U.S. patent application number 13/796766 was filed with the patent office on 2013-12-05 for polynucleotide primers.
This patent application is currently assigned to QIAGEN MANCHESTER LIMITED. The applicant listed for this patent is QIAGEN MANCHESTER LIMITED. Invention is credited to Paul Francis RAVETTO, Nicola Jo THELWELL, David Mark WHITCOMBE.
Application Number | 20130323736 13/796766 |
Document ID | / |
Family ID | 34586669 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323736 |
Kind Code |
A1 |
WHITCOMBE; David Mark ; et
al. |
December 5, 2013 |
POLYNUCLEOTIDE PRIMERS
Abstract
A polynucleotide primer comprising at least the final six
nucleotides of one of the following primer sequences, or a sequence
complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to
77.
Inventors: |
WHITCOMBE; David Mark;
(Manchester, GB) ; THELWELL; Nicola Jo;
(Manchester, GB) ; RAVETTO; Paul Francis;
(Manchester, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
QIAGEN MANCHESTER LIMITED; |
|
|
US |
|
|
Assignee: |
QIAGEN MANCHESTER LIMITED
Manchester
GB
|
Family ID: |
34586669 |
Appl. No.: |
13/796766 |
Filed: |
March 12, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11910511 |
Jun 2, 2008 |
|
|
|
PCT/GB2006/001227 |
Apr 4, 2006 |
|
|
|
13796766 |
|
|
|
|
Current U.S.
Class: |
435/6.12 ;
536/24.33 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6851 20130101; C12Q 1/6886 20130101; C12Q 1/6851 20130101;
C12Q 1/6883 20130101; C12Q 2537/155 20130101; C12Q 2600/16
20130101 |
Class at
Publication: |
435/6.12 ;
536/24.33 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 4, 2005 |
GB |
0506807.7 |
Claims
1. A polynucleotide comprising at least the final six nucleotides
of one of the following primer sequences, or a sequence
complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to
77.
2. A polynucleotide according to claim 1 wherein the polynucleotide
is less than 100 nucleotides long, preferably less than 80
nucleotides long, more preferably less than 60 nucleotides long,
more preferably less than 40 nucleotides, more preferably less than
30 nucleotides long.
3. A polynucleotide according to claim 1 comprising at least 75% of
the final 8, 10, 12, 14, 16, 17, 18 or 20 nucleotides, or the
entirety of one of the following primer sequences, or a sequence
complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to
77.
4. A polynucleotide according to claim 1 further comprising a
quencher group and a fluorophore group.
5. A polynucleotide according to claim 4 wherein the quencher group
and the fluorophore group are separated by a nucleotide tail
sequence comprising first and second regions, the nucleotides of
the first region being complementary to but in reverse order from
the nucleotides of the second region, such that hybridisation of
the first region to the second group results in the quencher group
to be sufficiently close to the fluorophore group to quench the
fluorophore group.
6. A polynucleotide according to claim 5 wherein the tail sequence
further comprises a third region having a sequence complementary to
a region of the EGFR gene.
7. A polynucleotide according to claim 6 comprising at least the
final six nucleotides of SEQ. ID NO. 3 or 10 and the tail sequence
comprises SEQ. ID NO. 19.
8. A polynucleotide according to claim 6 comprising at least the
final six nucleotides of SEQ. ID NOS. 6 or 12 and the tail sequence
comprises SEQ. ID NO. 20.
9. A polynucleotide according to claim 6 comprising at least the
final six nucleotides of SEQ. ID NO. 76 and the tail sequence
comprises SEQ. ID NO. 77.
10. A polynucleotide according to claim 4 wherein the quencher
group comprises black hole quencher 1 (BHQ1) and the fluorophore
group comprises FAM.
11. A polynucleotide according to claim 4 wherein the quencher
group comprises black hole quencher 2 (BHQ2) and the fluorophore
comprises Cal Red.
12. A kit comprising at least a pair of polynucleotides wherein the
pair of polynucleotides comprises at least four or five of the
final six nucleotides of one of the following pairs of primer
sequences, respectively, or sequences complementary thereto: SEQ.
ID NO. 1 and SEQ. ID NO. 15, or SEQ. ID NO. 2 and SEQ. ID NO 15, or
SEQ. ID NO. 3 and SEQ. ID NO. 15, or SEQ. ID NO. 4 and SEQ. ID NO.
15, or SEQ. ID NO. 5 and SEQ. ID NO. 15, or SEQ. ID NO. 6 and SEQ.
ID NO. 16, or SEQ. ID NO. 7 and SEQ. ID NO. 17, or SEQ. ID NO. 8
and SEQ. ID NO. 18, or SEQ. ID NO. 9 and SEQ. ID NO. 18, or SEQ. ID
NO. 10 and SEQ. ID NO. 15, or SEQ. ID NO. 11 and SEQ. ID NO. 15 or
SEQ. ID NO. 12 and SEQ. ID NO. 16, or SEQ. ID NO. 13 and SEQ. ID
NO. 17, or SEQ. ID NO. 14 and SEQ. ID NO. 18 or SEQ. ID NO. 21 and
SEQ. ID NO. 15, or SEQ. ID NO. 22 and SEQ. ID NO. 24, or SEQ. ID
NO. 23 and SEQ. ID NO. 24, or SEQ. ID NO. 25 and SEQ. ID NO. 41, or
SEQ. ID NO. 26 and SEQ. ID NO. 41, or SEQ. ID NO. 27 and SEQ. ID
NO. 41, or SEQ. ID NO. 28 and SEQ. ID NO. 41, or SEQ. ID NO. 29 and
SEQ. ID NO. 42, or SEQ. ID NO. 30 and SEQ. ID NO. 43, or SEQ. ID
NO. 31 and SEQ. ID NO. 44, or SEQ. ID NO. 32 and SEQ. ID NO. 44, or
SEQ. ID NO. 33 and SEQ. ID NO. 41, or SEQ. ID NO. 34 and SEQ. ID
NO. 45, or SEQ. ID NO. 35 and SEQ. ID NO. 41, or SEQ. ID NO. 36 and
SEQ. ID NO. 41, or SEQ. ID NO. 37 and SEQ. ID NO. 42, or SEQ. ID
NO. 38 and SEQ. ID NO. 43, or SEQ. ID NO. 39 and SEQ. ID NO. 44, or
SEQ. ID NO. 40 and SEQ. ID NO. 45, or SEQ. ID NO. 74 and SEQ. ID
NO. 76 or SEQ. ID NO. 75 and SEQ. ID NO. 76.
13. A kit according to claim 12 wherein the pair of polynucleotides
are each polynucleotides comprising at least the final six
nucleotides of one of the following primer sequences, or a sequence
complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to
77.
14. A kit comprising at least a set of three polynucleotides,
wherein the set of three polynucleotides comprises at least four or
five of the final six nucleotides of one of the following sets of
three primer sequences, respectively, or sequences complementary
thereto: SEQ. ID NOS. 1, 10 and 15, or SEQ. ID NOS. 2, 10 and 15,
or SEQ. ID NOS. 3, 10 and 15, or SEQ. ID NOS. 4, 11 and 15, or SEQ.
ID NOS. 5, 11 and 15, or SEQ. ID NOS. 6, 12 and 16, or SEQ. ID NOS.
7, 13 and 17, or SEQ. ID NOS. 8, 14 and 18, or SEQ. ID NOS. 9, 14
and 18 or SEQ. ID NOS. 21, 10 and 15, or SEQ. ID NOS. 22, 23 and
24, or SEQ. ID NOS. 25, 35 and 41, or SEQ. ID NOS. 26, 35 and 41,
or SEQ. ID NOS. 27, 36 and 41, or SEQ. ID NOS. 28, 36 and 41, or
SEQ. ID NOS. 29, 32 and 42, or SEQ. ID NOS. 30, 38 and 43, or SEQ.
ID NOS. 31, 39 and 44, or SEQ. ID NOS. 32, 39 and 44, or SEQ. ID
NOS. 33, 35 and 41, or SEQ. ID NOS. 34, 40 and 45, SEQ. ID NOS. 74,
75 and 76.
15. A kit according to claim 14 wherein the set of three
polynucleotides are each polynucleotides comprising at least the
final six nucleotides of one of the following primer sequences, or
a sequence complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or
74 to 77.
16. A kit according to claim 12 further comprising nucleotide
triphosphates, a polymerisation enzyme and/or a buffer
solution.
17. A method of detecting the presence or absence of a mutation in
the EGFR gene comprising the steps of: a) mixing a nucleic acid
sample comprising at least a fragment of the EGFR gene with a
polynucleotide complementary to a region of the fragment of the
EGFR gene; and b) detecting hybridisation of the polynucleotide to
the nucleic acid sample wherein hybridisation indicates the
presence or absence of a mutation.
