U.S. patent application number 09/916228 was filed with the patent office on 2003-01-09 for serial analysis of transcript expression using long tags.
Invention is credited to Kinzler, Kenneth W., Sparks, Andrew, Velculescu, Victor E., Vogelstein, Bert.
Application Number | 20030008290 09/916228 |
Document ID | / |
Family ID | 26915890 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008290 |
Kind Code |
A1 |
Velculescu, Victor E. ; et
al. |
January 9, 2003 |
Serial analysis of transcript expression using long tags
Abstract
Serial analysis of gene expression, SAGE, a method for the rapid
quantitative and qualitative analysis of transcripts, has been
improved to provide more genetic information about each analyzed
transcript. In SAGE, defined sequence tags corresponding to
expressed genes are isolated and analyzed. Sequencing of over 1,000
defined tags in a short period of time (e.g., hours) reveals a gene
expression pattern characteristic of the function of a cell or
tissue. Moreover, SAGE is useful as a gene discovery tool for the
identification and isolation of novel sequence tags corresponding
to novel transcripts and genes.
Inventors: |
Velculescu, Victor E.;
(Baltimore, MD) ; Sparks, Andrew; (Baltimore,
MD) ; Kinzler, Kenneth W.; (Baltimore, MD) ;
Vogelstein, Bert; (Baltimore, MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
26915890 |
Appl. No.: |
09/916228 |
Filed: |
July 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60221556 |
Jul 28, 2000 |
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60233431 |
Sep 18, 2000 |
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Current U.S.
Class: |
435/6.1 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6809 20130101;
C12Q 1/6809 20130101; C12Q 1/6809 20130101; C12Q 2521/313 20130101;
C12Q 2521/501 20130101; C12Q 2525/191 20130101; C12Q 2521/301
20130101; C12Q 2521/501 20130101; C12Q 2525/191 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Goverment Interests
[0002] This invention was made with support from National
Institutes of Health Grant Nos. CA43460 and CA57345. The Government
retains certain rights in this invention.
Claims
1. In a method for detecting expressed transcripts in which a first
defined nucleotide sequence tag is isolated from a first cDNA
oligonucleotide and a second defined nucleotide sequence tag is
isolated from a second cDNA oligonucleotide, and the first defined
nucleotide sequence tag is linked to a first oligonucleotide linker
thereby forming a first linked nucleic acid, wherein the first
oligonucleotide linker comprises a recognition site for a
restriction endonuclease that allows DNA cleavage at a site in the
first defined nucleotide sequence tag distant from the first
recognition site; and the second defined nucleotide sequence tag is
linked to a second oligonucleotide linker thereby forming a second
linked nucleic acid, wherein the second oligonucleotide linker
comprises a second recognition site for the restriction
endonuclease that allows DNA cleavage at a site in the first
defined nucleotide sequence tag distant from the second recognition
site; wherein the first and the second linked nucleic acids are
cleaved with said restriction endonuclease; wherein the first and
second tags are ligated to form ditags; and the nucleotide sequence
of at least one tag of the ditag is determined to detect gene
expression, the improvement comprising: using MmeI as the
restriction endonuclease to form 3' overhanging ends on said first
and second tags.
2. A method for the detection of transcript expression comprising:
producing complementary deoxyribonucleic acid (cDNA)
oligonucleotides; isolating a first defined nucleotide sequence tag
from a first cDNA oligonucleotide and a second defined nucleotide
sequence tag from a second cDNA oligonucleotide; linking the first
tag to a first oligonucleotide linker thereby forming a first
linked nucleic acid, wherein the first oligonucleotide linker
comprises a first recognition site for MmeI restriction
endonuclease; linking the second tag to a second oligonucleotide
linker thereby forming a second linked nucleic acid, wherein the
second oligonucleotide linker comprises a second recognition site
for MmeI restriction endonuclease; cleaving the first and the
second linked nucleic acids with MmeI restriction endonculease to
form 3' overhanging ends; ligating the first and second tags to
form a ditag: and determining the nucleotide sequence of at least
one tag of the ditag to detect transcript expression.
3. The method of claim 2 wherein the first oligonucleotide linker
comprises a first amplification primer hybridization sequence, and
the second oligonucleotide linker comprises a second amplification
primer hybridization sequence; said method further comprising the
step of amplifying the ditag oligonucleotide using primers which
hybridize to the first and second amplification primer
hybridization sequences.
4. The method of claim 2 further comprising producing concatemers
of the ditags prior to the step of determining.
5. The method of claim 4 wherein the concatemer consists of about 2
to 200 ditags.
6. The method of claim 2 wherein said 3' overhanging ends are not
removed to form blunt ends prior to said step of ligating.
7. Te method of claim 2 wherein the first and second
oligonucleotide linkers comprise the same nucleotide sequence.
8. Te method of claim 2 wherein the first and second
oligonucleotide linkers comprise different nucleotide
sequences.
9. The method of claim 4 wherein the concatemer consists of about 8
to 20 ditags.
10. The method of claim 2 wherein the ditag is about 38 to 60 base
pairs.
11. The method of claim 10 wherein the ditag is about 38 to 42 base
pairs.
12. The method of claim 3 wherein the step of amplifying is
performed by polymerase chain reaction (PCR).
13. The method of claim 2 further comprising the step of comparing
the nucleotide sequence determined to a database comprising
mammalian genomic sequences whereby matching sequences are
identified.
14. A method for detection of transcript expression comprising:
cleaving a cDNA sample with a first restriction endonuclease,
wherein the endonuclease cleaves the cDNA at a defined position in
the cDNA thereby producing defined sequence tags; isolating the
defined cDNA tags and forming a pool of tags; ligating the pool of
tags with oligonucleotide linkers having a recognition site for a
second restriction endonuclease which is MmeI which forms 3'
overhanging ends; cleaving the tags with MmeI restriction
endonuclease to form 3' overhanging ends; ligating the pool of tags
to produce at least one ditag; and determining the nucleotide
sequence of at least one ditag, wherein the nucleotide sequence of
the ditag corresponds to sequence from at least one expressed
transcripts.
15. The method of claim 14 further comprising amplifying the at
least one ditag.
16. The method of claim 14 wherein the 3' overhanging ends are not
removed to form blunt ends prior to said step of ligating.
17. The method of claim 14 wherein the first restriction
endonuclease has a four base pair recognition site.
18. The method of claim 17 wherein the first restriction
endonuclease is NlallI.
19. The method of claim 14 wherein the cDNA comprises a means for
capture.
20. The method of claim 19 wherein the means for capture is a
binding element.
21. The method of claim 20 wherein the binding element is
biotin.
22. The method of claim 14 wherein the oligonucleotide linkers
comprise a homogeneous population having a single nucleotide
sequence.
23. The method of claim 14 wherein the oligonucleotide linkers
comprise a first and second linker each having a distinct
nucleotide sequence.
24. The method of claim 14 wherein said 3' overhanging ends are
removed to form blunt ends prior to said step of ligating.
25. The method of claim 14 wherein the ditag is about 38 to 60 base
pairs.
26. The method of claim 14 further comprising ligating the ditags
to produce a concatemer.
27. The method of claim 26 wherein the concatemer consists of about
2 to 200 ditags.
28. The method of claim 27 wherein the concatemer consists of about
8 to 20 ditags.
29. The method of claim 15 wherein the amplifying is by polymerase
chain reaction (PCR).
30. The method of claim 14 wherein the oligonucleotide linkers
comprise an amplification primer hybridization sequence.
Description
[0001] This application claims the benefit of provisional
applications 60/221,556 filed Jul. 28, 2000 and 60/233,431 filed
Sep. 18, 2000.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of gene
and transcript expression and specifically to a method for the
serial analysis of a large number of transcripts by identification
of a defined region of a transcript which corresponds to a region
of an expressed gene.
BACKGROUND OF THE INVENTION
[0004] Determination of the genomic sequence of higher organisms,
including humans, is now a real and attainable goal. However, this
analysis only represents one level of genetic complexity. The
ordered and timely expression of genes represents another level of
complexity equally important to the definition and biology of the
organism.
[0005] The role of sequencing complementary DNA (cDNA), reverse
transcribed from mRNA, as part of the human genome project has been
debated as proponents of genomic sequencing have argued the
difficulty of finding every mRNA expressed in all tissues, cell
types, and developmental stages and have pointed out that much
valuable information from intronic and intergenic regions,
including control and regulatory sequences, will be missed by cDNA
sequencing (Report of the Committee on Mapping and Sequencing the
Human Genome, National Academy Press, Washington, D.C., 1988).
Sequencing of transcribed regions of the genome using cDNA
libraries has heretofore been considered unsatisfactory. Libraries
of cDNA are believed to be dominated by repetitive elements,
mitochondrial genes, ribosomal RNA genes, and other nuclear genes
comprising common or housekeeping sequences. It is believed that
cDNA libraries do not provide all sequences corresponding to
structural and regulatory polypeptides or peptides (Putney, et al.,
Nature, 302:718, 1983).
[0006] Another drawback of standard cDNA cloning is that some mRNAs
are abundant while others are rare. The cellular quantities of mRNA
from various genes can vary by several orders of magnitude.
