the primer sites, the joined paired tags were PCR
amplified using standard PCR conditions, gel purified, and digested
with BamHI to release the joined paired tags flanked by cut BamHI
sites. The released joined paired tags flanked by cut BamHI sites
were gel purified using PolyAcrylamide Gel Electrophoresis (PAGE)
followed by elution of the paired tags from the gel. The released
paired tags flanked by cut BamHI sites ("sticky ends") were
concatenated by ligation to each other via the BamHI sticky ends to
form concatemers of approximately 600-800 base pairs. The
concatemers were size-selected on an agarose gel and subsequently
purified. The purified, size-selected concatemers were ligated into
the BamHI site of pUC-based E. coli vector (e.g., pUC18), and the
construct transformed into E. coli using standard electroporation
procedures. Colonies containing the concatemer vectors were
selected, grown, and plasmid DNA was prepared from them for
sequencing using standard protocols.
[0137] The plasmid DNA was sequenced using cycle sequencing kits
from Applied Biosystems and separated on an Applied Biosystems
3730XL DNA analyzer. The readout sequences were then separated
using a PERL script which split the sequences based on the flanking
BamHI sites in order to identify the sequences of the paired tags.
Individual tags in the paired tag were determined by using the
substring of sequence on each side of a paired tag that contained
the BamHI recognition site plus 18 nucleotides. The BamHI
restriction site was subtracted from the tag sequence and the tag
sequence matched to a hash of all 18mers in the E. coli genome. As
will be understood by a person of skill in the art, a "hash" is a
standard computing term meaning an array of arrays, or a list of
lists in which the first list contains each 18mer in the genome as
separate entries and each 18mer entry has associated with it a list
of information, which in this case has information regarding where
the 18 mer is located in the genome and which strand it was
located. The hash of all 18 mers in the E. coli genome contained
information on the location and orientation of each 18 mer present
in the genome. Each tag was therefore assigned a location and
orientation in the genome. Some tags matched repetitive 18 mers in
the genome, i.e., a stretch of 18 nucleotides was found in multiple
locations in the genome. In these cases, half of the repetitive 18
mer tags sequenced were unambiguously placed based on the location
and orientation of their paired tag mate. Initial analysis of 45
unique paired tags obtained from the sequencing revealed
approximately 85% of them were correctly paired, i.e., the paired
tag sequences were both the expected 2-3 kb distance away from each
other and on opposite strands in the genome.
Example 5 Method for Generating Paired Genome Sequence Tags
[0138] This method generates paired tags from the ends of a set of
genomic DNA fragments by generating the tags by fragmentation means
other than by cleavage with restriction endonucleases that cleave
distally to their recognition sites. This method produces
end-sequence tags in which the tag length can be arbitrarily large,
thus accommodating longer sequence read lengths than the shorter
sequence read lengths (e.g., 18-27 bases) typically obtained by a
restriction endonuclease approach.
[0139] This method is particularly useful for generating DNA
sequence information from each of the two ends of a set of DNA
fragments in such a way that the pairing information is preserved.
Such information is useful for de novo sequencing and DNA sequence
assembly using genome assembly software such as Arachne, the
Paracel Genome assembler, Jazz or the Celera Assembler. It is also
useful for re-sequencing, polymorphism discovery and genotyping
applications to characterize the genetic differences between
genomes from different individuals, for example, between two human
genomes, to characterize a genome for the presence of known
mutations or polymorphisms, or to characterize associations between
specific sequences or polymorphisms with particular phenotypes,
predisposition to disease or with other traits of interest. An
illustration of the method is presented in FIG. 9.
