U.S. patent application number 11/390803 was filed with the patent office on 2006-10-05 for capturing sequences adjacent to type-iis restriction sites for genomic library mapping.
This patent application is currently assigned to Affymetrix, INC.. Invention is credited to Thomas R. Gingeras, Robert J. Lipshutz, Ronald J. Sapolsky.
Application Number | 20060223097 11/390803 |
Document ID | / |
Family ID | 26975981 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060223097 |
Kind Code |
A1 |
Sapolsky; Ronald J. ; et
al. |
October 5, 2006 |
Capturing sequences adjacent to type-IIS restriction sites for
genomic library mapping
Abstract
The present invention relates to novel methods for sequencing
and mapping genetic markers in polynucleotide sequences using
Type-IIs restriction endonucleases. The methods herein described
result in the "capturing" and determination of specific
oligonucleotide sequences located adjacent to Type-IIs restriction
sites. The resulting sequences are useful as effective markers for
use in genetic napping, screening and manipulation.
Inventors: |
Sapolsky; Ronald J.; (Palo
Alto, CA) ; Lipshutz; Robert J.; (Palo Alto, CA)
; Gingeras; Thomas R.; (Santa Clara, CA) |
Correspondence
Address: |
AFFYMETRIX, INC;ATTN: CHIEF IP COUNSEL, LEGAL DEPT.
3420 CENTRAL EXPRESSWAY
SANTA CLARA
CA
95051
US
|
Assignee: |
Affymetrix, INC.
Santa Clara
CA
|
Family ID: |
26975981 |
Appl. No.: |
11/390803 |
Filed: |
June 12, 2006 |
Related U.S. Patent Documents
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10942479 |
Sep 16, 2004 |
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11390803 |
Jun 12, 2006 |
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10696452 |
Oct 29, 2003 |
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10942479 |
Sep 16, 2004 |
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10193751 |
Jul 10, 2002 |
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10696452 |
Oct 29, 2003 |
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09940868 |
Aug 27, 2001 |
6509160 |
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10193751 |
Jul 10, 2002 |
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09412246 |
Oct 5, 1999 |
6291181 |
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09940868 |
Aug 27, 2001 |
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09008094 |
Jan 16, 1998 |
6027894 |
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09412246 |
Oct 5, 1999 |
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08485606 |
Jun 7, 1995 |
5710000 |
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09008094 |
Jan 16, 1998 |
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08307881 |
Sep 16, 1994 |
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08485606 |
Jun 7, 1995 |
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Current U.S.
Class: |
435/6.13 ;
435/91.2; 506/9 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 2600/156 20130101; C12Q 1/6809 20130101; C12Q 1/6855 20130101;
C12Q 1/6874 20130101; C12Q 1/6827 20130101; C12Q 1/6869 20130101;
C12Q 1/6806 20130101; C12Q 1/6855 20130101; C12Q 2565/501 20130101;
C12Q 1/683 20130101; C12Q 1/683 20130101; C12Q 1/6874 20130101;
C12Q 1/683 20130101; C12Q 1/6827 20130101; C12Q 2525/191 20130101;
C12Q 1/6855 20130101; C12Q 2525/131 20130101; C12Q 2521/313
20130101; C12Q 2525/143 20130101; C12Q 2525/191 20130101; C12Q
2565/501 20130101; C12Q 2525/179 20130101; C12Q 2525/191 20130101;
C12Q 2521/313 20130101; C12Q 2525/131 20130101; C12Q 2525/191
20130101; C12Q 2521/313 20130101; C12Q 2525/191 20130101; C12Q
2525/161 20130101; C12Q 2525/143 20130101; C12Q 2525/155 20130101;
C12Q 2525/179 20130101; C12Q 2521/313 20130101; C12Q 2521/313
20130101; C12Q 2521/313 20130101; C12Q 2525/191 20130101; C12Q
2525/155 20130101; C12Q 2565/501 20130101; C12Q 2525/191 20130101;
C12Q 2525/131 20130101; C12Q 2521/313 20130101; C12Q 2521/313
20130101; C12Q 2525/191 20130101; C12Q 2521/313 20130101; C12Q
2525/179 20130101; C12Q 1/6809 20130101; C12Q 1/6874 20130101; C12Q
1/6869 20130101; C12Q 1/6855 20130101; C12Q 1/683 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Goverment Interests
[0002] Research leading to the present invention was funded in part
by NIH grant Nos. 5-F32-HG00105 and RO1 HG00813-02, and the
government may have certain rights to the invention.
Claims
1. A method of identifying sequences in a polynucleotide sequence,
comprising: first cleaving the polynucleotide sequence with a first
type-IIs endonuclease; first ligating a first adapter sequence to
the polynucleotide sequence cleaved in said first cleaving step,
said first adapter having a recognition site for a second type-IIs
endonuclease; second cleaving the polynucleotide sequence resulting
from said first ligating step, with the second type-IIs
endonuclease; second ligating a second adapter sequence to the
polynucleotide sequence cleaved in said second cleaving step; and
determining the sequence of nucleotides of the polynucleotide
sequence between the first and second adapter sequences.
2. The method of claim 1, wherein: in said first cleaving step, the
first type-IIs endonuclease is selected from the group consisting
of BsmAI, EarI, MnlI, PleI, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI,
SapI, SfaNI, BseRI, HphI and MboII; and in said second cleaving
step, the second type-IIs endonuclease is selected from the group
consisting of HgaI, BbvI, BspMI, BsmFI and FokI.
3. The method of claim 2, wherein in said first cleaving step, the
first type-IIs endonuclease is EarI; and in said second cleaving
step, the second type-IIs endonuclease is HgaI.
4. The method of claim 1, wherein in said first and second ligating
steps, said first and second adapter sequences comprise primer
sequences.
5. The method of claim 4, wherein prior to said determining step,
the sequence of oligonucleotides in the polynucleotide between the
first and second adapter sequences in amplified.
6. The method of claim 1, wherein in said determining step, the
sequence of nucleotides between the first and second adapter
sequences is determined by hybridization to an oligonucleotide
probe.
7. The method of claim 6, wherein said oligonucleotide probe is a
positionally distinct probe on an oligonucleotide array, a position
of the probe being indicative of the sequence of the probe.
8. A method of generating an ordered map of a library of genomic
fragments, the method comprising: identifying sequences in each of
the genomic fragments in the library, according to the method of
claim 1; comparing the sequences identified in each fragment with
the sequences identified in each other fragment to obtain a level
of correlation between each fragment and each other fragment; and
ordering the fragments according to their level of correlation.
9. A method of identifying polymorphisms in a target polynucleotide
sequence, the method comprising: identifying sequences in a
wild-type polynucleotide sequence, according to the method of claim
1, repeating said identifying step on the target polynucleotide
sequence; and determining differences in the sequences identified
in each of said identifying steps, the differences being indicative
of a polymorphism.
10. The method of claim 1, wherein said sequences in a
polynucleotide sequence are proximal to a polymorphism.
11. A method of identifying a source of a biological sample, the
method comprising: identifying a plurality of sequences in a
polynucleotide sequence derived from the sample, according to the
method of claim 1; and comparing the plurality of sequences
identified in said identifying step with a plurality of sequences
identically identified from a polynucleotide derived from a known
source, identity of the plurality of sequences identified from the
sample with the plurality of sequences identified from the known
source being indicative that the sample was derived from the known
source.
12. A method of determining a relative location of a target
nucleotide sequence on a polynucleotide, the method comprising:
generating an ordered map of the polynucleotide according to the
method of claim 8; fragmenting the polynucleotide; determining
which fragment includes the target nucleotide sequence; correlating
a marker on the fragment with a marker on the ordered map to
identify the approximate location of the target nucleotide sequence
on the polynucleotide.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/307,881, filed Sep. 16, 1994, which is
hereby incorporated by reference in its entirety for all
purposes.
[0003] The present invention generally relates to novel methods for
isolating, characterizing and mapping genetic markers in
polynucleotide sequences. More particularly, the present invention
provides methods for mapping genetic material using Type-IIs
restriction endonucleases. The methods herein described result in
the "capturing" and determination of specific oligonucleotide
sequences located adjacent to Type-IIs restriction sites. The
resulting sequences are useful as effective markers for use in
genetic mapping, screening and manipulation.
BACKGROUND OF THE INVENTION
[0004] The relationship between structure and function of
macromolecules is of fundamental importance in the understanding of
biological systems. These relationships are important to
understanding, for example, the functions of enzymes, structural
proteins and signalling proteins, ways in which cells communicate
with each other, as well as mechanisms of cellular control and
metabolic feedback.
[0005] Genetic information is critical in continuation of life
processes. Life is substantially informationally based and its
genetic content controls the growth and reproduction of the
organism and its complements. The amino acid sequences of
polypeptides, which are critical features of all living systems,
are encoded by the genetic material of the cell. Further, the
properties of these polypeptides, e.g., as enzymes, functional
proteins, and structural proteins, are determined by the sequence
of amino acids which make them up. As structure and function are
integrally related, many biological functions may be explained by
elucidating the underlying structural features which provide those
functions, and these structures are determined by the underlying
genetic information in the form of polynucleotide sequences.