18. A method according to claim 17 wherein the polynucleotide is a
polynucleotide comprising at least the final six nucleotides of one
of the following primer sequences, or a sequence complementary
thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77 and comprises
at least four or five of the final six nucleotides of SEQ. ID NOS.
1 to 9, 21, 22, 25 to 34 or 75 or sequences complementary thereto
and step b) indicates the presence of a mutation.
19. A method according to claim 17 wherein the polynucleotide is a
polynucleotide comprising at least the final six nucleotides of one
of the following primer sequences, or a sequence complementary
thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77 and comprises
at least four or five of the final six nucleotides of SEQ. ID NOS.
10 to 14, 23, 35 to 40 or 74 or sequences complementary thereto;
and step b) indicates the absence of a mutation.
20. A method according to claim 17 further comprising the step of,
prior to step a), amplifying the number of copies of the fragment
of the EGFR gene using thermal cycling nucleic acid amplification,
preferably PCR.
21. A method according to claim 17, wherein step b) comprises
carrying out DNA polymerisation using the polynucleotide as a first
primer and detecting the extension product of polymerisation.
22. A method according to claim 20 wherein step b) comprises the
step of mixing the nucleic acid sample and the polynucleotide with
a second primer which corresponds to a region of the fragment of
the EGFR sequence downstream of the region to which the
polynucleotide is complementary and carrying out PCR on the
mixture.
23. A method according to claim 22 wherein the second primer
comprises: SEQ. ID NO. 15 and the polynucleotide comprises at least
four or five of the final six nucleotides of SEQ. ID NOS. 1 to 5,
10, 11 or 21; SEQ. ID NO. 16 and the polynucleotide comprises at
least four or five of the final six nucleotides of SEQ. ID NOS. 6
or 12; SEQ. ID NO. 17 and the polynucleotide comprises at least for
or five of the final six nucleotides of SEQ. ID NOS. 7 or 13; SEQ.
ID NO. 18 and the polynucleotide comprises at least four or five of
the final six nucleotides of SEQ. ID NOS. 8, 9 or 14; SEQ. ID NO.
24 and the polynucleotide comprises at least four or five of the
final six nucleotides of SEQ. ID NOS: 22 or 23; SEQ. ID NO. 41 and
the polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 25 to 28, 33, 35 or 36; SEQ. ID NO. 42
and the polynucleotide comprises at least four or five of the final
six nucleotides of SEQ. ID NOS. 29 or 37; SEQ. ID NO. 43 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 30 to 38; SEQ. ID NO. 44 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 31, 32 or 39; SEQ. ID NO. 45 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 34 or 40; or SEQ. ID NO. 76 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NO. 74 or 75.
24. A method according to claim 21 wherein step b) comprises the
step of mixing the nucleic acid sample and the polynucleotide with
a second primer which corresponds to a region of the fragment of
the EGFR sequence downstream of the region to which the
polynucleotide is complementary and carrying out PCR on the
mixture.
25. A method according to claim 24 wherein the second primer
comprises: SEQ. ID NO. 15 and the polynucleotide comprises at least
four or five of the final six nucleotides of SEQ. ID NOS. 1 to 5,
10, 11 or 21; SEQ. ID NO. 16 and the polynucleotide comprises at
least four or five of the final six nucleotides of SEQ. ID NOS. 6
or 12; SEQ. ID NO. 17 and the polynucleotide comprises at least for
or five of the final six nucleotides of SEQ. ID NOS. 7 or 13; SEQ.
ID NO. 18 and the polynucleotide comprises at least four or five of
the final six nucleotides of SEQ. ID NOS. 8, 9 or 14; SEQ. ID NO.
24 and the polynucleotide comprises at least four or five of the
final six nucleotides of SEQ. ID NOS: 22 or 23; SEQ. ID NO. 41 and
the polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 25 to 28, 33, 35 or 36; SEQ. ID NO. 42
and the polynucleotide comprises at least four or five of the final
six nucleotides of SEQ. ID NOS. 29 or 37; SEQ. ID NO. 43 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 30 to 38; SEQ. ID NO. 44 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 31, 32 or 39; SEQ. ID NO. 45 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 34 or 40; or SEQ. ID NO. 76 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NO. 74 or 75.
26. A method according to claim 21 wherein step a) comprises the
step of mixing the nucleic acid sample with a pair of a mutation
specific polynucleotide and a wild-type specific polynucleotide,
the pair being selected from at least four or five of the final six
sequences of: SEQ. ID NO: 1 and SEQ. ID NO: 10; SEQ. ID NO: 2 and
SEQ. ID NO: 10; SEQ. ID NO: 3 and SEQ. ID NO: 10; SEQ. ID NO: 4 and
SEQ. ID NO: 11; SEQ. ID NO: 5 and SEQ. ID NO: 11; SEQ. ID NO: 6 and
SEQ. ID NO: 12; SEQ. ID NO: 7 and SEQ. ID NO: 13; SEQ. ID NO: 8 and
SEQ. ID NO: 14; SEQ. ID NO: 9 and SEQ. ID NO: 14; SEQ. ID NO: 21
and SEQ. ID NO: 10; SEQ. ID NO: 22 and SEQ. ID NO: 23; SEQ. ID NO:
25 and SEQ. ID NO: 35; SEQ. ID NO: 26 and SEQ. ID NO: 35; SEQ. ID
NO: 27 and SEQ. ID NO: 36; SEQ. ID NO: 28 and SEQ. ID NO: 36; SEQ.
ID NO: 29 and SEQ. ID NO: 37; SEQ. ID NO: 30 and SEQ. ID NO: 38;
SEQ. ID NO: 31 and SEQ. ID NO: 39; SEQ. ID NO: 32 and SEQ. ID NO:
39; SEQ. ID NO: 33 and SEQ. ID NO: 35; SEQ. ID NO: 34 and SEQ. ID
NO: 40; or SEQ. ID NO. 74 and SEQ. ID NO. 75.
27. A method according to claim 26 wherein the nucleic acid sample
comprises wild-type sequences and mutated sequences and further
comprising step c) wherein the number of amplification cycles
required to amplify the wild-type sequences to a predetermined
quantity is compared with the number of amplification cycles
required to amplify the mutated sequences to the predetermined
quantity thereby providing an indication of the ratio of the wild
type sequences to mutated sequences in the sample.
28. A method according to claim 27 wherein the nucleic acid sample
comprises a portion of tumourous tissue and a portion of
non-tumourous tissue and wherein step c) further comprises the step
determining the ratio of tumourous tissue to non-tumourous tissue
in the sample.
29. A method according to claim 27 further comprising the step of,
prior to step a), enriching the nucleic acid sample to increase the
ratio of tumourous tissue to non-tumourous tissue in the
sample.
30. A method according to claim 28 further comprising the step of,
prior to step a), enriching the nucleic acid sample to increase the
ratio of tumourous tissue to non-tumourous tissue in the
sample.
31. A method according to claim 22 wherein step b) comprises
detecting if amplification of at least a portion of the EGFR gene
occurs.
32. A method according to claim 17 wherein the polynucleotide
comprise a quencher group and a fluorophore group and wherein step
b) comprises exposing the mixture to light of a wavelength to which
the fluorophore is responsive in the absence of the quencher group
and detecting light at the wavelength emitted by the fluorophore
group in the absence of the quencher group.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/910,511 filed Jun. 2, 2008, which in turn
is a 371 filing of PCT/GB2006/001227 filed Apr. 4, 2006, which
claims priority from GB 0506807.7, filed Apr. 4, 2005. These prior
applications are incorporated herein by reference.
SEQUENCE SUBMISSION
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is entitled
3974-102SequenceListing.txt, was created on 12 Mar. 2013 and is 15
kb in size. The information in the electronic format of the
Sequence Listing is part of the present application and is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to a polynucleotide, a kit
comprising a polynucleotide and a method for detecting the presence
or absence of mutations in a gene.
BACKGROUND ART
[0004] Epidermal Growth Factor (EGF) and its corresponding receptor
(EGFR) are responsible for modulating growth of many classes of
cells. Mutations of EGFR have been implicated in uncontrolled cell
proliferation and tumour growth.
[0005] Although there are many examples of nucleic acid changes
having potential as tumour markers, their value as clinical tools
in cancer diagnosis, staging or even screening, needs to be
demonstrated and two important criteria must be met. Firstly,
nucleic acids of adequate yield and quality must be extracted from
the clinical material; secondly, robust and accurate methods of
analysis are required. For reliable tumour genotyping to be useful
in disease staging any test has to be adequately validated and
there should be demonstrable benefits over current methods.
[0006] A number of studies have examined the association of lung
cancer and its response to gefinitib (AstraZeneca's IRESSA) with
mutations in the oncogene, EGFR. However, there have been
differences in the spectrum and reported frequencies of EGFR
mutations. One major issue is that in order to identify a wide
range of possible mutations, nucleotide sequencing of the kinase
domain of the EGFR gene has been undertaken in excised tumours. The
utility of this approach is limited by a number of factors, mainly:
[0007] 1. Not all of the biopsy sample is tumour [0008] 2. Not all
tumour carries the mutation
[0009] For example, as little as 10% of a specimen may be tumour,
the remainder being marginal non-tumour cells. Tumours are
notoriously heterogeneous in their genetic makeup and as little as
10% of the tumour cells may contain any particular genetic change.
