US2010330571A1PendingUtilityA1
Method of measuring adaptive immunity
Individually held — no corporate assignee on recordPriority: Jun 25, 2009Filed: Jun 4, 2010Published: Dec 30, 2010
Est. expiryJun 25, 2029(~2.9 yrs left)· nominal 20-yr term from priority
C12N 15/10C12Q 1/6881G16B 40/00C12Q 1/6883G06F 17/10C12Q 1/6874C12N 15/1065C12Q 2600/16C12Q 1/6869
57
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Claims
Abstract
A method of measuring immunocompetence is described. This method provides a means for assessing the effects of diseases or conditions that compromise the immune system and of therapies aimed to reconstitute it. This method is based on quantifying T-cell diversity by calculating the number of diverse T-cell receptor (TCR) beta chain variable regions from blood cells.
Claims
exact text as granted — not AI-modified1 . A composition comprising:
(a) a multiplicity of V-segment primers, wherein each primer comprises a sequence that is complementary to a single functional V segment or a small family of V segments; and (b) a multiplicity of J-segment primers, wherein each primer comprises a sequence that is complementary to a J segment;
wherein the V segment and J-segment primers permit amplification of a TCR or IG CDR3 region by a multiplex polymerase chain reaction (PCR) to produce a multiplicity of amplified DNA molecules sufficient to quantify the diversity of the TCR or IG genes.
2 . The composition of claim 1 , wherein each V-segment primer comprises a sequence that is complementary to a single Vγ segment or a family of similar Vγ segments, and each J segment primer comprises a sequence that is complementary to a Jγ segment, and wherein V segment and J-segment primers permit amplification of a TCRγ CDR3 region.
3 . The composition of claim 1 , wherein each V-segment primer comprises a sequence that is complementary to a single Vδ segment or a family of similar Vδ segments, and each J segment primer comprises a sequence that is complementary to a Jδ segment, and wherein V segment and J-segment primers permit amplification of a TCRα CDR3 region.
4 . The composition of claim 1 , wherein each V-segment primer comprises a sequence that is complementary to a single Vα segment or a family of similar Vα segments, and each J segment primer comprises a sequence that is complementary to a Jα segment, and wherein V segment and J-segment primers permit amplification of a TCRα CDR3 region.
5 . The composition of claim 1 , wherein each V-segment primer comprises a sequence that is complementary to a single Vβ segment or a family of similar Vβ segments, and each J segment primer comprises a sequence that is complementary to a Jβ segment, and wherein V segment and J-segment primers permit amplification of a TCRβ CDR3 region.
6 . The composition of claim 1 , wherein the V segment have similar annealing strength.
7 . The composition of claim 1 , wherein all J segment primers anneal to the same conserved framework region motif.
8 . The composition of claim 1 , wherein the amplified DNA molecule starts from said conserved motif and diagnostically identifies the J segment and includes the junction and into the V segment.
9 . The composition of claim 1 , further comprising a set of sequencing oligonucleotides, wherein the sequencing oligonucleotides hybridize to a regions within the amplified DNA molecules.
10 . The composition of claim 1 , wherein the amplified DNA spans a V-D-J junction.
11 . The composition of claim 1 , wherein the V-segment or J-segment are selected to contain a sequence error-correction by merger of closely related sequences.
12 . The composition of claim 1 , further comprising a universal C segment primer for generating cDNA from mRNA.
13 . The composition of claim 5 , wherein the V segment primer is anchored at position −43 in the Vβ segment relative to the recombination signal sequence (RSS).
14 . The composition of claim 5 , wherein the multiplicity of V segment primers consist of at least 14 primers specific to 14 different Vβ genes.
15 . The composition of claim 5 , wherein the V segment primers have sequences that are selected from the group consisting of SEQ ID NOS:1-45.
16 . The composition of claim 5 , wherein the V segment primers have sequences that are selected from the group consisting of SEQ ID NOS:58-102.
17 . The composition of claim 5 , wherein there is a V segment primer for each Vβ segment or family of Vβ segments.
18 . The composition of claim 5 , wherein the primers do not cross an intron/exon boundary.
19 . The composition of claim 5 , wherein the J segment primers hybridize with a conserved element of the Jβ segment, and have similar annealing strength.
20 . The composition of claim 5 , wherein the multiplicity of J segment primers consist of at least five primers specific to five different Jβ genes.
21 . The composition of claim 5 , wherein the J segment primers have sequences that are selected from the group consisting of SEQ ID NOS:46-57 and 483.
22 . The composition of claim 5 , wherein the J segment primers have sequences that are selected from the group consisting of SEQ ID NOS:103-113, 468 and 484.
23 . The composition of claim 5 , wherein there is a J segment primer for each Jβ segment.
24 . The composition of claim 5 , wherein the amplified Jβ gene segments each have a unique four base tag at positions +11 through +14 downstream of the RSS site.
25 . The composition of claim 24 , wherein the sequencing oligonucleotides hybridize adjacent to a four base tag within the amplified Jβ gene segments at positions +11 through +14 downstream of the RSS site.
