U.S. patent application number 10/889898 was filed with the patent office on 2005-03-31 for differentially methylated sequences in pancreatic cancer.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Goggins, Michael G., Ueki, Takashi.
Application Number | 20050069924 10/889898 |
Document ID | / |
Family ID | 23034863 |
Filed Date | 2005-03-31 |
United States Patent
Application |
20050069924 |
Kind Code |
A1 |
Goggins, Michael G. ; et
al. |
March 31, 2005 |
Differentially methylated sequences in pancreatic cancer
Abstract
The present invention provides a method for detecting a cellular
proliferative disorder in a subject. The method includes contacting
a nucleic acid-containing specimen from the subject with an agent
that provides a determination of the methylation state of at least
one gene or associated regulatory region of the gene and
identifying aberrant methylation of regions of the gene or
regulatory region, wherein aberrant methylation is identified as
being different when compared to the same regions of the gene or
associated regulatory region in a subject not having said cellular
proliferative, thereby detecting a cellular proliferative disorder
in the subject.
Inventors: |
Goggins, Michael G.;
(Baltimore, MD) ; Ueki, Takashi; (Sparks,
MD) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
|
Family ID: |
23034863 |
Appl. No.: |
10/889898 |
Filed: |
July 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10889898 |
Jul 8, 2004 |
|
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10084555 |
Feb 25, 2002 |
|
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60271268 |
Feb 23, 2001 |
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Current U.S.
Class: |
435/6.12 ;
435/199; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6886 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/199; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/47; C12N 009/22 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule selected from SEQ ID NO:1-42,
and regulatory sequences associated therewith.
2. The nucleic acid molecule of claim 1, wherein said associated
regulatory sequences contain CpG-rich regions.
3. The nucleic acid molecule of claim 2, wherein the state of
methylation of the CpG-rich regions is determinative of the
presence of a cellular proliferative disorder in a subject from
which the nucleic acid molecule is isolated.
4. The nucleic acid molecule of claim 2, wherein hypermethylation
of said CpG islands is indicative of the presence of a cellular
proliferative disorder in a subject from which said nucleic acid is
isolated.
5. The nucleic acid molecule of claim 1, wherein the molecule is
selected from SEQ ID NO:39-42.
6. A substantially purified polypeptide encoded by a polynucleotide
selected from SEQ ID NO:39-42.
7. A method for detecting a cellular proliferative disorder in a
subject comprising: a) contacting a nucleic acid-containing
specimen from the subject with an agent that provides a
determination of the methylation state of at least one gene or
associated regulatory region of the gene selected from MICP 1-42 of
Table 1 and combinations thereof; and b) identifying aberrant
methylation of regions of the gene or regulatory region, wherein
aberrant methylation is identified as being different when compared
to the same regions of the gene or associated regulatory region in
a subject not having said cellular proliferative, thereby detecting
a cellular proliferative disorder in the subject.
8. The method of claim 7, wherein the regions of said gene are
contained within CpG rich regions.
9. The method of claim 7, wherein the gene is SEQ ID NO:39, 40, 41
or 42.
10. The method of claim 7, wherein aberrant methylation comprises
hypermethylation when compared to the same regions of the gene or
associated regulatory regions in a subject not having the cellular
proliferative disorder.
11. The method of claim 10, wherein the regions comprise regulatory
regions of the gene.
12. The method of claim 7, wherein the agent is a pair of primers
that hybridize with a target sequence in the gene or associated
regulatory region of the gene.
13. The method of claim 7, wherein the nucleic acid-containing
specimen comprises a tissue selected from the group consisting of
brain, colon, urogenital, lung, renal, prostate, pancreas, liver,
esophagus, stomach, hematopoietic, breast, thymus, testis, ovarian,
and uterine.
14. The method of claim 7, wherein the nucleic acid-containing
specimen is selected from the group consisting of serum, urine,
saliva, blood, duodenal fluid, pancreatic fluid, cerebrospinal
fluid, pleural fluid, ascites fluid, sputum, stool, and biopsy
sample.
15. The method of claim 11, wherein said cellular proliferative
disorder is selected from the group consisting of low grade
astrocytoma, anaplastic astrocytoma, glioblastoma, medulloblastoma,
gastric cancer, colorectal cancer, colorectal adenoma, acute
myelogenous leukemia, lung cancer, renal cancer, leukemia, breast
cancer, prostate cancer, endometrial cancer and neuroblastoma.
16. A kit useful for the detection of a cellular proliferative
disorder in a subject comprising: a) carrier means
compartmentalized to receive a sample therein; b) one or more
containers comprising a first container containing a reagent which
modifies unmethylated cytosine and a second container containing
primers for amplification of a CpG-containing nucleic acid, wherein
the primer hybridizes with a target polynucleotide sequence having
the sequence selected from SEQ ID NO:1-42.
17. The kit of claim 16, further comprising a third container
containing a methylation sensitive restriction endonuclease.
18. The kit of claim 16, wherein said modifying reagent is
bisulfite.
19. The kit of claim 16, wherein the primer hybridizes with a
target polynucleotide sequence having a sequence as set forth in
SEQ ID NO:39, 40, 41 or 42.
20. Isolated oligonucleotide primer(s) for detection of a
methylated CpG-containing nucleic acid wherein the primer
hybridizes with a target polynucleotide sequence having the
sequence selected from the group consisting of SEQ ID NO:1-42.
21. Isolated oligonucleotide primer pairs selected from SEQ ID
NO:43-105.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Ser. No. 60/271,268,
filed Feb. 23, 2001, which is herein incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the regulation of
gene expression and more specifically to a method of determining
the DNA methylation status of CpG sites in a given locus and
correlating the methylation status with the presence of a cell
proliferative disorder.
BACKGROUND OF THE INVENTION
[0003] DNA methylases transfer methyl groups from the universal
methyl donor S-adenosyl methionine to specific sites on the DNA.
Several biological functions have been attributed to the methylated
bases in DNA. The most established biological function for
methylated DNA is the protection of DNA from digestion by cognate
restriction enzymes. The restriction modification phenomenon has,
so far, been observed only in bacteria. Mammalian cells, however,
possess a different methylase that exclusively methylates cytosine
residues that are 5' neighbors of guanine (CpG). This modification
of cytosine residues has important regulatory effects on gene
expression, especially when involving CpG rich areas, known as CpG
islands, located in the promoter regions of many genes.
[0004] Methylation has been shown by several lines of evidence to
play a role in gene activity, cell differentiation, tumorigenesis,
X-chromosome inactivation, genomic imprinting and other major
biological processes (Razin, A., H., and Riggs, R. D. eds. in DNA
Methylation Biochemistry and Biological Significance,
Springer-Verlag, New York, 1984). In eukaryotic cells, methylation
of cytosine residues that are immediately 5' to a guanosine, occurs
predominantly in CG poor regions (Bird, A., Nature, 321:209, 1986).
In contrast, CpG islands remain unmethylated in normal cells,
except during X-chromosome inactivation (Migeon, et al., supra) and
parental specific imprinting (Li, et al., Nature, 366:362, 1993)
where methylation of 5' regulatory regions can lead to
transcriptional repression. De novo methylation of the Rb gene has
been demonstrated in a small fraction of retinoblastomas (Sakai, et
al., Am. J. Hum. Genet., 48:880, 1991), and recently, a more
detailed analysis of the VHL gene showed aberrant methylation in a
subset of sporadic renal cell carcinomas (Herman, et al., Proc.
Natl. Acad. Sci., U.S.A., 91:9700, 1994). Expression of a tumor
suppressor gene can also be abolished by de novo DNA methylation of
a normally unmethylated CpG island (Issa, et al., Nature Genet.,
7:536, 1994; Herman, et al., supra; Merlo, et al., Nature Med.,
1:686, 1995; Herman, et al., Cancer Res., 56:722, 1996; Graff, et
al., Cancer Res., 55:5195, 1995; Herman, et al., Cancer Res.,
55:4525, 1995).
[0005] Human cancer cells typically contain somatically altered
nucleic acid, characterized by mutation, amplification, or deletion
of critical genes. In addition, the nucleic acid from human cancer
cells often displays somatic changes in DNA methylation (E. R.
Fearon, et al., Cell, 61:759, 1990; P. A. Jones, et al., Cancer
Res., 46:461, 1986; R. Holliday, Science, 238:163, 1987; A. De
Bustros, et al., Proc. Natl. Acad. Sci., USA, 85:5693, 1988); P. A.
Jones, et al., Adv. Cancer Res., 54:1, 1990; S. B. Baylin, et al.,
Cancer Cells, 3:383, 1991; M. Makos, et al., Proc. Natl. Acad.
Sci., USA, 89:1929, 1992; N. Ohtani-Fujita, et al., Oncogene,
8:1063, 1993). However, the precise role of abnormal DNA
methylation in human tumorigenesis has not been established.
Aberrant methylation of normally unmethylated CpG islands has been
described as a frequent event in immortalized and transformed
cells, and has been associated with transcriptional inactivation of
defined tumor suppressor genes in human cancers. In the development
of colorectal cancers (CRC), a series of tumor suppressor genes
(TSG) such as APC, p53, DCC and DPC4 are inactivated by mutations
and chromosomal deletions. Some of these alterations result from a
chromosomal instability phenotype described in a subset of CRC.
Recently, an additional pathway has been shown to be involved in a
familial form of CRC, hereditary non-polyposis colorectal cancer.
The cancers from these patients show a characteristic mutator
phenotype which causes microsatellite instability (MI), and
mutations at other gene loci such as TGF-.beta.-RII (Markowitz et
al., Science, 268(5215): 1336-8, 1995) and BAX. This phenotype
usually results from mutations in the mismatch repair (MMR) genes
hMSH2 and hMLH1. A subset of sporadic CRC also show MI, but
mutations in MMR genes appear to be less frequent in these
tumors.
[0006] Another molecular defect described in CRC is CpG island
(CGI) methylation. CGIs are short sequences rich in the CpG
dinucleotide and can be found in the 5' region of about half of all
human genes. Methylation of cytosine within 5' CGIs is associated
with loss of gene expression and has been seen in physiological
conditions such as X chromosome inactivation and genomic
imprinting. Aberrant methylation of CGIs has been detected in
genetic diseases such as the fragile-X syndrome, in aging cells and
in neoplasia. About half of the tumor suppressor genes which have
been shown to be mutated in the germline of patients with familial
cancer syndromes have also been shown to be aberrantly methylated
in some proportion of sporadic cancers, including Rb, VHL, p16,
hMLH1, and BRCA1. TSG methylation in cancer is usually associated
with (Antequera, et al., Proc. Natl. Acad. Sci. USA,
90:11995-11999, 1993) lack of gene transcription and (Baylin, et
al., Adv. Cancer Res., 72:141-196, 1998) absence of coding region
mutation. Thus it has been proposed that CGI methylation serves as
an alternative mechanism of gene inactivation in cancer.
[0007] The causes and global patterns of CGI methylation in human
cancers remain poorly defined. Aging could play a factor in this
process because methylation of several CGIs could be detected in an
age-related manner in normal colon mucosa as well as in CRC. In
addition, aberrant methylation of CGIs has been associated with the
MI phenotype in CRC as well as specific carcinogen exposures.
However, an understanding of aberrant methylation in CRC has been
somewhat limited by the small number of CGIs analyzed to date. In
fact, previous studies have suggested that large numbers of CGIs
are methylated in immortalized cell lines, and it is not well
understood whether this global aberrant methylation is caused by
the cell culture conditions or whether they are an integral part of
the pathogenesis of cancer.
[0008] Most of the methods developed to date for detection of
methylated cytosine depend upon cleavage of the phosphodiester bond
alongside cytosine residues, using either methylation-sensitive
restriction enzymes or reactive chemicals such as hydrazine which
differentiate between cytosine and its 5-methyl derivative. Genomic
sequencing protocols which identify a 5-MeC residue in genomic DNA
as a site that is not cleaved by any of the Maxam Gilbert
sequencing reactions have also been used, but still suffer
disadvantages such as the requirement for large amount of genomic
DNA and the difficulty in detecting a gap in a sequencing ladder
which may contain bands of varying intensity.
[0009] Mapping of methylated regions in DNA has relied primarily on
Southern hybridization approaches, based on the inability of
methylation-sensitive restriction enzymes to cleave sequences which
contain one or more methylated CpG sites. This method provides an
assessment of the overall methylation status of CpG islands,
including some quantitative analysis, but is relatively insensitive
and requires large amounts of high molecular weight DNA.
[0010] Another method utilizes bisulfite treatment of DNA to
convert all unmethylated cytosines to uracil. The altered DNA is
amplified and sequenced to show the methylation status of all CpG
sites. However, this method is technically difficult, labor
intensive and without cloning amplified products, it is less
sensitive than Southern analysis, requiring approximately 10% of
the alleles to be methylated for detection.
[0011] Identification of the earliest genetic changes in
tumorigenesis is a major focus in molecular cancer research.
Diagnostic approaches based on identification of these changes are
likely to allow implementation of early detection strategies and
novel therapeutic approaches targeting these early changes might
lead to more effective cancer treatment.
[0012] About half of all human genes have 5'CpG islands and these
islands are usually associated with the 5' regulatory regions of
genes (Antequera, et al., Proc. Natl. Acad. Sci. USA
90:11995-11999, 1993). The 5' CpG islands of most nonimprinted
genes are thought to remain unmethylated in normal cells but may
become methylated during aging or tumorigenesis. Through
interactions between methyl CpG binding proteins, histones and
histone deacetylase, 5' CpG island methylation can contribute to
changes in chromatin that cause transcriptional silencing (Baylin,
et al., Adv. Cancer Res. 72:141-196, 1998). Promoter methylation is
implicated in the transcriptional silencing of tumor suppressor and
mismatch repair genes (e.g. p16, Rb, VHL, hMLH1) in many cancers.
Although 13 hypermethylated genes and clones in pancreatic cancers
were previously identified (Ueki, et al., Cancer Res. 60:1835-1839,
2000), there almost certainly are others. Costello et al. have
estimated that .about.400 genes are aberrantly methylated in
cancers and have found evidence for tumor-specific pattern of
methylation (Costello, et al., Nat. Genet. 24:132-138, 2000). A
better description of the pattern of DNA methylation abnormalities
in cancer may improve an understanding of the role of DNA
methylation in tumorigenesis and identification of differentially
methylated CpG islands in cancer may lead to the discovery of novel
genes with tumor suppressor properties. Finally, identified genes
or loci could be utilized as cancer-specific markers for the early
detection of cancer (Belinsky, et al., Proc. Natl. Acad. Sci. USA
95:11891-11896, 1998).
[0013] Pancreatic cancer is the fourth leading cause of cancer
death in men and in women and each year .about.28,000 Americans die
of the disease (Greenlee, et al., CA Cancer J. Clin. 50:7-33,
2000). Frequent genetic changes such as mutational activation of
the K-ras oncogene and inactivation of the p16, DPC4, p53, MKK4,
STK11, TGFBR2, and TGFBR1 tumor suppressor genes have been
described in pancreatic cancer (Goggins, et al., Ann. Oncol.
10:4-8, 1999, Rozenblum, et al., Cancer Res. 57:1731-1734, 1997).
Although multiple tumor suppressor pathways have been shown to play
a role in pancreatic carcinogenesis, little is known about the
contribution of DNA methylation to inactivation of genes in these
pathways. Recently, a novel technique, methylated CpG island
amplification (MCA), was developed to enrich for methylated CpG
rich sequences. MCA coupled with RDA (MCA/RDA) can recover CpG
islands differentially methylated in cancer cells (Toyota, et al.,
Cancer Res. 59:2307-2312, 1997).
SUMMARY OF THE INVENTION
[0014] The present invention is based on the finding that several
genes are newly identified as being differentially methylated in
cancer. This seminal discovery is useful for cancer screening,
risk-assessment, prognosis, minimal-residual disease
identification, staging and identification of therapeutic targets.
The identification of new genes that are methylated in cancer,
aging or diseases associated with aging increases the likelihood of
finding genes methylated in a particular cancer; increases the
sensitivity and specificity of methylation detection; allows
methylation profiling using multiple genes; and allows
identification of new targets for therapeutic intervention. The
invention also provides a newly identified gene that is a target
for hypermethylation in human tumors.
[0015] In one embodiment, there are provided methods for detecting
a cellular proliferative disorder in a subject. The subject may
have or be at risk of having a cellular proliferative disorder. The
method of the invention is useful for diagnostic as well as
prognostic analyses. One method for detecting a cellular
proliferative disorder in a subject includes contacting a nucleic
acid-containing specimen from the subject with an agent that
provides a determination of the methylation state of at least one
gene or associated regulatory region of the gene; and identifying
aberrant methylation of regions of the gene or regulatory region,
wherein aberrant methylation is identified as being different when
compared to the same regions of the gene or associated regulatory
region in a subject not having the cellular proliferative, thereby
detecting a cellular proliferative disorder in the subject. The
method includes multiplexing by utilizing a combination of primers
for more than one loci, thereby providing a methylation "profile"
for more than one gene or regulatory region.
[0016] For the first time, the invention provides methylated forms
of genes and/or their associated regulatory sequences referred to
herein as MICP1-42. MICP39-42 have no homology to known human
sequences. Eleven clones matched human genes (MICP1-11); 10 clones
matched human ESTs (MICP12-21); 5 clones matched human CpG islands
(MICP22-26); and 12 clones matched human genome sequences
(MICP27-38). (see Table 1 as reference).
[0017] Invention methods include determining, in a nucleic
acid-containing specimen taken from a subject, the methylation
state of a gene or regulatory sequences associated therewith,
wherein the expression or non-expression of the gene is associated
with the presence of the cellular proliferative disorder, and
identifying as having a cellular proliferative disorder a subject
that has aberrant methylation of regions of the gene or associated
regulatory sequences when compared to the same regions of the gene
in a subject not having the cellular proliferative disorder. In one
aspect of this embodiment, the methylated regions of the gene and
associated regulatory sequences are contained within CpG islands
(i.e., CpG rich regions). In particular, the aberrant methylation
typically includes hypermethylation as compared with the same
regions of the gene or regulatory sequences in a subject not having
the cellular proliferative disorder.