In total therefore as little as 1% of a tumour sample for genetic
analysis may contain the specific variation of interest.
[0010] The detection limits for sequencing are of the order of
15-25%; that is sequencing can detect the rarer allele at levels no
worse than 1 in 4 to 1 in 6. As discussed above, the prevalence of
the mutation in a given tumour sample may be significantly below
such levels. Current approaches require that a degree of sample
enrichment be performed, namely an attempt to excise selectively
the tumour material from a paraffin-embedded tissue section. This
is expensive, time consuming and lacks sensitivity.
[0011] In the present invention we have now devised novel
diagnostic methods for the detection of EGFR mutations based on the
amplification refractory mutation system (ARMS) as disclosed in,
for example, EP-A-0332435. Validated tests for four EGFR point
mutations and four deletions have been developed and the tests have
been applied in an investigation of the incidence of the mutations
in tumours from patients. The ARMS technology is capable of
selectively amplifying specific sequence variants in a background
of alternative sequences.
[0012] ARMS is a simple and accurate method and has several
benefits over other PCR-based mutation detection systems.
Specifically, the technique does not require the use of
radioisotopes nor the multiple probing of immobilised PCR amplicons
nor the cloning of PCR amplicons.
[0013] ARMS avoids the need for DNA sequencing of single-strand
conformation polymorphism products, a procedure that could be
expected to be constrained by sequence under-representation as
discussed above. Similarly, under-represented mutant sequences
could go undetected using PCR in conjunction with restriction
fragment length polymorphism which is limited to low cycle numbers
for the PCR to avoid false positive results. Previously generated
amplicons therefore have the potential to cause carry-over
contamination when PCR is resumed. ARMS can be performed under
conditions in which carry-over contamination is avoided, as in the
present invention, allowing the use of high PCR cycle numbers and
resulting in exceptionally high detection sensitivity.
DISCLOSURE OF THE INVENTION
[0014] Thus, in general terms the present invention relates to a
diagnostic method for the detection of EGFR mutations in cancer,
particularly lung cancer, using the amplification refractory
mutation system (ARMS). The invention also relates to mutation
specific primers suitable for use in the method and to diagnostic
kits containing these primers.
[0015] According to one aspect of the present invention, there is
provided a polynucleotide comprising at least the final six
nucleotides of one of the following primer sequences, or a sequence
complementary thereto: SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77.
The term "the final six nucleotides" means the six nucleotides at
the 3' end of the polynucleotide.
[0016] Conveniently, the polynucleotide is less than 100
nucleotides long, preferably less than 80 nucleotides long, more
preferably less than 60 nucleotides long, more preferably less than
40 nucleotides, more preferably less than 30 nucleotides long.
[0017] Preferably, the polynucleotide comprises at least 75% and
more preferably 100% of the final 8, 10, 12, 14, 16, 17, 18 or 20
nucleotides, or the entirety of one of the following primer
sequences, or a sequence complementary thereto: SEQ. ID NOS. 1 to
18, 21 to 45 or 74 to 77.
[0018] Advantageously, the polynucleotide further comprises a
quencher group and a fluorophore group. "Fluorophore groups" are
those groups which are capable of absorbing light at a first
wavelength and in response emitting light at a second wavelength. A
"quencher group" is a group which, when in sufficiently close
proximity to a fluorophore group, is capable of preventing, or
"quenching", the emission of light from the fluorophore group.
Typically, a particular type of quencher group will only work with
respect to certain types of fluorophore group.
[0019] Conveniently, the quencher group and the fluorophore group
are separated by a nucleotide tail sequence comprising first and
second regions, the nucleotides of the first region being
complementary to but in reverse order from the nucleotides of the
second region, such that hybridisation of the first region to the
second group results in the quencher group to be sufficiently close
to the fluorophore group to quench the fluorophore group.
[0020] Preferably, the tail sequence further comprises a third
region having a sequence complementary to a region of the EGFR
gene.
[0021] Advantageously, the polynucleotide comprises at least the
final six nucleotides of SEQ. ID NO. 3 or 10 and the tail sequence
comprise SEQ. ID NO. 19.
[0022] Alternatively, the polynucleotide comprises at least the
final six nucleotides of SEQ. ID NOS. 6 or 12 and the tail sequence
comprises SEQ. ID NO. 20.
[0023] Alternatively, the polynucleotide comprises at least the
final six nucleotides of SEQ. ID NO: 76 and the tail sequence
comprises SEQ. ID NO: 77.
[0024] Conveniently, the quencher group comprises black hole
quencher 1 (BHQ1) and the fluorophore group comprises FAM.
[0025] Alternatively, the quencher group comprises black hole
quencher 2 (BHQ2) and the fluorophore comprises Cal Red.
[0026] Preferably, a blocking moiety is provided between the primer
sequence and the tail sequence to prevent polymerase mediated chain
extension of the tail sequence. A preferred blocking moiety is a
hexethylene glycol (HEG) monomer.
[0027] According to another aspect of the present invention, there
is provided a kit comprising at least a pair of polynucleotides,
wherein the pair of polynucleotides comprises at least four or five
of the final six nucleotides of one of the following pairs of
primer sequences, respectively, or sequences complementary thereto:
SEQ. ID NO. 1 and SEQ. ID NO. 15, or SEQ. ID NO. 2 and SEQ. ID NO
15, or SEQ. ID NO. 3 and SEQ. ID NO. 15, or SEQ. ID NO. 4 and SEQ.
ID NO. 15, or SEQ. ID NO. 5 and SEQ. ID NO. 15, or SEQ. ID NO. 6
and SEQ. ID NO. 16, or SEQ. ID NO. 7 and SEQ. ID NO. 17, or SEQ. ID
NO. 8 and SEQ. ID NO. 18, or SEQ. ID NO. 9 and SEQ. ID NO. 18, or
SEQ. ID NO. 10 and SEQ. ID NO. 15, or SEQ. ID NO. 11 and SEQ. ID
NO. 15 or SEQ. ID NO. 12 and SEQ. ID NO. 16, or SEQ. ID NO. 13 and
SEQ. ID NO. 17, or SEQ. ID NO. 14 and SEQ. ID NO. 18 or SEQ. ID NO.
21 and SEQ. ID NO. 15, or SEQ. ID NO. 22 and SEQ. ID NO. 24, or
SEQ. ID NO. 23 and SEQ. ID NO. 24, or SEQ. ID NO. 25 and SEQ. ID
NO. 41, or SEQ. ID NO. 26 and SEQ. ID NO. 41, or SEQ. ID NO. 27 and
SEQ. ID NO. 41, or SEQ. ID NO. 28 and SEQ. ID NO. 41, or SEQ. ID
NO. 29 and SEQ. ID NO. 42, or SEQ. ID NO. 30 and SEQ. ID NO. 43, or
SEQ. ID NO. 31 and SEQ. ID NO. 44, or SEQ. ID NO. 32 and SEQ. ID
NO. 44, or SEQ. ID NO. 33 and SEQ. ID NO. 41, or SEQ. ID NO. 34 and
SEQ. ID NO. 45, or SEQ. ID NO. 35 and SEQ. ID NO. 41, or SEQ. ID
NO. 36 and SEQ. ID NO. 41, or SEQ. ID NO. 37 and SEQ. ID NO. 42, or
SEQ. ID NO. 38 and SEQ. ID NO. 43, or SEQ. ID NO. 39 and SEQ. ID
NO. 44, or SEQ. ID NO. 40 and SEQ. ID NO. 45, or SEQ. ID NO. 74 and
SEQ. ID NO. 76 or SEQ. ID NO. 75 and SEQ. ID NO. 76. For example,
the pair of polynucleotides comprises a first polynucleotide
consisting of the final six nucleotides of SEQ ID NO. 1 and a
second polynucleotide consisting of the final six nucleotides of
SEQ ID NO. 15.
[0028] According to a further aspect of the present invention,
there is provided a kit comprising at least a set of three
polynucleotides wherein the set of three polynucleotides comprises
at least four or five of the final six nucleotides of one of the
following sets of three primer sequences, respectively, or
sequences complementary thereto: SEQ. ID NOS. 1, 10 and 15, or SEQ.
ID NOS. 2, 10 and 15, or SEQ. ID NOS. 3, 10 and 15, or SEQ. ID NOS.