Further, in addition to encoding polypeptides, polynucleotide
sequences also can be involved in control and regulation of gene
expression. It therefore follows that the determination of the
make-up of this genetic information has achieved significant
scientific importance.
[0006] Physical maps of genomic DNA assist in establishing the
relationship between genetic loci and the DNA fragments which carry
these loci in a clone library. Physical maps include "hard" maps
which are overlapping cloned DNA fragments ("contigs") ordered as
they are found in the genome of origin, and "soft" maps which
consist of long range restriction enzyme and cytogenetic maps
(Stefton and Goodfellow, 1992). In the latter case, the combination
of rare cutting restriction endonucleases (e.g., NotI) and pulse
gel electrophoresis allows for the large scale mapping of genomic
DNAs. These methods provide a low resolution or top down approach
to genomic mapping.
[0007] A bottom up approach is exemplified by construction of
contiguous or "contig" maps. Initial attempts to construct contig
maps for the human genome have been based upon ordering inserts
cloned into cosmids. More recent studies have utilized yeast
artificial chromosomes (YACs) which allow for cloning larger
inserts. The construction of contig maps require that many clones
be examined (4-5 genome equivalents) in order to assure that
sufficient overlap between clones is achieved. Currently, four
approaches are used to identify overlapping sequences.
[0008] The first method is restriction enzyme fingerprinting. This
method involves the electrophoretic sizing of restriction enzyme
generated DNA fragments for each clone and establishing a criterion
for clone overlap based on the similarity of fragment sets produced
for each clone. The sensitivity and specificity of this approach
has been improved by labelling of fragments using ligation, and
end-filling techniques. The detection of repetitive sequence
elements (e.g., [GT].sub.n) has also been employed to provide
characteristic markers.
[0009] The second method generally employed in mapping applications
is the binary scoring method. This method involves the
immobilization of members of a clone library to filters and
hybridization with sets of oligonucleotide probes. Several
mathematical models have been developed to avoid the need for large
numbers of the probe sets which are designed to detect the overlap
regions.
[0010] A third method is the Sequence Tagged Site ("STS") method.
This method employs PCR techniques and gel analysis to generate DNA
products whose lengths characterize them as being related to common
regions of sequence that are present in overlapping clones. The
sequence of the primary pairs and the characteristic distance
between them provides sufficient information to establish a single
copy landmark (SCL) which is analogous to single copy probes that
are unique in the entire genome.
[0011] A fourth method uses cross-hybridizing libraries. This
method involves the immobilization of two or more pools of cosmid
libraries followed by cross-hybridization experiments between pairs
of the libraries. This cross-hybridization demonstrates shared
cloned sequences between the library pairs. See, e.g., Kupfer, et
al., (1995) Genomics 27:90-100.
[0012] Although each of these methods is capable of generating
useful physical maps of genomic DNA, they each involve complex
series of reaction steps including multiple independent synthesis,
labelling and detection procedures.
[0013] Traditional restriction endonuclease mapping techniques,
i.e., as described above, typically utilize restriction enzyme
recognition/cleavage sites as genetic markers. These methods
generally employ Type-II restriction endonucleases, e.g., EcoRI,
HindIII and BamHI, which will typically recognize specific
palindromic nucleotide sequences, or restriction sites, within the
polynucleotide sequence to be mapped, and cleave the sequence at
that site. The restriction fragments which result from the cleavage
of separate fragments of the polynucleotide (i.e., from a prior
digestion) are then separated by size. Overlap is shown where
restriction fragments of the same size appear from Type-II
endonuclease digestion of separate polynucleotide fragments.
[0014] Type-IIs endonucleases, on the other hand, generally
recognize non-palindromic sequences. Further, these endonucleases
generally cleave outside of their recognition site, thus producing
overhangs of ambiguous base pairs. Szybalski, 1985, Gene
40:169-173. Additionally, as a result of their non-palindromic
recognition sequences, the use of Type-IIs endonucleases will
generate more markers per Kb than a, similar Type-II endonuclease,
e.g., approximately twice as often.
[0015] The use of Type-IIs endonucleases in mapping genomic markers
has been described in, e.g., Brenner, et al., P.N.A.S. 86:8902-8906
(1989). The methods described involved cleavage of genomic DNA with
a Type-IIs endonuclease, followed by polymerization with a mixture
of the four deoxynucleotides as well as one of the four specific
fluorescently labelled dideoxynucleotides (ddA, ddT, ddG or ddC).
Each successive unpaired nucleotide within the overhang of the
Type-IIs cleavage site would be filled by either a normal
nucleotide or the labelled dideoxynucleotide. Where the latter
occurred, polymerization stopped. Thus, the polymerization reaction
yields an array of double stranded fluorescent DNA fragments of
slightly different sizes. By reading the size from smallest size to
largest, in each of the nucleotide groups, one can determine the
specific sequence of the overhang. However, this method can be time
consuming and yields only the sequence of the overhang region.
[0016] Oligonucleotide probes have long been used to detect
complementary nucleic acid sequences in a nucleic acid of interest
(the "target" nucleic acid). In some assay formats, the
oligonucleotide probe is tethered, i.e., by covalent attachment, to
a solid support, and arrays of oligonucleotide probes immobilized
on solid supports have been used to detect specific nucleic acid
sequences in a target nucleic acid. See, e.g., U.S. patent
application Ser. No. 08/082,937, filed Jun. 25, 1993, which is
incorporated herein by reference. Others have proposed the use of
large numbers of oligonucleotide probes to provide the complete
nucleic acid sequence of a target nucleic acid but failed to
provide an enabling method for using arrays of immobilized probes
for this purpose. See U.S. Pat. Nos. 5,202,231 and 5,002,867.
[0017] The development of VLSIPS.TM. (Very Large Substrate
Immobilized Polymer Synthesis) technology has provided methods for
making very large combinations of oligonucleotide probes in very
small arrays. See U.S. Pat. No. 5,143,854 and PCT patent
publication Nos. WO 90/15070 and 92/10092, each of which is
incorporated herein by reference in its entirety for all purposes.
U.S. patent application Ser. No. 08/082,937, incorporated above,
also describes methods for making arrays of oligonucleotide probes
that can be used to provide the complete sequence of a target
nucleic acid and to detect the presence of a nucleic acid
containing a specific nucleotide sequence.
[0018] The construction of genetic linkage maps and the development
of physical maps are essential steps on the pathway to determining
the complete nucleotide sequence of the human or other genomes.
Present methods used to construct these maps rely upon information
obtained from a range of technologies including gel-based
electrophoresis, hybridization, polymerase chain reaction (PCR) and
chromosome banding. These methods, while providing useful mapping
information, are very time consuming when applied to very large
genome fragments or other nucleic acids. There is therefore a need
to provide improved methods for the identification and correlation
of genetic markers on a nucleic acid which can be used to rapidly
generate genomic maps. The present invention meets these and other
needs.
SUMMARY OF THE INVENTION
[0019] The present invention provides methods for identifying
specific oligonucleotide sequences using Type-IIs endonucleases in
sequential order to capture the ambiguous sequences adjacent to the
Type-IIs recognition sites. These ambiguous sequences can then be
probed sequentially with probes specific for the various
combinations of possible ambiguous base pair sequences. By
determining which probe hybridizes with an ambiguous sequence, that
sequence is thus determined. Further, because that sequence is
adjacent to a specific Type-IIs cleavage site that portion of the
sequence is also known. This contiguous sequence is useful as a
marker sequence in mapping genomic libraries.
[0020] In one embodiment, the present invention provides a method
of identifying sequences in a polynucleotide sequence. The method
comprises cleaving the polynucleotide sequence with a first
type-IIs endonuclease. A first adapter sequence, having a
recognition site for a second type-IIs endonuclease, is ligated to
the polynucleotide sequence cleaved in the first cleaving step. The
polynucleotide sequence resulting from the first ligating step, is
cleaved with the second type-IIs endonuclease, and a second adapter
sequence is ligated to the polynucleotide sequence cleaved in the
second cleaving step. The sequence of nucleotides of the
polynucleotide sequence between the first and second adapter
sequences is then determined.
[0021] In another embodiment, the present invention provides a
method of generating an ordered map of a library of genomic
fragments. The method comprises identifying sequences in each of
the genomic fragments in the library, as described above. The
identified sequences in each fragment are compared with the
sequences identified in each other fragment to obtain a level of
correlation between each fragment and each other fragment. The
fragments are then ordered according to their level of
correlation.
[0022] In a further embodiment, the present invention provides a
method of identifying polymorphisms in a target polynucleotide
sequence. The method comprises identifying sequences in a wild-type
polynucleotide sequence, according to the methods described above.
The identifying step is repeated on the target polynucleotide
sequence. The differences in the sequences identified in each of
the identifying steps are determined, the differences being
indicative of a polymorphism.