[0018] Determining the methylation state of the gene includes
contacting the nucleic acid-containing specimen with an agent that
modifies unmethylated cytosine, amplifying a CpG-containing nucleic
acid in the specimen by means of CpG-specific oligonucleotide
primers, wherein the oligonucleotide primers distinguish between
modified methylated and nonmethylated nucleic acid, and detecting
the methylated nucleic acid based on the presence-or absence of
amplification products produced in said amplifying step. The method
includes optionally contacting the amplification products with a
methylation sensitive restriction endonuclease. Other methods for
determining methylation status of a gene and/or regulatory
sequences are well known in the art and are described more fully
herein.
[0019] In another embodiment of the present invention there is
provided a kit useful for the detection of a cellular proliferative
disorder in a subject having or at risk for having a cellular
proliferative disorder. Invention kits include a carrier means
compartmentalized to receive a sample, one or more containers
comprising a first container containing a reagent which modifies
unmethylated cytosine and a second container containing primers for
amplification of a CpG-containing nucleic acid, wherein the primers
distinguish between modified methylated and nonmethylated nucleic
acid, and optionally, a third container containing a methylation
sensitive restriction endonuclease. In a preferred embodiment, the
cell proliferative disorder is pancreatic carcinoma.
[0020] To identify CpG islands differentially methylated in
pancreatic adenocarcinoma, methylated CpG island amplification
(MCA) was used, coupled with representational difference analysis
(MCA/RDA). Clones differentially methylated (termed MICP,
methylated in carcinoma of the pancreas) were isolated in a panel
of 8 pancreatic cancer cell lines compared to normal pancreas. 95%
of these clones were CpG islands and among these clones were 5' CpG
islands of several known genes, including Cyclin G and
Preproenkephalin (ppENK, encoding [Met5]-enkephalin). Seven of the
clones (Cyclin G, ppENK, MICP20, 23, 33, 35 and 36) were not
methylated in 14 normal pancreata by MSP while 15 primary
pancreatic adenocarcinomas were methylated in 7%, 87%, 13%, 53%,
33%, 40% and 0% of cases, respectively. Two of the 5 chronic
pancreatitis specimens harbored methylation of three (Cyclin G,
ppENK and MICP23) and one (ppENK) clones, respectively. There was
no identification of methylation of MICP33 and 35 in any normal
gastrointestinal tissues tested. Three clones (ppENK, MICP20 and
23) were variably methylated in normal gastric, duodenal and
colonic mucosae. Aberrant methylation of Cyclin G and ppENK in
methylated pancreatic cancer cell lines was associated with
transcriptional silencing that was reversible with
5-aza-2'-deoxycytidine treatment.
[0021] These data indicate that multiple CpG islands undergo de
novo methylation during pancreatic carcinogenesis. Methylation of
some CpG islands is cancer-specific while others show
tissue-specific patterns of methylation.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows the representative results of MCIPs isolated by
MCA/RDA. Dot blot analysis. 1A, An example of kinetic enrichment of
methylated sequences by RDA. MCA products from the driver and the
tester (PL8) and the PCR products from first (1st SA), second (2nd
SA) and third (3rd SA) competitive hybridization/selective
amplification were blotted onto the membrane and hybridized with a
labeled Cyclin G probe. 1B, Dot blot-analysis using MICPs isolated
from MCA/RDA as probes. First three MICPs were hybridized only to
the tester (either PL3 or PL8), whereas the next three MICPs were
weakly hybridized to the driver as well as the tester.
[0023] FIG. 2 shows a CpG plot across the 5' CpG islands of Cyclin
G and ppENK showing the relation between isolated clones and their
corresponding genes. The positions of MSP primers are also
indicated. The black boxes represent exons.
[0024] FIG. 3 shows a summary of the average level of methylation
of selected MCA/RDA MICPs by bisulfite-modified genomic sequencing.
The specimen number and the age of the patients are in the left
column. Empty oval, 0-10% methylation; white oval with black dots,
11-30% methylation; black oval with white dots, 31-70% methylation;
black oval, 71-100% methylation; .cndot., not determined. NP,
normal pancrease; PCa, primary pancreatic adenocarcinonma.
[0025] FIG. 4 shows MSP analyses of MICP36 (A) and MICP25 (B) in
primary pancreatic adenocarcinomas and normal tissues. The PCR
products in the lanes U and M indicate the presence of unmethylated
and methylated templates, respectively. PCa, primary pancreatic
carcinoma, NP, normal pancreas, NC, normal colonic mucosa.
Expression of Cyclin G (C) and ppENK (D) in pancreatic cancer cell
lines and normal pancreata by RT-PCR analysis. Cyclin G and ppENK
were coamplified with GAPDH to ensure the RNA integrity. Cyclin G
was expressed at low level in the methylated cell line PL8 compared
with unmethylated cell lines (PL3, CAPAN2, MiaPaca2, and Panc1) and
four normal pancreata. 5 Aza-dC treatment increased Cyclin G
expression in PL8. All methylated cell lines examined (PL3, CAPAN2,
MiaPaca2 and Panc1) lacked expression of ppENK, whereas all three
normal pancreata expressed ppENK. Treatment of four cell lines with
5 Aza-dC restored the ppENK expression. The expected sizes of the
PCR products are 306 bp for GAPDH, 207 bp for Cyclin G, and 179 bp
for ppENK.
[0026] FIG. 5 shows aberrant methylation of MICPs and
clinicopathological variables. Multivariate linear regression
analysis was used to determine the relationship between the number
of aberrantly methylated MICPs in a pancreatic adenocarcinoma and
the variables patient age (left plot) and tumor diameter (right
plot). For each decade increase in age, the average number of
methylated loci increases by 0.28, regardless of the tumor
diameter. For each increase in tumor diameter (in cm), the number
of mehtylated loci increases by 0.156 regardless of patient
age.
[0027] FIGS. 6a-6i show the nucleic acid sequence for SEQ ID
NO:1-42.
DETAILED DESCRIPTION OF THE INVENTION
[0028] It has been determined that an aberrant methylation state of
nucleic acids in certain genes, particularly regulatory sequences,
is diagnostic for the presence or potential development of a
cellular proliferative disorder in subjects bearing the aberrantly
methylated nucleic acids. More particularly, the hypermethylation
of certain nucleotides localized in CpG islands has been shown to
affect the expression of genes associated with the CpG islands;
typically such hypermethylated genes have reduced or abolished
expression, primarily due to down-regulated transcription. Using a
recently developed PCR-based technique called methylated CpG island
amplification (MCA), several nucleic acid molecules aberrantly
methylated in pancreatic cancer cells line were identified.
[0029] Of the 42 unique clones isolated using MCA/RDA, seven
(Cyclin G, ppENK, MICP20, 23, 33, 35 and 36) were CpG islands
aberrantly methylated in pancreatic carcinoma compared to normal
pancreas. Indeed, none of these 7 clones, were methylated in a
panel of normal pancreata using the highly sensitive method of MSP.
Two of these seven clones corresponded to the 5' CpG island of the
genes ppENK and Cyclin G. ppENK encodes opioid growth factor, also
known as [Met5]-enkephalin. This opioid peptide induces apoptosis
in lung cancer cell lines (Maneckjee & Minna, Cell Growth
Differ. 5:1033-1040, 1994) and several studies have demonstrated
that this peptide has a negative growth regulatory effect on
various kinds of cancers, including pancreatic cancer (Zagon, et
al., Int. J. Oncol. 14:577-584, 1999). Aberrant methylation of the
5' CpG island of ppENK was found in 87% of primary pancreatic
carcinomas. Hypermethylation was associated with transcriptional
silencing of this gene in pancreatic cancer cell lines. Although a
low level of methylation in other normal mucosae by MSP was also
observed, these results indicate that de novo methylation of 5' CpG
islands and transcriptional repression of ppENK might contribute to
pancreatic carcinogenesis.
[0030] Cyclin G is a target for transcriptional activation by p53
(Okamoto, et al., Embo. J. 13:48616-4822, 1994). Conflicting
functions for Cyclin G have been reported. When overexpressed
Cyclin G is associated with cell growth in vitro (Smith, et al.,
Exp. Cell. Res. 230:61-68, 1997), while increased expression of
Cyclin G also augments apoptosis of multiple cancer cell lines in
response to different stimuli (Okamoto & Prives, Oncogene
18:4606-4615, 1999). The data demonstrate the association between
hypermethylation of the 5'CpG island of Cyclin G and its
transcriptional repression in the pancreatic cancer cell line PL8
(FIG. 4C), suggesting that Cyclin G could have been selected for
silencing because of tumor suppressive functions. These data extend
previous findings demonstrating aberrant methylation of multiple
cancer-related genes including p16 and hMLH1 in pancreatic cancer
(Ueki, et al., Cancer Res. 60:1835-1839, 2000).
[0031] Methylation of several clones in pancreata with chronic
pancreatitis was also observed. Two of the 5 pancreata with chronic
pancreatitis harbored aberrant methylation and one of these two
pancreata contained a PanIN lesion and this latter sample displayed
methylation of 3 clones. Previous studies demonstrated that chronic
pancreatitis is a significant risk factor of development of
pancreatic cancer (Lowenfels, et al., N. Engl. J. Med.
328:1433-1437, 1993) and duct lesions (PanIN) often found in
chronic pancreatitis are considered as the precursors of
infiltrating pancreatic carcinoma (Hruban, et al., Clin. Cancer
Res. 6:2969-2972, 2000). The presence of aberrant methylation in
DNA from chronic pancreatitis suggests that de novo methylation of
CpG islands may be an early event in pancreatic cancer development
in this setting. Additional studies are needed to determine the
role and the timing of de novo methylation of CpG islands in
progression model of pancreatic cancer (Hruban, et al., Clin.
Cancer Res. 6:2969-2972, 2000). The absence of methylated templates
of MICP33 and 35 in any normal tissue examined raises the
possibility that MSP could be used to detect aberrant methylation
of these clones in clinical samples such as stool, blood, or
pancreatic fluid, for the early detection of pancreatic
cancers.
[0032] Three additional known genes containing methylated 5'CpG
islands in pancreatic cancers were identified (GAD1, ECEL1 and
PAX5). These genes were isolated by MCA/RDA because relatively
fewer DNA templates were methylated in normal pancreas. Some genes
that are methylated in cancer and in only a small percentage of
normal cells may appear to undergo selection during carcinogenesis
(Salem, et al., Cancer Res. 60:2473-2476, 2000), but are merely
unselected epigenetic markers of stem cells that have evolved to
cancers by other clonal selection events. This phenomenon is
important to be aware of when studying methylation in human cancer
and makes it much more difficult to assign causality to methylation
phenomena in cancers compared to genetic events such as homozygous
deletion. For normally unmethylated genes whose function is well
characterized such as hMLH1, or for genes that are methylated as a
second hit for a tumor suppressor gene (e.g. VHL, E-cadherin), or
for tumor suppressor genes alternatively targeted by genetic and
epigenetic inactivation (e.g. p16 and RB) (Baylin, et al., Adv.
Cancer Res. 72:141-196, 1998), the biological significance of
"aberrant methylation" is well accepted. As additional genes are
identified that are methylated in pancreatic cancer, it will be
important to rule out low-level methylation using sensitive
techniques such as MSP (Herman, et al., Proc. Natl. Acad. Sci. USA
93:9821-9826, 1996) before such genes are accepted as having
undergone selection through methylation.
[0033] Three CpG islands that were differentially methylated in
pancreatic cancer but were not located within 5' regions of their
corresponding genes were also identified, CSX, MCT3 and ICAM5. The
role of hypermethylation of non-5' CpG islands need to be defined,
but recent studies have shown that aberrant methylation in cancer
is not confined to the 5' region but can occur in internal exons
and 3' regions of genes (Costello, et al., Nat. Genet. 24:132-138,
2000, Liang, et al., Genomics 53:260-268, 1998). It is probable
that several of the unknown clones identified are genes whose
expression is repressed by aberrant methylation. For example, GRAIL
and GENSCAN programs suggest putative coding exons downstream of
MICP23 and MICP33.
[0034] In addition to identifying CpG islands with cancer-related
methylation, CpG islands that were methylated in both normal
pancreata and neoplastic tissues were isolated. Methylation of
non-neoplastic colorectal (Ahuja, et al., Cancer Res. 58:5489-5494,
1998, Toyota, et al., Proc. Natl. Acad. Sci. USA 96:8681-8686,
1999), bladder and prostate tissues (Liang, et al., Genomics
53:260-268, 1998) has been observed for several genes and CpG
islands. Methylation in normal tissues that is not the result of
imprinting is frequently "age-related." This has been best shown in
the colonic mucosa for genes such as ER (Ahuja, et al., Cancer Res.
58:5489-5494, 1998, Toyota, et al., Proc. Natl. Acad. Sci. USA
96:8681-8686, 1999). However, normal pancreata used in this study
were obtained from 14 patients (mean age of 62) and for at least 5
of these clones there was no evidence of age-related methylation
(FIG. 3). This suggests that the aberrant methylation observed in
the pancreatic cancers in this study is not simply a function of
age. Some of the clones listed in Table 1 could undergo age-related
methylation, but to demonstrate this would require many more normal
pancreata. Methylation of ppENK, MICP20 and 23 in a significant
percentage of the DNA templates within normal gastric, duodenal and
colonic mucosae highlights the tissue specific nature of DNA
methylation. This low level methylation of different normal tissues
might also explain some of the tumor-specific methylation patterns
observed by others (Costello, et al., Nat. Genet. 24:132-138,
2000). The data indicate that there are tissue-specific methylation
patterns in normal tissues (Liang, et al., Genomics 53:260-268,
1998) and age-related methylation changes are probably restricted
to certain genes and/or tissues (Ahuja, et al., Cancer Res.
58:5489-5494, 1998).
[0035] In this study, the MCA/RDA technique was modified and these
modifications may have improved the efficiency of the MCA/RDA
technique. Betaine was included in the PCR reaction and amplified
the methylated templates under a higher annealing temperature
(77.degree. C.). The combination of betaine and DMSO can uniformly
amplify a mixture of DNA with different GC content (Baskaran, et
al., Genome Res. 6:633-638, 1996). These modifications might have
enhanced the amplification of distinct clones instead of Alu
repetitive sequences that accounted for 60% of the recovered clones
using the original protocol (Toyota, et al., Cancer Res.
59:2307-2312, 1997). The subtractive and kinetic enrichment of
differentially methylated sequence by RDA as shown in this study
(FIG. 1A) may have advantages over other techniques to isolate
differentially methylated sequences between normal tissue and
cancer (Costello, et al., Nat. Genet. 24:132-138, 2000, Liang, et
al., Genomics 53:260-268, 1998). MCA/RDA, however, has limitations
for identifying differentially methylated sequences. First, MCA
only detects differentially methylated sequences with two
restriction enzyme sites (SmaI in this study). Second, MCA/RDA not
only identifies absolute differences in methylation between the
tester and the driver, it also will identify methylated sequences
both in the tester and the driver if for example, there is low
level methylation in the driver (normal pancreas). Third, some
clones that appeared to be unmethylated by dot-blot hybridization
were indeed methylated by bisulfite sequencing in normal pancreata
(MICP15, FIG. 1B and FIG. 4C). Finally, even if bisulfite
sequencing suggests that there is no methylation of a clone in
normal pancreata, it is important to rule out low-level methylation
in normal tissues using a sensitive technique, such as MSP.
[0036] The results indicate that aberrant methylation of CpG
islands is a common event in pancreatic carcinogenesis. Some de
novo methylated CpG islands could serve as cancer-specific markers
while others reflect tissue specific pattern of DNA
methylation.
[0037] Methylated nucleic acid sequences are also provided. For the
first time, the present invention provides methylated chemical
structures for MICP 1-42 (see Table 1). One of skill in the art can
now readily locate the CpG-rich sequences associated with these
genes and identify such methylated forms of the genes/regulatory
sequences by methods described herein (The gene sequences can be
identified in a gene database found at
http://www.ncbi.nim.nih.gov/UniGene/index.html). The invention
provides CpG-rich regions from the above genes as set forth in SEQ
ID Nos 1-42, equivalent to MICP 1-42, respectively.
[0038] The term "polynucleotide" or "nucleic acid sequence" refers
to a polymeric form of nucleotides at least 10 bases in length. An
"isolated polynucleotide" is a polynucleotide that is not
immediately contiguous with both of the coding sequences with which
it is immediately contiguous (one on the 5' end and one on the 3'
end) in the naturally occurring genome of the organism from which
it is derived. Thus, an isolated polynucleotide may include a
coding region with its associated regulatory sequences. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a cDNA)
independent of other sequences. The nucleotides of the invention
can be ribonucleotides, deoxyribonucleotides, or modified forms of
either nucleotide. Specifically, methylated forms of nucleotides in
a polynucleotide sequence are also included. The term includes
single and double forms of DNA.
[0039] As will be understood by those of skill in the art, when the
sequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ID
NO:1-42, are replaced by ribonucleotides A, G, C, and U,
respectively. Also included in the invention are fragments of the
above-described nucleic acid sequences that are at least 15 bases
in length, which is sufficient to permit the fragment to
selectively hybridize to DNA that encodes polypeptides encoded by
SEQ ID NO:1-42. The term "selectively hybridize" refers to
hybridization under moderately or highly stringent conditions (See,
Sambrook, as cited herein) which excludes non-related nucleotide
sequences.
[0040] The nucleic acid sequence includes the disclosed sequence
and sequences that encode conservative variations of the
polypeptides encoded by polynucleotides provided herein. The term
"conservative variation" as used herein denotes the replacement of
an amino acid residue by another, biologically similar residue.
Examples of conservative variations include the substitution of one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acid, or glutamine for asparagine, and the
like. The term "conservative variation" also includes the use of a
substituted amino acid in place of an unsubstituted parent amino
acid provided that antibodies raised to the substituted polypeptide
also immunoreact with the unsubstituted polypeptide.
[0041] Nucleic acid sequences of the invention can be expressed in
vitro by DNA transfer into a suitable host cell. "Host cells" are
cells in which a vector can be propagated and its DNA expressed.