[0140] Some steps of this procedure are optional, or could be
substituted by other specific methods. In a particular embodiment,
the method comprises circularizing a first set of DNA fragments
("1" in FIG. 9), produced by random fragmentation of the DNA to be
sequenced (e.g., a genomic DNA sample--this could be accomplished
by mechanical shearing, or by specific- or non-specific
endonuclease digestion, oxidative fragmentation by metal ions, or
any other fragmentation means) using a short DNA linker that has an
attached affinity tag (3 in FIG. 9) to allow subsequent binding and
recovery of DNA fragments containing the affinity tag. The method
shown in FIG. 9 specifies ligation of non-self-complementary
end-linkers (2 in FIG. 9) having a unique 5' or 3' overhang to the
starting set of fragments prior to circularization (after healing
the ends on the starting set of fragments to create blunt ends
using a combination of DNA polymerase(s), exonuclease(s) and/or
polynucleotide kinase). This approach improves the specificity of
the subsequent circularization step to ensure that the DNA
fragments do not self-ligate or circularize without incorporating
the affinity tag linker (3 in FIG. 9), which, in the above example
comprises a set of non-self-complementary ends that are
complementary to those on the end-linkers (2 in FIG. 9). After
circularization, the sample is treated with Bal31, (or with a
similar exonuclease, or with a mixture of an exonuclease plus a
single-stranded DNA specific exo- or endonuclease, or with a
commercial "plasmid-safe" DNAse product) to destroy any leftover
linear molecules including any unligated affinity tag. The
exonuclease is inactivated, and the remaining circles are
fragmented into smaller pieces to produce a second set of DNA
fragments. The pieces that contain the affinity tag are captured,
purified and are then sequenced: a) directly, using a single
molecule sequencing approach, b) after molecular cloning using
standard methods (e.g., after ligation into a selectable vector
followed by transformation and propagation of individual fragments
in E. coli or other suitable host), or c) after amplification by
PCR (with or without the addition of universal priming sites (4) by
ligation of oligonucleotides at the ends). One method for
amplification and sequencing, shown in the figure above, is by
clonal amplification on magnetic beads containing an
oligonucleotide complementary to the universal primers (4 in FIG.
9) in a water/oil emulsion (or by some other clonal amplification
means) such that the amplified fragments become bound to the beads
in both possible orientations. Alternate means of amplifying after
applying means for attachment of different primers to the two ends
of the sheared fragments could be applied to produce magnetic beads
with the final amplified fragments in one orientation only.
[0141] The affinity tag shown in FIG. 9 represents biotin attached
to one of the nucleotides, but many other types of affinity tags
could be used, such as digoxygenin or other small molecule for
which specific antibodies are available. Alternatively, a target
sequence for a DNA binding protein such as the E. coli lactose
repressor could be included in the linker, or a sequence capable of
being hybridized as a triple helix. In the example above, the DNA
fragments would be recovered by binding to a matrix (e.g., magnetic
beads) with immobilized streptavidin prior to ligation of the
universal primer oligonucleotides
[0142] The affinity tag oligonucleotide comprises sequences that
double as sequencing primers. A different primer is used to
sequence in each direction outward from the tag.
[0143] An advantage of this method over previous methods is that it
can generate tags of any desired length, up to several kilobases,
on average, if desired. Paired tag fragment lengths of several
hundred bases would be adequate to support sequence read lengths of
25-200 bases using conventional Sanger sequencing or revolutionary
sequencing by synthesis methodologies, such as Polony Technology as
developed by G. Chuch and colleagues, Pyrosequencing in picotiter
plates as developed by 454 corporation, or Massively Parallel
Signature Sequencing as developed by Lynx Therapeutics. The precise
size of the two sub-fragments flanking the affinity tag linker will
be variable, and unknown, but on average it will be about half of
the total fragment length). Thus for a 50 base read length and a
total fragment length of 580 bases with a 30 base affinity tag, 90%
of sequence reads originating from the affinity tag would be at
least 50 bases long. If longer read lengths are desired, a longer
paired tag fragment length can be generated.
[0144] One means for attachment of two different primers to the two
ends of the final fragments involves the use of a second
circularization step to introduce two different sequences capable
of being used as PCR primers instead of ligating a single universal
primer. The advantage of this implementation is that it avoids the
requirement for doing PCR on a support using a single primer by
"bridge PCR"--a method that is known to result in poor efficiency
of fragment amplification and which also results in both DNA
strands being attached to the support, which reduces sequencing
efficiency. The second circularization method is illustrated in the
lower half of FIG. 10.
