US2015376708A1PendingUtilityA1
Probes and methods for determining the presence or absence of genetic segments
Est. expiryJun 18, 2030(~3.9 yrs left)· nominal 20-yr term from priority
G06F 19/24G06F 19/20C12Q 2600/156C12Q 1/6883G16B 25/20G16B 40/10G16B 20/20C12Q 1/6876G16B 20/00G16B 40/00G16B 25/00
45
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
A method for determining the presence or absence of a genetic segment of interest, such as an exon, an intron or a promoter, in a DNA-containing sample, and probe sets for use in such methods, including probe sets comprising oligonucleotide probes having nucleotides sequences selected from those of SEQ ID NOS: 1-101.
Claims
exact text as granted — not AI-modified1 . A method for determining the presence or absence of a genetic segment of interest in a DNA-containing sample, the method comprising:
(i) bringing at least a first probe set comprising a plurality of replicates of at least one oligonucleotide probe that interrogates a first cognate sequence within said segment of interest into contact with (a) a plurality of reference samples in which the genetic segment of interest is absent, and (b) a plurality of reference samples in which the genetic segment of interest is present, under conditions that allow probe-cognate sequence hybridisation to occur; (ii) measuring the intensity of probe-sample hybridisation of each of the reference samples, thereby obtaining a first cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is absent and a second cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is present; and (iii) establishing a “no call” region of hybridisation intensity values that lies in the region between said first and second clusters and which is bounded by a lower no call boundary (“L NC ”) and an upper no call boundary (“U NC ”), wherein the L NC and U NC represent statistical confidence limits for assigning a hybridisation intensity value to said first and said second clusters, respectively; iv) contacting the first probe set with at least one DNA-containing test sample under conditions that allow probe-cognate sequence hybridisation to occur; v) measuring the intensity of probe-sample hybridisation of the at least one test sample; and vi) comparing the measured hybridisation intensity with the L NC and the U NC ,
wherein a measured hybridisation intensity below the L NC indicates that said genetic segment of interest is absent in the test sample and a measured hybridisation intensity above U NC indicates that said genetic segment of interest is present in the test sample.
2 . A method according to claim 1 , wherein said genetic segment of interest is selected from the group consisting of: an exon, an intron and a promoter.
3 . A method according to claim 1 , wherein said cognate sequence comprises a polymorphic site and wherein said first probe set comprises a plurality of replicates of at least two, at least three or at least four allele-specific oligonucleotide probes that distinguish the alleles of said polymorphic site.
4 . A method according to claim 3 , wherein said test sample comprises said genetic segment of interest, the method further comprising genotyping said test sample to identify an allele at said polymorphic site.
5 . A method according to claim 1 , wherein at least a second probe set is employed in addition to said first probe set and wherein the second probe set comprises a plurality of replicates of at least one oligonucleotide probe that interrogates a second cognate sequence within said segment of interest, the method further comprising establishing a second no call region bounded by L NC and U NC boundaries for the second probe set.
6 . A method according to claim 5 , wherein the test sample is classified as having or not having said segment of interest based on a strict consensus between the determinations of the first and second probe sets.
7 . A method according to claim 6 , wherein said strict consensus is a strict consensus as set forth in Table 1.
8 . A method according to claim 5 , wherein the test sample is classified as having or not having said segment of interest based on a majority consensus between the determinations of the first and second probe sets in the absence of contradictory determinations.
9 . A method according to claim 8 , wherein said majority consensus is a majority consensus as set forth in Table 2.
10 . A method according to claim 1 , wherein the oligonucleotide probes are attached to a solid support.
11 . A method according to claim 10 , wherein the oligonucleotide probes are attached to a substantially planar solid support in the form of an array.
12 . A method according to claim 10 , wherein the oligonucleotide probes are attached to one or more particles.
13 . A method according to claim 12 , wherein said particles are selected from the group consisting of: micrometer-sized beads, nanometer-sized beads and cylinders.