The cell may be prokaryotic or eukaryotic. 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 cells" is used. Methods of stable
transfer, meaning that the foreign DNA is continuously maintained
in the host, are known in the art.
[0042] In one aspect, the nucleic acid sequences may be inserted
into an expression vector. The term "expression vector" refers to a
plasmid, virus or other vehicle known in the art that has been
manipulated by insertion or incorporation of the sequence of
interest genetic sequences. Polynucleotide sequence which encode
sequence of interest can be operatively linked to expression
control sequences. "Operatively linked" refers to a juxtaposition
wherein the components so described are in a relationship
permitting them to function in their intended manner. An expression
control sequence operatively linked to a coding sequence is ligated
such that expression of the coding sequence is achieved under
conditions compatible with the regulatory or expression control
sequences. As used herein, the terms "regulatory sequences" and
"expression control sequences" refers to nucleic acid sequences
that regulate the expression of a nucleic acid sequence to which it
is operatively linked. Expression control sequences are operatively
linked to a nucleic acid sequence when the expression control
sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signal for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of mRNA, and stop codons. The terms "regulatory
sequences" and "expression control sequences" are intended to
included, at a minimum, components whose presence can influence
expression, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion
partner sequences. An example of an expression control sequence
includes a promoter.
[0043] A "promoter" is a minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell-type specific, tissue-specific, or
inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the gene. Both constitutive and
inducible promoters, are included in the invention (see, e.g.,
Bitter et al., Methods in Enzymology 153:516-544, 1987). For
example, when cloning in bacterial systems, inducible promoters
such as pL of bacteriophage plac, ptrp, ptac (ptrp-lac hybrid
promoter) and the like may be used. When cloning in mammalian cell
systems, promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g.,
the retrovirus long terminal repeat; the adenovirus late promoter;
the vaccinia virus 7.5K promoter) may be used. Promoters produced
by recombinant DNA or synthetic techniques may also be used to
provide for transcription of the nucleic acid sequences of the
invention.
[0044] In the present invention, the polynucleotide sequences may
be inserted into an expression vector which contains a promoter
sequence which facilitates the efficient transcription of the
inserted genetic sequence of the host. The expression 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,
but are not limited to the T7-based expression vector for
expression in bacteria (Rosenberg et al., Gene 56:125, 1987), the
pMSXND expression vector for expression in mammalian cells (Lee and
Nathans, J. Biol. Chem. 263:3521, 1988) and baculovirus-derived
vectors for expression in insect cells. The DNA segment can be
present in the vector operably linked to regulatory elements, for
example, a promoter (e.g., T7, metallothionein I, or polyhedron
promoters).
[0045] Polynucleotide sequences of the invention can be expressed
in either prokaryotes or eukaryotes. Hosts can include microbial,
yeast, insect and mammalian organisms. Methods of expressing DNA
sequences having eukaryotic or viral sequences in prokaryotes are
well known in the art. Biologically functional viral and plasmid
DNA vectors capable of expression and replication in a host are
known in the art. Such vectors are used to incorporate DNA
sequences of the invention.
[0046] "Transformation" means a genetic change induced in a cell
following incorporation of new DNA (i.e., DNA exogenous to the
cell). Where the cell is a mammalian cell, the genetic change is
generally achieved by introduction of the DNA into the genome of
the cell (i.e., stable).
[0047] Thus, a "transformed cell" is a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding sequence of interest.
Transformation of a host cell with recombinant DNA 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 CaCl2 method using procedures well
known in the art. Alternatively, MgCl2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired.
[0048] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransformed with DNA
sequences encoding the sequence of interest, and a second foreign
DNA molecule encoding a selectable phenotype, such as the herpes
simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein (see for example, Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
[0049] Isolation and purification of microbial expressed
polypeptide, or fragments thereof, provided by the invention, may
be carried out by conventional means including preparative
chromatography and immunological separations involving monoclonal
or polyclonal antibodies.
[0050] In one embodiment, the invention provides substantially
purified polypeptide encoded by polynucleotide sequences SEQ ID
NO:1-42. The term "substantially purified" as used herein refers to
a polypeptide which is substantially free of other proteins,
lipids, carbohydrates or other materials with which it is naturally
associated. One skilled in the art can purify a polypeptide
sequence using standard techniques for protein purification. The
substantially pure polypeptide will yield a single major band on a
non-reducing polyacrylamide gel. The purity of the polypeptide can
also be determined by amino-terminal amino acid sequence
analysis.
[0051] Minor modifications of the primary amino acid sequences may
result in proteins which have substantially equivalent activity as
compared to the unmodified counterpart polypeptide described
herein. Such modifications may be deliberate, as by site-directed
mutagenesis, or may be spontaneous. All of the polypeptides
produced by these modifications are included herein as long as the
biological activity still exists.
[0052] The polypeptides of the invention also include dominant
negative forms of the invention polypeptide which do not have the
biological activity of invention polypeptide sequence. A "dominant
negative form" of invention is a polypeptide that is structurally
similar to invention polypeptide but does not have wild-type
invention function. For example, a dominant-negative invention
polypeptide may interfere with wild-type invention function by
binding to, or otherwise sequestering, regulating agents, such as
upstream or downstream components, that normally interact
functionally with the invention polypeptide.
[0053] To identify genes differentially methylated in colorectal
cancer, methylated CpG island amplification was used followed by
representational difference analysis (Razin and Cedar, Cell 17:
473-476, 1994, herein incorporated by reference). One of the clones
recovered (MINT31, see U.S. patent application Ser. No. 09/309,175,
incorporated by reference herein in its entirety) mapped to human
chromosome 17q21 using a radiation hybrid panel. A Blast search
revealed this fragment to be completely identical to part of a BAC
clone (Genbank: AC004590) sequenced by high throughput genomic
sequencing. The region surrounding MINT31 fulfills the criteria of
a CpG island: GC content 0.67, CpG/GpC ratio 0.78 and a total of
305 CpG sites in a 4 kb region. Using this CpG island and 10 kb of
flanking sequences in a Blast analysis, several regions highly
homologous to the rat T-type calcium channel gene, CACNA1G, were
identified (Perez-Reyes et al., Nature 391: 896-900. 1998, herein
incorporated by reference). Several ESTs were also identified in
this region. Using Genscan, 2 putative coding sequences (G1, and
G2) were identified. Blastp analysis revealed that G1 has a high
homology to the EH-domain-binding protein, epsin, while G2 is
homologous to a C. elegans hypothetical protein (accession No.
2496828).
[0054] Due to the clear correlation between methylation of CpG
islands and cellular proliferative disorders, in another embodiment
of the present invention, there are provided methods for detecting
a cellular proliferative disorder in a subject having or at risk
for said cellular proliferative disorder. The method includes
assaying, in nucleic acid-containing specimen taken from said
subject, the methylation state of a gene or its associated
regulatory regions, wherein the expression state of the gene or its
associated regulatory regions is associated with the presence of
the cellular proliferative disorder, and identifying as having a
cellular proliferative disorder a subject that has aberrant
methylation of regions of said gene. The method provides for
detecting a cellular proliferative disorder in a subject having or
at risk for said cellular proliferative disorder by identifying
aberrantly methylation of regions of a gene when compared to the
same regions of the gene in a subject not having said cellular
proliferative disorder.
[0055] The aberrant methylation comprises hypermethylated CpG rich
regions (i.e., islands). In one aspect of the present invention,
the CpG rich regions are associated with the invention genes gene,
and affect the expression thereof in a methylation state-dependent
manner.
[0056] A "cell proliferative disorder" or "cellular proliferative
disorder" is any disorder in which the proliferative capabilities
of the affected cells is different from the normal proliferative
capabilities of unaffected cells. An example of a cell
proliferative disorder is neoplasia. Malignant cells (i.e., cancer)
develop as a result of a multistep process. Specific, non-limiting
examples of cell proliferative disorders associated with increased
methylation of CpG-islands are low grade astrocytoma, anaplastic
astrocytoma, glioblastoma, medulloblastoma, gastric cancer,
colorectal cancer, colorectal adenoma, acute myelogenous leukemia,
lung cancer, renal cancer, pancreatic cancer, leukemia, breast
cancer, prostate cancer, endometrial cancer and neuroblastoma.
[0057] A cell proliferative disorder as described herein may be a
neoplasm. Such neoplasms are either benign or malignant. The term
"neoplasm" refers to a new, abnormal growth of cells or a growth of
abnormal cells that reproduce faster than normal. A neoplasm
creates an unstructured mass (a tumor) which can be either benign
or malignant. For example, the neoplasm may be a head, neck, lung,
esophageal, stomach, small bowel, colon, bladder, kidney, or
cervical neoplasm. The term "benign" refers to a tumor that is
noncancerous, e.g. its cells do not proliferate or invade
surrounding tissues. The term "malignant" refers to a tumor that is
metastastic or no longer under normal cellular growth control.
[0058] A cell proliferative disorder may be an age-associated
disorder. Examples of age-associated disorders which are cell
proliferative disorders include colon cancer, lung cancer, breast
cancer, prostate cancer, and melanoma, amongst others.
[0059] A "nucleic acid containing specimen" includes any type of
material containing a nucleic acid to be subject to invention
methods. The nucleic acid may be contained in a biological sample.
Such samples include but are not limited to any bodily fluid, such
as a serum, urine, saliva, blood, cerebrospinal fluid, pleural
fluid, ascites fluid, sputum, stool, or a biopsy sample.
[0060] Samples or specimens include any CpG-rich DNA sequence,
whatever the origin, as long as the sequence is detectably present
in a sample. While routine diagnostic tests may not be able to
identify cancer cells in these samples, the method of the present
invention identifies neoplastic cells derived from the primary
tumor or from a metastases. The method includes non-invasive
sampling (e.g., bodily fluid) as well as invasive sampling (e.g.,
biopsy). The sample of DNA of the subject may be serum, plasma,
lymphocytes, urine, sputum, bile, stool, cervical tissue, saliva,
tears, pancreatic juice, duodenal juice, cerebral spinal fluid,
regional lymph node, histopathologic margins, and any bodily fluid
that drains a body cavity or organ. Therefore, the method provides
for the non-invasive detection of various tumor types including
head and neck cancer, lung cancer, esophageal cancer, stomach
cancer, small bowel cancer, colon cancer, bladder cancer, kidney
cancers, cervical cancer and any other organ type that has a
draining fluid accessible to analysis. For example, neoplasia of
regional lymph nodes associated with a primary mammary tumor can be
detected using the method of the invention. Regional lymph nodes
for head and neck carcinomas include cervical lymph nodes,
prelaryngeal lymph nodes, pulmonary juxta-esophageal lymph nodes
and submandibular lymph nodes. Regional lymph nodes for mammary
tissue carcinomas include the axillary and intercostal nodes.
Samples also include urine DNA for bladder cancer or plasma or
saliva DNA for head and neck cancer patients.
[0061] Any nucleic acid sample, in purified or nonpurified form,
can be utilized as the starting nucleic acid or acids in accordance
with the present invention, provided it contains, or is suspected
of containing, a nucleic acid sequence containing a target locus
(e.g., CpG-containing nucleic acid). In general, the CpG-containing
nucleic acid is DNA. However, invention methods may employ, for
example, samples that contain DNA, or DNA and RNA, including
messenger RNA, wherein DNA or RNA may be single stranded or double
stranded, or a DNA-RNA hybrid may be included in the sample. A
mixture of nucleic acids may also be employed. The specific nucleic
acid sequence to be detected may be a fraction of a larger molecule
or can be present initially as a discrete molecule, so that the
specific sequence constitutes the entire nucleic acid. It is not
necessary that the sequence to be studied be present initially in a
pure form; the nucleic acid may be a minor fraction of a complex
mixture, such as contained in whole human DNA. The nucleic
acid-containing sample used for detection of methylated CpG may be
from any source including, but not limited to, brain, colon,
urogenital, lung, renal, pancreas, liver, esophagus, stomach,
hematopoietic, breast, thymus, testis, ovarian, and uterine tissue,
and may be extracted by a variety of techniques such as that
described by Maniatis, et al. (Molecular Cloning: a Laboratory
Manual, Cold Spring Harbor, N.Y., pp 280, 281, 1982).
[0062] The nucleic acid of interest can be any nucleic acid where
it is desirable to detect the presence of a differentially
methylated CpG island. The CpG island comprises a CpG island
located in a gene or regulatory region for a gene. A "CpG island"
is a CpG rich region of a nucleic acid sequence. The nucleic acid
sequence may include, for example, MICP 1-42. However, any gene or
nucleic acid sequence of interest containing a CpG sequence can
provide diagnostic information (i.e., via its methylation state)
using invention methods.
[0063] Moreover, these markers can also be multiplexed in a single
amplification reaction to generate a low cost, reliable cancer
screening test for many cancers simultaneously.
[0064] A combination of DNA markers for CpG-rich regions of nucleic
acid may be amplified in a single amplification reaction. The
markers are multiplexed in a single amplification reaction, for
example, by combining primers for more than one locus. For example,
DNA from a urine sample can be amplified with three different
randomly labeled primer sets, such as those used for the
amplification of the MICP38-42 loci, in the same amplification
reaction. The reaction products are separated on a denaturing
polyacrylamide gel, for example, and then exposed to film for
visualization and analysis. By analyzing a panel of markers, there
is a greater probability of producing a more useful methylation
profile for a subject.
[0065] If the sample is impure (e.g., plasma, serum, stool,
ejaculate, sputum, saliva, cerebrospinal fluid, or blood or a
sample embedded in paraffin), it may be treated before
amplification with a reagent effective for lysing the cells
contained in the fluids, tissues, or animal cell membranes of the
sample, and for exposing the nucleic acid(s) contained therein.
Methods for purifying or partially purifying nucleic acid from a
sample are well known in the art (e.g., Sambrook et al., Molecular
Cloning: a Laboratory Manual, Cold Spring Harbor Press, 1989,
herein incorporated by reference).
[0066] In order to detect a differential methylation state for a
gene or CpG-containing region of interest, invention methods
include any means known in the art for detecting such differential
methylation. For example, detecting the differential methylation
may include contacting the nucleic acid-containing specimen with an
agent that modifies unmethylated cytosine, amplifying a
CpG-containing nucleic acid in the specimen by means of
CpG-specific oligonucleotide primers, wherein the oligonucleotide
primers distinguish between modified methylated and nonmethylated
nucleic acid, and detecting the methylated nucleic acid based on
the presence or absence of amplification products produced in said
amplifying step. This embodiment includes the PCR-based methods
described in U.S. Pat. No. 5,786,146, incorporated herein in its
entirety.
[0067] For the first time, the methylation state of a number of
genes has been correlated with cell proliferative disorders.
Examples of such genesand their NCBI accession numbers, including
the location of the clone, are set out in Table 1.
[0068] In another embodiment, detection of differential methylation
is accomplished by contacting a nucleic acid sample suspected of
comprising a CpG-containing nucleic acid with a methylation
sensitive restriction endonuclease that cleaves only unmethylated
CpG sites under conditions and for a time to allow cleavage of
unmethylated nucleic acid. The sample is further contacted with an
isoschizomer of the methylation sensitive restriction endonuclease,
that cleaves both methylated and unmethylated CpG-sites, under
conditions and for a time to allow cleavage of methylated nucleic
acid. Oligonucleotides are added to the nucleic acid sample under
conditions and for a time to allow ligation of the oligonucleotides
to nucleic acid cleaved by the restriction endonuclease, and the
digested nucleic acid is amplified by conventional methods such as
PCR wherein primers complementary to the oligonucleotides are
employed. Following identification, the methylated CpG-containing
nucleic acid can be cloned, using method well known to one of skill
in the art (see Sambrook et al., Molecular Cloning: a Laboratory
Manual, Cold Spring Harbor Press, 1989).
[0069] As used herein, a "methylation sensitive restriction
endonuclease" is a restriction endonuclease that includes CG as
part of its recognition site and has altered activity when the C is
methylated as compared to when the C is not methylated. Preferably,
the methylation sensitive restriction endonuclease has inhibited
activity when the C is methylated (e.g., SmaI). Specific
non-limiting examples of a methylation sensitive restriction
endonucleases include Sma I, BssHII, or HpaII. Such enzymes can be
used alone or in combination. Other methylation sensitive
restriction endonucleases will be known to those of skill in the
art and include, but are not limited to SacII, EagI, and BstUI, for
example. An "isoschizomer" of a methylation sensitive restriction
endonuclease is a restriction endonuclease which recognizes the
same recognition site as a methylation sensitive restriction
endonuclease but which cleaves both methylated and unmethylated
CGs. One of skill in the art can readily determine appropriate
conditions for a restriction endonuclease to cleave a nucleic acid
(see Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold
Spring Harbor Press, 1989). Without being bound by theory, actively
transcribed genes generally contain fewer methylated CGs than in
other genes.
[0070] In one embodiment of the invention, a nucleic acid of
interest is cleaved with a methylation sensitive endonuclease. In
one aspect, cleavage with the methylation sensitive endonuclease
creates a sufficient overhang on the nucleic acid of interest.
Following cleavage with the isoschizomer, the cleavage product can
still have a sufficient overhang. An "overhang" refers to nucleic
acid having two strands wherein the strands end in such a manner
that a few bases of one strand are not base paired to the other
strand. A "sufficient overhang" refers to an overhang of sufficient
length to allow specific hybridization of an oligonucleotide of
interest. In one embodiment, a sufficient overhang is at least two
bases in length. In another embodiment, the sufficient overhang is
four or more bases in length. An overhang of a specific sequence on
the nucleic acid of interest may be desired in order for an
oligonucleotide of interest to hybridize. In this case, the
isoschizomer can be used to create the overhang having the desired
sequence on the nucleic acid of interest.
[0071] In another aspect of this embodiment, the cleavage with a
methylation sensitive endonuclease results in a reaction product of
the nucleic acid of interest that has a blunt end or an
insufficient overhang. In this embodiment, an isoschizomer of the
methylation sensitive restriction endonuclease can create a
sufficient overhang on the nucleic acid of interest. "Blunt ends"
refers to a flush ending of two stands, the sense stand and the
antisense strand, of a nucleic acid.