[0007] Techniques based on cDNA subtraction or differential display
can be quite useful for comparing gene expression differences
between two cell types (Hedrick, et al., Nature, 308:149, 1984;
Liang and Pardee, Science, 257:967, 1992), but provide only a
partial analysis, with no direct information regarding abundance of
messenger RNA. The expressed sequence tag (EST) approach has been
shown to be a valuable tool for gene discovery (Adams, et al.,
Science 252:1656, 1991; Adams, et al., Nature, 355:632, 1992; Okubo
et al., Nature Genetics, 2:173, 1992), but like Northern blotting,
RNase protection, and reverse transcriptase-polymerase chain
reaction (RT-PCR) analysis (Alwine, et al., Proc. Natl. Acad Sci,
U.S.A., 74:5350, 1977; Zinn et al, Cell, 34:865, 1983; Veres, et
al., Science, 237:415, 1987), only evaluates a limited number of
genes at a time. In addition, the EST approach preferably employs
nucleotide sequences of 150 base pairs or longer for similarity
searches and mapping.
[0008] Sequence tagged sites (STSs) (Olson, et al., Science,
245:1434, 1989) have also been utilized to identify genomic markers
for the physical mapping of the genome. These short sequences from
physically mapped clones represent uniquely identified map
positions in the genome. In contrast, the identification of
expressed genes relies on expressed sequence tags which are markers
for those genes actually transcribed and expressed in vivo.
[0009] The restriction enzyme MmeI is a class II restriction
endonclease which is a monomeric protein of 101 kDa. It is derived
from Methylophilus methylotrophus. MmeI has a pI of 7.85 and is
active in the pH range of 6.5 to 10, with the optimum at 7 to 8.
MmeI cleaves DNA 20/18 nucleotides 3' of the asymmetric recognition
sequence (5'-TCCRAC-3'). See Tucholski et al., Gene, vol. 157, pp.
87-92, 1995.
[0010] There is a need for an improved method which allows rapid,
detailed analysis of thousands of expressed genes and/or expressed
transcripts for the investigation of a variety of biological
applications, particularly for establishing the overall pattern of
gene expression in different cell types or in the same cell type
under different physiologic or pathologic conditions.
Identification of different patterns of expression has several
utilities, including the identification of appropriate therapeutic
targets, candidate genes for gene therapy (e.g., gene replacement),
tissue typing, forensic identification, mapping locations of
disease-associated genes, and for the identification of diagnostic
and prognostic indicator genes. There is a need in the art for more
efficient methods of accomplishing these taks. There is a need in
the art for methods of determining correspondence between isolated
nucleic acids and genes and/or expressed transcripts identified in
genomic databases. There is a need in the art for methods of
identifying rare expressed genes not otherwise predicted as well as
for identifying non-translated RNA factors. There is a need in the
art for additional tools to assist in assigning function to genes
identified in the human genome.
SUMMARY OF THE INVENTION
[0011] The present invention provides a method for the rapid
analysis of numerous transcripts in order to identify the overall
pattern of transcript expression (transcriptome) in different cell
types or in the same cell type under different physiologic,
developmental or disease conditions. The method is based on the
identification of a "long" nucleotide sequence tag at a defined
position in a messenger RNA. The tag is used to identify the
corresponding transcript and/or gene from which it was transcribed.
By utilizing dimerized tags, termed a "ditag", the method of the
invention allows elimination of certain types of bias which might
occur during cloning and/or amplification and possibly during data
evaluation.
[0012] Concatemerization of these nucleotide sequence tags allows
the efficient analysis of transcripts in a serial manner by
sequencing multiple tags on a single DNA molecule, for example, a
DNA molecule inserted in a vector or in a single clone.
[0013] The method described herein is the serial analysis of
transcript expression, an approach which allows the analysis of a
large number of transcripts. To demonstrate this strategy, cDNA
sequence tags were generated from mRNA, randomly paired to form
ditags, concatenated, and cloned. Manual sequencing of 1,000 tags
revealed a characteristic gene expression pattern. Identification
of such patterns is important diagnostically and therapeutically,
for example. Moreover, the use of serial analysis as a transcript
discovery tool was documented by the identification and isolation
of new pancreatic corresponding to novel tags. This method provides
a broadly applicable means for the quantitative cataloging and
comparison of expressed transcripts in a variety of normal,
developmental, and disease states. "Long SAGE" of "Long SATE"
permits the ready and accurate identification of isolated tags with
genomic sequence data.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A & 1B shows a schematic of SAGE. The first
restriction enzyme, or anchoring enzyme, is NlaIII and the second
enzyme, or tagging enzyme, is FokI in this example. Sequences
represent primer derived sequences, and transcript derived
sequences with "X" and "O" representing nucleotides of different
tags.
[0015] FIG. 2 shows a comparison of transcript abundance. Bars
represent the percent abundance as determined by SAGE (dark bars)
or hybridization analysis (light bars). SAGE quantitations were
derived from Table 1 as follows: TRY 1/2 includes the tags for
trypsinogen 1 and 2, PROCAR indicates tags for procarboxypeptidase
A1, CHYMO indicates tags for chymotrypsinogen, and ELA/PRO includes
the tags for elastase IIIB and protease E. Error bars represent the
standard deviation determined by taking the square root of counted
events and converting it to a percent abundance (assumed Poisson
distribution).
[0016] FIGS. 3A & 3B shows the results of screening a cDNA
library with SAGE tags. P1 and P2 show typical hybridization
results obtained with 13 bp oligonucleotides as described in the
Examples. P1 and P2 correspond to the transcripts described in
Table 2. Images were obtained using a Molecular Dynamics
PhosphorImager and the circle indicates the outline of the filter
membrane to which the recombinant phage were transferred prior to
hybridization.
[0017] FIG. 4 is a block diagram of a tag code database access
system in accordance with the present invention.
[0018] FIG. 5 shows a schematic of Long SAGE. The first restriction
enzyme, or anchoring enzyme (AE) is NlaIII, and the second enzyme,
or tagging enzyme (TE) is MmeI in this example. Sequences represent
primer derived sequences, and transcript derived sequences are
represented with "X" and "O" representing nucleotides of different
tags.
[0019] FIG. 6 shows an analysis of chromosome 22 SAGE tags. As
shown in the bar graph, as the length of the tag increases the
accuracy of the process of matching a SAGE tag to a genomic
database increases dramatically. Tags in the range of 19-21
nucleotides are extemely accurate for matching to a genomic
database. See also Table 4 which provides theoretical probabilities
that a tag is unique in the human genome based on its size.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] The present invention provides an improvement to the SAGE
technique described in U.S. Pat. Nos. 5,695,937 and 5,866,330. The
SAGE technique as it was originally taught and as it has been
subsequently consistently practiced, uses the type IIS restriction
endonuclease BsmFI as the "tagging enzyme". 1 It has now been found
that longer tags of 20-22 nucleotides can be made which provide
sufficient information to uniquely identify genomic sequences in
the human genome. Surpisingly, not only genes expressed as protein
can be identified, but also biologically active transcribed RNA
which is not translated. Genomic regions that were previously
thought to represent the non-coding strand may also be identified
as transcriptionally active. Using MmeI as the tagging enzyme, 3'
overhanging ends are formed. These ends can be ligated without
removal of the overhanging ends and surprisingly this provides not
only longer tags but also increased efficiency of ditag formation.
1 Velculescu et al., Science 270:484-487, 1995; Virlon et al., PNAS
96:15286-15291, 1999; Angelastro et al., PNAS 97:10424-10429, 2000,
Wang et al. PNAS 95:11909-11914, 1998, Lee et al., PNAS
98:3340-3345,2001; Sun et al., British Journal of Psychiatry 178:
s137-s141, 2001; Hashimoto et al., Blood 96:2206-2214,2000; Takano
et al., British J. Cancer 83:1495-1502,2000; Boon et al., EMBO J.
20:1383-1393,2001; Matsumara et al., Plant J. 20:719-726, 1999; Ryo
et al., FEBS Lett. 462:182-186, 1999; Anisimov et al., Eur. J.
Heart Failure 3:271-281,2001; Suzuki et al, Blood
96:2584-2591,2000; Inoue et al., Glia 28:165-171, 1999; Chrast et
al., Genome Research 10:2006-2021, 2000.
[0021] Using longer tags, we have identified genomic sequences
which were previously not identified as transcribed. For example,
sequences have been identified as transcribed which appear to be
the reverse strand of known genes. Because the tags can be matched
to human genomic sequences, RT-PCR primers can be designed from the
matched human genomic sequences. Thus confirmation of the
biological relevance of the reverse strand transcripts has been
obtained.