4, 11 and 15, or SEQ. ID NOS. 5, 11 and 15, or SEQ. ID NOS. 6, 12
and 16, or SEQ. ID NOS. 7, 13 and 17, or SEQ. ID NOS. 8, 14 and 18,
or SEQ. ID NOS. 9, 14 and 18, or SEQ. ID NOS. 21, 10 and 15, or
SEQ. ID NOS. 22, 23 and 24, or SEQ. ID NOS. 25, 35 and 41, or SEQ.
ID NOS. 26, 35 and 41, or SEQ. ID NOS. 27, 36 and 41, or SEQ. ID
NOS. 28, 36 and 41, or SEQ. ID NOS. 29, 32 and 42, or SEQ. ID NOS.
30, 38 and 43, or SEQ. ID NOS. 31, 39 and 44, or SEQ. ID NOS. 32,
39 and 44, or SEQ. ID NOS. 33, 35 and 41, or SEQ. ID NOS. 34, 40
and 45 or SEQ. ID NOS. 74, 75, 76. For example, the set of three
polynucleotides comprises a first polynucleotide consisting of the
final six nucleotides of SEQ ID NO. 1, a second polynucleotide
consisting of the final six nucleotides of SEQ IS NO. 10 and a
third polynucleotide consisting of the final six nucleotides of SEQ
ID NO. 15.
[0029] Conveniently the polynucleotides in the kit are as described
above.
[0030] Preferably, the kit further comprises nucleotide
triphosphates, a polymerisation enzyme and/or a buffer
solution.
[0031] According to another aspect of the present invention, there
is provided the use of a polynucleotide or a kit as described above
or a polynucleotide comprising four or five of the final six
nucleotides of SEQ. ID NOS. 1 to 18, 21 to 45 or 74 to 77 or
sequences complementary thereto for detecting a mutation in a
nucleic acid sample containing at least a fragment of the EGFR
gene.
[0032] Advantageously, the fragment of the EGFR gene in the nucleic
acid sample is at least 10 nucleotides long, preferably 20
nucleotides long, more preferably 30 nucleotides long and more
preferably 40 nucleotides long.
[0033] According to a further aspect of the present invention,
there is provided a method of detecting the presence or absence of
a mutation in the EGFR gene comprising the steps of: [0034] a)
mixing a nucleic acid sample comprising at least a fragment of the
EGFR gene with a polynucleotide complementary to a region of the
fragment of the EGFR gene; and [0035] b) detecting hybridisation of
the polynucleotide to the nucleic acid sample wherein hybridisation
indicates the presence or absence of a mutation.
[0036] Advantageously, the polynucleotide is a polynucleotide as
described above and comprises at least for or five of the final six
nucleotides of SEQ. ID NOS. 1 to 9, 21, 22, 25 to 34 or 75 or
sequences complementary thereto and step b) indicates the presence
of a mutation.
[0037] Preferably, the polynucleotide is a polynucleotide as
described above and comprises at least for or five of the final six
nucleotides of SEQ. ID NOS. 10 to 14, 23, 35 to 40 or 74 or
sequences complementary thereto; and step b) indicates the absence
of a mutation.
[0038] Conveniently, the method further comprises the step of prior
to step a), amplifying the number of copies of the fragment of the
EGFR gene using thermal cycling nucleic acid amplification,
preferably PCR.
[0039] Preferably step b) comprises carrying out DNA polymerisation
using the polynucleotide as a first primer and detecting the
extension product of polymerisation.
[0040] Advantageously, the method step b) comprises the step of
mixing the nucleic acid sample and the polynucleotide with a second
primer which corresponds to a region of the fragment of the EGFR
sequence downstream of the region to which the polynucleotide is
complementary and carrying out PCR on the mixture.
[0041] Conveniently, the second primer comprises: SEQ. ID NO. 15
and the polynucleotide comprises at least four or five of the final
six nucleotides of SEQ. ID NOS. 1 to 5, 10, 11 or 21; SEQ. ID NO.
16 and the polynucleotide comprises at least the four or five final
six nucleotides of SEQ. ID NOS. 6 or 12; SEQ. ID NO. 17 and the
polynucleotide comprises at least the four or five final six
nucleotides of SEQ. ID NOS. 7 or 13; or SEQ. ID NO. 18 and the
polynucleotide comprises at least the four or five final six
nucleotides of SEQ. ID NOS. 8, 9 or 14; SEQ. ID NO. 24 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS: 22 or 23; SEQ. ID NO. 41 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 25 to 28, 33, 35 or 36; SEQ. ID NO. 42
and the polynucleotide comprises at least four or five of the final
six nucleotides of SEQ. ID NOS. 29 or 37; SEQ. ID NO. 43 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 30 to 38; SEQ. ID NO. 44 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 31, 32 or 39; or SEQ. ID NO. 45 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NOS. 34 or 40; SEQ. ID NO. 76 and the
polynucleotide comprises at least four or five of the final six
nucleotides of SEQ. ID NO. 74 or 75.
[0042] Preferably step a) comprises the step of mixing the nucleic
acid sample with a pair of a mutation specific polynucleotide and a
wild-type specific polynucleotide, the pair being selected from at
least four or five of the final six sequences of: SEQ. ID NO: 1 and
SEQ. ID NO: 10; SEQ. ID NO: 2 and SEQ. ID NO: 10; SEQ. ID NO: 3 and
SEQ. ID NO: 10; SEQ. ID NO: 4 and SEQ. ID NO: 11; SEQ. ID NO: 5 and
SEQ. ID NO: 11; SEQ. ID NO: 6 and SEQ. ID NO: 12; SEQ. ID NO: 7 and
SEQ. ID NO: 13; SEQ. ID NO: 8 and SEQ. ID NO: 14; SEQ. ID NO: 9 and
SEQ. ID NO: 14; SEQ. ID NO: 21 and SEQ. ID NO: 10; SEQ. ID NO: 22
and SEQ. ID NO: 23; SEQ. ID NO: 25 and SEQ. ID NO: 35; SEQ. ID NO:
26 and SEQ. ID NO: 35; SEQ. ID NO: 27 and SEQ. ID NO: 36; SEQ. ID
NO: 28 and SEQ. ID NO: 36; SEQ. ID NO: 29 and SEQ. ID NO: 37; SEQ.
ID NO: 30 and SEQ. ID NO: 38; SEQ. ID NO: 31 and SEQ. ID NO: 39;
SEQ. ID NO: 32 and SEQ. ID NO: 39; SEQ. ID NO: 33 and SEQ. ID NO:
35; SEQ. ID NO: 34 and SEQ. ID NO: 40; or SEQ. ID NO. 74 and SEQ.
ID NO. 75.
[0043] Advantageously the nucleic acid sample comprises wild-type
sequences and mutated sequences and further comprising step c)
wherein the number of amplification cycles required to amplify the
wild-type sequences to a predetermined quantity is compared with
the number of amplification cycles required to amplify the mutated
sequences to the predetermined quantity thereby providing an
indication of the ratio of the wild type sequences to mutated
sequences in the sample.
[0044] Conveniently the nucleic acid sample comprises a portion of
tumourous tissue and a portion of non-tumourous tissue and wherein
step c) further comprises the step determining the ratio of
tumourous tissue to non-tumourous tissue in the sample.
[0045] Preferably the method further comprises the step of, prior
to step a), enriching the nucleic acid sample to increase the ratio
of tumourous tissue to non-tumourous tissue in the sample.
[0046] Preferably step b) comprises detecting if amplification of
at least a portion of the EGFR gene occurs.
[0047] Advantageously the polynucleotide comprises a quencher group
and a fluorophore group and step b) comprises exposing the mixture
to light of a wavelength to which the fluorophore is responsive in
the absence of the quencher group and detecting light at the
wavelength emitted by the fluorophore group in the absence of the
quencher group.
[0048] Where reference is made in the specification to "at least
four or five of the final six sequences" of a reference sequence,
this means that, of the six nucleotides in the reference sequence,
either one or two of the nucleotides may be missing or replaced
with a different nucleotide. Of course, in some embodiments, the
sequence comprises all six of the nucleotides of the reference
sequence.
[0049] In preferred embodiments of the present invention, reference
to "EGFR" is to the human EGFR gene available as accession number
NC.sub.--000007 in Homo sapiens chromosome 7, region
55054219.55242525 as on 30 Mar. 2006. In some other embodiments,
reference is to the sequence as on 4 Mar. 2005. Both sequences are
incorporated herein by reference. In some embodiments, the EGFR
gene is not identical to these sequences but is at least 90%
identical to either or both of the sequences.
[0050] A reference in this specification is made to a percentage of
a polynucleotide compared with a reference polynucleotide, this can
be determined by algorithms known in the art.
[0051] For example the percentage identity between two sequences
can be determined using the BLASTP algorithm version 2.2.2
(Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer,
Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman
(1997), "Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs", Nucleic Acids Res. 25:3389-3402) using
default parameters.