[0072] Once a sufficient overhang is created on the nucleic acid of
interest, an oligonucleotide is ligated to the nucleic acid cleaved
of interest which has been cleaved by the methylation specific
restriction endonuclease. "Ligation" is the attachment of two
nucleic acid sequences by base pairing of substantially
complementary sequences and/or by the formation of covalent bonds
between two nucleic acid sequences. In one aspect of the present
invention, an "oligonucleotide" is a nucleic acid sequence of about
2 up to about 40 bases in length. It is presently preferred that
the oligonucleotide is from about 15 to 35 bases in length.
[0073] In one embodiment, an adaptor is utilized to create DNA ends
of desired sequence and overhang. An "adaptor" is a double-stranded
nucleic acid sequence with one end that has a sufficient
single-stranded overhang at one or both ends such that the adaptor
can be ligated by base-pairing to a sufficient overhang on a
nucleic acid of interest that has been cleaved by a methylation
sensitive restriction enzyme or an isoschizomer of a methylation
sensitive restriction enzyme. Adaptors can be obtained
commercially, or two oligonucleotides can be utilized to form an
adaptor. Thus, in one embodiment, two oligonucleotides are used to
form an adaptor; these oligonucleotides are substantially
complementary over their entire sequence except for the region(s)
at the 5' and/or 3' ends that will form a single stranded overhang.
The single stranded overhang is complementary to an overhang on the
nucleic acid cleaved by a methylation sensitive restriction enzyme
or an isoschizomer of a methylation sensitive restriction enzyme,
such that the overhang on the nucleic acid of interest will base
pair with the 3' or 5' single stranded end of the adaptor under
appropriate conditions. The conditions will vary depending on the
sequence composition (GC vs AT), the length, and the type of
nucleic acid (see Sambrook et al., Molecular Cloning: a Laboratory
Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview,
N.Y., 1998).
[0074] Following the ligation of the oligonucleotide, the nucleic
acid of interest is amplified using a primer complementary to the
oligonucleotide. Specifically, the term "primer" as used herein
refers to a sequence comprising two or more deoxyribo-nucleotides
or ribonucleotides, preferably more than three, and more preferably
more than eight, wherein the sequence is capable of initiating
synthesis of a primer extension product, which is substantially
complementary to a nucleic acid such as an adaptor or a ligated
oligonucleotide. Environmental conditions conducive to synthesis
include the presence of nucleoside triphosphates and an agent for
polymerization, such as DNA polymerase, and a suitable temperature
and pH. The primer is preferably single stranded for maximum
efficiency in amplification, but may be double stranded. If double
stranded, the primer is first treated to separate its strands
before being used to prepare extension products. In one embodiment,
the primer is an oligodeoxyribo-nucleotide. The primer must be
sufficiently long to prime the synthesis of extension products in
the presence of the inducing agent for polymerization. The exact
length of primer will depend on many factors, including
temperature, buffer, and nucleotide composition. The
oligonucleotide primer typically contains 12-20 or more
nucleotides, although it may contain fewer nucleotides.
[0075] Primers of the invention are designed to be "substantially"
complementary to each strand of the oligonucleotide to be amplified
and include the appropriate G or C nucleotides as discussed above.
This means that the primers must be sufficiently complementary to
hybridize with their respective strands under conditions which
allow the agent for polymerization to perform. In other words, the
primers should have sufficient complementarity with a 5' and 3'
oligonucleotide to hybridize therewith and permit amplification of
CpG containing nucleic acid sequence.
[0076] Primers of the invention are employed in the amplification
process which is an enzymatic chain reaction that produces
exponential quantities of target locus relative to the number of
reaction steps involved (e.g., polymerase chain reaction or PCR).
Typically, one primer is complementary to the negative (-) strand
of the locus and the other is complementary to the positive (+)
strand. Annealing the primers to denatured nucleic acid followed by
extension with an enzyme, such as the large fragment of DNA
Polymerase I (Klenow) and nucleotides, results in newly synthesized
+ and - strands containing the target locus sequence. Because these
newly synthesized sequences are also templates, repeated cycles of
denaturing, primer annealing, and extension results in exponential
production of the region (i.e., the target locus sequence) defined
by the primer. The product of the chain reaction is a discrete
nucleic acid duplex with termini corresponding to the ends of the
specific primers employed.
[0077] The oligonucleotide primers of the invention may be prepared
using any suitable method, such as conventional phosphotriester and
phosphodiester methods or automated embodiments thereof. In one
such automated embodiment, diethylphos-phoramidites are used as
starting materials and may be synthesized as described by Beaucage,
et al. (Tetrahedron Letters, 22:1859-1862, 1981). One method for
synthesizing oligonucleotides on a modified solid support is
described in U.S. Pat. No. 4,458,066.
[0078] Where the CpG-containing nucleic acid sequence of interest
contains two strands, it is necessary to separate the strands of
the nucleic acid before it can be used as a template for the
amplification process. Strand separation can be effected either as
a separate step or simultaneously with the synthesis of the primer
extension products. This strand separation can be accomplished
using various suitable denaturing conditions, including physical,
chemical, or enzymatic means, the word "denaturing" includes all
such means. One physical method of separating nucleic acid strands
involves heating the nucleic acid until it is denatured. Typical
heat denaturation may involve temperatures ranging from about
80.degree. to 105.degree. C. for times ranging from about 1 to 10
minutes. Strand separation may also be induced by an enzyme from
the class of enzymes known as helicases or by the enzyme RecA,
which has helicase activity, and in the presence of riboATP, is
known to denature DNA. The reaction conditions suitable for strand
separation of nucleic acids with helicases are described by Kuhn
Hoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and
techniques for using RecA are reviewed in C. Radding (Ann. Rev.
Genetics, 16:405-437, 1982).
[0079] When complementary strands of nucleic acid or acids are
separated, regardless of whether the nucleic acid was originally
double or single stranded, the separated strands are ready to be
used as a template for the synthesis of additional nucleic acid
strands. This synthesis is performed under conditions allowing
hybridization of primers to templates to occur. Generally synthesis
occurs in a buffered aqueous solution, generally at a pH of about
7-9. Preferably, a molar excess (for genomic nucleic acid, usually
about 108:1 primer:template) of the two oligonucleotide primers is
added to the buffer containing the separated template strands. It
is understood, however, that the amount of complementary strand may
not be known if the process of the invention is used for diagnostic
applications, so that the amount of primer relative to the amount
of complementary strand cannot be determined with certainty. As a
practical matter, however, the amount of primer added will
generally be in molar excess over the amount of complementary
strand (template) when the sequence to be amplified is contained in
a mixture of complicated long-chain nucleic acid strands. a large
molar excess is preferred to improve the efficiency of the
process.
[0080] The deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and
dTTP are added to the synthesis mixture, either separately or
together with the primers, in adequate amounts and the resulting
solution is heated to about 90.degree.-100.degree. C. from about 1
to 10 minutes, preferably from 1 to 4 minutes. After this heating
period, the solution is allowed to cool to approximately room
temperature, which is preferable for the primer hybridization. To
the cooled mixture is added an appropriate agent for effecting the
primer extension reaction (called herein "agent for
polymerization"), and the reaction is allowed to occur under
conditions known in the art. The agent for polymerization may also
be added together with the other reagents if it is heat stable.
This synthesis (or amplification) reaction may occur at room
temperature up to a temperature above which the agent for
polymerization no longer functions. Thus, for example, if DNA
polymerase is used as the agent, the temperature is generally no
greater than about 40.degree. C. Most conveniently the reaction
occurs at room temperature.
[0081] The agent for polymerization may be any compound or system
which will function to accomplish the synthesis of primer extension
products, including enzymes. Suitable enzymes for this purpose
include, for example, E. coli DNA polymerase I, Klenow fragment of
E. coli DNA polymerase I, T4 DNA polymerase, other available DNA
polymerases, polymerase muteins, reverse transcriptase, and other
enzymes, including heat-stable enzymes (i.e., those enzymes which
perform primer extension after being subjected to temperatures
sufficiently elevated to cause denaturation such as Taq DNA
polymerase, and the like). Suitable enzymes will facilitate
combination of the nucleotides in the proper manner to form the
primer extension products which are complementary to each locus
nucleic acid strand. Generally, the synthesis will be initiated at
the 3' end of each primer and proceed in the 5' direction along the
template strand, until synthesis terminates, producing molecules of
different lengths. There may be agents for polymerization, however,
which initiate synthesis at the 5' end and proceed in the other
direction, using the same process as described above.
[0082] Preferably, the method of amplifying is by PCR, as described
herein and as is commonly used by those of ordinary skill in the
art. However, alternative methods of amplification have been
described and can also be employed. PCR techniques and many
variations of PCR are known. Basic PCR techniques are described by
Saiki et al. (1988 Science 239:487-491) and by U.S. Pat. Nos.
4,683,195, 4,683,202 and 4,800,159, which are incorporated herein
by reference.
[0083] The conditions generally required for PCR include
temperature, salt, cation, pH and related conditions needed for
efficient copying of the master-cut fragment. PCR conditions
include repeated cycles of heat denaturation (i.e. heating to at
least about 95.degree. C.) and incubation at a temperature
permitting primer: adaptor hybridization and copying of the
master-cut DNA fragment by the amplification enzyme. Heat stable
amplification enzymes like the pwo, Thermus aquaticus or
Thermococcus litoralis DNA polymerases are commercially available
which eliminate the need to add enzyme after each denaturation
cycle. The salt, cation, pH and related factors needed for
enzymatic amplification activity are available from commercial
manufacturers of amplification enzymes.
[0084] As provided herein an amplification enzyme is any enzyme
which can be used for in vitro nucleic acid amplification, e.g. by
the above-described procedures. Such amplification enzymes include
pwo, Escherichia coli DNA polymerase I, Klenow fragment of E. coli
DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Thermus
aquaticus (Taq) DNA polymerase, Thermococcus litoralis DNA
polymerase, SP6 RNA polymerase, T7 RNA polymerase, T3 RNA
polymerase, T4 polynucleotide kinase, Avian Myeloblastosis Virus
reverse transcriptase, Moloney Murine Leukemia Virus reverse
transcriptase, T4 DNA ligase, E. coli DNA ligase or
Q.beta.replicase. Preferred amplification enzymes are the pwo and
Taq polymerases. The pwo enzyme is especially preferred because of
its fidelity in replicating DNA.
[0085] Once amplified, the nucleic acid can be attached to a solid
support, such as a membrane, and can be hybridized with any probe
of interest, to detect any nucleic acid sequence. Several membranes
are known to one of skill in the art for the adhesion of nucleic
acid sequences. Specific non-limiting examples of these membranes
include nitrocellulose (NITROPURE) or other membranes used in for
detection of gene expression such as polyvinylchloride, diazotized
paper and other commercially available membranes such as
GENESCREEN, ZETAPROBE (Biorad), and NYTRAN Methods for attaching
nucleic acids to these membranes are well known to one of skill in
the art. Alternatively, screening can be done in a liquid
phase.
[0086] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0087] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined empirically.
In general, conditions of high stringency are used for the
hybridization of the probe of interest.
[0088] The probe of interest 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 probe, or will be
able to ascertain such, using routine experimentation.
[0089] In one embodiment, representational difference analysis
(RDA, see Lisitsyn et al., Science 259:946-951, 1993, herein
incorporated by reference) can be performed on CpG-containing
nucleic acid following MCA. MCA utilizes kinetic and subtractive
enrichment to purify restriction endonuclease fragments present in
one population of nucleic acid fragments but not in another. Thus,
RDA enables the identification of small differences between the
sequences of two nucleic acid populations. RDA uses nucleic acid
from one population as a "tester" and nucleic acid from a second
population as a "driver" in order to clone probes for single copy
sequences present in (or absent from) one of the two populations.
In one embodiment, nucleic acid from a "normal" individual or
sample, not having a disorder such as a cell-proliferative disorder
is used as a "driver," and nucleic acid from an "affected"
individual or sample, having the disorder such as a cell
proliferative disorder is used as a "tester." In one embodiment,
the nucleic acid used as a "tester" is isolated from an individual
having a cell proliferative disorder such as low grade astrocytoma,
anaplastic astrocytoma, glioblastoma, medulloblastoma, gastric
cancer, colorectal cancer, colorectal adenoma, acute myelogenous
leukemia, leukemia, lung cancer, renal cancer, breast cancer,
prostate cancer, endometrial cancer and neuroblastoma. The nucleic
acid used as a "driver" is thus normal astrocytes, normal glial
cells, normal brain cells, normal gastric cells, normal colorectal
cells, normal leukocytes, normal lung cells, normal kidney cells,
normal breast cells, normal prostate cells, normal uterine cells,
and normal neurons, respectively. In an additional embodiment, the
nucleic acid used as a "driver" is isolated from an individual
having a cell proliferative disorder such as low grade astrocytoma,
anaplastic astrocytoma, glioblastoma, medulloblastoma, gastric
cancer, colorectal cancer, colorectal adenoma, acute myelogenous
leukemia, leukemia, lung cancer, renal cancer, breast cancer,
prostate cancer, endometrial cancer and neuroblastoma. The nucleic
acid used as a "tester" is thus normal astrocytes, normal glial
cells, normal brain cells, normal gastric cells, normal colorectal
cells, normal leukocytes, normal lung cells, normal kidney cells,
normal breast cells, normal prostate cells, normal uterine cells,
and normal neurons, respectively. One of skill in the art will
readily be able to identify the "tester" nucleic acid useful with
to identify methylated nucleic acid sequences in given "driver"
population.
[0090] The materials for use in the assay of the invention are
ideally suited for the preparation of a kit. Therefore, in
accordance with another embodiment of the present invention, there
is provided a kit it useful for the detection of a cellular
proliferative disorder in a subject having or at risk for said
cellular proliferative disorder. Invention kits include a carrier
means compartmentalized to receive a sample in close confinement
therein, one or more containers comprising a first container
containing a reagent which modifies unmethylated cytosine and a
second container containing primers for amplification of a
CpG-containing nucleic acid, wherein the primers distinguish
between modified methylated and nonmethylated nucleic acid, and
optionally, a third container containing a methylation sensitive
restriction endonuclease. Primers contemplated for use in
accordance with the invention include those that would amplify
sequences or fragments thereof as set forth in SEQ ID NOs:
1-42.
[0091] Carrier means are suited for containing one or more
container means such as vials, tubes, and the like, each of the
container means comprising one of the separate elements to be used
in the method. In view of the description provided herein of
invention methods, those of skill in the art can readily determine
the apportionment of the necessary reagents among the containiner
means. For example, one of the container means can comprise a
container containing an oligonucleotide for ligation to nucleic
acid cleaved by a methylation sensitive restriction endonuclease.
One or more container means can also be included comprising a
primer complementary to the oligonucleotide. In addition, one or
more container means can also be included which comprise a
methylation sensitive restriction endonuclease. One or more
container means can also be included containing an isoschizomer of
said methylation sensitive restriction enzyme.
[0092] It should be noted that as used herein and in the appended
claims, the singular forms "a," "and," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the restriction enzyme" includes reference to one
or more restriction enzymes and equivalents thereof known to those
skilled in the art, and so forth.
[0093] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0094] All publications mentioned herein are incorporated herein by
reference in full for the purpose of describing and disclosing the
methodologies which are described in the publications which might
be used in connection with the presently described invention. The
publications discussed above and throughout the text are provided
solely for their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior invention.
[0095] The following example is intended to illustrate but not to
limit the invention in any manner, shape, or form, either
explicitly or implicitly. While they are typical of those that
might be used, other procedures, methodologies, or techniques known
to those skilled in the art may alternatively be used.
EXAMPLE 1
Collection and Preparation of Pancreatic Cell Lines
[0096] The following pancreatic adenocarcinoma cell lines were
used: PL3 and PL8 (both CIMP-cell lines (CpG island methylator
phenotype) were from Dr. Elizabeth Jaffe, John Hopkins University);
CAPAN1, CAPAN2, Panc1, Hs766T, and MiaPaca2 (from the American Type
Culture Collection, Rockville, Md.) and Colo357 (from the European
Collection of Animal Cell Cultures, Salisbury, United Kingdom). In
addition, seventeen pancreatic cancer xenografts were selected at
random from a total of 90 xenografts, which were established from
the primary carcinomas as described in Ueki, et al., Cancer Res.,
60:1835-1839, 2000. Forty-seven primary pancreatic adenocarcinomas,
15 normal pancreata, 5 pancreata from patients with chronic
pancreatitis, and a panel of normal tissues were obtained from the
resected surgical specimens at The Johns Hopkins Medical
Institutions, Baltimore, Md. Frozen tissues or paraffin-embedded
tissues were microdissected to obtain >40% neoplastic
cellularity in the primary pancreatic adenocarcinomas, and 3 of the
15 frozen normal pancreatic tissues were also microdissected to
enrich the normal ductal epithelium. DNA was extracted from
microdissected primary pancreatic adenocarcinomas and normal
tissues as well as from lymphocytes of four cancer-free individuals
using standard methods.
EXAMPLE 2
Methylated CpG Island Amplification/Representational Difference
Analysis (MCA/RDA)
[0097] MCA/RDA was performed as described by Toyota et al., Cancer
Res., 59:2307-2312, 1999, modifying such procedure to increase the
efficiency by digesting 5 .mu.g of DNA with SmaI and XmaI (New
England Biolabs). The restriction fragments were then ligated to
RMCA adapter and amplified by PCR in 10 mM Tris-HCl (pH 8.3), 1.5
mM MgCl2, 50 mM KCl, 0.5 M betaine, 2% DMSO, 200 .mu.M each
deoxynucleotide triphosphate, 100 .mu.mol of RMCA 24mer primer and
15 units of Taq polymerase (Life Technologies, Inc.) in a final
reaction volume of 100 .mu.l. The reaction mixture was then
incubated at 72.degree. C. for 5 min and at 95.degree. C. for 3
min, and then subjected to 25 cycles of 1 min at 95.degree. C. and
3 min at 77.degree. C. followed by a final extension of 10 min at
77.degree. C. Betaine was included in the PCR reaction to help
amplify the methylated templates at a higher annealing temperature
(77.degree. C.). The combination of betaine and DMSO can uniformly
amplify a mixture of DNA with different GC content (Baskaran, et
al., Genome Res. 6:633-638, 1996). These modifications might have
enhanced the amplification of distinct MICPs (methylated in
carcinoma of the pancreas) instead of Alu repetitive sequences that
accounted for 60% of the recovered clones using the original
protocol (Toyota et al., Cancer Res. 59:2307-2312, 1999). The MCA
amplicon from either the pancreatic cancer cell line PL3 or PL8 was
used as the tester for RDA, and a MCA amplicon generated from a
mixture of DNA from the normal pancreata of six different patients
was used as the driver. RDA was performed on these MCA amplicons
using different adapters, JMCA and NMCA. Sequences of adapters used
for MCA/RDA are listed in Table 3A and 3B and are available at
http://pathology2.jhu.edu/pancreas/prim0425.htm, which website is
incorporated herein by reference. After the third round of
competitive hybridization and selective amplification, the RDA
difference products of second and third round amplifications were
cloned into pbluescript II plasmid vector (Stratagene).
