U.S. patent application number 15/133932 was filed with the patent office on 2016-08-11 for single cell analysis by polymerase cycling assembly.
The applicant listed for this patent is Adaptive Biotechnologies Corp.. Invention is credited to Malek Faham, Thomas Willis.
Application Number | 20160230236 15/133932 |
Document ID | / |
Family ID | 42153463 |
Filed Date | 2016-08-11 |
United States Patent
Application |
20160230236 |
Kind Code |
A1 |
Faham; Malek ; et
al. |
August 11, 2016 |
SINGLE CELL ANALYSIS BY POLYMERASE CYCLING ASSEMBLY
Abstract
The invention provides a method of making measurements on
individual cells of a population, particularly cells that have
identifying nucleic acid sequences, such as lymphoid cells. In one
aspect, the invention provides a method of making multiparameter
measurements on individual cells of such a population by carrying
out a polymerase cycling assembly (PCA) reaction to link their
identifying nucleic acid sequences to other cellular nucleic acids
of interest. The fusion products of such PCA reaction are then
sequenced and tabulated to generate multiparameter data for cells
of the population.
Inventors: |
Faham; Malek; (Pacifica,
CA) ; Willis; Thomas; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Adaptive Biotechnologies Corp. |
Seattle |
WA |
US |
|
|
Family ID: |
42153463 |
Appl. No.: |
15/133932 |
Filed: |
April 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13908813 |
Jun 3, 2013 |
9347099 |
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15133932 |
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13100395 |
May 4, 2011 |
8507205 |
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13908813 |
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12615263 |
Nov 9, 2009 |
8236503 |
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13100395 |
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61112693 |
Nov 7, 2008 |
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61332175 |
May 6, 2010 |
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61446822 |
Feb 25, 2011 |
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61452594 |
Mar 14, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/16 20130101;
C12N 15/1072 20130101; C12Q 1/6881 20130101; C12Q 2600/106
20130101; C12Q 1/6883 20130101; Y02A 90/10 20180101; C12Q 1/6809
20130101; C12Q 2600/158 20130101; C12Q 2600/118 20130101; C12Q
1/6827 20130101; C12Q 1/6886 20130101; C12Q 1/6869 20130101; C12Q
2600/156 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of analyzing a plurality target nucleic acids in each
cell of a population, the method comprising the steps of: providing
multiple reactors each containing a single cell in a polymerase
cycling assembly (PCA) reaction mixture comprising a pair of outer
primers and one or more pairs of linking primers specific for the
plurality of target nucleic acids; performing a PCA reaction in the
reactors to form fusion products of the target nucleic acids in the
reactors; and sequencing the fusion products from the reactors to
identify the target nucleic acids of each cell in the
population.
2. The method of claim 1 wherein said multiple reactors are aqueous
micelles of a water-in-oil emulsion.
3. The method of claim 3 wherein said water-in-oil emulsion is
generated by a microfluidics device.
4. The method of claim 1 wherein said population is a population of
B cells and/or T cells.
5. The method of claim 4 wherein at least one pair of primers from
said outer primers and said linking primers is specific for a
clonotype of said B cells and/or T cells.
6. The method of claim 5 wherein at least one pair of primers from
said outer primers and said linking primers is specific for a
nucleic acid sequence of said B cells and/or T cells that is a
cancer marker or encodes a cancer marker.
7. The method of claim 6 wherein said nucleic acid is an RNA that
indicates a cancerous state by over expression.
8. The method of claim 6 wherein said nucleic acid is a DNA that
indicates a cancerous state by excess copy number.
9. A method of distinguishing multiple subpopulations of
lymphocytes, the method comprising the steps of: providing multiple
reactors each containing a single lymphocyte in a polymerase
cycling assembly (PCA) reaction mixture comprising a pair of outer
primers and one or more pairs of linking primers, at least one pair
of such primers being specific for a nucleic acid containing a
clonotype and one or more pairs of such primers being specific for
one or more target nucleic acids characteristic of the multiple
subpopulations of lymphocytes; performing a PCA reaction in each
reactor to form a fusion product comprising the target nucleic
acids and a clonotype of the lymphocyte therein; sequencing the
fusion products from the reactors; and classifying each lymphocyte
into a subpopulation by the target nucleic acids associated with
its clonotype.
10. The method of claim 9 wherein said multiple reactors are
aqueous micelles of a water-in-oil emulsion.
11. The method of claim 10 wherein said water-in-oil emulsion is
generated by a microfluidics device.
12. The method of claim 9 wherein said nucleic acid containing said
clonotype and said one or more target nucleic acids are RNA and
wherein said step of classifying includes determining the relative
expression levels of said one or more target nucleic acids.
13. A method of detecting cross-lineage rearrangements in a
population of lymphocytes, the method comprising the steps of:
providing multiple reactors each containing a single lymphocyte in
a polymerase cycling assembly (PCA) reaction mixture comprising a
pair of outer primers and one or more pairs of linking primers, at
least one pair of such primers being specific for a nucleic acid
containing at least a portion of a B cell receptor gene and at
least one pair of such primers being specific for a nucleic acid
containing at least a portion of a T cell receptor gene; performing
a PCA reaction in each reactor to form a fusion product comprising
the target nucleic acids and a clonotype of the lymphocyte therein;
sequencing the fusion products from the reactors; and determining
the presence, absence or level of fusion products that comprise
both a portion of a B cell receptor gene and a portion of a T cell
receptor gene to detect cross-lineage rearrangements in the
population of lymphocytes.
14. The method of claim 13 wherein said multiple reactors are
aqueous micelles of a water-in-oil emulsion.
15. The method of claim 14 wherein said water-in-oil emulsion is
generated by a microfluidics device.