BRIEF DESCRIPTION OF DRAWINGS
[0052] In order that the present invention may be more readily
understood and so that further features thereof may be appreciated,
embodiments of the invention will now be described, by way of
example, with reference to the accompanying figures in which:
[0053] FIG. 1 is a graph showing the results of PCR amplification
carried out on a sample in the presence of a primer of the present
invention and control primers;
[0054] FIG. 2 is a graph showing the results of PCR amplification
carried out on a sample in the presence of a primer specific for
del S752-1759 (SEQ. ID NO. 5) and a corresponding wild-type
specific primer;
[0055] FIG. 3 is a graph showing the results of PCR amplification
carried out on a sample in the presence of a primer specific for
the L858R mutation (SEQ. ID NO. 6) and a corresponding wild-type
specific primer;
[0056] FIG. 4 is a graph (lower) showing the results of PCR
amplification carried out on a sample in the presence of a primer
specific to a mutation and a corresponding wild-type specific
primer, and a graph (upper) showing the results of sequencing of
the sample;
[0057] FIG. 5 is a graph (lower) showing the results of PCR
amplification carried out on a sample in the presence of a primer
specific to a mutation and a corresponding wild-type specific
primer, and a graph (upper) showing the results of sequencing of
the sample; and
[0058] FIG. 6 is a graph showing the results of PCR amplification
carried out on a set of samples, in the presence of a primer
specific to a mutation; and
[0059] FIG. 7 is a graph showing the results of PCR amplification,
using control primers, of the samples analysed in the results shown
in FIG. 6.
DETAILED DESCRIPTION
[0060] In general terms, the present invention provides a
diagnostic method of the detection of EGFR mutations in cancer,
which method comprises contacting a test sample of nucleic acid
with a diagnostic primer for a EGFR mutation in the presence of
appropriate nucleotide triphosphates and an agent for
polymerisation, such that the diagnostic primer is efficiently
extended only when a EGFR mutation is present in the sample; and
detecting the presence or absence of a EGFR mutation by reference
to the presence or absence of a diagnostic primer extension
product.
[0061] There are disclosed herein, primers (SEQ. ID NOS: 1 to 9,
21, 22 and 75) which can be used in the method of the
invention.
[0062] Each of the diagnostic primers detects the presence or
absence of one of the following EGFR mutations: [0063] 1) del
E746-A750 (two different mutations exist) [0064] 2) del L747-T751
insS [0065] 3) del L747-P753 insS [0066] 4) del S752-1759 [0067] 5)
Exon 21 L858R, [0068] 6) Exon 21 L861Q, [0069] 7) Exon 18 G719C
[0070] 8) Exon 18 G719S [0071] 9) Exon 20 T790M
[0072] Mutations 1) to 8) confer sensitivity of an individual to
gefinitib whereas mutation 9) confers resistance to gefinitib.
[0073] In some embodiments, diagnostic primers specific for the
corresponding mutations on the opposite DNA strand are used (SEQ.
ID NOS: 25 to 34).
[0074] The test sample of nucleic acid is conveniently a sample of
blood, faeces, sputum, colonic lavage, bronchial lavage or other
body fluid, or tissue obtained from an individual. The individual
is conveniently human, preferably Homo sapiens. It will be
appreciated that the test sample may equally be a nucleic acid
sequence corresponding to the sequence in the test sample. That is
to say that all or a part of the region in the sample nucleic acid
may firstly be amplified using any convenient technique such as PCR
or whole genome amplification (WGA) before use in the method of the
invention.
[0075] Any convenient enzyme for polymerisation may be used
provided that it does not affect the ability of the DNA polymerase
to discriminate between normal and mutant template sequences to any
significant extent. Examples of convenient enzymes include
thermostable enzymes which have no significant 3'-5' exonuclease
activity, for example Taq DNA polymerase, particularly "Ampli Taq
Gold".TM. DNA polymerase (PE Applied Biosystems), Stoffel fragment,
or other appropriately N-terminal deleted modifications of Taq or
Tth (Thermus thermophilus) DNA polymerases.
[0076] There are disclosed herein primers for the above EGFR point
mutations which have been shown to detect the specific mutations
reliably and robustly. Therefore in a further aspect of the
invention we provide diagnostic primers comprising SEQ. ID NOS: 1
to 9, 21, 22, 25 to 34 or 75 and derivatives thereof wherein 6-8 of
the nucleotides at the 3' end are identical to the sequences and
wherein up to 10, such as up to 8, 6, 4, 2, 1, of the remaining
nucleotides are optionally varied without significantly affecting
the properties of the diagnostic primer. Conveniently, the sequence
of the diagnostic primer is exactly as shown in any one of SEQ. ID
NOS: 1 to 9, 21, 22, 25 to 34 or 75.
[0077] It is to be appreciated that alternative versions of the
above described diagnostic methods are configured so that extension
of the diagnostic primer indicates the absence of the EGFR
mutation. For example, in the embodiments the primers comprise 6-8
nucleotides from the 3' end of any one of SEQ. ID NOS. 10 to 14, 23
or 74. In some embodiments, primers specific for the wild type
sequences present the corresponding section of the complementary
DNA strand are used (SEQ. ID NOS. 35 to 40).
[0078] In many embodiments, it is convenient to use a diagnostic
primer of the invention with a further amplification primer in one
or more cycles of PCR amplification. A convenient example of this
aspect is set out European patent number EP-A-1-0332435. The
further amplification primer is either a forward or a reverse
common primer. Examples of such common primers are SEQ. ID NOS: 15
to 18, 24 and 76 and, to be used in conjunction with the primers
for the complementary strand, SEQ. ID NOS: 41 to 45.
[0079] Any convenient control primer pair may be used. Control
primers from an unrelated region of the genome, namely part of the
human albumen gene, are used herein.
[0080] The diagnostic methods of the invention as outlined above
are conveniently effected in one or more reaction vessels in some
embodiments. Where more than one diagnostic mutation is to be
assayed, the diagnostic primer (and corresponding amplification
primer) are provided in individual tubes i.e. one tube per mutation
in certain embodiments. Alternatively, the reactions are
multiplexed, that is to say that all the diagnostic primers and
amplification primers are in one tube (see EP-A-1-0332435) in other
embodiments.
[0081] A variety of methods may be used to detect the presence or
absence of diagnostic primer extension products and/or
amplification products. These will be apparent to the person
skilled in the art of nucleic aid detection procedures. Preferred
methods avoid the need for radio-labelled reagents. Particular
detection methods include "TaqMan".TM. product detection, for
example as described in U.S. Pat. No. 5,487,972 & U.S. Pat. No.
5,210,015; Scorpions (WO-A-99/066071), which has particular
benefits because specific diagnostic primers may be linked to a
specific detector fluorophore, allowing the detection of multiple
mutation targets from the same region of the genome.
[0082] Conveniently, real-time detection is employed which allows
the quantitation of the mutation(s) within the sample. More
specifically, the number of cycles required to amplify a DNA sample
to a predetermined level using primers specific for the wild-type
sequences is compared with the number of cycles required using
primers specific for a mutant sequence. By the use of this
comparison and by reference to control amplifications containing
standardised proportions of mutant target, the ratio of the amount
of DNA in the sample containing the mutation relative to the amount
of wild type DNA is quantified. Of course this only relates to the
sample as presented and does not compensate for low tumour
representation within the sample. For this reason, it is preferred
to combine this quantitative allele specific approach with a method
to estimate the level of tumour within the sample. Alternatively,
the quantitative allele specific approach is combined with a method
to enrich the sample by specifically excising the tumour material
from the overall sample.
[0083] In some embodiments, one or more of the diagnostic primers
of the invention is conveniently packaged with instructions for use
in the method of the invention and appropriate packaging and sold
as a kit. The kits conveniently include one or more of the
following: appropriate nucleotide triphosphates, for example dATP,
dCTP, dGTP, dTTP, a suitable polymerase as previously described,
and a buffer solution.
[0084] The invention will now be illustrated but not limited by
reference to the following Examples.
Materials And Methods
Construction of Synthetic Templates
[0085] In the absence of validated and uniform genomic material
that carries each of the mutations, it was necessary to construct
synthetic templates. For EGFR deletion tests this was done using
long synthetic overlapping oligonucleotides, each comprising
approximately half the desired amplicon. The sequences of these
long oligonucleotides are shown in Table 1.