[0023] In still another embodiment, the present invention provides
a method of identifying a source of a biological sample. The method
comprises identifying a plurality of sequences in a polynucleotide
sequence derived from the sample, according to the methods
described herein. The plurality of sequences identified in the
identifying step are compared with a plurality of sequences
identically identified from a polynucleotide derived from a known
source. The identity of the plurality of sequences identified from
the sample with the plurality of sequences identified from the
known source is indicative that the sample was derived from the
known source.
[0024] In an additional embodiment, the present invention provides
a method of determining a relative location of a target nucleotide
sequence on a polynucleotide. The method comprises generating an
ordered map of the polynucleotide according to the methods
described herein. The polynucleotide is fragmented. The fragment
which includes the target nucleotide sequence is then determined,
and a marker on the fragment is correlated with a marker on the
ordered map to identify the approximate location of the target
nucleotide sequence on the polynucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows examples of combinations of Type-IIs
endonucleases useful in the present invention. Gaps in the sequence
illustrate the cleavage pattern of the first Type-IIs endonuclease,
shown to the left, whereas arrows illustrate the cleavage points of
the second Type-IIs endonuclease, shown to the right, when the
recognition site for that endonuclease is ligated to the first
cleaved sequence. FIG. 1 also shows the expected frequency of
cleavage of the first Type-IIs endonuclease, the number of
recognition sites in .lamda. DNA, and the size of the sandwiched
sequence.
[0026] FIG. 2 shows a schematic representation of an embodiment of
the present invention for capturing Type-IIs restriction sites
showing (1) a first cleavage with EarI, (2) followed by a ligation
to the 5' overhang of a first adapter sequence, (3) cleavage with
HgaI, (4) ligation to second adapter sequence followed by PCR
amplification (5).
[0027] FIG. 3 shows a schematic representation of a preferred
embodiment of the present invention using (1) a first cleavage with
EarI followed by DNA polymerization of the overhang to yield a
blunt end, (2) ligation to blunt end first adapter sequence, (3)
melting off the unligated adapter strand followed by DNA
polymerization to extend dsDNA across the first adapter strand, (4)
cleavage with HgaI at the EarI recognition site, (5) ligation of
second adapter sequence to target sequence, and (6)
amplification/transcription of the captured target sequence.
[0028] FIG. 4 shows the combinatorial design for an oligonucleotide
array used to probe a four nucleotide captured ambiguous sequence.
The probes upon the array are 15 mers having the sequence
3'C-T-G-C-G-w-x-y-z-C-T-T-C-T-C 5', where -w-x-y-z- are determined
by the probe's position on the array. For example, the probe
indicated by the darkened square on the array shown will have the
w-x-y-z sequence of -A-T-G-C-.
[0029] FIG. 5 shows the predicted and actual fluorescent
hybridization pattern of captured sequences from .lamda. DNA as
described in Example 1 upon an oligonucleotide array probe having
the combinatorial design of FIG. 4. Panel A shows the predicted
hybridization pattern where the darkened squares indicate expected
marker/probe hybridizations from captured sequences from .lamda.
DNA cut with EarI and captured with HgaI bearing adapter sequences.
The actual fluorescence of the hybridization is shown in panel
B.
[0030] FIG. 6 shows a portion of known map of a yeast chromosomal
library, illustrating the positions of each fragment of the library
within yeast chromosome IV.
[0031] FIG. 7A shows a plot of correlation coefficient scores among
hybridization patterns of yeast chromosomal fragments when using
Type-IIs and adjacent sequences as markers. FIG. 7B shows the
predicted "correlation" scores for EarI captured marker sequences
for fifty simulated sequences from yeast chromosome III. The inner
product scores for pair-wise comparison of the sequences is plotted
versus the percent overlap of the sequences. FIG. 7C shows the same
simulated correlation using BbsI captured marker sequences. FIG. 7D
shows a simulated correlation using HphI captured marker
sequences.
[0032] FIGS. 8A, 8B and 8C show a schematic representation of the
identification of polymorphic markers, using the methods of the
present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0033] In general, the present invention provides novel methods for
identifying and characterizing sequence based nucleic acid markers
as well as a method for determining their presence. The methods may
generally be used for generating maps for large, high molecular
weight nucleic acids, i.e., for mapping short clones, cosmids,
YACs, as well as in methods for genetic mapping for entire genomes.
Generally, the methods of the present invention involve the
capturing of ambiguous nucleic acid sequence segments using
sequential cleavage with restriction endonucleases. In particular,
the methods of the invention include a first cleavage which leaves
ambiguous sequences downstream from the recognition site of the
cleavage enzyme. A second type-IIs recognition site is ligated to
the target sequence, and a second cleavage, recognizing the second
site, cleaves upstream from the first cleavage site, within the
first recognition site, resulting in short sequences which contain
the recognition site and an ambiguous sequence "captured" from the
target sequence, between the two cleavage sites. The combination of
the recognition site and the captured sequences are particularly
useful as genetic markers for genomic mapping applications.
[0034] In one embodiment, the methods of the present invention
comprise the use of type-IIs endonucleases to capture sequences
adjacent to the type-IIs recognition site. These captured sequences
then become effective sequence based markers. More particularly,
this method comprises first treating the polynucleotide sequence
with a first Type-IIs endonuclease having a specific recognition
site on the sequence, thereby cleaving the sequence. A first
"adapter sequence" which comprises a second Type-IIs endonuclease
recognition site is ligated to the cleaved sequence. The resulting
heterologous sequence thus has an ambiguous sequence sandwiched
between two different Type-IIs endonuclease recognition sites. This
resulting sequence is then treated with a second Type-IIs
endonuclease specific for the ligated recognition site, thereby
cleaving the sequence. A second adapter sequence is then ligated to
this cleaved sequence. The sequence resulting from this ligation is
then probed to determine the sequence of the sandwiched, or
"captured", ambiguous sequence.
I. Type-IIs Endonucleases
[0035] Type-IIs endonucleases are generally commercially available
and are well known in the art. Like their Type-II counterparts,
Type-IIs endonucleases recognize specific sequences of nucleotide
base pairs within a double stranded polynucleotide sequence. Upon
recognizing that sequence, the endonuclease will cleave the
polynucleotide sequence, generally leaving an overhang of one
strand of the sequence, or "sticky end."
[0036] Type-II endonucleases, however, generally require that the
specific recognition site be palindromic. That is, reading in the
5' to 3' direction, the base pair sequence is the same for both
strands of the recognition site. For example, the sequence
TABLE-US-00001 .dwnarw. 5' -G-A-A-T-T-C- 3' 3' -C-T-T-A-A-G- 5'
.uparw.
is the recognition site for the Type-II endonuclease EcoRI, where
the arrows indicate the cleavage sites in each strand. This
sequence is palindromic in that both strands of the sequence, when
read in the 5' to 3' direction are the same.
[0037] The Type-IIs endonucleases, on the other hand, generally do
not require palindromic recognition sequences. Additionally, these
Type-IIs endonucleases also generally cleave outside of their
recognition sites. For example, the Type-IIs endonuclease EarI
recognizes and cleaves in the following manner: TABLE-US-00002
.dwnarw. -C-T-C-T-T-C-N-N-N-N-N- (SEQ ID NO:2)
-G-A-G-A-A-G-n-n-n-n-n .uparw.
where the recognition sequence is -C-T-C-T-T-C-, N and n represent
complementary, ambiguous base pairs and the arrows indicate the
cleavage sites in each strand. As the example illustrates, the
recognition sequence is non-palindromic, and the cleavage occurs
outside of that recognition site. Because the cleavage occurs
within an ambiguous portion of the polynucleotide sequence, it
permits the capturing of the ambiguous sequence up to the cleavage
site, under the methods of the present invention.
[0038] Specific Type-IIs endonucleases which are useful in the
present invention include, e.g., EarI, MnlI, PleI, AlwI, BbsI,
BsaI, BsmAI, BspMI, Esp3I, HgaI, SapI, SfaNI, BbvI, BsmFI, FokI,
BseRI, HphI and MboII. The activity of these Type-IIs endonucleases
is illustrated in FIG. 1, which shows the cleavage and recognition
patterns of the Type-IIs endonucleases.
II. Capturing Ambiguous Sequences Adjacent to Type-IIs Restriction
Sites
[0039] A general schematic of the capturing of the ambiguous
sequences is shown in FIGS. 2 and 3.
[0040] Treatment of the polynucleotide sequence sought to be mapped
with a Type-IIs endonuclease, results in a cleaved sequence having
a number of ambiguous, or unknown, nucleotides adjacent to a
Type-IIs endonuclease recognition site within the target sequence.
Additionally, within this ambiguous region, an overhang is created.