14 . A method according to claim 1 , wherein said test sample comprises DNA amplified from genomic DNA of a test subject, which DNA has been fragmented and/or labelled with a detectable label.
15 . A method according to claim 1 , wherein each of the reference samples comprises DNA amplified from genomic DNA of a reference subject, which has been fragmented and/or labelled with a detectable label.
16 . A method according to claim 1 , wherein the DNA of the reference samples and/or the test sample has been labelled with a fluorescent label and wherein measuring hybridisation intensity comprises measuring the fluorescence signal of the fluorescent label at each oligonucleotide probe location.
17 . A method according to claim 16 , wherein the hybridisation intensity for each probe set is measured as I/B, where:
I is determined as a measure of central tendency of the measured fluorescence signal of the replicates of each oligonucleotide probe; and B is determined as a measure of central tendency of the background fluorescence signal of the replicates of each oligonucleotide probe.
18 . A method according to claim 17 , wherein the measure of central tendency is selected from the group consisting of: the mean and the median.
19 . A method according to claim 17 , wherein said measured fluorescence signal is determined after trimming extreme readings.
20 . A method according to claim 17 , wherein said background fluorescence signal is determined after trimming extreme readings.
21 . A method according to claim 17 , wherein the L NC and U NC values are calculated according to the following formulae:
L
NC
=
T
I
/
B
·
[
2
·
a
(
I
/
B
)
n
-
aMedian
I
/
B
]
-
a
(
I
/
B
)
n
2
T
I
/
B
-
aMedian
I
/
B
U
NC
=
T
I
/
B
·
[
pMedian
I
/
B
-
2
·
p
(
I
/
B
)
1
]
+
p
(
I
/
B
)
1
2
pMedian
I
/
B
-
T
I
/
B
where:
T
I
/
B
=
pMedian
I
/
B
·
a
(
I
/
B
)
n
-
aMedian
I
/
B
·
p
(
I
/
B
)
1
pMedian
I
/
B
+
a
(
I
/
B
)
n
-
aMedian
I
/
B
-
p
(
I
/
B
)
1
aMedianI/B is a measure of central tendency of the measured I/B values of the reference samples in which the genetic segment of interest is absent;
pMedianI/B is a measure of central tendency of the measured I/B values of the reference samples in which the genetic segment of interest is present;
a(I/B) n is the greatest measured I/B value of the reference samples in which the genetic segment of interest is absent; and
p(I/B) 1 is the lowest measured I/B value of the reference samples in which the genetic segment of interest is present.
22 . A method according to claim 21 , wherein said measure of central tendency is selected from the group consisting of: the mean and the median.
23 . A method according to claim 1 , wherein the genetic segment of interest comprises an exon of the human RHD gene.
24 . A method according claim 23 , wherein the oligonucleotide probes are selected from the probes shown in Table 4.
25 . A method according to claim 24 , wherein the method further comprises genotyping the test sample to identify at least one allele at a site of single nucleotide polymorphism (“SNP”) in the human RHD gene.
26 . A probe set for determining the presence or absence in a DNA-containing test sample of at least one exon of the human RHD gene, the set comprising a plurality of oligonucleotide probes of between 10-50 nucleotides in length, each of the probes comprising or consisting of a contiguous nucleotide sequence selected from the sequences of SEQ ID NOS: 1-101.
27 . A probe set according to claim 26 , wherein the oligonucleotide probes are of between 15-40 nucleotides in length.
28 . A probe set according to claim 26 , wherein the oligonucleotide probes are of between 19-27 nucleotides in length.