TABLE-US-00001 TABLE 1 SEQ. ID Name Sequence NO. EGFRDEL15
AAAATTCCCGTCGCTATCAAAACATCTCCGAAAGCCAACAAGG 47 CONFOR
AAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCAT GGCTCTGAACCTCA
EGFRDEL15 AACATTTAGGATGTGGAGATGAGCAGGGTCTAGAGCAGAGCA 48 CONREV
GCTGCCAGACATGAGAAAAGGTGGGCCTGAGGTTCAGAGCC ATGGACC EGFRDEL18
CGCTATCAAGGAATCGAAAGCCAACAAGGAAATCCTCGATGT 49 CONFOR
GAGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCT CAGGCCCACCTTTTCT
EGFRDEL18 AGAAAGACATAGAAAGTGAACATTTAGGATGTGGAGATGAGC 50 CONREV
AGGGTCTAGAGCAGAGCAGCTGCCAGACATGAGAAAAGGTG GGCCTGAGGT EGFRDEL24
GCTATCAAGGAATTAAGAGAAGCAACACTCGATGTGAGTTTCT 51 CONFOR
GCTTTGCTGTGTGGGGGTCCATGGCTCTGAACCTCAGGCCCA CCTTTTCTCATGTCT
EGFRDEL24 AGAAAGACATAGAAAGTGAACATTTAGGATGTGGAGATGAGC 52 CONREV
AGGGTCTAGAGCAGAGCAGCTGCCAGACATGAGAAAAGGTG GGC EGFRDEL12
AATTCCCGTCGCTATCAAGGAACCATCTCCGAAAGCCAACAA 53 CONFOR
GGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTC CATGGCTCTGAACCTC
EGFRDEL12 GTGAACATTTAGGATGTGGAGATGAGCAGGGTCTAGAGCAGA 54 CONREV
GCAGCTGCCAGACATGAGAAAAGGTGGGCCTGAGGTTCAGA GCCATGGACC
[0086] In order to construct the targets, the two overlapping
1/2-amplicons were mixed in equimolar concentrations in the
presence of polymerase, buffer and dNTPs. The primer mix was then
incubated for 25 cycles of 1 minute each with an increment of
1.degree. C. per cycle from 50.degree. C. to 75.degree. C.
Synthetic templates were subsequently diluted 1 in 1 million into
genomic DNA for use as controls in PCR reactions.
[0087] For SNP tests synthetic cassettes were produced using PCR to
incorporate required mutations into two half cassettes. Half
cassettes were then mixed in equimolar concentrations and used as a
template for the construction of full length cassettes containing
the required SNP target mutation. Specific primer sequences for the
construction of cassettes are shown in Table 2.
TABLE-US-00002 TABLE 2 SEQ. ID Name Sequence NO. EGFR Ex18 AU
CGCCATGCACAACTTCCCTAC 55 EGFR Ex18 AL TCCAGAATTTAATGATGCTGCGTCT 56
EGFR Ex18 ML GAACGCACCGGAGCACA 57 EGFR Ex18 BU
TTGGTGACATGTTGGTACATCCATC 58 EGFR Ex18 MU TTCAAAAAGATCAAAGTGCTGTGC
59 EGFR Ex18 BL CGTTAACTGGCAATTGTGAGATGGT 60 EGFR Ex21 AU
AGTCCAGTAAGTTCAAGCCCAGGTC 61 EGFR Ex21 AL GTTCCTTAGGTGTCCTTGACAGCAG
62 EGFR Ex21 CCAGCAGTTTGGCCCGC 63 L858R ML EGFR Ex21 BU
CAGAGATTTCAATTGCAGCGAGATT 64 EGFR Ex21 AGATCACAGATTTTGGGCGGG 65
L858R MU EGFR Ex21 BL TAGGTTTCTGAGCACCCTCTGTGTC 66 EGFR Ex21
TCTCTTCCGCACCCAGCTGT 67 L861Q ML EGFR Ex21 TTGGGCTGGCCAAACAGC 68
L861Q MU EGFR Ex 20 ACCGTGCAGCTCATCATGC 46 T790M MU EGFR Ex 20
GCTGTGAAATACCTGGCTTGTTGTT 69 T790M BL EGFR Ex 20
GAAGGGCATGAGCTGCATG 70 T790M BU EGFR Ex 20
CCAGGCAGCCTTTAGTCACTGTAGA 71 T790M ML EGFR Ex 20
GGCCTCTCTGTCATGGGGAAT 72 T790M AU EGFR Ex 20
ACCTGCTCCACTCCACCACTATCAC 73 T790M AL
Variant Specific PCR
[0088] Specific primer sequences for each ARMS reaction and control
are shown in Tables 3 and 4.
[0089] Reactions were performed in 0.2 ml vessels (single tubes,
strips or plates). Reactions (25 .mu.l) typically contained: [0090]
250 nM diagnostic primer [0091] 250 nM reverse primer [0092] 250 nM
each of control primers (where used) [0093] 200 .mu.M each dNTP
[0094] 10 mM Tris-HCl (pH 8.3) [0095] 50 mM KCl [0096] 2.5-4 mM
MgCl2
[0097] Some reactions performed better in the presence of Qiagen
Multiplex Reaction buffer that contains a proprietary mixture of
Ammonium Sulphate, MgCl.sub.2, and volume excluders such as Poly
(ethylene glycol) MW 8000, and/or Dextran (MW 50,000); see
US-A-2004-0115712. For Real Time Quantitative PCR, YO-PRO-1
(Molecular Probes) was included at a final concentration of 1
.mu.M. This dye binds double-stranded DNA and fluoresces with high
efficiency, enabling a general detection of PCR product in a
reaction. Alternatively, and more preferred, we used Scorpions (a
molecule that combines a primer and fluorogenic probe), which
offers specific detection of target amplicons.
TABLE-US-00003 TABLE 3 Allele Specific primers (1) WILD TYPE
PRIMERS MUTANT PRIMERS COMMON PRIMERS SEQ SEQ SEQ ID ID ID Test
NAME NO. SEQUENCE NAME NO. SEQUENCE NAME NO. SEQUENCE del L747-
EGFR 10 CGT CGC TAT CAA EGFR DEL 1 CCC GTC GCT ATC EGFR 15 GAG ATG
AGC T751 insS WTA GGA ATT AAG AGA 12 AAG GA CCA EX19 AGG GTC TAG
AGC REV AGC AGA G del E746- EGFR 10 CGT CGC TAT CAA EGFR DEL 2 TTA
AAA TTC CCG EGFR 15 GAG ATG AGC A750 WTA GGA ATT AAG AGA 15 TCG CTA
TCA AAA C EX19 AGG GTC TAG AGC REV AGC AGA G del E746- EGFR 19 FAM
CCGCGG EGFR DEL 19 CAL RED CCGCGG EGFR 15 GAG ATG AGC A750 WTA and
GATTTCCTTGTTGGCTTTCG 15 and GATTTCCTTGTTGGCTT EX19 AGG GTC TAG
Scorpions Scorpion 10 CCGCGG BHQ1 HEG Scorpions 3 TCG CCGCGG BHQ2
REV AGC AGA G CGTCGCTATCAAGGAATTAA HEG GAGAAGC TTAAAATTCCCGTCGCT
ATCAAAAC del L747- EGFR 11 GAG AG CA CAT EGFR DEL 4 CCG TCG CTA TCA
EGFR 15 GAG ATG AGC P753 insS WT CTC CGA AAG CC 18 AGG AAT CGA EX19
AGG GTC TAG REV AGC AGA G del S752- EGFR 11 GAG AG CA CAT EGFR DEL
5 CAA GGA ATT AAG EGFR 15 GAG ATG AGC 1759 WT CTC CGA AAG CC 24 AGA
AGC AAC ACT EX19 AGG GTC TAG CGA REV AGC AGA G Exon 21 EGFR 12 CAT
GTC AAG ATC EGFR 6 CAT GTC AAG ATC EGFR 16 GCT GAC CTA L858R EX21
ACA GAT TTT GGC EX21 ACA GAT TTT GGG EX21 AAG CCA CCT L858R CT
L858R AG L858R CCT TAC T WT MUT COM Exon 21 EGFR 20 FAM CCGCGG EGFR
20 CAL RED CCGCGG EGFR 16 GCT GAC CTA L858R EX21 and
ATTCTTTCTCTTCCGCACCC EX21 and ATTCTTTCTCTTCCGCA EX21 AAG CCA CCT
Scorpions L858R 12 CCGCGG BHQ1 HEG L858R 6 CCC CCGCGG BHQ2 L858R
CCT TAC T WT CATGTCAAGATCACAGATTTT MUT HEG COM Scorpion GGCCT
Scorpions CATGTCAAGATCACAGA TTTTGGGAG Exon 21 EGFR 13 TTT CTC TTC
CGC EGFR 7 TTT CTC TTC CGC EGFR 17 CTG TTT CAG L861Q EX21 ACC CAC
CA EX21 ACC CAG AT EX21 GGC ATG AAC L861Q L861Q L861Q TAC TTG G WT
MUT COM Exon 18 EGFR 14 CTG AAT TCA AAA EGFR 8 CTG AAT TCA AAA EGFR
18 CCT TTG GTC G719C EX18 AGA TCA AAG TGC EX18 AGA TCA AAG TGC EX18
TGT GAA TTG G719C TCG G719C CGT COM GTC TCA C WT MUT
TABLE-US-00004 TABLE 4 Allele Specific primers (2) WILD TYPE
PRIMERS MUTANT PRIMERS COMMON PRIMERS SEQ SEQ SEQ ID ID ID Test
NAME NO. SEQUENCE NAME NO. SEQUENCE NAME NO. SEQUENCE Exon EGFR 14
CTG AAT TCA AAA EGFR 9 CTG AAT TCA AAA EGFR 18 CCT TTG GTC 18 EX18
AGA TCA AAG TGC EX18 AGA TCA AAG TGC EX18 TGT GAA TTG G719S G719S
TCG G719S CGA COM GTC TCA C WT MUT EGFR EGFR 10 CGT CGC TAT CAA
EGFR 21 TTAAAATTCCCGTCGC EGFR 15 GAG ATG AGC del WTA GGA ATT AAG
AGA del TATCAAGACA EX19 AGG GTC TAG 15 2 AGC 15 2 REV AGC AGA G For
EGFR EGFR 23 ACCTCCACCGTGCAGCTCAT EGFR 22 ACCTCCACCGTGCAGC EGFR 24
ATGGCAAACTCTT T790M T790M AAC T790M TCATCCT T790M GCTATCCCAGGA FC
FT Reverse EGFR T790M 74 TCCACCGTGCAGCTCATCTC T790M 75
TCCACCGTGCAGCTCA EGFR 76 TTGTCTTTGTGTT T790M A B TCTT T790M
CCCGGACAT Reverse II EGFR T790M 74 TCCACCGTGCAGCTCATCTC T790M 75
TCCACCGTGCAGCTCA EGRR 76 FAM- T790M A B TCTT T790M and CCGCGGCTCATG
Reverse 77 CCCTTCGGCTCC II GCGG-DABCYL- Scorpions HEG-
TTGTCTTTGTGTT CCCGGACAT-3'
[0098] PCRs were performed in Real Time PCR cyclers (Mx4000 and
Mx3000P from Stratagene), using standard conditions: [0099]
95.degree. C. for 10-15 minutes to activate the hot-start enzyme,
followed by up to 50 cycles of: [0100] 95.degree. C., 60 s [0101]
65.degree. C., 60 s--Deletion tests
[0102] For SNP tests, the anneal/extend step was modified to
60.degree. C., for 60 s.