The recognition site and the ambiguous nucleotides are termed the
"target sequence." The overhang may be 2, 3, 4 or 5 or more
nucleotides in length while the ambiguous sequence may be from 4 to
9 or more nucleotides in length, both of which will depend upon the
Type-IIs endonucleases used. Examples of specific Type-IIs
endonucleases for this first cleavage include BsmAI, EarI, MnlI,
PleI, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI, SapI, SfaNI, BseRI,
HphI and MboII. Again, these first Type-IIs endonucleases and their
cleavage patterns are shown in FIG. 1, where the shaded region to
the left illustrates the recognition site of the first Type-IIs
endonuclease, and gaps in the sequence illustrate the cleavage
pattern of the enzyme. Cleavage of high molecular weight DNA with
EarI leaves an overhang of three ambiguous base pairs, as shown in
FIGS. 2 and 3, step 1. The recognition site of EarI is indicated by
the bar. Thus, EarI cleavage of the target nucleic acid will
produce a sequence having the following cleavage end:
TABLE-US-00003 -C-T-C-T-T-C-N- (SEQ ID NO:2)
-G-A-G-A-A-G-n-n-n-n
[0041] The overhanging bases are then filled in. This is preferably
carried out by treatment of the target sequence with a DNA
polymerase, such as Klenow fragment or T4 DNA polymerase, resulting
in a blunt end sequence as shown in FIG. 3, step 1. Alternatively
however, the overhang may be filled by the hybridization of this
overhang with an adapter sequence having an overhang complementary
to that of the target sequence, as shown in FIG. 2, step 2. A
tagging scheme, similar to this latter method has been described.
See, D.R. Smith, PCR Meth. and Appl. 2:21-27 (1992).
[0042] Following cleavage and fill in of the overhang portion, an
adapter sequence is typically ligated to the cleavage end. The
adapter sequences described in the present invention generally are
specific polynucleotide sequences prepared for ligation to the
target sequence. In preferred embodiments, these sequences will
incorporate a second type-IIs restriction site. Ligation of an
adapter including a HgaI recognition site is shown in FIGS. 2 and
3, step 2. The adapter sequences are generally prepared by
oligonucleotide synthesis methods generally well known in the art,
such as the phosphoramidite or phosphotriester methods described
in, e.g., Gait, oligonucleotide Synthesis: A Practical Approach,
IRL Press (1990).
[0043] An adapter sequence prepared to include a second type-IIs
recognition site, for example, the HgaI recognition site
3'-C-G-C-A-G-5' would be ligated to the cleaved target sequence to
provide a cleavage site on the other end of the ambiguous sequence.
For example, ligation of the HgaI adapter to the target sequence
would produce the following sequence having the cleavage pattern
shown: TABLE-US-00004 .dwnarw. -C-T-C-T-T-C-N-N-N-N-G-C-G-T-C-
-G-A-G-A-A-G-n-n-n-n-C-G-C-A-G- .uparw.
[0044] In addition to the Type-IIs recognition sites, preferred
adapter sequences will also generally include PCR primers and/or
promoter sequences for in vitro transcription, thereby facilitating
amplification and labeling of the target sequence.
[0045] The method of ligation of the first adapter sequence to the
target sequence may be adapted depending upon the particular
embodiment practiced. For example, where ligation of the first
adapter sequence is to the overhang of the target sequence, as
shown in FIG. 2, step 2, the adapter sequence will generally
comprise an overhang which is complementary to the overhang of the
target sequence. For this embodiment, a mixture of adapter
sequences would generally be used wherein all possible permutations
of the overhang are present. For example, the number of specific
probe sequences will typically be about 4.sup.m where m is the
number of overhanging nucleotides. For example, where the target
sequence after the first cleavage has a 4 base pair overhang of
ambiguous nucleotides, the mixture of sequences would typically
comprise adapters having upwards of 4.sup.4, or 256 different
overhang sequences. Where the overhang in question includes greater
numbers of nucleotides, the adapters would generally be provided in
two or more separate mixtures to minimize potential ligation of the
adapters within each mixture. For example, one set of adapters may
incorporate a pyrimidine nucleotide in a given position of the
overhang for all adapters in the mixture whereas the other set will
have a purine nucleotide in that position. As a result, ligation of
the adapters to adapters in the same mixture will be substantially
reduced. For longer overhang sequences, it may often be desirable
to provide additional separate mixtures of adapters. Ligation of
the adapter sequence to the target sequence is then carried out
using a DNA ligase according to methods known in the art.
[0046] Where the overhang of the target sequence is filled in by
Klenow fragment polymerization, as in FIG. 3, step 1, a blunt end
adapter sequence is ligated to the target sequence. See, FIG. 3,
step 2. Because a blunt end ligation is used rather than an
overhang, a mixture of hybridizable sequences is unnecessary, and a
single adapter sequence is used. Further, this method avoids any
hybridization between the overhangs in the mixture of adapter
sequences.
[0047] Using this method, the polymerized target sequence will be
phosphorylated on only the 5' strand. Further, as the adapter will
have only 3' and 5' hydroxyls for ligation, only the 3' end of the
adapter will be ligated to the blunt, phosphorylated 5' end of the
target sequence, leaving a gap in the other strand. The unligated
strand of the adapter sequence may then be melted off and the
remaining polynucleotide again treated with DNA polymerase, e.g.,
Klenow or E. coli DNA polymerase, as shown in FIG. 3, step 3,
resulting in a double-stranded, heterologous polynucleotide. This
polynucleotide has the ambiguous nucleotide sequence sandwiched
between the first Type-IIs endonuclease recognition site ("site
A"), and the second, ligated Type-IIs recognition site ("site B").
One skilled in the art will recognize that approximately half of
the adapter sequences will ligate to the target sequence in an
inverted orientation. However, this does not affect the results of
the methods of the present invention due to the inability of the
second type-IIs enzyme to cleave the target sequence in those cases
where the adapter is inverted. This is discussed in greater detail,
below.
[0048] The polynucleotide resulting from ligation of this first
adapter sequence to the target sequence is then treated with a
second Type-II endonuclease specific for the ligated recognition
site B. This second endonuclease treatment cleaves the remainder of
the original polynucleotide from the target sequence. In preferred
aspects, the second type-IIs endonuclease will be selected, or the
second recognition site will be positioned within the adapter
sequence, whereby the cleavage pattern of the second Type-IIs
endonuclease results in the second cleavage substantially or
entirely overlapping the first recognition site A, i.e., the
cleavage of each strand is within or adjacent to the first
recognition site (site A). FIG. 2, step 3, and FIG. 3, step 4 show
the cleavage of the polynucleotide using HgaI (the HgaI recognition
site is shown by the bar). Where the adapter sequence is ligated in
a reverse orientation, as previously noted, no cleavage will occur
within the first recognition site, as the recognition site will be
at the distal end of the adapter sequence. Further, any primer
sequences present within this adapter will be inverted preventing
subsequent amplification. By selecting a second Type-IIs
endonuclease different from the first, recleavage of the first
cleavage site is avoided. Selection of an appropriate type-IIs
endonuclease for the second cleavage, and thus, the appropriate
recognition site for the first adapter sequence, may often depend
upon the first endonuclease used, or as described above, the
position of the recognition site within the adapter. In preferred
aspects, the first and second type-IIs endonucleases are selected
whereby the second endonuclease cleaves entirely within the first
endonucleases recognition sequence. Examples of Type-IIs
endonucleases for the second cleavage generally include those
described above, and are typically selected from HgaI, BbvI, BspMI,
BsmFI and FokI. Particularly preferred combinations of Type-IIs
endonucleases for the first and second cleavages, as well as their
cleavage patterns are shown in FIG. 1. Continuing with the previous
example, HgaI cleavage of the sample target sequence would produce
the following sequence having the ambiguous base pairs captured by
the first adapter sequence: TABLE-US-00005
-C-T-C-T-T-C-N-N-N-N-G-C-G-T-C- -G-n-n-n-n-C-G-C-A-G-
[0049] Depending upon the type-IIs endonucleases used in each step,
the sequence of the overhang is known. For example, in the above
example, the HgaI cleavage site for the second endonuclease is
within the first endonuclease's recognition site, e.g., the EarI
site. An example of a known overhang sequence is demonstrated in
FIGS. 2 and 3, steps 4 and 5, respectively.
[0050] As noted, in the preferred aspects the second cleavage site
substantially or entirely overlaps the first recognition site A.
Accordingly, the number of possible hybridizing sequences for this
ligation step is rendered unique. The specific recognition site A
of the first Type-IIs endonuclease is known. Thus, where the second
cleavage occurs entirely within the first recognition site A, only
the unique sequences hybridizing to that sequence would be used. On
the other hand, where the second cleavage occurs to some extent
outside of the first recognition site A, a mixture of specific
adapter sequences hybridizable to all possible permutations of
nucleotides outside of site A is used. For example, where cleavage
incorporates one nucleotide outside of the first recognition site,
the four variations to the known sequence are possible and a
mixture of adapter sequences hybridizable to all four is used (See,
e.g., MnlI-HgaI enzyme pairing in FIG. 1). The number of bases
included in the second cleavage which fall outside the first
recognition site is readily determinable from the endonucleases
used.
[0051] As with the first adapter sequence, the second adapter
sequence may comprise a PCR primer sequence and/or a promoter
sequence for in vitro transcription.