EXAMPLE 3
DNA Sequencing of Clones and Dot Blot Hybridization
[0098] The clones recovered from each cell line after MCA/RDA were
amplified with T3 and T7 primers and then sequenced using KS primer
as recommended by the manufacturer (Sequitherm Excel; Epicentre
Technologies). To determine the methylation status of MCA/RDA MICPs
in pancreatic cancer and normal pancreas, MICPs were screened by
hybridizing them to a dot blot of MCA products of pancreatic
cancers and normal pancreata. Plasmid DNA containing each
independent clone was prepared and digested with SmaI. DNA
fragments were recovered from agarose gel and used as a probe for
dot blot hybridization. Aliquots (1 .mu.l) of the mixture of
10.times.SSC and MCA products from the driver and from the tester
(PL3 and PL8) both before and after each of the three rounds of RDA
competitive hybridization/selective amplification were blotted onto
nylon membranes in duplicate. Similarly, MCA products from six
pancreatic cell lines (CAPAN1, CAPAN2, Panc1, Hs766T, MiaPaca2, and
Colo357) and from eight other normal pancreata were also blotted
onto the membranes. The membranes were hybridized with 32P-labeled
probes overnight, washed, and exposed to a Kodak X-ray film.
EXAMPLE 4
Bisulfite Modification, Bisulfite-Modified Genomic Sequencing, and
Methylation-Specific PCR (MSP)
[0099] The bisulfite treatment was carried out for 16 h at
50.degree. C. using 1 .mu.g of genomic DNA, as described in Ueki,
et al., Cancer Res., 60:1835-1839, 2000. Genomic sequencing was
performed on bisulfite-treated DNA to examine the methylation
status of 10-20 CpG dinucleotides located in and/or around SmaI
sites of each clone in 22 pancreatic tissues (8 cancer cell lines,
6 primary adenocarcinomas, and 8 normal pancreata; Ueki, et al.,
Cancer Res. 60:1835-1839, 2000). Genomic sequencing of the coding
sequence of cyclin G was also performed in PL8. The level of
methylation of each clone was determined by quantifying the level
of methylation of each CpG site by comparing the intensity of
unconverted cytosine with that of cytosine plus thymidine.
Generally, in pancreatic cancer cell lines, the level of
methylation observed at each CpG dinucleotide was consistent
throughout the CpG island. Therefore, the average level of
methylation of each sequence was graded and placed into one of 4
grades: 0-10%, 11-30%, 31-70%, and 71-100%. MSP was performed as
described in Herman, et al., Proc. Natl. Acad. Sci. USA
93:9821-9826, 1996, and to acquire optimal specificity, each primer
pair contained four to six CpG sites, and high specific annealing
temperatures were used. The primers and the specific annealing
temperatures for each clone are listed in Table 3 and are available
at http://pathology2jhu.edu/pancreas/prim042- 5.htm, which website
is incorporated herein by reference. If validated MSP primers sets
specific for methylated and unmethylated templates revealed that
there was only amplification of methylated templates, the samples
are presumed to be 100% methylated. Methylated and unmethylated
templates were identified by bisulfite-modified sequencing. In
describing MSP results performed on CpG islands that were normally
unmethylated in non-neoplastic pancreas, a cancer sample was termed
"methylated" if MSP yielded any methylated templates.
EXAMPLE 5
Reverse Transcription-PCR (RT-PCR) and 5-aza-2'-deoxycytidine
(5Aza-dC) Treatment
[0100] Five pancreatic cancer cell lines (PL3, PL8, CAPAN2, Panc1,
and MiaPaca2) and four normal pancreata were used for RT-PCR
analysis. The cell lines were treated with demethylating agent
5Aza-dC (Sigma Chemical Co.) at a final concentration of 1 .mu.M
for 5 days. Total RNA was prepared using TRIzol (Life Technologies,
Inc.), reverse-transcribed and amplified. As a control for cDNA
integrity, glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH)
was also amplified. Primer sequences for RT-PCR are listed in Table
3 and are available at http://pathology2
jhu.edu/pancreas/prim0425.htm, which website is incorporated herein
by reference.
EXAMPLE 6
Statistics
[0101] The primary outcome variable was the observed number of 7
MICP loci found to be methylated in 64 pancreatic cancers.
Wilcoxon's rank-sum test compared the observed number of methylated
loci by tumor differentiation (poorly versus well or moderately
differentiated), lymph node status (0 or 1 versus>1 node
positive), and prior CIMP classification (CIMP positive versus CIMP
negative). Simple linear regression assessed the relationship
between the observed number of methylated loci and these
covariates: age, age squared, and tumor diameter (in cm).
Multivariate linear regression assessed the simultaneous
contribution of the clinicopathological and demographic variables
to the observed number of methylated loci. All of the tests were
two-sided. A P of <0.05 signified statistical significance.
EXAMPLE 7
Patient Population and Tissue Samples
[0102] Normal and tumor specimens were obtained from pancreatic
adenocarcinomas resected at The Johns Hopkins Hospital. Pancreatic
cancer xenografts were established from the primary carcinomas as
previously described in Caldas, et al., Nature Genet. 8:27-31,
1994, and carcinoma and normal tissues were stored at -70.degree.
C. Thirty-two xenografts were selected at random. Three MSI
carcinoma xenografts were added, as reported previously, as well as
another MSI primary carcinoma (Goggins, et al., Am. J. of Pathology
152:1501-1507, 1998). Genomic DNA was prepared from 35 xenografts
and 9 pancreatic adenocarcinoma cell lines (BxPc3, Capan1, Capan2,
Panc1, CFPAC1, MiaPaca2, Hs766T (all from ATCC, Rockville, Md.),
Co10357 (from ECACC, Salisbury, UK) and PL45, a low passage cell
line established in the Goggin Laboratory at John Hopkins
University School of Medicine). Where available, DNA was obtained
from primary pancreatic cancer tissue frozen at -80 C since Whipple
resection. Frozen tissues were microdissected to obtain DNA from
normal pancreatic tissue and the pancreatic carcinoma.
[0103] Patient records were reviewed to determine a history of
cigarette smoking, diabetes mellitus, and prognosis, and a family
history of pancreatic cancer. These data were reviewed in the
context of the DNA methylation data.
EXAMPLE 8
Bisulfite Modification and Genomic Sequencing
[0104] The bisulfite treatment was carried out for 16 h at
56.degree. C. on 1 .mu.g of genomic DNA, according to the procedure
of Herman et al, Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996.
Modified DNA was purified and eluted into 50 .mu.l of LoTE buffer.
The primers used for genomic sequencing of bisulfite-treated DNA
are listed in Table 3 and are available at http://pathology2
jhu.edu/pancreas/prim0425.htm, which website is incorporated herein
by reference. PCR was performed on 1-2 .mu.l of bisulfite-treated
DNA, and prior to sequencing, PCR reactions were incubated with
exonuclease I and shrimp alkaline phosphatase (Amersham, according
to the manufacturer recommendations). Sequencing of PCR products
was performed in microtiter plates as recommended by the
manufacturer (Sequitherm Excel, Epicentre Technologies, Madison,
Wis.
EXAMPLE 9
Methylation-Specific PCR (MSP) Assay
[0105] The methylation status of each gene was determined by MSP as
described by Herman et al, Proc. Natl. Acad. Sci. USA 93:9821-9826,
1996, in which the sequence difference of bisulfite-treated DNA was
detected by PCR using primers specific for either the methylated or
for the unmethylated DNA. Primer sequences and the specific
annealing temperatures for 13 genes are listed in Table 3 and are
available at http://pathology2 jhu.edu/pancreas/prim0425.htm, which
website is incorporated herein by reference. MSP was performed on 1
.mu.l of bisulfite-treated DNA under the conditions as follows:
95.degree. C. for 3 min; then 35-40 cycles of 95.degree. C. for 30
s, the specific annealing temperature for 30 s, and 72.degree. C.
for 30 s; and a final extension of 4 min at 72.degree. C. Five
.mu.l of each PCR product were directly loaded onto 3% agarose gels
or 10% acrylamide gels, stained with ethidium bromide and
visualized under UV illumination. All PCR reactions were performed
with positive controls for both unmethylated and methylated
alleles, and no DNA control. Finally, 3-6 CpG sites were included
in each primer pair and the specific annealing temperatures were
used for each gene to obtain optimal specificity.
EXAMPLE 10
Identification of Differentially Methylated Sequences
[0106] The strategy of MCA/RDA has been previously reported
(Toyota, et al., Cancer Res. 59:2307-2312, 1997). Ninety-six
randomly selected clones recovered from each cell line were
subjected to DNA sequencing and 66 clones were revealed to be
independent. Only 3 clones contained Alu-repetitive sequences. The
subsequent probing of labeled clones to MCA products of tester and
driver by dot-blot hybridization revealed that 43 of 66 clones
(MICP1-43) were differentially methylated in the tester compared to
the driver (FIG. 1). These 43 clones were also variably methylated
in the other 6 pancreatic cancer cell lines examined. A description
of these 43 differentially methylated clones is shown in Table 1.
All of the 43 clones had a GC content of greater than 50% and 41
(95%) had a sequence uniqueness sustaining the criteria of CpG
island (Gardiner-Garden & Frommer, J. Mol. biol. 196:261-282,
1987). The DNA homology search of each clone with the BLAST program
(National Center for Biotechnology Information) demonstrated that
84% (36) of the 43 clones had significant homologies to known human
sequences, including 8 clones matched to human gene sequences and 9
clones matched to human ESTs. Five clones were also matched or
contained a part of CpG islands isolated previously (Toyota, et
al., Cancer Res. 59:2307-2312, 1997, Cross, et al., Nat. Genet.,
6:236-244, 1994) and 14 clones had significant homology to
High-Throughput Genome Sequences in the three International
Nucleotide Sequence databases: DDBJ, EMBL and GenBank. The
remaining 7 had no significant homology to known sequences. MICP1
corresponded to the 3' noncoding region of the human homeobox gene
CSX, MICP2 corresponded to exon 1 and intron 1 of the human Cyclin
G, MICP3 corresponded to 5' region of the human
endothelin-converting enzyme-like I gene ECEL1, MICP4 corresponded
to intron 1 of the human glutamate decarboxylase 1 gene GAD I,
MICP5 corresponded to exon 7 to exon 9 of the human ICAM5, MICP6
corresponded to intron 2 to intron 3 of the human monocarboxylate
transporter 3 gene MCT3, MICP7 corresponded to the 5' region of the
human B-cell specific transcriptional factor gene PAX5, MICP8
corresponded to intron 1 and exon 2 of the human Preproenkephalin
gene (ppENK) (FIG. 2). Interestingly, 3 clones (MICP1, 17 and 22)
matched to CpG islands originally recovered from colorectal cancer
cell line using the same technique (named MINT 23, 20 and 32,
respectively) (Toyota, et al., Cancer Res. 59:2307-2312, 1997).
EXAMPLE 11
Characterization of the Sequences
[0107] For 30 of the 43 clones, methylation was detected in 2 or
more out of 8 normal pancreata by dot blot analysis, suggesting
that these clones could be frequently methylated in normal
pancreas. Therefore, only the remaining 13 clones were further
analyzed by bisulfite sequencing. For seven of the clones (Cyclin
G, ppENK, MICP20, 23, 33, 35 and 36), methylation was restricted to
pancreatic cancers (FIGS. 4A and 4B). In the case of 5 clones
(MCT3, PAX5, MICP15, 16 and 38), methylation was detectable in DNA
from normal pancreata as well as DNA from cancer tissues (FIG. 3C).
In the thirteenth clone (MICP42), cytosines at CpG sites were
similarly methylated in both pancreatic cancers and normal
pancreata. The sequence uniqueness of this clone also did not
satisfy the criteria for CpG islands. A summary of the level of
methylation of each clone is shown in FIG. 3D. There was little
individual variation in the level of methylation of these-6 clones
(MCT3, PAX5, MICP15, 16, 38 and 42) in normal pancreata from 8
patients at the age from 34 to 84 years old (FIG. 3 and data not
shown). Interestingly, the methylation of these CpG islands in
normal pancreata was sometimes heterogeneous. For example, although
the SmaI site of MICP 15 was not methylated in normal pancreata, 2
CpG sites near the SmaI site were methylated (FIG. 3C). A summary
of the results of MCA/RDA and bisulfite sequencing is also provided
in FIG. 4.
[0108] In order to identify low level of methylation of identified
clones in pancreatic tissues, MSP primers were designed for the 7
clones (Cyclin G, ppENK, MICP20, 23, 33, 35 and 36) differentially
methylated in pancreatic cancers by bisulfite sequencing. The
methylation status of these clones was examined in 15 primary
pancreatic adenocarcinomas, 5 DNA samples from pancreata with
chronic pancreatitis as well as 14 normal pancreata including three
specimens enriched in normal ductal epithelium. For MICP36, MSP
detected methylation in DNA of three pancreatic cancer cell lines
but not in any other specimens examined. Aberrant methylation of
the remaining 6 clones was detected in 7% to 87% of primary
pancreatic adenocarcinomas, while MSP confirmed a lack of
methylation of these 6 clones in DNA from 14 normal pancreata (FIG.
5 and Table 2A and 2B). DNA from one chronic pancreatitis tissue
containing pancreatic intraductal neoplasia (PanIN) (Hruban, et
al., Clin. Cancer Res. 6:2969-2972, 2000) harbored methylated
templates of Cyclin G, ppENK and MICP20 and DNA from another
chronic pancreatitis sample also had methylated templates of
ppENK.
[0109] Because tissue-specific methylation differences can be found
in normal tissues (Liang, et al., Genomics 53:260-268, 1998), the
clones found that were aberrantly methylated in pancreatic cancer,
as compared to normal pancreas, were also analyzed to see if they
are methylated in other normal tissues. By MSP, MICPs 33 and 35
were not methylated in any normal gastrointestinal tissues, while
ppENK, MICP20 and 23 were methylated in DNA samples from normal
gastric, duodenal and colonic mucosae (FIG. 5 and Table 2A and 2B).
Amplification of methylated templates of these clones was always
weak in normal mucosae compared to the primary pancreatic
adenocarcinomas (FIG. 3), suggesting that there were few methylated
DNA templates in these mucosae.
EXAMPLE 12
Expression of Cyclin G and ppENK in Pancreatic Cancer
[0110] To determine whether methylation of these clones resulted in
loss or decrease of gene expression, Cyclin G and ppENK were
further examined using RT-PCR in 5 and 4 pancreatic cell lines,
respectively. Partial methylation (-50%) of the 5' CpG island of
Cyclin G in PL8 (FIG. 4C) was associated with decreased expression
of Cyclin G by RT-PCR. The 5' CpG island of Cyclin G was not
methylated in a panel of normal pancreata. Cyclin G was expressed
in 4 normal pancreata by RT-PCR. Treatment with 5Aza-dC restored
the expression of Cyclin G in PL8 (FIG. 4C). Using RT-PCR, it was
found that ppENK was expressed in normal pancreata but not in any
of the 4 pancreatic cancer cell lines examined in which 5'CpG
island of this gene was methylated. 5Aza-dC treatment restored
ppENK expression in all 4 cell lines (FIG. 4D). Thus,
hypermethylation of the 5' CpG islands of Cyclin G and ppENK is
coincides with decreased expression in pancreatic cancers.
EXAMPLE 13
Determination of Methylated ppENK by Methylation Specific PCR of
Pancreatic Fluid as a Specific Marker of Pancreatic Cancer
[0111] Pancreatic fluid obtained from patients undergoing
pancreatic surgical resection from 38 patients with pancreatic
cancer, 7 with chronic pancreatitis, 4 with miscellaneous tumors
(islet cell, serous cyst adenoma, and lymphoma), 9 with ampullary
cancers, 9 with IPMNs (intraductal papillary mucinous neoplasms)
and 3 from bile duct cancers was analyzed. Methylated ppENK was
detected in 55% of pancreatic cancer fluids, none in the chronic
pancreatitis or other miscellaneous tumor group, 2 of 9 with IPMN,
2 of 3 with bile duct cancer, and 4 of 9 with ampullary cancer,
suggesting that the specificity of methylated ppENK in pancreatic
fluid is useful as a diagnostic marker.
[0112] 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.