[0145] The first steps in this procedure are the same as before. A
first set of DNA fragments (1 in FIG. 10) produced by fragmentation
of genomic DNA are circularized using a short DNA linker that has
an attached affinity tag (3 in FIG. 10) to allow subsequent binding
and recovery of DNA fragments containing the affinity tag. As,
before, the method employs initial ligation of
non-self-complementary end-linkers (2 in FIG. 10) having a unique
5' or 3' overhang to the starting set of fragments prior to
circularization (after healing the ends on the starting set of
fragments to create blunt ends using a combination of DNA
polymerase(s), exonuclease(s) and/or polynucleotide kinase). The
capping step improves the specificity of the subsequent
circularization step to ensure that the DNA fragments do not
self-ligate or circularize without incorporating the affinity tag
linker (3 in FIG. 10), which, in the above example comprises a set
of non-self-complementary ends that are complementary to those on
the end-linkers (2 in FIG. 10). The length of the affinity tag
linker is at least 20 nucleotides, such that two unique primer
sequences (A and B) can be accommodated, which will allow
hybridization of primers to both DNA strands to allow priming of
DNA synthesis in either direction, as seen in FIG. 11.
[0146] After circularization, the sample is treated with Bal31, (or
with a similar exonuclease, or with a mixture of an exonuclease
plus a single-stranded DNA specific exo- or endonuclease, or with a
commercial "plasmid-safe" DNAse product) to destroy any leftover
linear molecules including any unligated affinity tag. The
endonuclease is inactivated and the remaining circles are
fragmented into smaller pieces to produce a second set of DNA
fragments. The pieces that contain the affinity tag are captured at
this point, by mixing with streptavidin coated magnetic beads, for
example. After healing the ends on the starting set of fragments to
create blunt ends using a combination of DNA polymerase(s),
exonuclease(s) and/or polynucleotide kinase), a capping linker (2
in FIG. 11) is added to the linear DNA fragments. As before, the
preferred capping linker has non-self-complementary ends with a
unique 5' or 3' overhang. This approach improves the specificity of
the subsequent circularization step to ensure that the DNA
fragments do not self-ligate or circularize without incorporating
the inversion linker (4 in FIG. 11), which, in the above example
comprises a set of non-self-complementary ends that are
complementary to those on the end-linkers (2 in FIG. 11). The
inversion linker (4 in FIG. 11) comprises two unique sequences (C
and D) that can be used subsequently to prime DNA synthesis in a
PCR reaction, and a rare-cutting endonuclease site between the C
and D sequences. After circularization, the sample is treated with
Bal31, (or with a similar exonuclease, or with a mixture of an
exonuclease plus a single-stranded DNA specific exo- or
endonuclease, or with a commercial "plasmid-safe" DNAse product) to
destroy any leftover linear molecules including any unligated
inversion tag. The nuclease is inactivated and the remaining
circles are linearized using the rare-cutting Endonuclease (or
other rare cleavage means), in the case illustrated the
endonuclease is I-SceI. If desired, a second round of enrichment
for fragments containing the affinity tag could be performed at
this stage.
[0147] As before, sequencing could be done: a) directly, using a
single molecule sequencing approach, b) after molecular cloning
using standard Sanger methods (e.g., after ligation into a
selectable vector followed by transformation and propagation of
individual fragments in E. coli or other suitable host), c) after
amplification by PCR, or d) after clonal amplification using polony
technology, emulsion PCR, bridge PCR, or some other clonal
amplification means. A preferred method for amplification and
sequencing is by clonal amplification on magnetic beads containing
an oligonucleotide complementary to one end of the molecules to be
amplified (in a water/oil emulsion, for example) such that the
amplified fragments become bound to the beads in only one
orientation. For example, if the "C" primer is covalently attached
to the beads through a chemical linker at the 5' end (or
non-covalently using 5' biotin and streptavidin beads--or by some
other high affinity non-covalent attachment means). Molecules
amplified on beads will then become attached as shown in FIG.