26 . The composition of claim 24 , wherein the sequencing oligonucleotides are selected from the group consisting of SEG ID NOS:470-482.
27 . A composition comprising:
(a) a multiplicity of V segment primers, wherein each V segment primer comprises a sequence that is complementary to a single functional V segment or a small family of V segments; and (b) a multiplicity of J segment primers, wherein each J segment primer comprises a sequence that is complementary to a J segment;
wherein the V segment and J segment primers permit amplification of antibody heavy chain (IGH) V H region by a multiplex polymerase chain reaction (PCR) to produce a multiplicity of amplified DNA molecules sufficient to quantify the diversity of antibody heavy chain genes.
28 . A composition comprising:
(a) a multiplicity of V segment primers, wherein each V segment primer comprises a sequence that is complementary to a single functional V segment or a small family of V segments; and (b) a multiplicity of J segment primers, wherein each J segment primer comprises a sequence that is complementary to a J segment;
wherein the V segment and J segment primers permit amplification of antibody light chain (IGL) V L region by a multiplex polymerase chain reaction (PCR) to produce a multiplicity of amplified DNA molecules sufficient to quantify the diversity of antibody light chain genes.
29 . A method comprising:
(a) selecting a multiplicity of V segment primers, wherein each V segment primer comprises a sequence that is complementary to a single functional V segment or a small family of V segments; and (b) selecting a multiplicity of J segment primers, wherein each J segment primer comprises a sequence that is complementary to a J segment; (c) combining the V segment and J segment primers with a sample of genomic DNA to permit amplification of a TCR CDR3 region by a multiplex polymerase chain reaction (PCR) to produce a multiplicity of amplified DNA molecules sufficient to quantify the diversity of the TCR genes.
30 . The method of claim 29 , wherein each V segment primer comprises a sequence that is complementary to a single Vβ segment or a family of Vβ segments, and each J segment primer comprises a sequence that is complementary to a Jβ segment; and wherein combining the V segment and J segment primers with a sample of genomic DNA permits amplification of a TCRB CDR3 region by a multiplex polymerase chain reaction (PCR) and produces a multiplicity of amplified DNA molecules.
31 . The method of claim 30 , further comprising a step of sequencing the amplified DNA molecules.
32 . The method of claim 31 , wherein the sequencing step utilizes a set of sequencing oligonucleotides, that hybridize to a defined region within the amplified DNA molecules.
33 . The method of claim 32 , further comprising a step of calculating the total diversity of TCRβ CDR3 sequences among the amplified DNA molecules.
34 . The method of claim 33 , wherein the method shows that the total diversity of a normal human subject is greater than 1*10 6 sequences.
35 . The method of claim 33 , wherein the method shows that the total diversity of a normal human subject is greater than 2*10 6 sequences.
36 . The method of claim 33 , wherein the method shows that the total diversity of a normal human subject is greater than 3*10 6 sequences.
37 . A method of diagnosing immunodeficiency in a human patient, comprising measuring the diversity of TCR CDR3 sequences of the patient, and comparing the diversity of the subject to the diversity obtained from a normal subject.
38 . The method of claim 37 , wherein measuring the diversity of TCR sequences comprises the steps of:
(a) selecting a multiplicity of V segment primers, wherein each V segment primer comprises a sequence that is complementary to a single functional V segment or a small family of V segments; and (b) selecting a multiplicity of J segment primers, wherein each J segment primer comprises a sequence that is complementary to a J segment; (c) combining the V segment and J segment primers with a sample of genomic DNA to permit amplification of a TCR CDR3 region by a multiplex polymerase chain reaction (PCR) to produce a multiplicity of amplified DNA molecules; (d) sequencing the amplified DNA molecules; (e) calculating the total diversity of TCR CDR3 sequences among the amplified DNA molecules.
39 . The method of claim 38 , wherein comparing the diversity is determined by calculating using the following equation:
Δ
(
t
)
=
∑
x
E
(
n
x
)
measurement
1
+
2
-
∑
x
E
(
n
x
)
measurement
2
=
S
∫
0
∞
-
λ
(
1
-
-
λ
t
)
G
(
λ
)
wherein G(λ) is the empirical distribution function of the parameters λ 1 , . . . , λ s , n x is the number of clonotypes sequenced exactly x times, and
E
(
n
x
)
=
S
∫
0
∞
(
-
λ
λ
x
x
!
)
G
(
λ
)
.
40 . The method of claim 38 , wherein the diversity of at least two samples of genomic DNA are compared.
41 . The method of claim 40 , wherein one sample of genomic DNA is from a patient and the other sample is from a normal subject.
42 . The method of claim 40 , wherein one sample of genomic DNA is from a patient before a therapeutic treatment and the other sample is from the patient after treatment.
43 . The method of claim 40 , wherein the two samples of genomic DNA are from the same patient at different times during treatment.
44 . The method of claim 40 , in which a disease is diagnosed based on the comparison of diversity among the samples of genomic DNA.
45 . The method of claim 40 , wherein the immunocompetence of a human patient is assessed by the comparison.Join the waitlist — get patent alerts
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