[0022] Long tags that match sequences on the opposite strand of
previously annotated or predicted transcripts can be tested for
their validity by using the following protocol. Genomic sequence
data is obtained for approximately 200 base pairs surrounding the
SAGE tag (100 base pairs on both the 5' and 3' ends). Primers of 20
base pairs derived from sense (coding strand of the previously
annotated transcript) and antisense strands are designed to PCR
amplify a region approximately 100 base pairs long, inclusive of
the SAGE tag. Source RNA (any RNA derived from specific tissue or
cells expected to encode the reverse strand transcript) is reverse
transcribed, in the presence or absence of reverse transcriptase
(RT) into first strand cDNA using the sense primer. PCR reactions
are performed on the first strand cDNA. Amplification of a specific
DNA band of appropriate size from the sense-primed (+RT) cDNA
suggests the existence of an authentic reverse strand transcript so
long as the sense-primed (-RT) cDNA does not also produce the same
size DNA band.
[0023] SAGE is a rapid, quantitative process for determining the
abundance and nature of transcripts corresponding to expressed
genes. SAGE is based on the identification of and characterization
of partial, defined sequences of transcripts corresponding to gene
segments. These defined transcript sequence "tags" are markers for
genes which are expressed in a cell, a tissue, or an extract, for
example.
[0024] SAGE is based on several principles. First, as has now been
amply demonstrated, a short nucleotide sequence tag (9 to 10 bp)
contains sufficient information content to uniquely identify a
transcript, for example, from a database of cDNAs, provided it is
isolated from a defined position within the transcript. For
example, a sequence as short as 9 bp can distinguish 262,144
transcripts (4.sup.9) given a random nucleotide distribution at the
tag site, whereas estimates suggest that the human genome encodes
about 80,000 to 200,000 transcripts (Fields, et al., Nature
Genetics, 7:345 1994). The size of the tag can be shorter for lower
eukaryotes or prokaryotes, for example, where the number of
transcripts encoded by the genome is lower. For example, a tag as
short as 6-7 bp may be sufficient for distinguishing transcripts in
yeast. However, such short tags are typically not sufficient for
identifying sequences in a human genomic database. According to the
present invention, however, longer tags are obtained which are
particularly useful for matching to genomic databases. Tags as long
as 17-19, 19-21, 22-25, 26-30 nucleotides can be generated which
provide sufficient information to uniquely identify a genomic human
sequence, for example. As shown in TABLE 4, a 21-nucleotide tag has
a 99.83% chance of identifying a unique sequence in the human
genome.
[0025] Second, random dimerization of tags allows a procedure for
reducing bias (caused by amplification and/or cloning). Third,
concatenation of these sequence tags allows the efficient analysis
of transcripts in a serial manner by sequencing multiple tags
within a single vector or clone. As with serial communication by
computers, wherein information is transmitted as a continuous
string of data, serial analysis of the sequence tags requires a
means to establish the register and boundaries of each tag. All of
these principles may be applied independently, in combination, or
in combination with other known methods of sequence
identification.
[0026] SAGE provides a method for the detection of gene expression
in a particular cell or tissue, or cell extract, for example,
including at a particular developmental stage or in a particular
disease state. The method comprises producing complementary
deoxyribonucleic acid (cDNA) oligonucleotides, isolating a first
defined nucleotide sequence tag from a first cDNA oligonucleotide
and a second defined nucleotide sequence tag from a second cDNA
oligonucleotide, linking the first tag to a first oligonucleotide
linker, wherein the first oligonucleotide linker comprises a first
sequence for hybridization of an amplification primer and linking
the second tag to a second oligonucleotide linker, wherein the
second oligonucleotide linker comprises a second sequence for
hybridization of an amplification primer, and determining the
nucleotide sequence of the tag(s), wherein the tag(s) correspond to
an expressed gene. Preferably the linkers are distinct to eliminate
the possibility of formation of hairpin loops.
[0027] FIGS. 1A & 1B shows a schematic representation of the
analysis of messenger RNA (mRNA) using SAGE as described in the
method of the invention. mRNA is isolated from a cell or tissue of
interest for in vitro synthesis of a double-stranded DNA sequence
by reverse transcription of the mRNA. The double-stranded DNA
complement of mRNA formed is referred to as complementary
(cDNA).
[0028] The term "oligonucleotide" as used herein refers to primers
or oligomer fragments comprised of two or more deoxyribonucleotides
or ribonucleotides, preferably more than three. The exact size will
depend on many factors, which in turn depend on the ultimate
function or use of the oligonucleotide.
[0029] The method further includes ligating the first tag linked to
the first oligonucleotide linker to the second tag linked to the
second oligonucleotide linker and forming a "ditag". Each ditag
represents two defined nucleotide sequences of at least one
transcript, representative of at least one gene. Typically, a ditag
represents two transcripts from two distinct genes. The presence of
a defined cDNA tag within the ditag is indicative of expression of
a gene having a sequence of that tag. The first tag linked to the
first oligonucleotide linker and the second tag linked to the
second oligonucleotide linker can be ligated directly using
overhanging ends if they are formed by the tagging enzyme. The ends
can also be trimmed-back using an exonuclease to form blunt ends
for blunt ended ligation. It has been found that ligation of 2 bp
3'-overhanging ends formed using MmeI is significantly more
efficient than the blunt-end ligation previously employed in
conventional SAGE. We have found that using such overhanging ends
that there is less contamination by linker sequences in the ditags.
Thus when tags are concatamerized and/or cloned, a higher yield of
information is achieved because there are more tags per clone and
more relevant sequence on a per nucleotide basis.
[0030] The analysis of ditags, formed prior to any amplification
step, provides a means to eliminate potential distortions
introduced by amplification, e.g., PCR. The pairing of tags for the
formation of ditags is a random event. The number of different tags
is expected to be large, therefore, the probability of any two tags
being coupled in the same ditag is small, even for abundant
transcripts. Therefore, repeated ditags potentially produced by
biased standard amplification and/or cloning methods are excluded
from analysis by the method of the invention.
[0031] The term "defined" nucleotide sequence, or "defined"
nucleotide sequence tag, refers to a nucleotide sequence derived
from either the 5' or 3' terminus of a transcript. The sequence is
defined by cleavage with a first restriction endonuclease, and
represents nucleotides either 5' or 3' of the first restriction
endonuclease site, depending on which terminus is used for capture
(e.g. 3' when oligo-dT is used for capture as described
herein).
[0032] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes which bind to a
specific double-stranded DNA sequence termed a recognition site or
recognition nucleotide sequence, and cut double-stranded DNA at or
near the specific recognition site.
[0033] The first endonuclease, termed "anchoring enzyme" or "AE" in
FIG. 1, is selected by its ability to cleave a transcript at least
one time and therefore produce a defined sequence tag from either
the 5' or 3' end of a transcript. Preferably, a restriction
endonuclease having at least one recognition site and therefore
having the ability to cleave a majority of cDNAs is utilized. For
example, as illustrated herein, enzymes which have a 4 base pair
recognition site are expected to cleave every 256 base pairs
(4.sup.4) on average while most transcripts are considerably
larger. Restriction endonucleases which recognize a 4 base pair
site include NlaIII, as exemplified in the EXAMPLES of the present
invention. Other similar endonucleases having at least one
recognition site within a DNA molecule (e.g., cDNA) will be known
to those of skill in the art (see for example, Current Protocols in
Molecular Biology, Vol. 2, 1995, Ed. Ausubel, et al., Greene
Publish. Assoc. & Wiley Interscience, Unit 3.1.15; New England
Biolabs Catalog, 1995).
[0034] After cleavage with the anchoring enzyme, the most 5' or 3'
region of the cleaved cDNA can then be isolated by binding to a
capture medium. For example, as illustrated in the present
EXAMPLES, streptavidin beads are used to isolate the defined 3'
nucleotide sequence tag when the oligo dT primer for cDNA synthesis
is biotinylated. In this example, cleavage with the first or
anchoring enzyme provides a unique site on each transcript which
corresponds to the restriction site located closest to the poly-A
tail. Likewise, the 5' cap of a transcript (the cDNA) can be
utilized for labeling or binding a capture means for isolation of a
5' defined nucleotide sequence tag. Those of skill in the art will
know other similar capture systems (e.g., biotin/streptavidin,
digoxigenin/anti-digoxigenin) for isolation of the defined sequence
tag as described herein. Alternatively, the entire process can be
carried out while cDNA is attached to a bead. In fact, the cDNA can
be synthesized on the bead by binding mRNA to a bead which has one
or more oligo (dT) molecules coated or attached and reverse
transcribing the mRNA attached to the bead by hybridization via a
poly(A) tract. Subsequent digestion with the anchoring enzyme can
be done on the beads as well.
[0035] The invention is not limited to use of a single "anchoring"
or first restriction endonuclease. It may be desirable to perform
the method of the invention sequentially, using different enzymes
on separate samples of a preparation, in order to identify a
complete pattern of transcription for a cell or tissue. In
addition, the use of more than one anchoring enzyme provides
confirmation of the expression pattern obtained from the first
anchoring enzyme. Therefore, it is also envisioned that the first
or anchoring endonuclease may rarely cut cDNA such that few or no
cDNA representing abundant transcripts are cleaved. Thus,
transcripts which are cleaved represent "unique" transcripts.
Restriction enzymes that have a 7-8 bp recognition site for
example, would be enzymes that would rarely cut cDNA. Similarly,
more than one tagging enzyme, described below, can be utilized in
order to identify a complete pattern of transcription.