29 . A probe set according to claim 26 , wherein the probe set is for determining the presence or absence in a DNA-containing test sample of at least two exons of the human RHD gene, the set comprising two or more of (i)-(x):
(i) one or more RHD exon 1 probes of SEQ ID NOS: 1-5; (ii) one or more RHD exon 2 probes of SEQ ID NOS: 6-11; (iii) one or more RHD exon 3 probes of SEQ ID NOS: 12-17; (iv) one or more RHD exon 4 probes of SEQ ID NOS: 18-25; (v) one or more RHD exon 5 probes of SEQ ID NOS: 26-29; (vi) one or more RHD exon 6 probes of SEQ ID NOS: 30-79; (vii) one or more RHD exon 7 probes of SEQ ID NOS: 80-87; (viii) one or more RHD exon 8 probes of SEQ ID NOS: 88-91; (ix) one or more RHD exon 9 probes of SEQ ID NOS: 92-99; and (x) one or more RHD exon 10 probes of SEQ ID NOS: 100-101.
30 . A probe set according to claim 26 , wherein said plurality of oligonucleotide probes are attached to a solid support.
31 . A probe set according to claim 27 , wherein the oligonucleotide probes are attached to a substantially planar solid support in the form of an array.
32 . A probe set according to claim 27 , wherein the oligonucleotide probes are attached to one or more particles.
33 . A probe set according to claim 32 , wherein the one or more particles are selected from the group consisting of: micrometer-sized beads, nanometer-sized beads and cylinders.
34 . A kit comprising:
(a) a probe set comprising a plurality of oligonucleotide probes of between 10-50 nucleotides in length, each of the probes comprising or consisting of a contiguous nucleotide sequence selected from the sequences of SEQ ID NOS: 1-101; and (b) one or more of the following components:
(I) reagents and/or PCR primers suitable for amplifying human genomic DNA that comprises all or part of the RHD gene;
(II) a fluorescent label suitable for labelling DNA; and
(III) instructions for performing a method, the method comprising the following steps:
(i) bringing at least a first probe set comprising a plurality of replicates of at least one oligonucleotide probe that interrogates a first cognate sequence within said segment of interest into contact with (a) a plurality of reference samples in which the genetic segment of interest is absent, and (b) a plurality of reference samples in which the genetic segment of interest is present, under conditions that allow probe-cognate sequence hybridisation to occur; (ii) measuring the intensity of probe-sample hybridisation of each of the reference samples, thereby obtaining a first cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is absent and a second cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is present; and (iii) establishing a “no call” region of hybridisation intensity values that lies in the region between said first and second clusters and which is bounded by a lower no call boundary (“L NC ”) and an upper no call boundary (“U NC ”), wherein the L NC and U NC represent statistical confidence limits for assigning a hybridisation intensity value to said first and said second clusters, respectively; iv) contacting the first probe set with at least one DNA-containing test sample under conditions that allow probe-cognate sequence hybridisation to occur; v) measuring the intensity of probe-sample hybridisation of the at least one test sample; and vi) comparing the measured hybridisation intensity with the L NC and the U NC ,
wherein a measured hybridisation intensity below the L NC indicates that said genetic segment of interest is absent in the test sample and a measured hybridisation intensity above U NC indicates that said genetic segment of interest is present in the test sample.
35 . A method for selecting at least one optimal probe set, the method comprising:
(i) providing a plurality of candidate probe sets, each candidate probe set comprising a plurality of replicates of at least one oligonucleotide probe that interrogates a cognate sequence within a genetic segment of interest; (ii) bringing each of the candidate probe sets into contact with (a) a plurality of reference samples in which the genetic segment of interest is absent, and (b) a plurality of reference samples in which the genetic segment of interest is present, under conditions that allow probe-cognate sequence hybridisation to occur; (iii) measuring the intensity of probe-sample hybridisation of each of the reference samples, thereby obtaining for each candidate probe set a first cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is absent and a second cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is present; and (iv) scoring each of the candidate probe sets by dividing a measure of the interval between the first and second clusters by the sum of a measure of central tendency of the first and second clusters, thereby obtaining a probe set score for each of the candidate probe sets,
wherein a higher probe set score indicates that the candidate probe set is more likely to be optimal for determining the presence or absence of the genetic segment of interest.