1TABLE 1A The 42 MICPs identified by MCA/RDA in pancreatic cancers*
CpG MICP Blast homology Size (bp) Islands** Chromosome 1 CSX 414
yes 5q35 2 Cyclin G 392 yes 5q32-34 3 FLJ00083 479 no not known 4
GADI 309 yes 2 5 HLH 179 yes 20 6 ICAM5 630 yes 19p13.2 7 MCT3 308
yes 22q12.3-q31.2 8 PAX5 548 yes 9p13 9 ppENK 255 yes 8q23-q24 10
SMO 229 yes 7q34-q36 11 ZBP 399 yes 5q22 12 Human EST 431 yes Xp22
13 Human EST 600 yes 3p 14 Human EST 510 yes 5 15 Human EST 359 yes
not known 16 Human EST 370 yes 12q 17 Human EST 324 yes 6q 18 Human
EST 415 yes 11q 19 Human EST 392 yes not known 20 Human EST 359 yes
11q14 21 Human EST 545 yes 19q13.3 22 Human CpG island 386 yes
7q34-q36 23 Human CpG island 464 yes 20q13 24 Human CpG island 526
yes 15 25 Human CpG island 280 yes 6p22.1-23 26 Human CpG island
422 yes 1 27 Human genomic DNA 257 yes 17 28 Human genomic DNA 350
yes 7q34-q36 29 Human genomic DNA 336 yes 10 30 Human genomic DNA
293 yes 2q36-q37 31 Human genomic DNA 427 yes 13 32 Human genomic
DNA 470 no 21q22.3 33 Human genomic DNA 264 yes Xq22.1-23 34 Human
genomic DNA 371 yes 20p11.21- 11.23 35 Human genomic DNA 445 yes 4
36 Human genomic DNA 357 yes 19q13.1 37 Human genomic DNA 312 yes
7p14-15 38 Human genomic DNA 372 yes 5 39 No homology 307 yes not
known 40 No homology 331 yes not known 41 No homology 304 yes not
known 42 No homology 307 yes not known *7 of these 42 MICPs were
methylated in pancreatic cancer and not normal pancreas (see text)
**minimal length, 200 Bp; GC content, >50%; CpG/GpC,
>0.5(11)
[0113]
2TABLE 1B MICP* Size % GC CpG/GpC CpG Islands? NCBI Acc. #**
Location of clone*** 1 414 64.3 0.811 yes AF135523 1-414 2 392 62.8
0.613 yes D86077 35-426 3 479 63.9 0.444 no AK024484 1645-2107 4
309 64.4 0.727 yes AC007405 72554-72862 5 179 68.3 1.330 yes
AL121673 66270-66448 6 630 65.6 0.625 yes AF082802 5502-6131 7 308
66.6 0.704 yes AF132611 1082-1389 8 548 60.4 0.683 yes AL161781
129660-130226 9 255 67.8 0.867 yes V00509 622-876 10 229 58.2 0.927
yes AC083866 37615-37843 11 399 59.1 0.800 yes AC016663
106325-106723 12 431 68.4 0.909 yes AC002365 159360-159790 13 600
65.5 0.661 yes AC024167 62307-62906 14 510 63.7 0.771 yes AF135520
1-510 15 359 65.5 0.972 yes AW028607 259-376 16 370 64.9 1.030 yes
AC026336 11182-11568 17 324 67.6 0.816 yes AC068247 66045-66347 18
415 61.9 0.767 yes AP002781 190243-190537 19 392 55.1 1.214 yes
AI557774 193-392 20 359 55.6 0.731 yes AP002354 58648-59006 21 545
65.0 1.023 yes AC007785 13354-13898 22 386 66.3 0.605 yes AC005998
48200-48585 23 464 65.7 0.975 yes AF135532 1-464 24 526 64.2 0.645
yes AC074201 59279-59804 25 280 68.1 0.929 yes AL136305
115130-115409 26 422 63.0 0.767 yes AL137797.1 38754-39186 27 257
65.8 0.852 yes AC004706 88868-89124 28 350 64.9 0.900 yes AC004897
60876-61125 29 336 64.4 0.606 yes AC005661 43755-44090 30 293 67.0
0.760 yes AC012033 113181-113473 31 427 66.7 0.884 yes AC013721
31973-32339 32 470 59.6 0.292 no AJ239326 83971-84440 33 264 63.3
0.580 yes AL035088 102596-102859 34 371 63.0 0.722 yes AL080312
56645-57015 35 445 64.0 0.884 yes AC023868 130032-030491 36 357
68.9 0.800 yes AC002133 31977-32333 37 312 67.5 0.885 yes AC005826
151694-152005 38 372 65.6 0.778 yes AC011376 56983-57313 39 307
68.7 0.844 yes no homology 40 331 70.7 0.707 yes no homology 41 304
68.8 0.794 yes no homology 42 307 68.7 0.875 yes no homology *Total
4 clones with no homology to known human sequences (MICP38-42) and
38 clones matched $$ 11 clones matched to the human genes
(MICP1-11) 10 clones matched to human ESTs (MICP12-21)
[0114]
3TABLE 2A Tumor no. CpG Island Sample age Diam.(cm) nodes
methylated ppENK MICP27 MICP38 MICP25 MICP36 CyclInG zbp AsPC1 3 1
2 3 BxPC3 3 4 5 6 CAPAN1 3 7 8 9 CAPAN2 3 10 11 12 CFPAC 2 13 14
Colo357 5 15 16 17 18 19 20 Hs766T 3 21 22 23 MlaPaca2 4 24 25 26
27 28 Panc1 2 29 30 PL3 5 31 32 33 34 35 PL8 4 36 37 38 39 40 41
Px17 69 4.4 + 2 42 43 44 Px29 66 2.5 + 3 45 46 47 48 Px30 57 3.0 +
0 49 Px64 76 2.5 + 1 50 Px65 63 2.2 + 1 51 Px74 83 3.5 + 2 52 53
Px75 63 5.0 0 2 54 55 56 Px76 64 2.2 + 3 57 58 59 Px102 47 3.0 0 1
60 Px108 76 3.0 + 2 61 62 Px120 70 5.0 + 4 63 64 65 66 Px132 61 3.6
+ 3 67 68 69 Px143 54 2.0 + 1 70 Px184 59 4.0 + 4 71 72 73 74 Px186
55 4.0 + 2 75 76 Px195 67 4.0 + 0 Px282 79 3.0 0 2 77 78 1 64 4.7 0
1 79 2 67 3.0 + 2 80 81 4 54 2.7 + 2 82 83 6 69 4.5 + 2 84 85 9 85
8.0 + 4 86 87 88 89 11 65 2.5 + 0 12 81 10.0 + 2 90 91 13 53 + 1 92
14 57 3.0 + 2 93 94 95 196 84 6.0 + 4 96 97 98 99 198 76 4.0 + 3
100 101 102 240 50 3.5 0 0 248 67 2.0 + 1 103 281 36 6.0 + 1 104
287 72 2.5 0 1 105 666 34 8.0 + 4 106 107 108 109 5350 44 4.0 + 0
11632 66 6.5 + 2 110 111 12616 58 8x 1 112 13671 58 6.0 + 1 113
13653 63 14.5 + 3 114 115 116 17496 73 3.5 + 3 117 118 119 20852 78
5.0 + 2 120 121 21190 82 3.5 + 3 122 123 124 21889 60 4.0 + 3 125
126 127 22384 74 2.0 + 3 128 129 130 24494 78 3.5 + 1 131 24721 71
2.5 + 2 132 133 25392 57 3.0 + 1 134 26183 56 1.5 0 2 135 136 30011
65 4.0 + 3 137 138 139 30845 76 2.6 + 3 140 141 142 31742 66 1.5 0
1 143 32006 61 0 2 144 145 35083 59 1.5 + 1 146 35583 75 3.0 + 1
147 36378 74 5.2 + 2 148 149 37680 43 4.0 0 1 150 42946 71 3.5 + 2
151 152 45885 53 8x 3 153 154 155 46391 79 2.3 + 2 156 157 49126 49
4.0 + 1 158 49390 79 2.5 + 3 159 160 161 52150 72 7.0 0 3 162 163
164 165 52225 58 3.5 + 3 166 167 168 53413 85 3.5 + 2 169 170 55090
71 4.5 + 1 171
[0115]
4TABLE 2B Results of MSP CLONE 2 CLONE 8 n* Cyclin G PPENK CLONE 20
CLONE 23 CLONE 33 CLONE 35 CLONE 36 Primary pancreatic carcinoma 15
7% 87% 13% 53% 33% 40% 0% Normal pancreata 14 0% 0% 0% 0% 0% 0% 0%
Chronic pancreatitis 5 20% 40% 20% 0% 0% 0% ND Normal gastirc
mucosae 6 ND ND 50% 17% 0% 0% ND Normal duodenal mucoase 8 ND
100%** 50% 75% 0% 0% ND Normal colonic mucosae 6 ND 100%*** 33%
100% 0% 0% ND Normal lymphocytes 4 0% 0% 0% 0% 0% 0% ND *number
examined **only 4 samples examined ***only 3 samples examined
[0116]
5TABLE 3A PRIMER SEQUENCE FOR BISULFITE-SEQUENCE Annealing Clone
Orientation Sequence Temperature SEQ ID NO: RAR.beta. Forward
5'-GAGTTGGTGATGTTAGATTAG-3' 56 43 Reverse
5'-TTCCCAAAAAAATCCCAAATTC-3' 44 Sequence
5'-CTCCTTCCAAATAAATACTTAC-3' 45 THBS1 Forward
5'-AGAGAGGAGTTTAGATTGG-3' 54 46 Reverse 5'-CAAAAAAACTAAAACCTCAAC--
3' 47 Sequence Forward primer CACNA1G Forward 5'- 55, 53, 51, 48
TGGATAAAGGATGTTTGGGGTTTG- 49* 3' Reverse
5'-CCCTCCCCTTACCCCTAAATCC-3' 49 Sequence 5'-ACTCCCCCTCACTTTATTC--
3' 50 hMLH1 Forward 5'- 58 51 ATTATTTTAGTAGAGGTATATAAG- 3' Reverse
5'-CCAACCCCACCCTTCAAC-3' 52 Sequence Forward primer MINT1 Forward
5'-AAGAGAGGGTTGGAGAGTAG-3' 62 53 Reverse 5'- 54
CCCCTAAAAAAAAAATCAAAAATC- 3' Sequence 5'-GGGTTGGAGAGTAGGGGAGTT-3'
55 MINT2 Forward 5'- 60, 58, 56, 56 YGTTATGATTTTTTTGTTTAGTTAAT 54**
-3' Reverse 5'-TACACCAACTACCCAACTACCTC- 3' Sequence
5'-ACTTCCATTAAAAACAACTAC-3' 57 MINT31 Forward 5'- 58 58
TTTATTTATATAATTTTGTGTATGG- 3' Reverse 5'-CACCCCTCACTTTACTAAAAC-3'
59 Sequence Reverse primer MINT32 Forward
5'-TTTGGGAGGTAAATTYGTTGATT- 58, 56, 54, 60 3' 52*** Reverse 5'- 61
ACCRAACAAAAAACCTAAAAAAAC- 3' Sequence Forward primer *55 (5
cycles), 53 (5 cycles), 51 (5 cycles), 49 (26 cycles) **60 (3
cycles), 58 (4 cycles), 56 (5 cycles), 54 (26 cycles) ***58 (3
cycles), 56 (4 cycles), 54 (5 cycles), 52 (26 cycles)
[0117]
6TABLE 3B PRIMER SEQUENCES FOR MSP SEQ Orientation & Annealing
ID Clone Methylation Sequence Temperature NO: P16 Unmethylated F
5'-TTATTAGAGGGTGGGGTGGA- TTGT-3' 60 62 R
5'-CAACCCCAAACCCACAACCATAA-3' Methylated F
5'-TTATTAGAGGGTGGGGCGGATCGC-3' 65 63 R 5'-GACCCCCGAACCGCGACCCT-
AA-3' RAR.beta. Unmethylated F 5'-AGGATTGGGATGTTGAGAATG-3' 58 64 R
5'-TTACAAAAAACCTTCCAAATACA-3' Methylated F
5'-GGATTGGGATGTCGAGAAC-3' 64 65 R 5'-TACAAAAAACCTTCCGAATACG-3'
CACNA1G Unmethylated F 5'-GTTTTTTTTTGGATTTTTGTTTTTTG-3' 60 66 R
5'-TTTATTCCAACTTCTTCACTTCA-3' Methylated F
5'-GTTTTTTCGGGGCGGTTTC-3' 62 67 R 5'-TTCCGACTTCTTCGCTTCG-3" TIMP-3
Unmethylated F 5'- 59 68 TTTTGTTTTGTTATTTTTTGTTTTTGGTT- TT-3 R
5'-CCCCCCAAAAACCCCACCTCA-3' Methylated F 5'- 59 69
CGTTTCGTTATTTTTTGTTTTCGGTTTTC- 3' R 5'-CCGAAAACCCCGCCTCG-3' THBS1
Unmethylated F 5'-GTTTGGTTGTTGTTTATTGGTTG-3' 62 70 R
5'-CCTAAACTCACAAACCAACTCA-- 3' 71 Methylated F
5'-TGCGAGCGTTTTTTTAAATGC-3' 62 72 R 5'-TAAACTCGCAAACCAACTCG-3' 73
HMLH1 Unmethylated F 5'-TTAATAGGAAGAGTGGATAGTG-3' 56 74 R
5'-TCTATAAATTACTAAATCTCTTCA- -3' 75 Methylated F
5'-TTAATAGGAAGAGCGGATAGC-3' 58 76 R 3'-CTATAAATTACTAAATCTCTTCG-3'
77 E-Cad Unmethylated F 5'-TAATTTTAGGTTAGAGGGTTATTGT-3' 53 78 R
5'-CACAACCAATCAACAACACA-- 3' 79 Methylated F
5'-TTAGGTTAGAGGGTTATCGCGT-3' 57 80 R 5'-TAACTAAAAATTCACCTACCGAC-3'
81 DAPK Unmethylated F 5'-GGAGGATAGTTGGATTGAGTTAATGTT- 60 82 3' R
5'-CAAATCCCTCCCAAACACCAA-3' 83 Methylated F
5'-GGATAGTCGGATCGAGTTAACGTC-3' 60 84 R 5'-CCCTCCCAAACGCCGA-3' 85
MGMT Unmethylated F 5'- 59 86 TTTGTGTTTTGATGTTTGTAGGT- TTTTGT- 3' R
5'- 87 AACTCCACACTCTTCCAAAAACAA- AACA- 3' Methylated F
5'-TTTCGACGTTCGTACCTTTTCGC-3' 59 88 R 5'-GCACTCTTCCGAAAACGAAACG-3'
89 MINT1 Unmethylated F 5'-GGGGTTGAGGTTTTTTGTTAGT-3' 64 90 R
5'-TTCACAACCTCAAATCTACTTC- A-3' 91 Methylated F
5'-GGGTTGAGGTTTTTTGTTAGC-3' 64 92 R 5'-CTACTTCGCCTAACCTAACG-3' 93
MINT2 Unmethylated F 5'-GGTGTTGTTAAATGTAAATAATTTG-3' 58 94 R
5'-AAAAAAAAACACCTAAAACTC- A-3' 95 Methylated F
5'-AATCGAATTTGTCGTCGTTTC-3' 60 96 R 5'-AAATAAATAAATAAAAAAAAACGCG-3'
97 MINT31 Unmethylated F 5'-GAATTGAGATGATTTTAATTTTTTGT-3' 64 98 R
5'-CTAAAACCATCACCCCTAAA- CA-3' 99 Methylated F
5'-TTGAGACGATTTTAATTTTTTGC-3' 62 100 R 5'-AAAACCATCACCCCTAAACG-3'
101 MINT32 Unmethylated F 5'-GAGTGGTTAGAGGAATTTAGGT-3' 62 102 R
5'-CTAAAAAAACAAACAAAACATCC- A-3' 103 Methylated F
5'-GTGGTTAGAGGAATTTAGGC-3' 64 104 R 5'-AAAACGAACGAAACGTCCG-3'
105
[0118]
Sequence CWU 1
1
118 1 414 DNA Homo sapiens 1 cccgggcgcc cggccctggc tcgcggaatg
ggcggccaga tctcaggccc tgcgtgcccg 60 agctcagtcc cagttccaac
cgggggtgcc catggactct cggagggcac tcctgggggg 120 acagctaaga
caccaggctg caggatcact cattgcacgc tgcataatcg ccgccacaaa 180
ctctcccgtg cgcaagaaca aacgcgcgtg ggacagaaaa agttcctagg tctccgcagg
240 agtgaatgca aaatccaggg gactcagggt catgttggga gccccttctc
cccccgagag 300 tcagggagct gttgaggtgg gatcggtgag ggtcgcgcca
cgcgggtccc ttccctacca 360 ggctcggata ccatgcagcg tggacactcc
cgagttgctc tgcggaatcc cggg 414 2 392 DNA Homo sapiens 2 cccggggagg
gtggttaccg ctgaggagct gcagtctctg tcaaggtgag tgggactgcg 60
cgagagttga ccgccagtgc gggtggggag ctgggttggg ggcgcggggc gaggagtagg
120 tctggcccgc gcccttttcc acactaaact ctaccgctgt tgtgagcaca
agcccaggct 180 agtccgaggc tggaggggcg gagcggatcc ggcctcctga
ggtgcctttc gtgtctgccg 240 acccagtccc agggactagc ctggggagga
agaatggaac ccctgcagtt agaggttcct 300 cacatgacta gctctgaaga
cctcctgcct tcctgtcttt agttggtgtg ggagggacct 360 tccatgtatc
cagggcttag cttgtgcccg gg 392 3 298 DNA Homo sapiens 3 cccggggaga
gctgatcccg gacaggggtt ccgaggaggt aaaggaagct ccctggtgaa 60
ggtgagctct gcgcaggcgc tgtgcgggca gcccaggcac tccgtcagcg ccctctcaat
120 ccttaggagg agtcctccct gggtggtgct ccagggctgt gggctggacc
atgacggccc 180 ccagctgcgt agctccagct cctttccaac cggttggtgg
gcggttccga gtggggacac 240 gggatcggag gggactcctg ccgggtgtac
ccgctaccct cactagtccc gggatgcg 298 4 309 DNA Homo sapiens 4
cccgggacag agagattgcg ggctaatctg ggtagatcga ggaccccaca gagaagggcc
60 caccggccat cgcctccaca ccctccctcc gacctcactc ctagccccgc
gcgcgcagtc 120 gcacagcaac tcgggcagcg ggtcgactac ggcccctgga
aaagtaacag gttaccgttc 180 ctagtagcgc cttcggctgc tgctgcccag
gcgccctttg gagggacagg cgctcgcagc 240 atggaagctc aagggaaaat
cgctcttgcc ccacttcaac tagagcctca gcctccgtgc 300 ttccccggg 309 5 630
DNA Homo sapiens 5 cccgggagca tgcgggcact taccgctgcg aagccaccaa
ccctcggggc tctgcggcca 60 aaaatgtggc cgtcacggtg gaatgtgagt
aggggcaccg cggagttagg caggatctgt 120 gggacaaccc cggctggact
tcctggcccc cgtgtgagcc cctgcaatcc tgtttcccag 180 atggccccag
gtttgaggag ccgagctgcc ccagcaattg gacatgggtg gaaggatctg 240