[0036] In one embodiment of the invention, classical SAGE data and
long SAGE data are correlated. The classical and long SAGE methods
use different tagging enzymes (or the same tagging enzyme used
under different conditions) to generate different length tags. The
classical and long SAGE can either use the same or different
anchoring enzymes. If the same anchoring enzyme is used, the short
tags will nest within the long tags. This is advantageous for using
the large amount of expression data generated with short tags and
linking it to the genome using the long tags. Thus the long tags
serve to "anchor" the short tags to the genome. An example of such
anchoring is shown below.
[0037] Ten DLD1 colon cancer SAGE short tags that do not match to
any entries in Unigene (build132) are shown below. Long tag data
from a long SATE analysis performed on DLD1 colon cancer cells was
able to extend the given short tags. These 17 base tags are located
uniquely within the human genome and fall within the gene
descriptions noted.
1 Unigene Short Tag Match LongTag Description ATCACGCGCT None
ATCACGCCCTCATAATC Hypothetical protein TCACCCAGGG None
TCACCCAGGGACCCATT Ribosomal protein S4. X-linked TTGGTGATAC None
TTGGTGATACCCCCCGG RDBD AAACAAATCA None AAACAAATCACCATCCT KIAA0026
CCGTGGTAGC None CCGTGGTAGCCAATGTT Kinesin-related GAAGGAGATG None
GAAGGAGATGGCGAAAG Hypothetical protein GCCGCTCTC None
GCCGCTCTCCCGGACC Catenin-vinculin-related TCTTACCATA None
TCTTACCATACACACTG Hypothetical protein TGGATAATTC None
TGGATAATTCAAACAAA Hypothetical protein TTCCAGCCAA None
TTCCAGCCAATGGATGA Hypothetical protein
[0038] The term "isolated" as used herein includes polynucleotides
substantially free of other nucleic acids, proteins, lipids,
carbohydrates or other materials with which it is naturally
associated. cDNA is not naturally occurring as such, but rather is
obtained via manipulation of a partially purified naturally
occurring mRNA. Isolation of a defined sequence tag refers to the
purification of the 5' or 3' tag from other cleaved cDNA.
[0039] In one embodiment, the isolated defined nucleotide sequence
tags are separated into two pools of cDNA, when the linkers have
different sequences. Each pool is ligated via the anchoring, or
first restriction endonuclease site to one of two linkers. When the
linkers have the same sequence, it is not necessary to separate the
tags into pools. The first oligonucleotide linker comprises a first
sequence for hybridization of an amplification primer and the
second oligonucleotide linker comprises a second sequence for
hybridization of an amplification primer. In addition, the linkers
further comprise a second restriction endonuclease site, also
termed the "tagging enzyme" or "TE". Long SATE employs a TE which
cleaves at least 17, 18, 19, 20, or 21 nucleotides from its
recognition site. The method of the invention does not require, but
preferably comprises amplifying the ditag oligonucleotide after
ligation.
[0040] The second restriction endonuclease (TE) cleaves at a site
distant from or outside of the recognition site. For example, the
second restriction endonuclease can be a type IIS restriction
enzyme. Type IIS restriction endonucleases cleave at a defined
distance up to 2-13 nt away from their 4-7 bp asymmetric
recognition sites and include BbvI, BbvII, BinI FokI, HgaI, HphI,
MboII, MnlI, SfaNI, TaqII TthlllII, as reviewed in Szybalski, W.,
Gene, 40:169, 1985. Examples of type IIS restriction endonucleases
include BsmFI and FokI. Other similar enzymes will be known to
those of skill in the art (see, Current Protocols in Molecular
Biology, supra). A particularly preferred tagging enzyme, according
to the invention is an enzyme which cleaves 20/18 nucleotides 3' of
its recognition site forming 3' overhanging ends, such as MmeI. Any
other suitable enzyme known in the art can be used. In addition,
restriction endonucleases with desirable properties can be
artificially evolved, i.e., subjected to selection and screening,
to obtain an enzyme which is useful as a tagging enzyme for long
SATE. Desirable enzymes cleave at least 18-21 nucleotides distant
from their recognition sites. Artificial restriction endonucleases
can also be used. Such endonucleases are made by protein
engineering. For example, the endonuclease FokI has been engineered
by insertions so that it cleaves one nucleotide further away from
its recognition site on both strands of the DNA substrates. See Li
and Chandrasegaran, Proc. Nat. Acad. Sciences USA 90:2764-8, 1993.
Such techniques can be applied to generate restriction
endonucleases with desirable recognition sequences and desirable
distances from recognition site to cleavage site.
[0041] The first and second "linkers" which are ligated to the
defined nucleotide sequence tags are oligonucleotides having the
same or different nucleotide sequences. The linkers are designed so
that cleavage of the ligation products with the second restriction
enzyme, or tagging enzyme, results in release of the linker having
a defined nucleotide sequence tag (e.g., 3' of the restriction
endonuclease cleavage site as exemplified herein). The defined
nucleotide sequence tag may be from about 6 to 30 base pairs. In
classical SAGE, the tag is typically about 9 to 15 base pairs. In
long SATE the tag is 19-30 base pairs. Therefore, a ditag is from
about 12 to 60 base pairs, and preferably from 38 to 42 base
pairs.
[0042] The pool of defined tags ligated to linkers having the same
sequence, or the two pools of defined nucleotide sequence tags
ligated to linkers having different nucleotide sequences, are
randomly ligated to each other "tail to tail". The portion of the
cDNA tag furthest from the linker is referred to as the "tail". The
sticky tail ends formed by digestion with the tagging enzyme can in
some cases be filled-in with a DNA polymerase or removed by
nuclease digestion prior to ligation. Alternatively, no filling-in
may be done. As illustrated in FIG. 1, the ligated tag pair, or
ditag, has a first restriction endonuclease site upstream (5') and
a first restriction endonuclease site downstream (3') of the ditag;
a second restriction endonuclease cleavage site upstream and
downstream of the ditag, and a linker oligonucleotide containing
both a second restriction enzyme recognition site and an
amplification primer hybridization site upstream and downstream of
the ditag. In other words, the ditag is flanked by the first
restriction endonuclease site, the second restriction endonuclease
cleavage site and the linkers, respectively.
[0043] The ditag can be amplified by utilizing primers which
specifically hybridize to one strand of each linker. Preferably,
the amplification is performed by standard polymerase chain
reaction (PCR) methods as described (U.S. Pat. No. 4,683,195).
Alternatively, the ditags can be amplified by cloning in
prokaryotic-compatible vectors or by other amplification methods
known to those of skill in the art.
[0044] The term "primer" as used herein refers to an
oligonucleotide, whether occurring naturally or produced
synthetically, which is capable of acting as a point of initiation
of synthesis when placed under conditions in which synthesis of
primer extension product which is complementary to a nucleic acid
strand is induced, i.e., in the presence of nucleotides and an
agent for polymerization such as DNA polymerase and at a suitable
temperature and pH. The primer is preferably single stranded for
maximum efficiency in amplification. Preferably, the primer is an
oligodeoxy ribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
agent for polymerization. The exact lengths of the primers will
depend on many factors, including temperature and source of
primer.
[0045] The primers herein are selected to be "substantially"
complementary to the different strands of each specific sequence to
be amplified. This means that the primers must be sufficiently
complementary to hybridize with their respective strands.
Therefore, the primer sequence need not reflect the exact sequence
of the template. In the present invention, the primers can be
substantially or completely complementary to the oligonucleotide
linkers.
[0046] Cleavage of the amplified PCR product with the first
restriction endonuclease allows isolation of ditags which can be
concatenated by ligation. After ligation, it may be desirable to
clone the concatemers, although it is not required in the method of
the invention. Analysis of the ditags or concatemers, whether or
not amplification was performed, is by standard sequencing methods.
Concatemers generally consist of about 2 to 200 ditags and
preferably from about 8 to 20 ditags. While these are preferred
concatemers, it will be apparent that the number of ditags which
can be concatenated will depend on the length of the individual
tags and can be readily determined by those of skill in the art
without undue experimentation. After formation of concatemers,
multiple tags can be cloned into a vector for sequence analysis, or
Iternatively, ditags or concatemers can be directly sequenced
without cloning by methods known to those of skill in the art.
[0047] Among the standard procedures for cloning the defined
nucleotide sequence tags of the invention is insertion of the tags
into vectors such as plasmids or phage. The ditag or concatemers of
ditags produced by the method described herein are cloned into
recombinant vectors for further analysis, e.g., sequence analysis,
plaque/plasmid hybridization using the tags as probes, by methods
known to those of skill in the art.
[0048] The term "recombinant vector" refers to a plasmid, virus or
other vehicle known in the art that has been manipulated by
insertion or incorporation of the ditag genetic sequences. Such
vectors contain a promoter sequence which facilitates the efficient
transcription of the a marker genetic sequence for example. The
vector typically contains an origin of replication, a promoter, as
well as specific genes which allow phenotypic selection of the
transformed cells. Vectors suitable for use in the present
invention include for example, pBlueScript (Stratagene, La Jolla,
Calif.); pBC, pSL301 (Invitrogen) and other similar vectors known
to those of skill in the art. Preferably, the ditags or concatemers
thereof are ligated into a vector for sequencing purposes.