36 . A method according to claim 35 , wherein the reference samples are labelled with a fluorescent label and wherein the hybridisation intensity is measured as I/B, where:
I is determined as a measure of central tendency of the measured fluorescence signal of the replicates of each oligonucleotide probe and B is determined as a measure of central tendency of the background fluorescence signal of the replicates of each oligonucleotide probe
and wherein the probe set score (S) for each candidate probe set is calculated according to the following formulae:
S
=
A
M
=
p
(
I
/
B
)
1
-
a
(
I
/
B
)
n
pMedian
I
/
B
+
aMedian
I
/
B
where:
aMedianl/B is a measure of central tendency of the measured I/B values of the reference samples in which the genetic segment of interest is absent; pMedianl/B is a measure of central tendency of the measured I/B values of the reference samples in which the genetic segment of interest is present;
a(I/B) n is the greatest measured I/B value of the reference samples in which the genetic segment of interest is absent; and
p(I/B) 1 is the lowest measured I/B value of the reference samples in which the genetic segment of interest is present.
37 . A method according to claim 36 , wherein said measure of central tendency is selected from the group consisting of: the mean and the median.
38 . A method according to claim 1 , wherein at least said first probe set is a probe set that has been selected using a method which comprises the following steps:
(i) providing a plurality of candidate probe sets, each candidate probe set comprising a plurality of replicates of at least one oligonucleotide probe that interrogates a cognate sequence within a genetic segment of interest; (ii) bringing each of the candidate probe sets into contact with (a) a plurality of reference samples in which the genetic segment of interest is absent, and (b) a plurality of reference samples in which the genetic segment of interest is present, under conditions that allow probe-cognate sequence hybridisation to occur; (iii) measuring the intensity of probe-sample hybridisation of each of the reference samples, thereby obtaining for each candidate probe set a first cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is absent and a second cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is present; and (iv) scoring each of the candidate probe sets by dividing a measure of the interval between the first and second clusters by the sum of a measure of central tendency of the first and second clusters, thereby obtaining a probe set score for each of the candidate probe sets,
wherein a higher probe set score indicates that the candidate probe set is more likely to be optimal for determining the presence or absence of the genetic segment of interest.
39 . A method for determining a lower no call boundary (“L NC ”) and an upper no call boundary (“U NC ”) for at least a first probe set, the method comprising:
(i) bringing at least said first probe set comprising a plurality of replicates of at least one oligonucleotide probe that interrogates a first cognate sequence within a genetic segment of interest into contact with (a) a plurality of reference samples in which the genetic segment of interest is absent, and (b) a plurality of reference samples in which the genetic segment of interest is present, under conditions that allow probe-cognate sequence hybridisation to occur;
(ii) measuring the intensity of probe-sample hybridisation of each of the reference samples, thereby obtaining a first cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is absent and a second cluster of hybridisation intensity values for the reference samples in which the genetic segment of interest is present; and
(iii) establishing a “no call” region of hybridisation intensity values that lies in the region between said first and second clusters and which is bounded by L NC and U NC , wherein the L NC and U NC represent statistical confidence limits for assigning a hybridisation intensity value to said first and said second clusters, respectively.
40 . A method for determining the presence or absence of a genetic segment of interest in a DNA-containing sample, the method comprising:
(i) bringing at least a first probe set comprising a plurality of replicates of at least one oligonucleotide probe that interrogates a first cognate sequence within said segment of interest into contact with at least one DNA-containing test sample under conditions that allow probe-cognate sequence hybridisation to occur; (ii) measuring the intensity of probe-sample hybridisation of the at least one test sample; and (iii) comparing the measured hybridisation intensity with a pre-determined lower no call boundary (“L NC ”) and a pre-determined upper no call boundary (“U NC ”),
and wherein a measured hybridisation intensity below the L NC indicates that said genetic segment of interest is absent in the test sample and a measured hybridisation intensity above U NC indicates that said genetic segment of interest is present in the test sample.Join the waitlist — get patent alerts
Track US2015376708A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.