ggcgcctgtt ttcctgtgag gtcgatggga agccacagcc aagcgtgaag tgcgtgggct
300 ccgggggcac cactgagggg gtgctgctgc cgctggcacc cccagaccct
agtcccagag 360 ctcccagaat ccctagagtc ctggcacccg gtatctacgt
ctgcaacgcc accaaccgcc 420 acggctccgt ggccaaaaca gtcgtcgtga
gcgcggagtg tgagcgaggc ccaggcgggt 480 agggagcagg gtgccccacg
gtccaggcac tccctgacat cccccatggc tgttttgcag 540 cgccaccgga
gatggatgaa tctacctgcc caagtcacca gacgtggctg gaaggggctg 600
aggcttccgc gctggcctgc gccgcccggg 630 6 308 DNA Homo sapiens 6
cccgggtggt gaccctgccc tgcccacagg ccccgtgtcc agcatcctcg tgacccgctt
60 tggctgtcgc ccggtgatgc tggcgggtgg gctgctggct tccgcgggca
tgatcctagc 120 ttcctttgcc acgcgccttc tggagctcta cctgaccgct
ggggtgctca caggtgaggg 180 ccccctggtc tcctctccgc tgggttgggg
gtcgggggtt cttgctgcaa gatctgtcct 240 cggtttccct atgagggaca
gtcttcgaag tccctcggct gggttcccgg atctgctggg 300 ttcccggg 308 7 548
DNA Homo sapiens 7 cccgggaatc cacagccaca tttccgattg tggcgaaatc
tgctcagtgg ctctgcgatg 60 tccagttgcc gccgggggcc cccctttcct
cgagctctag gctcttcccc ctaggttgcg 120 acccagcctc gtgaccaccc
cctccaaaaa aacaaacaac actcttgctg aggacgattc 180 actctccaaa
actgcattgt ccggcgggcc aggagtctct acggacgcgt cccgcctgag 240
acttccctcg agccacccgc tgcaggccca cggcttcagt ggctaggccc agcagctgaa
300 ccaactcaag gctggggggg aacaggaggg aggcttgagt ctggcccgaa
gagagagggc 360 tggacatgcc acacctctgc tcggtctctg tggatctgat
ttcctctctg gaatcaagtc 420 ctggggctct gggactccac aacgtctcag
ggctcgaggg caatgcgatt ccacttacgg 480 gccggggtaa ggtgtctgga
actcccgcca atctccagaa actactgaga tgttctgctc 540 tgcccggg 548 8 255
DNA Homo sapiens 8 cccgggaacc gcgaggcgat ctgagtcgcc tccacgtcta
cctaaaagct gtcggccggg 60 agggcggggc cccagaaagg agcattcctg
cgggcttttg ctcgacgatc ccctgctgag 120 gctgtcgcgg cgagggtcct
gccgagggac cccgttctgc gcccaggcag gctcgaagca 180 cgcgtccctc
tctcctcgca gtccatggcg cggttcctga cactttgcac ttggctgctg 240
ttgctcggcc ccggg 255 9 431 DNA Homo sapiens 9 cccgggcaga gctcggagcg
cctccatccc cggaaccagg ggactccctg gagtgctccg 60 gtccaggcta
cgatcgaggc gcccccatcc cttgggccca gggagaggat cggagacacc 120
aggaggccct cggggctggg tgaagatctt tggttccggg ggtccgggag aggatccacc
180 ctcccaatac cccgactccc agggctctga ccaagaatgg aggtgcccct
tctccaggcc 240 tcgagccctc tgagcgccga ggccggccgc ctacaggtcc
cccgccgctg ggcggaccct 300 ctcattcggt tccctcacgt cacccgctgt
ccggcgcctg ggaactgggc tcctggaatt 360 tcctctcctg gggctgacag
atggccctct tttcctttct ctgcggcagc ctcgcccatc 420 ccggtcccgg g 431 10
600 DNA Homo sapiens 10 cccggggacg gggagggagg agggctgccg ggatgtgaac
cggggaaggc agctggggct 60 ggagagcagc gcggaaaggg ggcccaggga
gctggaaaga gggccaagag gagggcaagg 120 aaggtggcgg gcgacgggga
gaggaaagaa aaagggtgtc ttggcggtgg ccttggtaag 180 agaaaggggc
aaggggtata attgacaagg cactgaaagt attgaagtca gagccttggg 240
aaggatctac cgaactctcg gcggtccacg cggggacaga cctcagcccg tgagccttga
300 gctccacgcg gggacagacc tcagcccgtg agccttgagc tccacgcggg
gacagacctc 360 agcccgtgag ccttgagctc cacgcgggga cagacctcag
cccgtgagcc ttgagctcca 420 cgcggggaca gacctcagcc cgtgagcctt
gagctccacg cggggacaga cctcagcccg 480 tgagccttga gcccagaagg
agtggcagcc tcaggacgtt tgccaggtgg cctggaatgt 540 gagggaagcc
tcagccccgc caggaacaga gctggcgctg agttcccggc tcggcccggg 600 11 359
DNA Homo sapiens 11 cccggggcgc acgggcgtga gggtggggtc tcatcgcagg
ggcgccggga gcctccccgc 60 tccgctagct caaccaagga ccgctcagag
gggctctcac cctgaacctc ggcttttcta 120 aaggagcgag accagattcc
cttctcttct cgacgtcgtt tggtgttttc tcgttctttt 180 cgactgagca
cggcaatgcg cagacgtcga cgtctctcac tgctcatccc gatctgtaac 240
ctgaggtgag cccgaagacc gctgccgccg gcggccaccc cagcgcgggt ccgctgagga
300 tggaaacagc aagtgcgcgc cggccaggcc gccacctctc cctcctccaa
cagcccggg 359 12 370 DNA Homo sapiens 12 cccgggaggt agcgggagat
tgcacacgcg atcctgcgag tttccgaact ttggaagatc 60 gtgacccgga
gagacccttg ggaggagagg gccggccacc tcctaggggt gctgtttttt 120
taagggtcaa cccaggacgc tacgggaagc acctggcgca tccttggaac agtgggcttg
180 gtggcccacc gcaacgcctg gcgggaagga atggcggggc atcgtgtgcc
taatggaccc 240 cgtcacagac ccgccccaat ccgaggggcg gatgagctca
gaggacctgc ccaggacgct 300 ccttctccac tttccaggaa aaccgacggc
gtgcgcgcct ccgtgtcctc gcggagctgg 360 ggtccccggg 370 13 324 DNA Homo
sapiens 13 cccgggcctg ggctgtgcca gcccggcctg ccagtctcgg cccccattct
cgtacggagg 60 gatgcggcgc ctggggcctc gaagctccgg gcggttttgg
agaagttgaa gctcagccgc 120 gatgatatct ccacggcggc ggggatggtg
aaaggggttg tggaccacct gctgctcaga 180 ctgaagtgcg actccgcgtt
cagaggcgtc gggctgctga acaccgggag ctactacgag 240 cacgtgaagg
tgagctgctt ggcgccctcc cgccgagccc cgctgctcgg ccttccgcaa 300
tccgcagtcc ctaccttccc cggg 324 14 392 DNA Homo sapiens 14
cccgggaaag acctcgagag accttcttca acacgtctcc aggccagatt cccctaccat
60 ggctcgggag caatgacgcg ctccccctcg ccactcgcat ggagacccga
cttccctggc 120 gccccacgag aggctcactg acctcgccgt cgtacctcgt
gagagaccgc acctgggccg 180 cgtcgagaca cccgagattc cccgtcaccg
agagcttgag gccttcgtct cctgcatggc 240 ctagagacca atctcgcgac
ctgtctccaa acgccctcag gaggcttgac tcccttgagt 300 ccgcccagtg
agctccaaga gatacccgtc gcgattcgag agcagagcgg ggttctttgc 360
ttccactcga gatgaatgcc tgtctccccg gg 392 15 358 DNA Homo sapiens 15
cccgggagag gcgacagcct catccgttta tttcctccct tgaccatttg ttcagcgact
60 ctcccctccg ttcagcatcc aggttcctta cggctacagt gccccagccc
cgcctcacca 120 gcgcgacatt ctgccctgcc tacccactca gacacagtgc
ccttttcggt tcttcaaact 180 tgctaagcgt tttcctatcg atatctgcag
gtaacagatg gcacgctctc aaatagggta 240 atcggaggag ggtctaataa
aggaactatt ttcaacagcg gagtaggcgt tagggactcc 300 agtaggagta
gggctgtgtc ccaggatact aacagcaggg cgccttggcc gccccggg 358 16 545 DNA
Homo sapiens 16 cccgggctgt gcggagatgc gcaggcttgg ggcggcgttc
aggagggact gggaaaggga 60 tcgtgcccta gggtctcctg gtgcgaaagg
gtgcggcgca gcaggtggga tcagggtgag 120 gtccgctggc atttatgggg
tgggtggtgg aaaattggaa agagtttccg gggagttgtg 180 gaagactccg
gaaagaaggg tctgttgaag gcggtgtgtt gaaaggatgt aggggaatga 240
cagaggggtg ttttaggggt ccaattgggt gaggtctggg ggggaagata tcgagagggt
300 catgggccca ggagtgcacg tctaggagtt gatggggtag gcctgagggt
tcggagaagg 360 tgcggtcggg gaggagtctc gcgattgagt ttgtcggggc
gggcgggaag ctgacagctg 420 cctctgtggt ctcaggaggc ggactccgcc
ggtgcagccg cccccgacgc gggaggaccg 480 ggcctcatcg tcccgggagc
gctccgcgtc gcgaggccgc ggcgccgcgc gctcctcatc 540 ccggg 545 17 510
DNA Homo sapiens 17 cccggggggc gctgaggttg ccttctcagg cggaggaggc
gaaccctgta gcccgacaac 60 gccgggcttc gattttgagg agcttcttcc
gggatgtcgc tatactggcc ggagacgggc 120 cttgcgtgtc cattggggga
tggcgatggg gaccagtttc cgggtagaga aggagataac 180 tcgatacagg
aacgcacagg cagcctgaaa gcagcccagg atctcgacag gggaagggaa 240
gaccctgctc cctttgccca aatcctcccg ccctgtcctt gccttctgtc ccagatccca
300 agcccctcgt acctgcactc gggactcggt cagcccgata cgcagtgcca
gctcctcacg 360 cataaagatg tcggggtagt gagtcttggc gaagctcctc
tccaactcgt tgagctgtgc 420 gggggtgaag cgcgtccggt ggcgcttctg
cttctgttgg ccctgctgct ggccggcttg 480 gctggggttc gggccgccct
ggggcccggg 510 18 386 DNA Homo sapiens 18 cccgggaaac agttcaggac
gctcaagacc agaagcggga gcaaacccaa aaggagctcc 60 aaggaggtgt
gtgtggggag agccaggggg acgcaggact aggctctttc ctgcgcaagg 120
ggtggggaaa cccgcgaaag ccagggagtc gcgcgcactc acgccctcgc gccaccaggc
180 agagccaccg ctgcaaggag cccacgggtg cgcgctcgct ccagggcgga
tctttccaca 240 cccccctcac cctcaaaagc tcaggctgga gcggtcatca
gtgcggactc cggcacccca 300 cccacccagc aggggttaag gagggactgg
cgcccactct tgcctacagc tcctgcgcag 360 ggctccagcc gccaaatctt cccggg
386 19 514 DNA Homo sapiens misc_feature (415)..(415) n is any
nucleotide 19 cccgggctga atggtagacg ttctggcgcc gggcagcggc
caccggctgg ttcccacttc 60 cgcgcgcacc ccttaaactg tgttctagag
gccccagcct cgccttgcag cgcctcacta 120 gctcctgagg actagggact
cggcggtgag gcgggttggc ggctcgaacg agctgggcgt 180 cttcgttctc
tctcgctgcc tggctggctc agctggccct ccacagctcg gagcaaggcc 240
atagcaggga gtggaggtat attgggctgt cacctccttg ctggccggag ttatttgtag
300 actacagact cggaagaaca gacgcgccac gctctcgctt ggcattgctt
cggatcgcag 360 ctcctccttg gggtgcccca gcttggcgtt tatttgcctg
cgccaggctc tggcnacggt 420 caccgggcca gcggggaggg acggacggca
ggtgaccagc ctctgctgtg aagaaattcc 480 tgcgcgcccg gagctgtccc
taatgcatcc cggg 514 20 279 DNA Homo sapiens misc_feature (27)..(27)
n is any nucleotide 20 ccggggatcg ggagcttgga gtgaggngct cggcgtgacc
cgtgaggagc cccgcgggta 60 gagcggcgct gccccttcgc tccggggtga
actgaaactt tgctagggga gagggtcggc 120 gccagcctcg cggggttcgg
agaagaccca gcgctgtgcg aggtcggggc cgggcagggc 180 agagcagggg
tgaaaggaga gacctgtaat gacggcggga tttggggtgc ggagggttgc 240
gagggagggg ccgcaaccct gaacaattgc attcccggg 279 21 422 DNA Homo
sapiens 21 cccgggtctg gcttggtctc gcccgcgcag atcccgttca aactcagctg
ccaccaagtg 60 cgccttttct ctctggattg cgattctgca cgaattttcc
agttgagggt ggttcggcgc 120 tcagccagcc tctgccctcg aagacgcggc
cttggtctag gaaccttcag gtgggtgttt 180 gggcgcagtg gccctagttc
cctagaattc gttttgcctc ccgcggcctc agccgcgtgg 240 tcagcgagct
cgcgagaacg cagctgggca gtcccggacg gctctcgggc gcttctaggg 300
agcagtcaaa gggttgcgag gactgccagg gtccttcctc cctaggcttc ccacactgga
360 tggccgtaac tcagtcgttg acggcgacag ccagggccct gatggactgt
gcaggtcccg 420 gg 422 22 464 DNA Homo sapiens 22 cccgggtacc
tgcacagctc gctccctccc atccttcggg tcttcgctcg aacgtccgct 60
cctcggtgag gccttccctg gacaacgcat ttgaaacgta accccaaggc aagaagccac
120 cttccaggcg cgcagccgaa gcccagtgcc aaggaggccg gagactcggg
tgcccgcgca 180 tcccgaaaac agcctctgag gggtcctctg agcatccttc
cagcgtgttt gggaggcaaa 240 ctcgttgact agctcttgag aggagtggct
agaggaatcc aggcggggaa ggggacggtg 300 gactccagga gagtgtaatt
tacaaaggcg gggggcgggg acgcccaggt ccgagtccca 360 ggactctgcg
ccggacgctt cgcccgccct ttcaggtccc ctgcccggtc ctcgtacccg 420
cgcgggtccg gagaacctct gagcaccggc ccccagcccc cggg 464 23 257 DNA
Homo sapiens 23 cccgggatcg gtgcccgtta gtgggcacag gacgccgggt
gtccgaaggg ctgcctggag 60 atgcaactga ccttggcggg catcagaatg
acgagtggca gcagcaggag cggggtgacg 120 aacaagatca cgaaggactt
gaacttggag acatagctca gcgccgaggc catcgcgcgg 180 gagggagact
ggcgggcgag acgagtgagg ggcagctaga ggcgccgcgg gcttaagaag 240
gggccacagt ccccggg 257 24 350 DNA Homo sapiens 24 cccggggatg
gcgcgccagg taaggcaggc ggacgcgcgg acccctcggc gcagtgcggc 60
cccgaaggcg gtagggcgag aaagagctgg gacccccccg caacttcggg atcggctggg
120 tgaggctggg caggcctgat gctcaggggt tcgtgccggg aagttcaggt
cctcggacaa 180 cctctctcca gctctccgcc gccggtaccc acggcccagt
ctccacctgg ggaaaccccc 240 ttggcgtggc ttgtttcgtt acaagttatc
ctggtagagt gggcatgaag gcctcggagg 300 cagtgagtaa atctcatact
tctgttcttg gtggaatcct ggatcccggg 350 25 337 DNA Homo sapiens 25
ccccgggctg ccggctggag cccggcagat tcgctgcgca gcactgcccc ctggtatcca
60 gcgccgaaag tgcccccctc cgcagctgca aggctcctcc ctgggctgcg
cgggacagat 120 tttttctcct ttcctggcta cacgcctaac agagaagcta
tcccgaggga cctcaagaag 180 tccccccaag ccgtactcaa aagcctttct
ccctcctcct caagcgctca cttccccaaa 240 gaggacccgg acccctgact
gcctgagcca ggtccccagc atggtccgca acccttctcg 300 actccggcat
ccacctccag gctgacgtct acccggg 337 26 229 DNA Homo sapiens 26
cccgggggaa agagatgagt ggaaatcgtg ggccggacct gcaaggagag actcggcggg
60 caccttgctt ggtctgaggt cgtctgcagg aagcggactt ttctcctggc
tcaggatggg 120 aaagacaggg gatgcctgaa gtcaacgggg acttctgttc
catctctgcc ccgttctcca 180 ggcccgccag tttttcctgc tttggttaga
ttttccaacg tatcccggg 229 27 427 DNA Homo sapiens 27 cccggggctg
ctgaccgagc cccagggcag cccgcccttc cccctcctga agcccgacgt 60
cttcttcatg gctccgtcta ccggcttcgg agacccgctg gacttttcgc cgcctccgga
120 accctatatg aggaagcaaa tcgcgtccgc cacagcctcc aactaggaaa
ctccgcgact 180 ctcagcccct cagaagagaa acggagacgc gccaagcaaa
gccgttacac ggactgtgca 240 cgcgcctccg gtgtccctgc gcgtgacaca
aatttggccc cgagggagct ccatgtgcct 300 gagtcccagg agccctagat
gccagcgaca gcttgtcacc aggcctgcga cgccaatggg 360 cgggagtcgg
cggagctcag gacactgacg acgggcctgg gggaaagcgg tccccacaca 420 gcccggg
427 28 470 DNA Homo sapiens 28 cccggggaca gggggacccc cagatgctgc
acggctgaca ggccaacgtg gcagaagctc 60 cagcttcaca ggaagccagt
gaccatgaga gtctgtagct gtaacgaagc cacagagctg 120 tggctttctt
tccccttcag ctctaggaaa ggttatctgc cctgcacaga tctccggagg 180
cctggctggg ctctgagagc atcagactga ttatcgtaag aaaataatct ctgcagacac
240 attccttgct agaagcaggg gacaaagccc agcttcaaag acaattccac
acacgccctc 300 cctgccctgc acagctgcct gccgggtggg agcagagccc