[0049] Vectors in which the ditags are cloned can be transferred
into a suitable host cell. "Host cells" are cells in which a vector
can be propagated and its DNA expressed. The term also includes any
progeny of the subject host cell. It is understood that all progeny
may not be identical to the parental cell since there may be
mutations that occur during replication. However, such progeny are
included when the term "host cell" is used. Methods of stable
transfer, meaning that the foreign DNA is continuously maintained
in the host, are known in the art.
[0050] Transformation of a host cell with a vector containing
ditag(s) may be carried out by conventional techniques as are well
known to those skilled in the art. Where the host is prokaryotic,
such as E. coli, competent cells which are capable of DNA uptake
can be prepared from cells harvested after exponential growth phase
and subsequently treated by the CaCl.sub.2 method using procedures
well known in the art. Alternatively, MgCl.sub.2 or RbCl can be
used. Transformation can also be performed by electroporation or
other commonly used methods in the art.
[0051] The ditags present in a particular clone can be sequenced by
standard methods (see for example, Current Protocols in Molecular
Biology, supra, Unit 7) either manually or using automated
methods.
[0052] In another embodiment, the present invention provides a kit
useful for detection of gene expression wherein the presence of a
defined nucleotide tag or ditag is indicative of expression of a
gene having a sequence of the tag. The kit comprises: a first
container containing an oligonucleotide linker having a sequence
useful for hybridization to an amplification primer; the
oligonucleotide linker further comprises a restriction endonuclease
recogition site for an enzyme which cleaves 18-20 nucleotides
distant from its recognition sequence; and a second container
having a nucleic acid primer for hybridization to the
oligonucleotide linker. Other containers may comprise the
restriction endonuclease and/or a DNA polymerase for amplification.
A particularly preferred retriction endonuclease is MmeI, an enzyme
which forms 3' overhanging ends.
[0053] In yet another embodiment, the invention provides an
oligonucleotide concatamer having at least two defined nucleotide
sequence tags, wherein at least one of the sequence tags
corresponds to at least one expressed gene. The concatamer consists
of about 1 to 200 ditags, and preferably about 8 to 20 ditags. Such
concatamers are useful for the analysis of gene expression by
identifying the defined nucleotide sequence tag corresponding to an
expressed gene in a cell, tissue or cell extract, for example.
[0054] It is envisioned that the identification of differentially
expressed transcripts using the SATE technique of the invention can
be used in combination with other genomics techniques. For example,
individual tags, and preferably ditags, can be hybridized with
oligonucleotides immobilized on a solid support (e.g.,
nitrocellulose filter, glass slide, silicon chip). Such techniques
include "parallel sequence analysis" or PSA, as described below.
The sequence of the ditags formed by the method of the invention
can also be determined using limiting dilutions by methods
including clonal sequencing (CS).
[0055] Briefly, PSA is performed after ditag preparation, wherein
the oligonucleotide sequences to which the ditags are hybridized
are preferably unlabeled and the ditag is preferably detectably
labeled. Alternatively, the oligonucleotide can be labeled rather
than the ditag. The ditags can be detectably labeled, for example,
with a radioisotope, a fluorescent compound, a bioluminescent
compound, a chemiluminescent compound, a metal chelator, or an
enzyme. Those of ordinary skill in the art will know of other
suitable labels for binding to the ditag, or will be able to
ascertain such, using routine experimentation. For example, PCR can
be performed with labeled (e.g., fluorescein tagged) primers.
Preferably, the ditag contains a fluorescent end label.
[0056] The labeled or unlabeled ditags are separated into
single-stranded molecules which are preferably serially diluted and
added to a solid support (e.g., a silicon chip as described by
Fodor, et al., Science, 251:767, 1991) containing oligonucleotides
representing, for example, every possible permutation of a 10-mer
(e.g., in each grid of a chip). The solid support is then used to
determine differential expression of the tags contained within that
support (e.g., on a grid on a chip) by hybridization of the
oligonucleotides on the solid support with tags produced from cells
under different conditions (e.g., different stage of development,
growth of cells in the absence and presence of a growth factor,
normal versus transformed cells, comparison of different tissue
expression, etc). In the case of fluoresceinated end labeled
ditags, analysis of fluorescence is indicative of hybridization to
a particular 10-mer. When the immobilized oligonucleotide is
fluoresceinated for example, a loss of fluorescence due to
quenching (by the proximity of the hybridized ditag to the labeled
oligo) is observed and is analyzed for the pattern of gene
expression. An illustrative example of the method is shown in
Example 4 herein.
[0057] The SATE method of the invention is also useful for clonal
sequencing, similar to limiting dilution techniques used in cloning
of cell lines. For example, ditags or concatemers thereof, are
diluted and added to individual receptacles such that each
receptacle contains less than one DNA molecule per receptacle. DNA
in each receptacle is amplified and sequenced by standard methods
known in the art, including mass spectroscopy. Assessment of
differential expression is performed as described above for
SAGE.
[0058] Those of skill in the art can readily determine other
methods of analysis for ditags or individual tags produced by SATE
as described in the present invention, without resorting to undue
experimentation. The concept of deriving a defined tag from a
sequence in accordance with the present invention is useful in
matching tags of samples to a sequence database, in particular a
database of genomic sequences, such as humans, mice, cows, pigs,
horses, etc. In the preferred embodiment, a computer method is used
to match a sample sequence with known sequences.
[0059] One of the primary strengths of using a restriction
endonuclease as a tagging enzyme which cuts at least 17 or 18
nucleotides distant from its recognition site is the ability to
unambiguously identify a location in the genome from which a long
tag is derived. Thus, it is significantly easier and more accurate
to determine the identity of the gene or genomic region that gave
rise to a tag, particularly if one is dealing with an organism for
which significant genomic data but only limited cDNA sequence
information is available. Table 4 shows a computation of the
probability that tags of differing length will be unique in the
human genome. In addition, a comparison of the number of times long
tags vs their cognate short tags "hit" the human genome is shown in
FIG. 5. This analysis is based on theoretical tags derived from
known genes on Chromosome 22.
[0060] In one embodiment, a sequence tag for a sample is compared
to corresponding information in a sequence database to identify
known sequences that match the sample sequence. Preferably the
database is genomic sequence, more preferably human genomic
sequence. One or more tags can be determined for each sequence in
the sequence database as the N base pairs adjacent to each
anchoring enzyme site within the sequence. However, in the
preferred embodiment, only the first anchoring enzyme site from the
3' end is used to determine a tag. In the preferred embodiment, the
adjacent base pairs defining a tag are on the 3' side of the
anchoring enzyme site, and N is preferably 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, or 29.
[0061] A linear search through such a database may be used.
However, in the preferred embodiment, a sequence tag from a sample
is converted to a unique numeric representation by converting each
base pair (A, C, G, or T) of an N-base tag to a number or "tag
code" (e.g., A=O, C=1, G=2, T=3, or any other suitable mapping). A
tag is determined for each sequence of a sequence database as
described above, and the tag is converted to a tag code in a
similar manner. In the preferred embodiment, a set of tag codes for
a sequence database is stored in a pointer file. The tag code for a
sample sequence is compared to the tag codes in the pointer file to
determine the location in the sequence database of the sequence
corresponding to the sample tag code. (Multiple corresponding
sequences may exist if the sequence database has redundancies).
[0062] FIG. 4 is a block diagram of a tag code database access
system in accordance with the present invention. A sequence
database 10 (e.g., the Human Genome Sequence Database) is processed
as described above, such that each sequence has a tag code
determined and stored in a pointer file 12. A sample tag code X for
a sample is determined as described above, and stored within a
memory location 14 of a computer. The sample tag code X is compared
to the pointer file 12 for a matching sequence tag code. If a match
is found, a pointer associated with the matching sequence tag code
is used to access the corresponding sequence in the sequence
database 10.
[0063] The pointer file 12 may be in any of several formats. In one
format, each entry of the pointer file 12 comprises a tag code and
a pointer to a corresponding record in the sequence database 12.
The sample tag code X can be compared to sequence tag codes in a
linear search. Alternatively, the sequence tag codes can be sorted
and a binary search used. As another alternative, the sequence tag
codes can be structured in a hierarchical tree structure (e.g., a
B-tree), or as a singly or doubly linked list, or in any other
conveniently searchable data structure or format.
[0064] In the preferred embodiment, each entry of the pointer file
12 comprises only a pointer to a corresponding record in the
sequence database 10. In building the pointer file 12, each
sequence tag code is assigned to an entry position in the pointer
file 12 corresponding to the value of the tag code. For example, if
a sequence tag code was "1043", a pointer to the corresponding
record in the sequence database 10 would be stored in entry #1043
of the pointer file 12. The value of a sample tag code X can be
used to directly address the location in the pointer file 12 that
corresponds to the sample tag code X, and thus rapidly access the
pointer stored in that location in order to address the sequence
database 10.