12.
[0148] All molecules on any one bead will contain the same
sequences, in the same orientation. The unattached strand can then
be removed by denaturation. On some beads (I. in FIG. 12), the
orientation of the affinity-tag linker will be such that a primer
complementary to the A sequence will hybridize to attached DNA
strand. On other beads (II. in FIG. 12), the orientation of the
affinity-tag linker will be such that a primer complementary to the
B sequence will hybridize to attached DNA strand. By performing a
sequencing by synthesis procedure on a mixture of clonally
amplified templates (e.g., a mixture of beads on a polony slide)
sequentially with two sets of primers, sequences from both ends of
the starting set of DNA fragments (1 in FIG. 12) can be acquired.
To do this, sequences are first generated by priming with the D
primer (each bead with clonally amplified molecules will produce a
unique sequence). Next, sequences are generated by priming with a
mixture of the A and B primers (each bead with clonally amplified
molecules will produce a unique sequence, primed from either the A
or B primer only).
[0149] The starting set of fragments (1 in FIG. 12) can be of any
length up to several hundred kilobases, but preferably will be in
the range of 2 to 200 kb. The second set of DNA fragments (produced
after circularization with the affinity tag) can be of any length
up to the size of the first set of fragments, but preferably will
be in the range of 0.1 to 2 kb. The average read length that can be
obtained from each end will be equal to 1/2 the length of the
second set of fragments minus the length of the affinity tag
linker.
[0150] The relevant teachings of all the references, patents and
patent applications cited herein are incorporated herein by
reference in their entirety.
[0151] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
33110DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Oligonucleotide linkermodified_base(1)..(2)a, c, t or
gmodified_base(9)..(10)a, c, t or g 1nnatgatgnn 10236DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2agcttccatg gatttggagt acaccaaatc catggt
3636DNAArtificial SequenceDescription of Artificial Sequence
Synthetic adapter-top strand oligonucleotide 3ccgaca
6410DNAArtificial SequenceDescription of Artificial Sequence
Synthetic adapter-bottom strand oligonucleotide 4tgtcggatcc
10510DNAArtificial SequenceDescription of Artificial Sequence
Synthetic top strand oligonucleotide ASCI
linkermodified_base(9)..(10)a, c, t or g 5ggcgcgccnn
10610DNAArtificial SequenceDescription of Artificial Sequence
Synthetic bottom strand oligonucleotide ASCI
linkermodified_base(9)..(10)a, c, t or g 6ggcgcgccnn
10768DNAArtificial SequenceDescription of Artificial Sequence
Synthetic pBT Lox oligonucleotide linkermodified_base(49)..(68)a,
c, t or g 7ataacttcgt ataatgtatg ctatacgaac ggtagcgatc gctccaacnn
nnnnnnnnnn 60nnnnnnnn 68816PRTArtificial SequenceDescription of
Artificial Sequence Synthetic pBT Lox peptide linker 8Ile Thr Ser
Tyr Asn Val Cys Tyr Thr Asn Gly Ser Asp Arg Ser Asn 1 5 10 15
968DNAArtificial SequenceDescription of Artificial Sequence
Synthetic pTRG Lox oligonucleotide linkermodified_base(49)..(68)a,
c, t or g 9ataacttcgt atagcataca ttatacgaac ggtagcgatc gctccaacnn
nnnnnnnnnn 60nnnnnnnn 681016PRTArtificial SequenceDescription of
Artificial Sequence Synthetic pTRG Lox peptide linker 10Tyr Arg Ser
Tyr Ser Ile His Tyr Thr Asn Gly Ser Asp Arg Ser Asn 1 5 10 15
1140DNAArtificial SequenceDescription of Artificial Sequence
Synthetic vector insert oligonucleotide 11atgatccatg gatttggagt
acaccaaatc catggatgca 40129DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 12gtcggatcc
91337DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide linkermodified_base(19)..(19)a, c, t or g
13atgatccatg gatccgacng tcggatccat ggatgca 371437DNAArtificial
SequenceDescription of Artificial Sequence Synthetic cloned
oligonucleotide tagmodified_base(19)..(19)a, c, t or g 14atgatccatg
gatccgacng tcggatccat ggatgca 371513DNAArtificial
SequenceDescription of Artificial Sequence Synthetic top-strand
oligonucleotide paired tagmodified_base(9)..(9)a, c, t or g
15gatccgacng tcg 131613DNAArtificial SequenceDescription of