Measurement of Extracted DNA
[0103] The amount of DNA extracted from each tumour sample was
measured by real time amplification of a control locus. Standard
curves for this reaction were generated using known concentrations
of high quality DNA extracted from cell lines (ECACC, Wiltshire,
UK).
Sequencing of Controls and Positive Samples
[0104] Sequencing was performed on amplicons generated from exon
specific primers, by standard cycle sequencing, using big dye
terminators. Reactions were separated using an ABI 3100 capillary
sequencing instrument, according to the manufacturer's
instructions.
Results and Discussion
ARMS Tests Development and Validation
[0105] ARMS tests specific for each of the deletions and SNPs were
tested against: [0106] 1. "normal" genomic DNA (gDNA) [0107] 2.
mismatched synthetic targets [0108] 3. synthetic target diluted in
buffer [0109] 4. synthetic target diluted in gDNA (to mimic the
tumour situation) [0110] 5. No template (water instead of
template)
[0111] Reactions included the fluorescent DNA binding dye YO-PRO-1
and were monitored in real time. In such reactions, the point at
which amplification becomes visible above baseline (known as the
Ct) is an indicator of the quantity of target.
[0112] Primers which are specific for the same mutations but on the
complementary DNA strand have also been developed and these are
shown in Tables 5 and 6.
TABLE-US-00005 TABLE 5 Allele Specific primers for Reverse Strand
(I) WILD TYPE PRIMERS MUTANT PRIMERS COMMON PRIMERS SEQ SEQ SEQ ID
ID ID Test NAME NO. SEQUENCE NAME NO. SEQUENCE NAME NO. SEQUENCE
del EGFR 35 GGCTTTCGGAGATGTTGCTTCTC EGFR 25 CTTGTTGGCTTTCGGAGAT
EGFR 41 CTGGTAACATCCAC L747- WT del GGT Ex 19 CCAGATCACTG T751 Rev
12 For insS del rev 15 + 12 del EGFR 35 GGCTTTCGGAGATGTTGCTTCTC
EGFR 26 TTGTTGGCTTTCGGAGATG EGFR 41 CTGGTAACATCCAC E746- WT del
TTTTG Ex 19 CCAGATCACTG A750 Rev 151 For del rev 15 + 12 del EGFR
36 GCTTCTCTTAATTCCTTGATAGCG EGFR 27 GGATTTCCTTGTTGGCTTTC EGFR 41
CTGGTAACATCCAC L747- WT ACG del GAT Ex 19 CCAGATCACTG P753 Rev 18
For insS 18 + rev 24 del EGFR 36 GCTTCTCTTAATTCCTTGATAGCG EGFR 28
CAAAGCAGAAACTCACATC EGFR 41 CTGGTAACATCCAC S752- WT ACG del GAGTG
Ex 19 CCAGATCACTG 1759 Rev 24 For 18 + rev 24 Exon EGFR 37
CTCTTCCGCACCCAGCAGTTTGG EGFR 29 CTCTTCCGCACCCAGCAGT EGFR 42
TTCCCATGATGATCT 21 L858R TCA L858R TTGGCAC L858R GTCCCTCACAGCA
L858R RT RG Forward Exon EGFR 38 ATCACAGATTTTGGGCTGGCCAA EGFR 30
ATCACAGATTTTGGGCTGG EGFR 43 GAGCTCACCCAGAA 21 L861Q TCA L861Q
CCAATAA L861Q TGTCTGGAGAGCAT L861Q FA FT Reverse Exon EGFR 39
TATACACCGTGCCGAACGCACCG EGFR 31 TATACACCGTGCCGAACGC G719 44
GGGCTGAGGTGAC 18 G719 GAGAC G719C ACCGGAACA Forward CCTTGTCTCTGTGTT
G719C R RT WT Exon EGFR 39 TATACACCGTGCCGAACGCACCG EGFR 32
TATACACCGTGCCGAACGC G719 44 GGGCTGAGGTGAC 18 G719 GAGAC G719S
ACCGGATCT Forward CCTTGTCTCTGTGTT G719S R RA WT EGFR EGFR 35
GGCTTTCGGAGATGTTGCTTCTC EGFR 33 TTGTTGGCTTTCGGAGATG EGFR 41
CTGGTAACATCCAC del WT del TCT Ex 19 CCAGATCACTG 15 2 Rev 15 2 For
Reverse del rev 15 + 12
TABLE-US-00006 TABLE 6 Allele Specific primers for Reverse Strand
(II) WILD TYPE PRIMERS MUTANT PRIMERS COMMON PRIMERS SEQ SEQ SEQ ID
ID ID Test NAME NO SEQUENCE NAME NO. SEQUENCE NAME NO. SEQUENCE
EGFR EGFR 40 AGCCGAAGGGCATGAGCTTCA EGFR 34 AGCCGAAGGGCATGAGCTG EGFR
45 GCACAGCTTTTCCT T790M T790M T790M AG T790M CCATGAGTACG Reverse RT
RT Forward WT
Example 1
[0113] The mutation specific primer EGFR Del 12 was tested against
matched synthetic target and three mismatched mutant synthetic
targets (del 15 (1), del 18 and del 24), as well as normal DNA and
a water control. The results are shown in FIG. 1.
[0114] It is clear that in this reaction the primer shows absolute
specificity for its own target and does not detect wild type
sequences nor any of the other deletion mutations found in the same
genomic region.
Example 2
Use of ARMS Tests on Tumour Samples
[0115] Forty-two DNA samples from Non-Small Cell Lung Cancers and
cell lines were analysed using each of the mutation specific
primers SEQ. ID NOS: 1 to 9. Tumour DNA had been extracted from
paraffin embedded tissue on slides.
[0116] A number of samples were found to be positive using this
method: nine for the L858R SNP and one for the 15 base pair
deletion del E746-A750.
[0117] Confirmatory sequencing was performed for the region
surrounding the putative mutations.
[0118] FIG. 2 shows a typical negative result for a batch of 8
samples tested with the del S752-1759 specific primers. It is clear
that the wild type specific reactions perform efficiently while the
mutation specific primers (SEQ. ID NO: 5) do not amplify indicating
the absence of this mutation in these samples.
[0119] FIG. 3 shows the L858R analysis for 8 positive samples. In
each case, the reaction using the mutation primers (SEQ. ID NO: 6)
is positive as is the wild type reaction. The fact that the mutant
reaction appears several cycles later than the corresponding wild
type reaction indicates that the mutation is not the majority
sequence in the samples.
[0120] FIG. 4 shows more detailed analysis of the allele specific
PCR analysis, combined with sequencing of the same exon from this
sample. In this sample, the sequencing approach would have failed
to detect the mutation that was clearly identified by the ARMS
approach (no peak is visible at the circled position).