[0065] Because only four values are needed to represent all
possible base pairs, using binary coded decimal (BCD) numbers for
tag codes in conjunction with the preferred pointer file 12
structure leads to a "sparse" pointer file 12 that wastes memory or
storage space. Accordingly, the present invention transforms each
tag code to number base 4 (i.e., 2 bits per code digit), in known
fashion, resulting in a compact pointer file 12 structure. For
example, for tag sequence "AGCT", with A=00.sub.2, C=01.sub.2,
G=10.sub.2, T=11.sub.2, the base four representation in binary
would be "00011011".
[0066] In contrast, the BCD representation would be "00000000
00000001 00000010 000000011". Of course, it should be understood
that other mappings of base pairs to codes would provide equivalent
function.
[0067] The concept of deriving a defined tag from a sample sequence
in accordance with the present invention is also useful in
comparing different samples for similarity. In the preferred
embodiment, a computer method is used to match sequence tags from
different samples. For example, in comparing materials having a
large number of sequences (e.g., tissue), the frequency of
occurrence of the various tags in a first sample can be mapped out
as tag codes stored in a distribution or histogram-type data
structure. For example, a table structured similar to pointer file
12 in FIG. 4 can be used where each entry comprises a frequency of
occurrence value. Thereafter, the various tags in a second sample
can be generated, converted to tag codes, and compared to the table
by directly addressing table entries with the tag code. A count can
be kept of the number of matches found, as well as the location of
the matches, for output in text or graphic form on an output
device, and/or for storage in a data storage system for later
use.
[0068] The tag comparison aspects of the invention may be
implemented in hardware or software, or a combination of both.
Preferably, these aspects of the invention are implemented in
computer programs executing on a programmable computer comprising a
processor, a data storage system (including volatile and
non-volatile memory and/or storage elements), at least one input
device, and at least one output device. Data input through one or
more input devices for temporary or permanent storage in the data
storage system includes sequences, and may include previously
generated tags and tag codes for known and/or unknown sequences.
Program code is applied to the input data to perform the functions
described above and generate output information. The output
information is applied to one or more output devices, in known
fashion.
[0069] Each such computer program is preferably stored on a storage
media or device (e.g., ROM or magnetic diskette) readable by a
general or special purpose programmable computer, for configuring
and operating the computer when the storage media or device is read
by the computer to perform the procedures described herein. The
inventive system may also be considered to be implemented as a
computer-readable storage medium, configured with a computer
program, where the storage medium so configured causes a computer
to operate in a specific and predefined manner to perform the
functions described herein.
[0070] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLES
[0071] For exemplary purposes, the SAGE method of the invention was
used to characterize gene expression in the human pancreas. NlaIII
was utilized as the first restriction endonuclease, or anchoring
enzyme, and BsmFI as the second restriction endonuclease, or
tagging enzyme, yielding a 9 bp tag (BsmFI was predicted to cleave
the complementary strand 14 bp 3' to the recognition site GGGAC and
to yield a 4 bp 5' overhang (New England BioLabs). Overlapping the
BsmFI and NlaIII (CATG) sites as indicated (GGGACATG) would be
predicted to result in a 11 bp tag. However, analysis suggested
that under the cleavage conditions used (37.degree. C.), BsmFI
often cleaved closer to its recognition site leaving a minimum of
12 bp 3' of its recognition site. Therefore, only the 9 bp closest
to the anchoring enzyme site was used for analysis of tags.
Cleavage at 65.degree. C. results in a more consistent 11 bp
tag.
[0072] Computer analysis of human transcripts from Gen Bank
indicated that greater than 95% of tags of 9 bp in length were
likely to be unique and that inclusion of two additional bases
provided little additional resolution. Human sequences (84,300)
were extracted from the GenBank 87 database using the Findseq
program provided on the IntelliGenetics Bionet on-line service. All
further analysis was performed with a SAGE program group written in
Microsoft Visual Basic for the Microsoft Windows operating system.
The SAGE database analysis program was set to include only
sequences noted as "RNA" in the locus description and to exclude
entries noted as "EST", resulting in a reduction to 13,241
sequences. Analysis of this subset of sequences using NlaIII as
anchoring Enzyme indicated that 4,127 nine bp tags were unique
while 1,511 tags were found in more than one entry. Nucleotide
comparison of a randomly chosen subset (100) of the latter entries
indicated that at least 83% were due to redundant data base entries
for the same gene or highly related genes (>95% identity over at
least 250 bp). This suggested that 5381 of the 9 bp tags (95.5%)
were unique to a transcript or highly conserved transcript family.
Likewise, analysis of the same subset of GenBank with an 11 bp tag
resulted only in a 6% decrease in repeated tags (1511 to 1425)
instead of the 94% decrease expected if the repeated tags were due
to unrelated transcripts.
Example 1
[0073] As outlined above, mRNA from human pancreas was used to
generate ditags. Briefly, five ug mRNA from total pancreas
(Clontech) was converted to double stranded cDNA using a BRL cDNA
synthesis kit following the manufacturer's protocol, using the
primer biotin-5'T.sub.18 -3'. The cDNA was then cleaved with NIaIII
and the 3' restriction fragments isolated by binding to magnetic
streptavidin beads (Dynal). The bound DNA was divided into two
pools, and one of two linkers was ligated to each pool.
[0074] After extensive washing to remove unligated linkers, the
linkers and adjacent tags were released by cleavage with BsmFI. The
resulting overhangs were filled in with T4 polymerase and the pools
combined and ligated to each other. The desired ligation product
was then amplified for 25 cycles. The PCR reaction was then
analyzed by polyacrylamide gel electrophoresis and the desired
product excised. An additional 15 cycles of PCR were then performed
to generate sufficient product for efficient ligation and
cloning.
[0075] The PCR ditag products were cleaved with NlaIII and the band
containing the ditags was excised and self-ligated. After ligation,
the concatenated ditags were separated by polyacrylamide gel
electrophoresis and products greater than 200 bp were excised.
These products were cloned into the SphI site of pSL301
(Invitrogen). Colonies were screened for inserts by PCR using T7
and T3 sequences outside the cloning site as primers. Clones
containing at least 10 tags (range 10 to 50 tags) were identified
by PCR amplification and manually sequenced as described (Del Sal,
et al., Biotechniques 7:514, 1989). Sequence files were analyzed
using the SAGE software group which identifies the anchoring enzyme
site with the proper spacing and extracts the two intervening tags
and records them in a database. The 1,000 tags were derived from
413 unique ditags and 87 repeated ditags. The latter were only
counted once to eliminate potential PCR bias of the quantitation.
The function of SAGE software is merely to optimize the search for
gene sequences.
[0076] Table 1 shows analysis of the first 1,000 tags. Sixteen
percent were eliminated because they either had sequence
ambiguities or were derived from linker sequences. The remaining
840 tags included 351 tags that occurred once and 77 tags that were
found multiple times. Nine of the ten most abundant tags matched at
least one entry in GenBank R87. The remaining tag was subsequently
shown to be derived from amylase. All ten transcripts were derived
from genes of known pancreatic function and their prevalence was
consistent with previous analyses of pancreatic RNA using
conventional approaches (Han, et al., Proc. Natl. Acad. Sci. U.S.A.
83:110, 1986; Takeda, et al., Hum. Mol. Gen., 2:1793, 1993).
2TABLE 1 Pancreatic SAGE Tags Tag Gene N Percent GAGCACACC
Procarboxypeptidase A1 (X67318) 64 7.6 TTCTGTGTG Pancreatic
Trypsinogen 2 (M27602) 46 5.5 GAACACAAA Chymotrypsinogen (M24400)
37 4.4 TCAGGGTGA Pancreatic Trypsin 1 (M22612) 31 3.7 GCGTGACCA
Elastase 111B (M18692) 20 2.4 GTGTGTGCT Protease E (D00306) 16 1.9
TCATTGGCC Pancreatic Lipase (M93285) 16 1.9 CCAGAGAGT
Procarboxypeptidase B (M81057) 14 1.7 TCCTCAAAA No Match, See Table
2, P1 14 1.7 AGCCTTGGT Bile Salt Stimulated Lipase (X54457) 12 1.4
GTGTGCGCT No Match 11 1.3 TGCGAGACC No Match, See Table 2, P2 9 1.1
GTGAAACCC 21 Alu entries 8 1.0 GGTGACTCT No Match 8 1.0 AAGGTAACA
Secretary Trypsin Inhibitor (M11949) 6 0.7 TCCCCTGTG No Match 5 0.6
GTGACCACG No Match 5 0.6 CCTGTAATC M91159, M29366,11 Alu entries 5
0.6 CACGTTGGA No Match 5 0.6 AGCCCTACA No Match 5 0.6 AGCACCTCC
Elongation Factor 2 (Z11692) 5 0.6 ACGCAGGGA No Match, See Table 2,
P3 5 0.6 AATTGAAGA No Match, See Table 2, P4 5 0.6 TTCTGTGGG No
Match 4 0.5 TTCATACAC No Match 4 0.5 GTGGCAGGC NF-kB(X61499), Mu
entry (S94541) 4 0.5 GTAAAACCC TNF receptor 11 (M55994), Alu entry
(X01448) 4 0.5 GAACACACA No Match 4 0.5 CCTGGGAAG Pancreatic Mucin
(J05582) 4 0.5 CCCATCGTC (SEQ ID NO:8-37) Mitochondrial CytC
Oxidase (X15759) 4 0.5 Summary SAGE tags occurring Greater than
three times 380 45.2 Occurring Three times (15 .times. 3 = ) 45 5.4
Two times (32 .times. 2 = ) 64 7.6 Onetime 351 41.8 Total SAGE Tags
840 100.0 "Tag" indicates the 9 bp sequence unique to each tag,
adjacent to the 4 bp anchoring NlaIII site. "N" and "Percent"
indicates the number of times the tag was identified and its
frequency, respectively. "Gene" indicates the accession number and
description of GenBank R87 entries found to match the indicated tag
using the SAGE software group with the following exceptions. When
multiple entries were #identified because of duplicated entries,
only one entry is listed. In the cases of chymotrypsinogen, and
trypsinogen 1, other genes were identified that were predicted to
contain the same tags, but subsequent hybridization and sequence
analysis identified the listed genes as the source of the tags.