[0052] The resulting target sequence will thus have the target
sequence, specifically, an ambiguous sequence attached to a portion
or all of the first recognition site, sandwiched or captured
between the two adapter sequences. For example, the resulting
target sequence will generally have the general sequence: (Adapter
sequence/A)-(Ambiguous sequence)-(B/Adapter sequence)
[0053] where A is a portion or all of the recognition site for the
first Type-IIs endonuclease, and B is the recognition site for the
second Type-IIs endonuclease. Again, applying the previous example,
the resulting target sequence would appear as follows:
TABLE-US-00006 Adapter 2 2 -C-T-C-T-T-C-N-N-N-N-G-C-G-T-C- Adapter
1 Adapter 2' -G-A-G-A-A-G-n-n-n-n-C-G-C-A-G- Adapter 1'
[0054] The sequence -C-T-C-T-T-C-N-N-N-N- is captured from the
original target sequence and sandwiched between the two adapter
sequences.
[0055] Prior to probing, the target sequence will generally be
amplified to increase the detectability of the sequence.
Amplification is generally carried out by methods well known in the
art. See FIGS. 2 and 3, steps 5 and 6, respectively. For example,
amplification may be performed by way of polymerase chain reaction
(PCR) using methods generally well known in the art. See, e.g.,
Recombinant DNA Methodology, Wu, et al., ed., Academic Press
(1989), Sambrook, et al., Molecular Cloning: A Laboratory Manual
(2nd ed.), vols. 1-3, Cold spring Harbor Laboratory, (1989),
Current Protocols in Molecular Biology, F. Ausubel, et al., ed.,
Greene Publishing and Wiley Interscience, New York (1987 and
periodic updates). As described earlier, this amplification may be
facilitated by the incorporation of specific primer sequences or
complements within the adapters. Further, such amplification may
also incorporate a label into the amplified target sequence. In a
preferred embodiment, the target sequence may be amplified using an
asymmetric PCR method whereby only the strand comprising the
appropriate recognition site A is amplified. Asymmetric
amplification is generally carried out by use of primer which will
initiate amplification of the appropriate strand of the target
sequence, i.e., the target sequence.
[0056] The amplified target sequence may then be probed using
specific oligonucleotide probes capable of hybridizing to the
(A)-(ambiguous sequence)-(B) target sequence. As both the A and B
sequences are set by the capturing method and are known, the probes
need only differ with respect to the ambiguous portion of the
sequence to be probed. For example, using the example sequence
provided above, assuming that one is probing with the top strand,
e.g., the bottom strand was amplified by appropriate selection of
primers, etc., the probes would generally have the sequence
C-T-C-T-T-C-n-n-n-n-G-C-G-T-C, where n denotes every possible base
at the particular position, e.g., A, T, G, C. The preparation of
oligonucleotide probes is performed by methods generally known in
the art. See, Gait, Oligonucleotide Synthesis: A Practical
Approach, IRL Press (1990). Additionally, these oligonucleotide
probes may be labelled, i.e., fluorescently or radioactively, so
that probes which hybridize with target sequences can be detected.
In preferred aspects, however, the probes will be immobilized, and
it will be the target that is labelled. Labelling of the target
sequence may be carried out using known methods. For example,
amplification of the target sequence can incorporate a label into
the amplified target sequence, e.g., by use of a labelled PCR
primer or by incorporating a label during in vitro transcription of
either strand.
[0057] In the preferred embodiment of the present invention, the
target sequence is probed using an oligonucleotide array. Through
the use of these oligonucleotide arrays, the specific hybridization
of a target sequence can be tested against a large number of
individual probes in a single reaction. Such oligonucleotide arrays
employ a substrate, comprising positionally distinct sequence
specific recognition reagents, such as polynucleotides, localized
at high densities. A single array can comprise a large number of
individual probe sequences. Further, because the probes are in
known positionally distinct orientations on the substrate, one need
only examine the hybridization pattern of a target oligonucleotide
on the substrate to determine the sequence of the target
oligonucleotide. Use and preparation of these arrays for
oligonucleotide probing is generally described in PCT patent
publication Nos. WO 92/10092, WO 90/15070, U.S. patent application
Ser. Nos. 08/143,312 and 08/284,064. Each of these references is
hereby incorporated by reference in its entirety for all
purposes.
[0058] As noted, the target sequence will have the general
sequence: (Adapter sequence/A)-(N.sub.k)-(B/Adapter sequence) where
N.sub.k denotes the ambiguous sequence of nucleotides of length k,
and the nucleotide sequence of each adapter sequence is known and
the sequence of sites A and B are known. Only the nucleotide
sequence of the ambiguous portion of the target sequence, N.sub.k
is not known. Thus, the number of probes required on the array
substrate is generally related to the number of ambiguous
nucleotides in the target sequence. In one embodiment, the number
of potential sequences for an ambiguous sequence is 4.sup.k, where
k is the number of ambiguous bases within the sequence. For
example, where there are four ambiguous nucleotides within the
target sequence, the array would generally include about 4.sup.4 or
256 or more separate probes, where each probe will include the
general sequence: (A')-(N'.sub.k)-(B') where "A'" and "B'" are the
complements to site-A and site-B of the target sequence,
respectively and are constant throughout the array, and "N'.sub.k"
generally represents all potential sequences of the length of the
ambiguous sequence of the target sequence. Thus, where the
ambiguous sequence contains, e.g., 4 nucleotides, "N'.sub.k" would
typically include, for example, 4.sup.4 different sequences, at
least one of which will hybridize with the target sequence. On an
oligonucleotide array, this is accomplished through a simple
combinatorial array like that shown in FIG. 4. Typically, as the
size of the ambiguous sequence increases, the number of probes on
the array will also increase, e.g., where the ambiguous sequence is
8 bases long, their will typically be about 4.sup.8 or 65,536
probes on the array.
[0059] In the case of high molecular weight nucleic acids, the
original polynucleotide sequence will generally comprise more than
one and even several specific Type-IIs endonuclease
recognition/cleavage sites, e.g., EarI sites. As a result, a number
of ambiguous sequence segments will be captured for a given
polynucleotide. Upon probing with an oligonucleotide array, the
sequence will hybridize with a number of probes which are
complementary to all of the captured sequences, producing a
distinctive hybridization pattern for the given polynucleotide
sequence. The specific hybridization pattern of the target sequence
upon the array will generally indicate the ambiguous sequences
adjacent to all of the cleavage sites as was described above.
III. Mapping Genomic Libraries
A. Physical Maps
[0060] A further embodiment of the present invention provides a
method for the ordered mapping of genomic libraries. Typically, the
term "genomic library" is defined as a set of sequence fragments
from a larger polynucleotide fragment. Such larger fragments may be
whole chromosomes, subsets thereof, plasmids, or other similar
large polynucleotides. Specifically, the methods of the present
invention are useful for mapping high molecular weight
polynucleotides including chromosomal fragments, cosmids and Yeast
Artificial Chromosomes (YACs).
[0061] Mapping techniques typically involve the identification of
specific genetic markers on individual polynucleotide fragments
from a genomic library. Comparison of the presence and relative
position of specific markers on fragments generated by different
cleavage patterns allows for the assembly of a contiguous genomic
map, or "contig".
[0062] Accordingly, in a particularly preferred aspect of the
present invention methods of genomic mapping are provided utilizing
the sequence capturing methods already described. In particular,
the methods of the present invention comprise identifying the
Type-IIs and adjacent sequences (target sequences) on the
individual fragments of a genomic library using the methods
described above. FIG. 6 shows a genomic map for a portion of a
yeast chromosome library, showing the overlap between the various
fragments of the library.
[0063] The individual fragments of the library are treated using
the above methods to capture the Type-IIs restriction sites and
their adjacent ambiguous sequences. These captured sequences are
then used as genetic markers, as described above, and a contig of
the particular library may be assembled. In the preferred aspects,
the captured Type-IIs and adjacent sequences will be hybridized to
specific positionally oriented probes on the array. By determining
the various probe sequences to hybridize with the captured
sequences, these captured sequences are thereby determined.
[0064] The combination of these mapping techniques with
oligonucleotide arrays provides the capability of identifying a
large number of genetic markers on a particular sequence.
Typically, a genomic fragment will have more than one, and even
several Type-IIs restriction sites within its sequence. Thus, when
probed with an oligonucleotide array, the captured sequences from a
particular genomic fragment will hybridize with a number of probes
on the array, producing a distinctive hybridization pattern. Each
hybridization pattern will generally comprise hybridization signals
which correspond to each of the captured sequence markers in the
fragment.
[0065] When repeated on separate fragments from the library, each
fragment will generally produce a distinctive hybridization
pattern, which reflects the sequences captured using the specific
type-IIs capture method. These hybridization patterns may be
compared with hybridization patterns from differentially generated
fragments. Where a specific marker is present in both fragments, it
is an indication of potential overlap between the fragments. Two
fragments that share several of the same Type-IIs sequences, e.g.,
overlapping fragments, will show similar hybridization patterns on
the oligonucleotide array.