ttgcagccgg gctcaggggc 360 ctgggcaggg acagcgtgtg gcaggggcac
agctgagaca ggagcctcaa agcgacacca 420 acccgacgtg aagctacagt
tgaggagaca cagctgcccc cattcccggg 470 29 264 DNA Homo sapiens 29
cccgggcggc ggccggattg cgggtgggtg agaggcagca gacgccgtgt ttacagctct
60 ctcgctagtt cgccacctca gccgcggctc tagggctgag ccagtcgcct
ccttctttaa 120 gattctggtc acagcagggg ctgggtttct aaggcaggtt
tctaaggtgt cttcctacag 180 acaccgctgc tgctaccttg ctaccttcag
cgctgggcac agccaggggc agcgcgagag 240 ggaggcaacg agagggttcc cggg 264
30 371 DNA Homo sapiens 30 cccgggaccc caatgccagg gaggggcctg
caggacccca gcggtgggcg agttgtgtcc 60 tgggtcacct tgtgtttcgc
agcgtggcgg tggcaggagc ccagcgcggg aggacatttt 120 catagcctcc
tacagtgaga aacgcccccc acccgacgct gcgctcatct gtgtccccgc 180
tgttgccggg gctctggtat ccacttgcgc gccctatgtg gtggggatcc acccagagcc
240 cagcgtcaag ttatacgggc gcttcactca gcgtcagcca agaccaggga
agcgcttctt 300 gccgtttagg agacgtctgc aagagataaa aagctagccc
acgatccacc cacaatcctc 360 gtgtccccgg g 371 31 179 DNA Homo sapiens
31 cccgggaact cgcggcaccc actgggtatt gtcgggaccc agcaagtcta
ggaacggggg 60 tgggtagagc atccttcggg cactgccgtt cgtccccaaa
agaagaccac cgcggggtcc 120 cagggccacg gcgaggacgg gcactggtca
gattccggac aggcggtcct ggccccggg 179 32 445 DNA Homo sapiens 32
cccgggctgt gaccagcgaa ttcgggcccc gcaggtgcag ctgataggag aaatccggct
60 ccgggagcga acccagcggc ggaaaggcgg gctccgcgcc caggcgggcc
ttggactgca 120 agaaggcgag gatgcgcgcg tacttcgtgt ccttggtctc
atcgtcacgt gtgagtatcg 180 accaggtcat catcgcacgt ggtaccatag
tggaagtagt tggcaaactc gctagagtct 240 gctggaggaa cgagcccgcc
gtaggacgga cacacctgag tgcccctccc acgcgagccc 300 aaagcgggtg
cagggcacct cccaccacat ttctggccaa agttcccatt tgaggcccgc 360
cctctcctct gcgcagtctt agagactggc gaggcacgcg caaacgccct cttccctgag
420 acctgacccc acccacccac ccggg 445 33 357 DNA Homo sapiens 33
cccgggggag ccccggaccc cgcatccccc agggcgcgga aactggcgag gccccaggag
60 ctcccattta tagctcagtt tccactgagc gcagtccctc taggacctgg
gctgagcaag 120 tttcttccac tctctccctt ccctcctcct caccccttgc
ctgcccctca accccggcag 180 ggcgcaggtg tccaacccag ccgggacccc
ctccctcctc gaacccaggt gttccggctc 240 ccagacccca attgagctgg
gggcgcccac ccgccggggg atcccgccct gcgtccccca 300 ttcatccgcg
tctcagccgc gggagtttct caacgggaag agggcggagc tcccggg 357 34 312 DNA
Homo sapiens 34 cccgggagga gagttggggc ttgggggacg ccgtgaactc
catggtcccc agcacgcggt 60 cctggccagg gacggggtcg tccgaactgc
cgtccagatt ccccaaggga gacaaagacc 120 cgaaacacag ctcaaagttt
ccgagagcag tcacagcggg gccagggact ccagaagtgt 180 cagctccaac
gactccagag ctgcacactg gcctctattc cccaccgcaa agccccagag 240
ccgcagagac ttcgaaggca gccggagagg agagggccca ccgagcacta cggcgggtgc
300 gcacgccccg gg
312 35 372 DNA Homo sapiens 35 cccgggagcc ggctcgctgg cggcgccagg
ccacgctctc tcattaacat cccgctcccg 60 gtggcgcagg ggagccggcc
aaagttcctc gcaaagtggc gagcgaagga gcgctgagca 120 ctgacgtctg
ggctggggag gagcgggtcc gagcgaggac ggagagggga cagagggaaa 180
gggaggcggg tgtcttcctc aggaatttga gctggggatc tgcatcctgg ccattgcagt
240 cctttagcat cctcgccgcg ccctgagcgc gctggaggct cgcaggctgc
gccctcccag 300 ggctgatgcc gcgtcctgct ccgccgttct gggacgtcgg
ggacaaaagt ggaggagacg 360 ggagagcccg gg 372 36 399 DNA Homo sapiens
36 cccgggctgc gagcgcggct cctggattcc agcctcccgc ccttcccagg
cgctggaatg 60 gacacggacg cccacagtgg cgggccaggt agtgccggag
tcgggggccc aggccgcggc 120 gccccgcgcc tcatcactta ccttgccttt
agctatcaat tccatgatgt agccaaattc 180 actcatctcc ccagactccg
acatgtttac accccttcac aaactctgga ggaccgacgc 240 gggtgtatcg
aatttgtcct ttcttttctc tttttctgtt tttagtctga gttttgccga 300
gctccccgcc cataagctgt taaccaggaa aagaggggaa gcgccgggga aagcaagaag
360 cgggcttggg tgaaatgaag gccatcgagg gctcccggg 399 37 307 DNA Homo
sapiens 37 cccgggcccg gtgcgcaccg gtgccgactt ggcagccgcc ctgtgcgctc
gacgaaaggg 60 tgagaaggag gcaggagtgc aggcggaagg agtgggcaat
cagcggcggg gacgagagtg 120 tgtcttcggg gaaaccaagt ctgagtgagc
gctgaagggg agtgtgcgga gcgtgccgtg 180 caccccgagc cccccgcctc
attgcctctc gcctctctcc acctgcccca tgatctgcgc 240 cagggaccgg
tcctctcccg tccgcaggct gtctaggtgg ccgttctggt ttgctgggac 300 ccccggg
307 38 331 DNA Homo sapiens 38 cccggggcca cctctgaggc atgaacccag
agacgcgcgc cctggtctgg gaaagcagga 60 ccgctgcgcc cagcgcctca
ggggtagagg cgggaacagg cccgcggtcg ctttgctggc 120 ggcggggaag
ggcgatctga cgatcaggga gttgcgcccc tctctctggg cctcgtgaag 180
gaacaagagc aattacagcg ctgggccggc cacgtagtcc tggggctagg tgggcccaat
240 gctccgggcc gcggggctgg agcgcggagg ctggagaggg aggaggaccc
tccgcggctc 300 caggtctccc agctggaggc tcacgcccgg g 331 39 304 DNA
Homo sapiens 39 ccgggcccgg tgcgcaccgg tgccgacttg gcagccgccc
tgtgcgctcg acgaaagggt 60 gagaaggagg caggagtgca ggcggaagga
gtgggcaatc agcggcggga cgagagtgtg 120 tcttcgggga aaccaagtct
gagtgagcgc tgaaggggag tgtgcggagc cgtgccgtgc 180 accccgagcc
ccccgcctca ttgcctctcg cctctctcca cctgccccat gatctgcgcc 240
agggagccgg tcctctcccg tccgcagctg tctaggtggc cgttctggtt tgctgggccc
300 cggg 304 40 307 DNA Homo sapiens 40 ccgggggtcc cagcaaacca
gaacggccac ctagacagcc tgcggacggg agaggaccgg 60 ttccctggcg
cagatcatgg ggcaggtgga gagaggcgag aggcaatgag gcgggggggc 120
tcggggtgca cggcacgctc cgcacactcc cctccagcgc tcactcagac ttggtttccc
180 cgaagacaca ctctcgtccc cgccgcgtga ttgcccactc cttccgcctg
cactccagcc 240 tccttctcac cctttcgtcg agcgcacagg cggctgccaa
gtcggcaccg gtgcgcaccg 300 gcccggg 307 41 304 DNA Homo sapiens 41
ccgggcccgg tgcgcaccgg tgccgacttg gcagccgccc tgtgcgctcg acgaaagggt
60 gagaaggagg caggagtgca ggcggaagga gtgggcaatc agcggcggga
cgagagtgtg 120 tcttcgggga aaccaagtct gagtgagcgc tgaaggggag
tgtgcggagc cgtgccgtgc 180 accccgagcc ccccgcctca ttgcctctcg
cctctctcca cctgccccat gatctgcgcc 240 agggagccgg tcctctcccg
tccgcagctg tctaggtggc cgttctggtt tgctgggccc 300 cggg 304 42 479 DNA
Homo sapiens misc_feature (1)..(479) n is any nucleotide 42
cccgggttcc tggcttgaac cctgtttctc cctgttctgc caggcatgct ggtccggaag
60 gtgtgtgtng ctgtnggctt taggtgggtg cagcccgtcc cacgtcacgg
cgagctctgt 120 ttcctgggct ggggacagtg aggtcatcgc tgcccatcct
ggagctctgg ctcctttcgg 180 gtacctgttc cctctcccag agagaccccc
agctgcatgc aggcctagtg ggctccacgg 240 cggagctggt tcccaggcta
cctgggttgc cacctctgtg ggtcccggct gccctctcgc 300 agccgccgct
acttcctcac cctcttggcc ctgcatttcc acgtctcatg gagccaacga 360
gagcaggggg tttgagccct tgtggaaatc tggggaggca ctgcttctcc ctccatgtga
420 gcagcttcac ccagcctggg gtcagtgctt acgctccacg cggcctggcc
ttccccggg 479 43 21 DNA Artificial sequence PCR primer 43
gagttggtga tgttagatta g 21 44 22 DNA Artificial sequence PCR primer
44 ttcccaaaaa aatcccaaat tc 22 45 22 DNA Artificial sequence PCR
primer 45 ctccttccaa ataaatactt ac 22 46 19 DNA Artificial sequence
PCR primer 46 agagaggagt ttagattgg 19 47 21 DNA Artificial sequence
PCR primer 47 caaaaaaact aaaacctcaa c 21 48 24 DNA Artificial
sequence PCR primer 48 tggataaagg atgtttgggg tttg 24 49 22 DNA
Artificial sequence PCR primer 49 ccctcccctt acccctaaat cc 22 50 19
DNA Artificial sequence PCR primer 50 actccccctc actttattc 19 51 24
DNA Artificial sequence PCR primer 51 attattttag tagaggtata taag 24
52 18 DNA Artificial sequence PCR primer 52 ccaaccccac ccttcaac 18
53 20 DNA Artificial sequence PCR primer 53 aagagagggt tggagagtag
20 54 24 DNA Artificial sequence PCR primer 54 cccctaaaaa
aaaaatcaaa aatc 24 55 21 DNA Artificial sequence PCR primer 55
gggttggaga gtaggggagt t 21 56 26 DNA Artificial sequence PCR primer
56 ygttatgatt tttttgttta gttaat 26 57 23 DNA Artificial sequence
PCR primer 57 tacaccaact acccaactac ctc 23 58 25 DNA Artificial
sequence PCR primer 58 tttatttata taattttgtg tatgg 25 59 21 DNA
Artificial sequence PCR primer 59 cacccctcac tttactaaaa c 21 60 23
DNA Artificial sequence PCR primer 60 tttgggaggt aaattygttg att 23
61 24 DNA Artificial sequence PCR primer 61 accraacaaa aaacctaaaa
aaac 24 62 24 DNA Artificial sequence PCR primer 62 ttattagagg
gtggggtgga ttgt 24 63 24 DNA Artificial sequence PCR primer 63
ttattagagg gtggggcgga tcgc 24 64 21 DNA Artificial sequence PCR
primer 64 aggattggga tgttgagaat g 21 65 19 DNA Artificial sequence
PCR primer 65 ggattgggat gtcgagaac 19 66 26 DNA Artificial sequence
PCR primer 66 gttttttttt ggatttttgt tttttg 26 67 19 DNA Artificial
sequence PCR primer 67 gttttttcgg ggcggtttc 19 68 31 DNA Artificial
sequence PCR primer 68 ttttgttttg ttattttttg tttttggttt t 31 69 29
DNA Artificial sequence PCR primer 69 cgtttcgtta ttttttgttt
tcggttttc 29 70 23 DNA Artificial Sequence PCR primer 70 gtttggttgt
tgtttattgg ttg 23 71 22 DNA Artificial sequence PCR primer 71
cctaaactca caaaccaact ca 22 72 21 DNA Artificial sequence PCR
primer 72 tgcgagcgtt tttttaaatg c 21 73 20 DNA Artificial sequence
PCR primer 73 taaactcgca aaccaactcg 20 74 22 DNA Artificial
sequence PCR primer 74 ttaataggaa gagtggatag tg 22 75 24 DNA
Artificial sequence PCR primer 75 tctataaatt actaaatctc ttca 24 76
21 DNA Artificial sequence PCR primer 76 ttaataggaa gagcggatag c 21
77 23 DNA Artificial sequence PCR primer 77 ctataaatta ctaaatctct
tcg 23 78 25 DNA Artificial sequence PCR primer 78 taattttagg
ttagagggtt attgt 25 79 20 DNA Artificial sequence PCR primer 79
cacaaccaat caacaacaca 20 80 22 DNA Artificial sequence PCR primer
80 ttaggttaga gggttatcgc gt 22 81 23 DNA Artificial sequence PCR
primer 81 taactaaaaa ttcacctacc gac 23 82 27 DNA Artificial
sequence PCR primer 82 ggaggatagt tggattgagt taatgtt 27 83 21 DNA
Artificial sequence PCR primer 83 caaatccctc ccaaacacca a 21 84 24
DNA Artificial sequence PCR primer 84 ggatagtcgg atcgagttaa cgtc 24
85 16 DNA Artificial sequence PCR primer 85 ccctcccaaa cgccga 16 86
29 DNA Artificial sequence PCR primer 86 tttgtgtttt gatgtttgta
ggtttttgt 29 87 28 DNA Artificial sequence PCR primer 87 aactccacac
tcttccaaaa acaaaaca 28 88 23 DNA Artificial sequence PCR primer 88
tttcgacgtt cgtacctttt cgc 23 89 22 DNA Artificial sequence PCR
primer 89 gcactcttcc gaaaacgaaa cg 22 90 22 DNA Artificial sequence
PCR primer 90 ggggttgagg ttttttgtta gt 22 91 23 DNA Artificial
sequence PCR primer 91 ttcacaacct caaatctact tca 23 92 21 DNA
Artificial sequence PCR primer 92 gggttgaggt tttttgttag c 21 93 20
DNA Artificial sequence PCR primer 93 ctacttcgcc taacctaacg 20 94
25 DNA Artificial sequence PCR primer 94 ggtgttgtta aatgtaaata
atttg 25 95 22 DNA Artificial sequence PCR primer 95 aaaaaaaaac
acctaaaact ca 22 96 21 DNA Artificial sequence PCR primer 96
aatcgaattt gtcgtcgttt c 21 97 25 DNA Artificial sequence PCR primer
97 aaataaataa ataaaaaaaa acgcg 25 98 26 DNA Artificial sequence PCR
primer 98 gaattgagat gattttaatt ttttgt 26 99 22 DNA Artificial
sequence PCR primer 99 ctaaaaccat cacccctaaa ca 22 100 23 DNA
Artificial sequence PCR primer 100 ttgagacgat tttaattttt tgc 23 101
20 DNA Artificial sequence PCR primer 101 aaaaccatca cccctaaacg 20
102 22 DNA Artificial sequence PCR primer 102 gagtggttag aggaatttag
gt 22 103 24 DNA Artificial sequence PCR primer 103 ctaaaaaaac
aaacaaaaca tcca 24 104 20 DNA Artificial sequence PCR primer 104
gtggttagag gaatttaggc 20 105 19 DNA Artificial sequence PCR primer
105 aaaacgaacg aaacgtccg 19 106 21 DNA Artificial sequence PCR
primer 106 acttccatta aaaacaacta c 21 107 23 DNA Artificial
sequence PCR primer 107 caaccccaaa cccacaacca taa 23 108 22 DNA
Artificial sequence PCR primer 108 gacccccgaa ccgcgaccct aa 22 109
23 DNA Artificial sequence PCR primer 109 ttacaaaaaa ccttccaaat aca
23 110 22 DNA Artificial sequence PCR primer 110 tacaaaaaac
cttccgaata cg 22 111 23 DNA Artificial sequence PCR primer 111
tttattccaa cttcttcact tca 23 112 19 DNA Artificial sequence PCR
primer 112 ttccgacttc ttcgcttcg 19 113 21 DNA Artificial sequence
PCR primer 113 ccccccaaaa accccacctc a 21 114 17 DNA Artificial
sequence PCR primer 114 ccgaaaaccc cgcctcg 17 115 21 DNA Artificial
sequence Amplification primer 115 ttgtgtgggg agttattgag t 21 116 23
DNA Artificial sequence Amplification primer 116 caccttcaca
aaaaaaatca atc 23 117 18 DNA Artificial sequence Amplification
primer 117 tgtggggagt tatcgagc 18 118 19 DNA Artificial sequence
Amplification primer 118 gccttcgcga aaaaaatcg 19
* * * * *
References