"Alu entry" indicates a match with a GenBank entry for a transcript
that contained at least one copy of the alu consensus sequence
(Deininger, et al., J Mol. Biol., 151:17, 1981).
Example 2
[0077] The quantitative nature of SAGE was evaluated by
construction of an oligo-dT primed pancreatic cDNA library which
was screened with cDNA probes for trypsinogen 1/2,
procarboxpeptidase A1, chymotrypsinogen and elastase I-IIB/protease
E. Pancreatic mRNA from the same preparation as used for SAGE in
Example 1 was used to construct a cDNA library in the ZAP Express
vector using the ZAP Express cDNA Synthesis kit following the
manufacturer's protocol (Stratagene). Analysis of 15 randomly
selected clones indicated that 100% contained cDNA inserts. Plates
containing 250 to 500 plaques were hybridized as previously
described (Ruppert, et al., Mol. Cell. Biol. 8:3104, 1988). cDNA
probes for trypsinogen 1, trypsinogen 2, procarboxypeptidase A1,
chymotrypsinogen, and elastase IIIB were derived by RT-PCR from
pancreas RNA. The trypsinogen 1 and 2 probes were 93% identical and
hybridized to the same plaques under the conditions used. Likewise,
the elastase IIIB probe and protease E probe were over 95%
identical and hybridized to the same plaques.
[0078] The relative abundance of the SAGE tags for these
transcripts was in excellent agreement with the results obtained
with library screening (FIG. 2). Furthermore, whereas neither
trypsinogen 1 and 2 nor elastase IIIB and protease E could be
distinguished by the cDNA probes used to screen the library, all
four transcripts could readily be distinguished on the basis of
their SAGE tags (Table 1).
Example 3
[0079] In addition to providing quantitative information on the
abundance of known transcripts, SAGE could be used to identify
novel expressed genes. While for the purposes of the SAGE analysis
in this example, only the 9 bp sequence unique to each transcript
was considered, each SAGE tag defined a 13 bp sequence composed of
the anchoring enzyme (4 bp) site plus the 9 bp tag. To illustrate
this potential, 13 bp oligonucleotides were used to isolate the
transcripts corresponding to four unassigned tags (P1 to P4), that
is, tags without corresponding entries from GenBank R87 (Table 1).
In each of the four cases, it was possible to isolate multiple cDNA
clones for the tag by simply screening the pancreatic cDNA library
using 13 bp oligonucleotide as hybridization probe (examples in
FIG. 3).
[0080] Plates containing 250 to 2,000 plaques were hybridized to
oligonucleotide probes using the same conditions previously
described for standard probes except that the hybridization
temperature was reduced to room temperature. Washes were performed
in 6.times.SSC/0.1% SDS for 30 minutes at room temperature. The
probes consisted of 13 bp oligonucleotides which were labeled with
.gamma.- .sup.2P-ATP using T4 polynucleotide kinase. In each case,
sequencing of the derived clones identified the correct SAGE tag at
the predicted 3' end of the identified transcript. The abundance of
plaques identified by hybridization with the 13-mers was in good
agreement with that predicted by SAGE (Table 2). Tags P1 and P2
were found to correspond to amylase and preprocarboxypeptidase A2,
respectively. No entry for preprocarboxypeptidase A2 and only a
truncated entry for amylase was present in GenBank R87, thus
accounting for their unassigned characterization. Tag P3 did not
match any genes of known function in GenBank but did match numerous
EST's, providing further evidence that it represented a bona fide
transcript. The cDNA identified by P4 showed no significant
homology, suggesting that it represented a previously
uncharacterized pancreatic transcript.
3TABLE 2 Characterization of Unassigned SAGE Tags Abundance SAGE
TAG SAGE 13mer Hyb Tag Description P1 TCCTCAAAA 1.7% 1.5% + 3' end
of Pancreatic Amylase (6/388) (M28443) P2 TGCGAGACC 1.1% 1.2% + 3'
end of Preprocarboxypeptidase A2 (43/3700) (U19977) P3 ACGCAGGGA
0.6% 0.2% + EST match (R45808) (5/2772) P4 AATTGAAGA 0.6% 0.4% + no
match (6/1587) "Tag" and "SAGE Abundance" are described in Table 1;
"13mer Hyb" indicates the results obtained by screening a cDNA
library with a 13mer, as described above. The number of positive
plaques divided by the total plaques screened is indicated in
parentheses following the percent abundance. A positive in the
"SAGE Tag" column indicates that the expected SAGE tag sequence was
identified near the 3' #end of isolated clones. "Description"
indicates the results of BLAST searches of the daily updated
GenBank entries at NCBI a of June 9, 1995 (Altschul, et al., J Mol.
Biol., 215:403, 1990). A description and Accession number are given
for the most significant matches. P1 was found to match a truncated
entry for amylase, and P2 was found to match an unpublished entry
for preprocarboxypeptidase A2 which was entered after GenBank
R87.
Example 4
[0081] Ditags produced by SAGE can be analyzed by PSA or CS, as
described in the specification. In a preferred embodiment of PSA,
the following steps are carried out with ditags: Ditags are
prepared, amplified and cleaved with the anchoring enzyme as
described in the previous examples.
4 OOOOOOOOOOXXXXXXXXXXCATG-3' 3'-GTACOOOOOOOOOOXXXXXXXXXX
[0082] Four-base oligomers containing an identifier (e.g., a
fluorescent moiety, FL) are prepared that are complementary to the
overhangs, for example, FL-CATG. The FL-CATG oligomers (in excess)
are ligated to the ditags as shown below:
5 5'-FL-CATGOOOOOOOOOOXXXXXXXXXXCATG
GTACOOOOOOOOOOXXXXXXXXXXGTAC-FL-5'
[0083] The ditags are then purified and melted to yield
single-stranded DNAs having the formula:
6 5'-FL-CATGOOOOOOOOOOOXXXXXXXXXCATG and
GTACOOOOOOOOOOXXXXXXXXXXGTAC-FL-5',
[0084] for example. The mixture of single-stranded DNAs is
preferably serially diluted. Each serial dilution is hybridized
under appropriate stringency conditions with solid matrices
containing gridded single-stranded oligonucleotides; all of the
oligonucleotides contain a half-site of the anchoring enzyme
cleavage sequence. In the example used herein, the oligonucleotide
sequences contain a CATG sequence at the 5' end:
7 CATGOOOOOOOOOO, CATGXXXXXXXXXX, etc. (or alternatively a CATG
sequence at the 3' end: OOOOOOOOOCATG)
[0085] The matrices can be constructed of any material known in the
art and the oligonucleotide-bearing chips can be generated by any
procedure known in the art, e.g. silicon chips containing
oligonucleotides prepared by the VLSIP procedure (Fodor et al.,
supra).
[0086] The oligonucleotide-bearing matrices are evaluated for the
presence or absence of a fluorescent ditag at each position in the
grid.
[0087] In a preferred embodiment, there are 4.sup.10, or 1,048,576,
oligonucleotides on the grid(s) of the general sequence
CATGOOOOOOOOOO, such that every possible 10-base sequence is
represented 3' to the CATG, where CATG is used as an example of an
anchoring enzyme half site that is complementary to the anchoring
enzyme half site at the 3' end of the ditag. Since there are
estimated to be no more than 100,000 to 200,000 different expressed
genes in the human genome, there are enough oligonucleotide
sequences to detect all of the possible sequences adjacent to the
3'-most anchoring enzyme site observed in the cDNAs from the
expressed genes in the human genome.
[0088] In yet another embodiment, structures as described above are
amplified, cleaved with tagging enzyme and thereafter with
anchoring enzyme to generate tag complements, which can then be
labeled, melted, and hybridized with oligonucleotides on a solid
support.