[0066] The greater the similarity or correlation between two
fragments, the higher the probability that these fragments share an
overlapping sequence. By correlating the hybridization pattern of
each fragment in the library against each other fragment in the
library, a single contiguous map of the particular library can be
constructed.
[0067] In practice, each fragment is correlated to each other
fragment, and a correlation score is given based upon the number of
probes which cross-hybridize with the Type-IIs and adjacent
sequences of both the first and second fragment. High scores
indicates high overlap. For example, a perfect overlap, i.e., the
comparison of two identical sequences would produce a correlation
score of 1. Similarly, sequences sharing no overlapping sequence
would, ideally, produce a correlation score of 0. However, in
practice, sequences that do not overlap will generally have
correlation scores above zero, due to potential non-specific
hybridizations, e.g., single base mismatches, background
hybridization, duplicated sequences, which may provide some
baseline correlations between otherwise unrelated fragments. As a
result, a cutoff may be established below which correlation scores
are not used. The precise cutoff may vary depending upon the level
of nonspecific hybridizations for the particular application. For
example, by using capture methods that cut less frequently, and/or
capture a greater number of sequences, the potential for duplicated
markers is substantially reduced, and the cutoff may be lower.
Correlation scores among all of the fragments may then be
extrapolated to provide approximate percent overlap among the
various fragments, and from this data, a contiguous map of the
genomic library can be assembled (FIG. 7A). Additionally, one of
skill in the art will appreciate that a more stringent
determination of cosmid overlap may be obtained by repeating the
capture and correlation methods using a different enzyme system,
thereby generating additional, different markers and overlap
data.
[0068] The combined use of sequence based markers and
oligonucleotide arrays, as described herein, provides a method for
rapidly identifying a large number of genetic markers and mapping
very large nucleic acid sequences, including, e.g., cosmids,
chromosome fragments, YACs and the like.
[0069] The present invention also provides methods for diagnosing a
genetic disorder wherein said disorder is characterized by a
mutation in a sequence adjacent to a known Type-IIs endonuclease
restriction site using the methods described above. Specifically,
sequences adjacent to Type-IIs restriction sites are captured and
their sequence is determined according to the methods described
above. The determined sequence is then compared to a "normal"
sequence to identify mutations.
A. Genetic Linkage Mapping
[0070] Genetic linkage markers are defined as highly polymorphic
sequences which are uniformly distributed throughout a genome. In
an additional embodiment, the methods of the present invention are
used to identify and define these polymorphic markers. Because
these markers are identified and defined by their proximity to
type-IIs restriction sites, they are referred to herein as
restriction site sequence polymorphisms ("RSSPs"). In general,
these RSSP markers are identified by comparing captured sequences
among two genomes. The methods of the present invention may
generally be used to identify these RSSPs in a number of ways. For
example, a polymorphism within the recognition site of the type-IIs
endonuclease will result in the presence of a captured sequence in
one genome where it is absent in the other. This is generally the
result where the polymorphism lies within the type-IIs recognition
site, thereby eliminating the recognition site in the particular
sequence, and, as a result, the ability to capture the adjacent
sequences. It will be appreciated that the inverse is also true,
that a polymorphism may account for the presence of a recognition
site where one does not exist in the wild type. Second, a
polymorphism may be identified which lies within the captured
ambiguous sequence. These polymorphisms will typically be detected
as a sequence difference between the compared genomes.
[0071] A wide variety of polymorphic markers may be identified for
any given genome, based upon the type-IIs enzymes used for the
first and second cleavages. For example, first cleavage enzymes
which recognize distinct sequences will typically also define a
number of distinct proximal polymorphisms.
[0072] The above described methods may be further modified, for
example, using methods similar to those reported by Nelson, et al.,
Nature Genetics (1993) 4:11-18. Nelson, et al. report the
identification of polymorphic markers using a system of genetic
mismatch scanning. In the method of Nelson, at al., the genomes to
be compared, e.g., grandchild and grandparent genomes, are first
digested with an endonuclease which produces a 3' overhang, i.e.,
PstI. One of the two genomes is methylated at all GATC sites in the
sequence (DAM+) while the other remains unmethylated (DAM-). The
genomic fragments from each group are denatured, mixed with each
other, and annealed, resulting in a mixture of homohybrids and
heterohybrids. In the homohybrids, both strands will be either
methylated or unmethylated, while in the heterohybrids, one strand
will be methylated. The mixture is then treated with nucleases
which will not cleave the hemimethylated nucleic acid duplexes, for
example DpmI and HboI. Next, the mixture is treated with a series
of mismatch repair enzymes, e.g., MutH, MutL and MutS, which
introduce a single strand nick on the duplexes which possess single
base mismatches. The mixture is then incubated with ExoIII, a 3' to
5' exonuclease which is specific for double stranded DNA, and which
will degrade the previously digested homohybrids and the nicked
strand of the mismatched heterohybrids, from the 3' side.
Purification of the full dsDNA is then carried out using methods
known in the art, e.g., benzoylated naphthoylated DEAE cellulose at
high salt concentrations, which will bind ssDNA but not dsDNA. As a
result, only the full-length, unaltered (perfectly matched)
heterohybrids are purified. The recovered dsDNA fragments which
indicate "identity by descent" (or "i.b.d.") are labelled and used
to probe genomic DNA to identify sites of meiotic
recombination.
[0073] An adaptation of the above method can be applied to the
capture methods of the present invention. In particular, the
methods of the present invention can be used to capture sequences
in the region of polymorphisms in a particular polynucleotide
sequence. FIGS. 8A, 8B and 8C show a schematic representation of
the steps used in practicing one embodiment of this aspect of the
present invention. Specifically, a subset of genomic DNA which is
identified by the presence of a type-IIs recognition site is
amplified (FIG. 8A), DNA containing polymorphisms within the
amplified subset are isolated (FIG. 8B), and the sequences adjacent
to the type-IIs recognition site in the isolated
polymorphism-containing sequences are identified and characterized
(FIG. 8C).
[0074] Initially, polynucleotides from different sources which are
to be compared, e.g., grandparent-grandchild, etc., are treated
identically in parallel systems. These polynucleotides are each
cleaved with a first type-IIs endonuclease, as is described in
substantial detail above. In FIG. 8A, step (a), for example, this
first cleavage is shown using BseR1. The specific Type-IIs enzyme
used in this first cleavage may again vary depending upon the
desired frequency of cleavage, the length of the target sequence,
etc.
[0075] As previously described, a first adapter bearing a second
type-IIs endonuclease recognition site is ligated to the cleaved
polynucleotides (FIG. 8A, step (b)). In the example of FIG. 8A,
steps (a), (b) and (c), this recognition site is that of the
type-IIs endonuclease FokI. The polynucleotides are then cleaved
with an endonuclease which will cleave upstream from the captured
sequence and ligated first adapter, such as a type II endonuclease,
e.g., HaeIII (see FIG. 8A, step (d)). Typically, this second
cleavage enzyme will be selected whereby it cleaves more frequently
than the first Type-IIs enzyme. A second adapter sequence may then
be ligated to this new cleavage site (FIG. 8A, step (e)). The
entire sequence, including the two adapter sequences is then
typically amplified (FIG. 8A, step (f)). The amplification is
facilitated in preferred aspects by incorporating a primer sequence
within the adapter sequences.
[0076] The amplified polynucleotides from each source is isolated
(FIG. 8B, step (g)). The polynucleotide from one source is then
methylated (FIG. 8B, step (h)). Both the methylated polynucleotide
from the first source and the un-ethylated polynucleotide from the
second source are mixed together, heated to denature duplex DNA,
and reannealed (FIG. 8B, step (i)). This generally results in a
mixture of hemimethylated heterohybrids having one strand from each
source, homohybrids of unmethylated dsDNA and homohybrids of fully
methylated dsDNA. At this point, unlike the method of Nelson, et
al. (DpmI and MboI additions are omitted), the mixture is treated
with the mismatch repair enzymes, e.g., MutLSH, which will nick
only hemimethylated, mismatched hybrids, leaving the homohybrids
and perfectly matched heterohybrids untouched (FIG. 8B, step (j)).
The nicked DNA is then digested, as in Nelson, et al., with an
exonuclease, e.g., ExoIII (FIG. 8B, step (k)). The mixture will
then contain dsDNA which is fully methylated, i.e., homohybrids of
DNA from one source, dsDNA which is unmethylated, i.e., homohybrids
of DNA from the other source, heterohybrids of dsDNA from both
sources, but which are perfectly matched, i.e., contains no
mismatches or polymorphisms, and ssDNA, i.e., the DNA which is left
from the heterohybrid, mismatched or polymorphic dsDNA. This ssDNA
reflects the polymorphism and may then be purified from the dsDNA
using the methods described in Nelson, et al., e.g., purification
over benzoylated naphthoylated DEAE cellulose in high salt (FIG.
8C, step (l)).