Artificial Sequence Synthetic bottom-strand oligonucleotide paired
tagmodified_base(9)..(9)a, c, t or g 16gatccgacng tcg
13178DNAArtificial SequenceDescription of Artificial Sequence
Synthetic restriction site oligonucleotide 17gcgatcgc
8188DNAArtificial SequenceDescription of Artificial Sequence
Synthetic restriction site oligonucleotide 18gcggccgc
8198DNAArtificial SequenceDescription of Artificial Sequence
Synthetic restriction site oligonucleotide 19ttaattaa
82023DNAArtificial SequenceDescription of Artificial Sequence
Synthetic top strand oligonucleotide linker 20aaaaaattag cggccgctcc
gac 232119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic bottom strand oligonucleotide
linkermisc_feature(1)..(1)5' phosphatemisc_feature(19)..(19)3'
methyl 21gtcggagcgg ccgctaatt 192223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic top strand
oligonucleotide linkermisc_feature(1)..(1)5'
phosphatemisc_feature(23)..(23)3' methyl 22aaaaaatgat taattaatcc
gac 232319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic bottom strand oligonucleotide linker 23gtcggattaa
ttaatcatt 192410042DNAArtificial SequenceDescription of Artificial
Sequence Synthetic top strand polynucleotide cloned
fragmentmodified_base(24)..(10023)a, c, t or g 24aaaaaattag
cggccgctcc gacnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900nnnnnnnnnn nnnnnnnnnn
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1920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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10020nnngtcggag cggccgctaa tt 100422510042DNAArtificial
SequenceDescription of Artificial Sequence Synthetic bottom strand
polynucleotide cloned fragmentmodified_base(24)..(10023)a, c, t or
g 25aaaaaattag cggccgctcc gacnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn 9840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 9900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9960nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10020nnngtcggag
cggccgctaa tt 100422610020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic top strand polynucleotide cloned
fragmentmodified_base(13)..(10012)a, c, t or g 26ggccgctccg
acnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn 480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
9300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
9600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9780nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9840nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
9900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nngtcggagc 100202710020DNAArtificial SequenceDescription
of Artificial Sequence Synthetic bottom strand polynucleotide
cloned fragmentmodified_base(13)..(10012)a, c, t or g 27ggccgctccg
acnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn 480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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6120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
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nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nngtcggagc
100202822DNAArtificial SequenceDescription of Artificial Sequence
Synthetic paired oligonucleotide tagmodified_base(1)..(1)a, c, t or
gmodified_base(22)..(22)a, c, t or g 28ngtcggagcg gccgctccga cn
222940DNAArtificial SequenceDescription of Artificial Sequence
Synthetic vector insert oligonucleotide 29tgcatccatg gatttggtgt
actccaaatc catggtacat 403040DNAArtificial SequenceDescription of
Artificial Sequence Synthetic vector insert oligonucleotide
30tgcatccatg gatttggtgt actccaaatc catggtacat 403137DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide linkermodified_base(19)..(19)a, c, t or g
31tgcatccatg gatccgacng tcggatccat ggtacat 373237DNAArtificial
SequenceDescription of Artificial Sequence Synthetic cloned
oligonucleotide tagmodified_base(19)..(19)a, c, t or g 32tgcatccatg
gatccgacng tcggatccat ggtacat 373322DNAArtificial
SequenceDescription of Artificial Sequence Synthetic paired
oligonucleotide tagmodified_base(1)..(1)a, c, t or
gmodified_base(22)..(22)a, c, t or g 33ngtcggagcg gccgctccga cn
22
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