[0089] A determination is made of differential expression by
comparing the fluorescence profile on the grids at different
dilutions among different libraries (representing differential
screening probes). For example:
8 Library A, Ditags Diluted Library B, Ditags Diluted 1:10 1:10 A B
C D E A B C D E 1 FL 1 FL 2 FL 2 FL FL 3 FL FL 3 FL FL 4 FL 4 5 FL
5 FL FL Library A, Ditags Diluted Library A, Ditags Diluted 1:50
1:100 A B C D E A B C D E 1 FL 1 FL 2 2 3 FL 3 FL 4 FL 4 FL 5 FL 5
FL Library B, Ditags Diluted Library B, Ditags Diluted 1:5 1:100 A
B C D E A B C D E 1 FL 1 FL 2 FL 2 FL 3 FL FL 3 FL 4 4 5 5
[0090] The individual oligonucleotides thus hybridize to ditags
with the following characteristics:
9TABLE 3 1:10 1:50 1:100 Dilution Lib A Lib B Lib A Lib B Lib A Lib
B 1A + + + + + + 2C + + + 2E + + 3B + + + + + + 3C + + + 4D + + +
5A + + + + 5E +
[0091] Table 3 summarizes the results of the differential
hybridization. Tags hybridizing to 1A and 3B reflect highly
abundant nRNAs that are not differentially expressed (since the
tags hybridize to both libraries at all dilutions); tag 2C
identifies a highly abundant mRNA, but only in Library B. 2E
reflects a low abundance transcript (since it is only detected at
the lowest dilution) that is not found to be differentially
expressed; 3C reflects a moderately abundant transcript (since it
is expressed at the lower two dilutions) in Library B that is
expressed at low abundance in Library A. 4D reflects a
differentially-expressed, high abundance transcript restricted to
Library A; 5A reflects a transcript that is expressed at high
abundance in Library A but only at low abundance in Library B; and
5E reflects a differentially-expressed transcript that is
detectable only in Library B.
[0092] In another PSA embodiment, step 3 above does not involve the
use of a fluorescent or other identifier; instead, at the last
round of amplification of the ditags, labeled dNTPs are used so
that after melting, half of all molecules are labeled and can serve
as probes for hybridization to oligonucleotides fixed on the
chips.
[0093] In yet another PSA embodiment, instead of ditags, a
particular portion of the transcript is used, e.g., the sequence
between the 3' terminus of the transcript and the first anchoring
enzyme site. In that particular case, a double-stranded cDNA
reverse transcript is generated as described in the Detailed
Description. The transcripts are cut with the anchoring enzyme, a
linker is added containing a PCR primer and amplification is
initiated (using the primer at one end and the poly A tail at the
other) while the transcripts are still on the strepavidin bead. At
the last round of amplification, fluoresceinated dNTPs are used so
that half of the molecules are labeled. The linker-primer can be
optionally removed by use of the anchoring enzyme at this point in
order to reduce the size of the fragments. The soluble fragments
are then melted and captured on solid matrices containing
CATGOOOOOOOOOO, as in the previous example. Analysis and scoring
(only of the half of the fragments which contain fluoresceinated
bases) is as described above.
[0094] For use in clonal sequencing, ditags or concatemers would be
diluted and added to wells of multiwell plates, for example, or
other receptacles so that on average the wells would contain,
statistically, less than one DNA molecule per well (as is done in
limited dilution for cell cloning). Each well would then receive
reagents for PCR or another amplification process and the DNA in
each receptacle would be sequenced, e.g., by mass spectroscopy. The
results will either be a single sequence (there having been a
single sequence in that receptacle), a "null" sequence (no DNA
present) or a double sequence (more than one DNA molecule), which
would be eliminated from consideration during data analysis.
Thereafter, assessment of differential expression would be the same
as described herein.
[0095] These results demonstrate that SAGE provides both
quantitative and qualitative data about gene expression. The use of
different anchoring enzymes and/or tagging enzymes with various
recognition elements lends great flexibility to this strategy. In
particular, since different anchoring enzymes cleave cDNA at
different sites, the use of at least 2 different Aes on different
samples of the same cDNA preparation allows confirmation of results
and analysis of sequences that might not contain a recognition site
for one of the enzymes.
Example 5
[0096] The Long SAGE method was performed using the standard SAGE
protocol (available from http://www.sagenet.org/sage_protocol.htm)
with the following modifications. Linkers containing the MmeI
recognition site were ligated to 3' cDNA ends after NlaIII
digestion (Linker 1A
5'-TTTGGATTTGCTGGTGCAGTACAACTAGGCTTAATATCCGACATG-3' and Linker 1B
5'-TCGGATATTAAGCCTAGTTGTACTGCACCAGCAAATCC Amino Modified C7-3' were
annealed together and ligated to half the cDNA population, and
Linker 2A 5'-TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGTCCGACATG-3' and
Linker 2B 5'-TCGGACGTACATCGTTAGAAGCTTGAATTCGAGCAG Amino Modified
C7-3' were annealed together and ligated to the remaining half of
the cDNA). Linker tag molecules were released from the cDNA using
the MmeI type IIS restriction endonuclease. (University of Gdansk
Center for Technology Transfer, Gdansk, Poland). Digestion was
performed at 37.degree. C. for 2.5 hrs using 40U MmeI in 300 .mu.L
of 10 mM HEPES, pH 8.0, 2.5 mM KOAc, 5 mM MgOAc, 2 mM DTT, and 40
.mu.M S-adenosylmethionine. To maximize the information content of
the LSAGE tags the 2 bp 3' overhang created by digestion with MmeI
was not polished, and the Linker 1 tag and Linker 2 tag molecules
were ligated together in a 6 .mu.l reaction containing 4 U T4 DNA
ligase (GIBCO BRL) in the supplied buffer for 2.5 hours at
16.degree. C. The SAGE software was modified to allow extraction of
21 bp tags from sequences of concatemer clones. A detailed protocol
of the LSAGE method and the LSAGE software group is available at
http://www.sagenet.org/LongSAGE.htm.
[0097] As efforts to fully characterize the genome near completion,
SATE should allow a direct readout of expression in any given cell
type or tissue. In the interim, a major application of SATE will be
the comparison of gene expression patterns in among tissues and in
various developmental and disease states in a given cell or tissue.
One of skill in the art with the capability to perform PCR and
manual sequencing could perform SAGE for this purpose. Adaptation
of this technique to an automated sequencer would allow the
analysis of over 1,000 transcripts in a single 3 hour run. An ABI
377 sequencer can produce a 451 bp readout for 36 templates in a 3
hour run (45 lbp/11 bp per tag.times.36=1476 tags). The appropriate
number of tags to be determined will depend on the application. For
example, the definition of genes expressed at relatively high
levels (0.5% or more) in one tissue, but low in another, would
require only a single day. Determination of transcripts expressed
at greater than 100 mRNA's per cell (0.025% or more) should be
quantifiable within a few months by a single investigator. Use of
two different Anchoring Enzymes will ensure that virtually all
transcripts of the desired abundance will be identified. The genes
encoding those tags found to be most interesting on the basis of
their differential representation can be positively identified by a
combination of data-base searching, hybridization, and sequence
analysis as demonstrated in Table 2. Obviously, SATE could also be
applied to the analysis of organisms other than humans, and could
direct investigation towards genes expressed in specific biologic
states.
10TABLE 4 Theoretical Matching of Tags to Genome. Probability
Complexity* tag is unique in genome.sup.+ Tag length (N bp) C =
4.sup.(N-4) P(u) = [(C-1)/C].sup.30,000,000 14 1,048,576 0.00% 15
4,194,304 0.08% 16 16,777,216 16.73% 17 67,108,864 63.95% 18
268,435,456 89.43% 19 1,073,741,824 97.24% 20 4,294,967,296 99.30%
21 17,179,869,184 99.83% *Complexity of tags is determined using a
tag length comprised of a constant 4 bp sequence representing the
restriction site at which the transcript was cleaved, followed by N
bp derived from the adjacent sequence in each transcript. .sup.+The
probability that a tag is unique in the genome is determined
assuming the genome contains .about.30 .times. 10.sup.6 Nlalll
derived tags and is comprised of random sequence.
[0098] SATE, as described herein, allows comparison of expression
of numerous genes among tissues or among different states of
development of the same tissue, or between pathologic tissue and
its normal counterpart. Such analysis is useful for identifying
therapeutically, diagnostically and prognostically relevant genes,
for example. Among the many utilities for SATE technology, is the
identification of appropriate antisense or triple helix reagents
which may be therapeutically useful. Further, gene therapy
candidates can also be identified by the SATE technology. Other
uses include diagnostic applications for identification of
individual genes or groups of genes whose expression is shown to
correlate to predisposition to disease, the presence of disease,
and prognosis of disease, for example. An abundance profile, such
as that depicted in Table 1, is useful for the above described
applications. SATE is also useful for detection of an organism
(e.g., a pathogen) in a host or detection of infection-specific
genes expressed by a pathogen in a host.
[0099] The ability to identify a large number of expressed genes in
a short period of time, as described by SATE in the present
invention, provides unlimited uses. Although the invention has been
described with reference to the presently preferred embodiment, it
should be understood that various modifications can be made without
departing from the spirit of the invention. Accordingly, the
invention is limited only by the following claims.
* * * * *
References