[0077] The purified single stranded DNA is then reamplified to
dsDNA using methods well know in the art, e.g., PCR (FIG. 8C, step
(m)). The amplified DNA may then cleaved with a second type-IIs
endonuclease which recognizes the site incorporated into the first
adapter sequence, as described above (FIG. 8C, step (n)), followed
by ligation of another adapter sequence to the cleavage end (FIG.
8C, step (o)). The captured sequence thus identifies a polymorphism
is which lies between the captured sequence and the upstream
cleavage site. The captured sequence may then be determined
according to the methods described herein, e.g., amplification,
labelling and probing (FIG. 8C, step (p)).
IV. Applications
[0078] The methods described herein are useful in a variety of
applications. For example, as is described above, these methods can
be used to generate ordered physical maps of genomic libraries, as
well as genetic linkage maps which can be used in the study of
genomes of varying sources. The mapping of these genomes allows
further study and manipulation of the genome in diagnostic and
therapeutic applications, e.g., gene therapy, diagnosis of genetic
predispositions for particular disorders and the like.
[0079] In addition to pure mapping applications, the methods of the
present invention may also be used in other applications. In a
preferred embodiment, the methods described herein are used in the
identification of the source of a particular sample. This
application would include forensic analysis to determine the origin
of a particular tissue sample, such as analyzing blood or other
evidence in criminal investigations, paternity investigations, etc.
Additionally, these methods can also be used in other
identification applications, for example, taxonomic study of
plants, animals, bacteria, fungi, viruses, etc. This taxonomic
study includes determination of the particular identity of the
species from which a sample is derived, or the interrelatedness of
samples from two separate species.
[0080] The various identification applications typically involve
the capturing and identification of sequences adjacent specific
type-IIs restriction sites in a sample to be analyzed, according to
the methods already described. These sequences are then compared to
sequences identically captured and identified from a known source.
Where sequences captured from both the sample and the source are
identical or highly similar, it is indicative that the sample was
derived from the source. Where the sequences captured from the
sample and known source share a large number of identical
sequences, it is indicative that the sample is related to the known
source. However, where the sample and source share few like
sequences, it is indicative of a low probability of
interrelation.
[0081] Precise levels of interrelation to establish a connection
between source and sample, i.e., captured sequence homology, will
typically be established based upon the interrelation which is
being proved or disproved, the identity of the known source, the
precise method used, and the like. Establishing the level of
interrelation is well within the ordinary skill in the art. For
example, in criminal investigations, a higher level of homology
between sample and known source sequences will likely be required
to establish the identity of the sample in question. Typically, in
the criminal context, interrelation will be shown where there is
greater than 95% captured sequence homology, preferably greater
then 99% captured sequence homology, and more preferably, greater
than 99.9% captured sequence homology. For other identification
applications, interrelation between sample and known source may be
established by a showing of, e.g., greater than 50% captured
sequence homology, and typically greater than 75% captured sequence
homology, preferably greater than 90% captured sequence homology,
and more preferably greater than 95 to 99% captured sequence
homology.
[0082] The level of interrelation will also typically vary
depending upon the portion of a genome or nucleic acid sequence
which in used for comparison. For example, in attempting to
identify a sample as being derived from one member of a species as
opposed to another member of the same species, it will generally be
desirable to capture sequences in a region of the species' genetic
material which displays a lower level of homology among the various
members of the same species. This results in a higher probability
of the captured sequences being specific to one member of the
species. The opposite can be true for taxonomic studies, i.e., to
identify the genus and species of the sample. For example, it may
generally be desirable to select a portion of the genetic material
of the known genus or species which is highly conserved among
members of the genus and/or species, thereby permitting
identification of the particular sample to that genus or
species.
[0083] The present invention is further illustrated by the
following examples. These examples are merely to illustrate aspects
of the present invention and are not intended as limitations of
this invention. The methods used generally employ commercially
available reagents or reagents otherwise known in the art.
EXAMPLES
Example 1
1. Digesting High Molecular Weight DNA with EarI
[0084] 4 .mu.g of .lamda. DNA was treated with 4 units of EarI in
10 .mu.l at 37.degree. C. for 4 hours. The reaction was then heated
to 70.degree. C. for 10 minutes. Cleavage was verified by running 5
.mu.l of the sample on an agarose gel to determine complete
cleavage. The remaining 5 .mu.l was brought to 40 .mu.l (final
concentration of 50 ng/.mu.l .lamda. DNA).
2. Klenow Fill-In Reaction
[0085] 4 .mu.l of the digested .lamda. DNA was added to 0.5 .mu.l
of 10.times. Klenow Buffer, 0.5 .mu.l 2 mM dNTPs, and 0.05 .mu.l of
0.25 units of Klenow fragment. The reaction mixture was incubated
for 20 minutes at 25.degree. C., followed by 10 minutes at
75.degree. C. Similar results were also obtained using T4 DNA
polymerase for the fill-in reaction.
3. Preparing Adapter Sequences
[0086] Two separate adapter sequences were prepared, adapter
sequence 1 and adapter sequence 2. Adapter sequence 1 is used in
the first ligation reaction whereas adapter 2 is used for the
second. As each adapter and its ligation are somewhat different,
they are addressed separately.
[0087] Double stranded adapter 1 comprising the second Type-IIs
endonuclease restriction site 3' C-G-C-A-G- . . . 5' and a T7
promoter sequence was prepared by adding 10 .mu.l each of 10 .mu.M
unphosphorylated T7 strand and its complement, heating the mixture
to 95.degree. C., then cooling over 20 minutes to anneal the
strands. The strands were prepared using DNA synthesis methods
generally well known in the art. The resulting mixture had a final
dsDNA adapter concentration of 5 .mu.M.
[0088] Adapter 2 comprising the overhang complementary to that
created by the HgaI digestion of the target sequence, as well as a
T3 promoter sequence was prepared by first creating the overhang
region. A single stranded oligonucleotide of the sequence 3' . . .
-G-A-G-A-A 5' was synthesized on a single-stranded T3 promoter
sequence. The final concentration of reagents is shown in
parentheses. The 5' end of this sequence was then phosphorylated as
follows: 10 .mu.l of 10 .mu.M the oligonucleotide (5 .mu.M), 2
.mu.l of 10.times. kinase buffer (1.times.), 2 .mu.l 10 mM ATP (1
mM), 5 .mu.l water and 1 .mu.l T4 polynucleotide kinase (10 units)
were added. The reaction was incubated at 37.degree. C. for 60
minutes, then at 68.degree. C. for 10 minutes and cooled.
[0089] To the T3/overhang ssDNA strand was added 10 .mu.l of 10
.mu.M appropriate antistrand and 3.33 .mu.l of buffer. This mixture
was heated to 95.degree. C. and cooled over 20 minutes to anneal
the two strands.
4. Ligation of First Adapter to Target Sequence
[0090] At least a 50:1 molar ratio of first adapter to cleavage
ends was desired and an approximate ratio of 100:1 adapters to
cleavage ends was targeted. An .lamda. DNA digested with EarI is
known to result in 34 pairs of cleavage ends, a 3400:1 mole ratio
of adapters to .lamda. DNA was used.
[0091] In 11 .mu.l total reaction mixture, the following were
combined, 5 .mu.l from the fill-in reaction (approx. 40 nmoles
target DNA), 4 .mu.l of 5 .mu.M first adapter (2 .mu.M final
concentration), 1.1 .mu.l 10.times. ligation buffer (1.times. final
concentration), and 1 .mu.l of T4 DNA ligase (400 units final
concentration).
[0092] The reaction was incubated at 25.degree. C. for 2 hours,
then incubated at 75.degree. C. for 10 minutes to inactivate the
ligase as well as dissociate unligated adapter strand.
5. Second Klenow Fill-In Reaction
[0093] Filling in the single stranded portion of the target
sequence/first adapter created by dissociation of the unligated
strand in step 4 above, was accomplished using the Klenow fragment
DNA polymerase.
[0094] In 14 .mu.l total was added 11 .mu.l of DNA to which the
first adapter had been ligated (approx. 34.4 nM total adapted
ends), 1.5 .mu.l 10.times. Klenow buffer (1.times.), 1.5 .mu.l 2 mM
dNTPs (50 .mu.M each dNTP) and 0.05 .mu.l Klenow fragment (0.25
units). This mixture was incubated at 37.degree. C. for 30 minutes,
then heated to 75.degree. C. for 10 minutes. Again, similar results
were obtained using E. coli DNA polymerase.
6. Second Digestion with HgaI
[0095] To the 14 .mu.l reaction mixture of step 6 was added 1 .mu.l
of HgaI (2 units). The reaction was incubated at 25.degree. C. for
3 hours. 1.6 .mu.l of 5 M NaCl (0.5 M) was then added to raise the
melting point of the target sequence to above 70.degree. C. The
reaction mixture was then heated to 65.degree. C. for 20
minutes.
7. Ligation of Second Adapter to Target Sequence
[0096] The 16 .mu.l reaction mixture from step 7 is expected to
have an approximate concentration of 4.4 nM target sequence with
compatible ends for the second ligation. This number is halved from
the expected concentration of total target sequence. This was to
account for the blunt end ligation of adapter 1 in the reverse
orientation such that HgaI cleavage would not occur.