[0121] FIG. 5 shows more detailed analysis of the allele specific
PCR analysis, combined with sequencing of the same exon from this
sample. In this sample, the sequencing approach could have detected
the mutation that was clearly identified by the ARMS approach (a
very weak peak is visible at the circled position). The
differential (.DELTA.Ct) between the wild type and mutant specific
reactions was smaller in this sample, indicating that the mutation
was relatively more prevalent than in the sample whose analysis is
shown in FIG. 4.
Example 3
[0122] FIG. 6 shows the results of PCR amplification of samples
containing DNA with, and without the T790M mutation. Five DNA
samples were provided, each comprising 0%, 1%, 10%, 50% or 100% DNA
with the T790M mutation. The samples were amplified using the
mutant primer of SEQ. ID NO. 75 and the reverse Scorpion primer of
SEQ. ID NOS. 76 and 77. The results show that the mutant primer was
specific for DNA having the T780M mutation because the sample
without any DNA having the mutation showed no amplification.
Furthermore, detection was shown to be quantitative in that the Ct
(threshold cycle) was related directly to the relative amount of
matched target, even in the presence of excess unmatched
target.
[0123] FIG. 7 shows the results of a control assay carried out on
the samples which were tested and reported in FIG. 6. PCR
amplification was carried out using control primers. FIG. 7
confirms that each sample had the same total amount of target since
the threshold cycles for each sample were the same in this control
amplification.
Sequence CWU 1
1
77120DNAHomo sapiens 1cccgtcgcta tcaaggacca 20225DNAHomo sapiens
2ttaaaattcc cgtcgctatc aaaac 25325DNAHomo sapiens 3ttaaaattcc
cgtcgctatc aaaac 25421DNAHomo sapiens 4ccgtcgctat caaggaatcg a
21527DNAHomo sapiens 5caaggaatta agagaagcaa cactcga 27626DNAHomo
sapiens 6catgtcaaga tcacagattt tgggag 26720DNAHomo sapiens
7tttctcttcc gcacccagat 20827DNAHomo sapiens 8ctgaattcaa aaagatcaaa
gtgccgt 27927DNAHomo sapiens 9ctgaattcaa aaagatcaaa gtgccga
271027DNAHomo sapiens 10cgtcgctatc aaggaattaa gagaagc 271121DNAHomo
sapiens 11gagagcacat ctccgaaagc c 211226DNAHomo sapiens
12catgtcaaga tcacagattt tggcct 261320DNAHomo sapiens 13tttctcttcc
gcacccacca 201427DNAHomo sapiens 14ctgaattcaa aaagatcaaa gtgctcg
271525DNAHomo sapiens 15gagatgagca gggtctagag cagag 251625DNAHomo
sapiens 16gctgacctaa agccacctcc ttact 251725DNAHomo sapiens
17ctgtttcagg gcatgaacta cttgg 251825DNAHomo sapiens 18cctttggtct
gtgaattggt ctcac 251932DNAArtificial SequenceScorpions primer tail
19ccgcgggatt tccttgttgg ctttcgccgc gg 322032DNAArtificial
SequenceScorpions primer tail 20ccgcggattc tttctcttcc gcacccccgc gg
322126DNAHomo sapiens 21ttaaaattcc cgtcgctatc aagaca 262223DNAHomo
sapiens 22acctccaccg tgcagctcat cct 232323DNAHomo sapiens
23acctccaccg tgcagctcat aac 232425DNAHomo sapiens 24atggcaaact
cttgctatcc cagga 252522DNAHomo sapiens 25cttgttggct ttcggagatg gt
222624DNAHomo sapiens 26ttgttggctt tcggagatgt tttg 242723DNAHomo
sapiens 27ggatttcctt gttggctttc gat 232824DNAHomo sapiens
28caaagcagaa actcacatcg agtg 242926DNAHomo sapiens 29ctcttccgca
cccagcagtt tggcac 263026DNAHomo sapiens 30atcacagatt ttgggctggc
caataa 263128DNAHomo sapiens 31tatacaccgt gccgaacgca ccggaaca
283228DNAHomo sapiens 32tatacaccgt gccgaacgca ccggatct
283322DNAHomo sapiens 33ttgttggctt tcggagatgt ct 223421DNAHomo
sapiens 34agccgaaggg catgagctga g 213523DNAHomo sapiens
35ggctttcgga gatgttgctt ctc 233627DNAHomo sapiens 36gcttctctta
attccttgat agcgacg 273726DNAHomo sapiens 37ctcttccgca cccagcagtt
tggtca 263826DNAHomo sapiens 38atcacagatt ttgggctggc caatca
263928DNAHomo sapiens 39tatacaccgt gccgaacgca ccggagac
284021DNAHomo sapiens 40agccgaaggg catgagcttc a 214125DNAHomo
sapiens 41ctggtaacat ccacccagat cactg 254228DNAHomo sapiens
42ttcccatgat gatctgtccc tcacagca 284328DNAHomo sapiens 43gagctcaccc
agaatgtctg gagagcat 284428DNAHomo sapiens 44gggctgaggt gacccttgtc
tctgtgtt 284525DNAHomo sapiens 45gcacagcttt tcctccatga gtacg
254619DNAArtificial Sequenceprimer 46accgtgcagc tcatcatgc
1947100DNAArtificial SequencePrimer 47aaaattcccg tcgctatcaa
aacatctccg aaagccaaca aggaaatcct cgatgtgagt 60ttctgctttg ctgtgtgggg
gtccatggct ctgaacctca 1004890DNAArtificial SequencePrimer
48aacatttagg atgtggagat gagcagggtc tagagcagag cagctgccag acatgagaaa
60aggtgggcct gaggttcaga gccatggacc 9049100DNAArtificial
SequencePrimer 49cgctatcaag gaatcgaaag ccaacaagga aatcctcgat
gtgagtttct gctttgctgt 60gtgggggtcc atggctctga acctcaggcc caccttttct
1005093DNAArtificial SequencePrimer 50agaaagacat agaaagtgaa
catttaggat gtggagatga gcagggtcta gagcagagca 60gctgccagac atgagaaaag
gtgggcctga ggt 9351100DNAArtificial SequencePrimer 51gctatcaagg
aattaagaga agcaacactc gatgtgagtt tctgctttgc tgtgtggggg 60tccatggctc
tgaacctcag gcccaccttt tctcatgtct 1005286DNAArtificial
SequencePrimer 52agaaagacat agaaagtgaa catttaggat gtggagatga
gcagggtcta gagcagagca 60gctgccagac atgagaaaag gtgggc
8653100DNAArtificial SequencePrimer 53aattcccgtc gctatcaagg
aaccatctcc gaaagccaac aaggaaatcc tcgatgtgag 60tttctgcttt gctgtgtggg
ggtccatggc tctgaacctc 1005493DNAArtificial SequencePrimer
54gtgaacattt aggatgtgga gatgagcagg gtctagagca gagcagctgc cagacatgag
60aaaaggtggg cctgaggttc agagccatgg acc 935521DNAArtificial
SequencePrimer 55cgccatgcac aacttcccta c 215625DNAArtificial
SequencePrimer 56tccagaattt aatgatgctg cgtct 255717DNAArtificial
SequencePrimer 57gaacgcaccg gagcaca 175825DNAArtificial
SequencePrimer 58ttggtgacat gttggtacat ccatc 255924DNAArtificial
SequencePrimer 59ttcaaaaaga tcaaagtgct gtgc 246025DNAArtificial
SequencePrimer 60cgttaactgg caattgtgag atggt 256125DNAArtificial
SequencePrimer 61agtccagtaa gttcaagccc aggtc 256225DNAArtificial
SequencePrimer 62gttccttagg tgtccttgac agcag 256317DNAArtificial
SequencePrimer 63ccagcagttt ggcccgc 176425DNAArtificial
SequencePrimer 64cagagatttc aattgcagcg agatt 256521DNAArtificial
SequencePrimer 65agatcacaga ttttgggcgg g 216625DNAArtificial
SequencePrimer 66taggtttctg agcaccctct gtgtc 256720DNAArtificial
SequencePrimer 67tctcttccgc acccagctgt 206818DNAArtificial
SequencePrimer 68ttgggctggc caaacagc 186925DNAArtificial
SequencePrimer 69gctgtgaaat acctggcttg ttgtt 257019DNAArtificial
SequencePrimer 70gaagggcatg agctgcatg 197125DNAArtificial
SequencePrimer 71ccaggcagcc tttagtcact gtaga 257221DNAArtificial
SequencePrimer 72ggcctctctg tcatggggaa t 217325DNAArtificial
SequencePrimer 73acctgctcca ctccaccact atcac 257420DNAHomo sapiens
74tccaccgtgc agctcatctc 207520DNAHomo sapiens 75tccaccgtgc
agctcatctt 207622DNAHomo sapiens 76ttgtctttgt gttcccggac at
227728DNAArtificial SequencePrimer 77ccgcggctca tgcccttcgg ctccgcgg
28
* * * * *