[0097] To the 16 .mu.l reaction mixture from step 7, was added 5
.mu.l of 3 .mu.M second adapter prepared in step 3, above (0.3
.mu.M), 5 .mu.l 10.times. ligation buffer (1.times.), 23.5 .mu.l
water and 0.5 .mu.l T4 DNA ligase (200 units). The reaction mixture
was incubated at 37.degree. C. for 30 minutes then heated to
65.degree. C. for 10 minutes.
8. PCR Amplification
[0098] 5 .mu.l of the captured target sequence from step 7 is used
as the template for PCR amplification (approx. 440 pM total; 14.7
pM each end). To this was added 1.25 .mu.l each of 10 .mu.M T7
primer, and 10 .mu.M T3 primer (0.25 .mu.M primer), 5 .mu.l
10.times. PCR buffer (1.times.), 5 .mu.l 4.times. 2 mM dNTPs (200
.mu.M each dNTP), 24.5 .mu.l water and 0.5 .mu.l Taq polymerase
(2.5 units).
[0099] PCR was carried out for 40 cycles of 94.degree. C. for 30
seconds, 55.degree. C. for 30 seconds and 72.degree. C. for 30
seconds. Controls were run using water, .lamda. DNA cut with EarI
and uncut .lamda. DNA subjected to steps 1-7. 2 .mu.l from the
reaction was run on a 4% NuSieve.RTM. Agarose gel, indicating a
62-bp amplicon which is carried into the next step.
9. Labelling--Asymmetric PCR
[0100] The 62-bp amplicon produced in step 8 is next labeled with a
5'-F label by asymmetric PCR.
[0101] 44 .mu.l of the PCR amplicon from step 8 (150 fmoles) is
mixed with 5 .mu.l of 10 .mu.M T7-5'F primer (1 .mu.M primer), 2
.mu.l of 10.times. PCR buffer (1.times. buffer), 3 .mu.l of 100 mM
MgCl.sub.2 (5 mM), 5 .mu.l of 4.times. 2 mM dNTPs (200 .mu.M each
dNTP) and 0.5 .mu.l Taq polymerase (2.5 units).
[0102] PCR was carried out for 40 cycles as described in step 8. 3
.mu.l from this reaction was the run on 4% NuSieve.RTM. Agarose gel
and compared to the amplicon from step 8 to confirm florescent
labelling.
9. Results
[0103] The florescent captured sequence was heated to 95.degree. C.
briefly, then buffered with 6.times. SSPE, 10 mM CTAB and 0.2%
Triton X-100. the captured sequence was then probed on an
oligonucleotide array having the combinatorial array shown in FIG.
4. FIG. 5, panel A shows the expected hybridization pattern of
.lamda. DNA to the array of FIG. 4 an denoted by the blackened
regions on the array. FIG. 5, panel B illustrates the actual
hybridization pattern of captured Type-IIs sites from .lamda. DNA
on an array as shown in FIG. 4. The close correlation between
expected and actual hybridization is evident.
Example 2
[0104] The above capture methods were applied to a genomic library
of 12 known cosmids from yeast chromosome IV. The clones have been
previously physically mapped using EcoRI-HindIII fragmentation. The
specific library, including known zap positions and overlap of the
12 cosmids, is illustrated in FIG. 6.
[0105] The twelve genomic clones were constructed in a pHC79
vector, in E. coli host HB101. Cosmid DNA was prepared from 3 ml
cultures by an alkaline lysis miniprep method. The miniprep DNA was
digested with EcoRI and HindIII to confirm the known fingerprint of
the large cloned inserts. Cosmid DNA was treated with linear
DNAase, Plasmid-Safe.TM. DNAse, at 37.degree. C. for 15 minutes,
followed by heat inactivation. The DNAse treatment was carried out
to remove any potential spurious EarI digested sites resulting from
contaminating bacterial DNA. This leaves cosmid DNA substantially
untouched. After confirming the presence of clean banding cosmid
DNA, the resulting cosmids were then subjected to the capture
methods described above. The pCH79 vector, without a yeast insert,
was transformed into HB101 and isolated as a miniprep, to serve as
a control.
[0106] The data from the array was normalized an follows. First,
the probe array was normalized for background intensity by
subtracting the background scan (hybridization buffer with no
target). Second, the data was normalized to the specific vector
used in producing the cosmids. Normalization to the vector had two
parts: first the average intensity of four hybridizing markers
present in pHC79 vector was calculated for each scan, for use as an
internal control in that scan. This intensity was divided into all
intensities in that scan, and second the overall background
intensity of the pCH79 vector in a bacterial host, absent a yeast
insert, was subtracted. The array signal was normalized for
relative hybridization of the probes on the array, by using
equimolar target mixtures for each probe. Finally, the four values
corresponding to the pCH79 markers were discarded.
[0107] The resulting hybridization patterns were then correlated,
pair-wise, between all cosmids. Specifically, the signal intensity
for each probe was compared among the same probe's intensity for
all other fragments. Where the signals were the same, there was
some correlation. The more signals that were the same, the higher
the correlation score.
[0108] These correlation scores are plotted against the known
percent overlap for these cosmids as determined from the
EcoRI/HindIII physical map. This plot is shown in FIG. 7A. As is
apparent, the correlation of hybridization scores between fragments
is readily correlatable to percent overlap of the fragments.
EXAMPLE 3
Simulated Annealing
[0109] The correlation scores from yeast chromosome IV, above, were
used to construct a best fitting contig, using the simulated
annealing process as described by Cuticchia, et al., The use of
simulated annealing in chromosome reconstruction experiments based
on binary scoring, Genetics (1992) 132:591-601. A global maximum
was sought for the sum of correlation coefficient scores for a
given sequence of cosmids in the randomly constructed and
permutated contig. The resulting high scoring contigs for all 12
cosmids and for the 10 "strong-signal" cosmids are shown below.
Each cosmid was assigned a rank based upon the known position of
that cosmid, and these are as follows: TABLE-US-00007 TABLE 1
Cosmid Number Cosmid Rank 9371 A 8552 B 8087 1 9481 2 9858 3 9583 4
8024 5 8253 6 9509 7 9460 8 8064 9 9831 10
[0110] Simulated annealing of all twelve cosmids produced the
following ordering: (1 2 3 4) (7 6 5) A B (8 9 10)
[0111] Inclusion of the weaker signal cosmids, A and B, results in
some shuffling of the predicted order of the cosmids. Removal of
cosmids A and X, the "weak-signal" cosmids, produced the following
ordered map of the remaining ten cosmids: (1 2 3 4 5 6 7) (8 9 10)
which reflects the proper ordering and indicates the existence of
the two "islands" of cosmids as seen in the physical map.
[0112] As can be seen, the inclusion of the weaker signal cosmids A
and D, 8552 and 9731, inverts the order of clones in the center
positions (5, 6 and 7), and improperly places
Example 4
Simulated Mapping of Yeast Chromosome III
[0113] To determine how well the distribution of points in FIG. 7A
matches the distribution of scores expected for a random set of
yeast cosmids, a random set of fifty 35 to 40 kb sequences from
yeast chromosome III ("YCIII") were simulated. A list of perfect
matches corresponding to EarI associated tetramers was also
generated. Due to the difficulty in assigning simulated intensity
scores for these markers, the marker probes were scored as 1, and 0
for non marker probe. Inner product scores were used instead of
correlation coefficients to determine the similarity of the marker
sets in 1225 comparisons of the fifty simulated YCIII cosmids. The
scores were plotted against expected overlap, and this in shown in
FIG. 7B. Even when perfect information regarding marker identities
in the tetramer sets is compared, a certain amount of scatter is
seen in the plot. Additionally, comparison of sequences with no
overlap generate inner product scores ranging from 0.05 to 0.4.
These two features are characteristic of the actual data shown in
FIG. 7A.
[0114] The simulation was repeated using BbsI and HphI as the first
cleaving enzyme, and the results are shown in FIGS. 7C and 7D,
respectively. From this data, it can be seen that the amount of
scatter in a particular plot is a function of the inverse of the
frequency of cleavage sites (e.g., number of markers) in the target
sequence. In particular, using HphI as the first cleaving enzyme
would produce 564 markers in YCIII, whereas BbsI would yield 212
and EarI would yield 274. The scatter for the more frequently
cutting HphI enzyme is substantially less than that for BbsI and
EarI. Additionally, as noted previously, the Y intercept is also
affected by the number of markers in the target sequence, as well
as the frequency of a particular marker (e.g., marker duplication).
Both of these factors may be influenced by the choice of capture
methods and enzymes.
[0115] The above description is illustrative and not restrictive.
Many variations of the invention will become apparent to those of
skill in the art upon review of this disclosure. The scope of the
invention should, therefore, be determined not with reference to
the above description, but instead should be determined with
reference to the appended claims along with their full scope of
equivalents. All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
Sequence CWU 1
1
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