Description
[0001] This application is a continuation-in-part of co-pending
U.S. application Ser. No. 12/615,263 filed 9 Nov. 2009, and claims
priority from co-pending U.S. provisional application Ser. No.
61/332,175 filed 6 May 2010, Ser. No. 61/446,822 filed 25 Feb.
2011, and Ser. No. 61/452,594 filed 14 Mar. 2011, all of which are
incorporated herein by reference in their entireties.
BACKGROUND
[0002] Cytometry plays an indispensable role in many medical and
research fields. Image-based and flow cytometers have found
widespread use in these fields for counting cells and measuring
their physical and molecular characteristics, e.g. Shapiro,
Practical Flow Cytometry, 4th Edition (Wiley-Liss, 2003). In
particular, flow cytometry is a powerful technique for rapidly
measuring multiple parameters on large numbers of individual cells
of a population enabling acquisition of statistically reliable
information about the population and its subpopulations. The
technique has been important in the detection and management of a
range of diseases, particularly blood-related diseases, such as
hematopoietic cancers, HIV, and the like, e.g. Woijciech, Flow
Cytometry in Neoplastic Hematology, Second Edition (Informa
Healthcare, 2010); Brown et al, Clinical Chemistry, 46: 8(B):
1221-1229 (2000). Despite this utility, flow cytometry has a number
of drawbacks, including limited sensitivity in rare cell detection,
e.g. Campana et al, Hematol. Oncol. Clin. North Am., 23(5):
1083-1098 (2009); limitations in the number of cell parameters that
can be practically measured at the same time; and costly
instrumentation.
[0003] In view of the above, it would be advantageous to many
medical and research fields if there were available alternative
methods and systems for making multiparameter measurements on large
numbers of individual cells that overcame the drawbacks of current
cytometric approaches.
SUMMARY OF THE INVENTION
[0004] The present invention is directed to methods for making
multiparameter measurements of target nucleic acids in single cells
of a population, particularly cells such as lymphocytes that
contain cell-specific recombined sequences. Aspects of the present
invention are exemplified in a number of implementations and
applications, some of which are summarized below and throughout the
specification.
[0005] In one aspect the invention includes a method of analyzing a
plurality target nucleic acids in each cell of a population
comprising the steps of: (a) providing multiple reactors each
containing a single cell in a polymerase cycling assembly (PCA)
reaction mixture comprising a pair of outer primers and one or more
pairs of linking primers specific for the plurality of target
nucleic acids; (b) performing a PCA reaction in the reactors to
form fusion products of the target nucleic acids in the reactors;
and (c) sequencing the fusion products from the reactors to
identify the target nucleic acids of each cell in the
population.
[0006] In another aspect the invention includes a method of
distinguishing multiple subpopulations of lymphocytes comprising
the steps of: (a) providing multiple reactors each containing a
single lymphocyte in a polymerase cycling assembly (PCA) reaction
mixture comprising a pair of outer primers and one or more pairs of
linking primers, one or more pairs of such primers being specific
for one or more target nucleic acids and at least one pair of such
primers being specific for a nucleic acid containing a clonotype;
(b) performing a PCA reaction in each reactor to form a fusion
product comprising the target nucleic acids and a clonotype of the
lymphocyte therein; (c) sequencing the fusion products from the
reactors; and (d) classifying each lymphocyte into a subpopulation
by the target nucleic acids associated with its clonotype.
[0007] These above-characterized aspects, as well as other aspects,
of the present invention are exemplified in a number of illustrated
implementations and applications, some of which are shown in the
figures and characterized in the claims section that follows.
However, the above summary is not intended to describe each
illustrated embodiment or every implementation of the present
invention.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0008] FIG. 1 illustrates steps of one embodiment of the method of
the invention.
[0009] FIGS. 2A-2C illustrate a PCA scheme for linking target
sequences where pairs of internal primers have complementary
tails.
[0010] FIGS. 3A-3C illustrate a PCA scheme for linking target
sequences where only one primer of each pair of internal primers
has a tail that is complementary to an end of a target
sequence.
[0011] FIGS. 4A-4C illustrate a PCA scheme for linking target
sequences where pairs of internal primers have complementary tails
and external primers have tails for continued amplification of an
assembled product by PCR.
[0012] FIGS. 5A-5F illustrate a multiplex of pairwise assemblies of
target sequences.
[0013] FIGS. 6A-6E illustrate a method of using PCA to link
together three sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, molecular biology (including recombinant
techniques), cell biology, and biochemistry, which are within the
skill of the art. Such conventional techniques include, but are not
limited to, sampling and analysis of blood cells, nucleic acid
sequencing and analysis, and the like. Specific illustrations of
suitable techniques can be had by reference to the example herein
below. However, other equivalent conventional procedures can, of
course, also be used. Such conventional techniques and descriptions
can be found in standard laboratory manuals such as Genome
Analysis: A Laboratory Manual Series (Vols. I-IV); PCR Primer: A
Laboratory Manual; and Molecular Cloning: A Laboratory Manual (all
from Cold Spring Harbor Laboratory Press); and the like.
[0015] The invention provides a method of making measurements on
individual cells of a population, particularly cells that have
identifying nucleic acid sequences, such as lymphoid cells. In one
aspect the invention provides a method of making multiparameter
measurements on individual cells of such a population. An objective
of assays of the invention is to carry out a polymerase cycling
assembly (PCA) reaction on individual cells to link their
identifying nucleic acid sequences to other cellular nucleic acids
of interest (referred to herein as "target nucleic acids"), the
products of such linking being referred to herein as "fusion
products." After their generation, fusion products can be sequenced
and tabulated to generate data, especially multiparameter data, for
each cell of a population. In one aspect, such data may include
gene expression data, data on the presence or absence of one or
more predetermined genomic sequences, gene copy number data, or
combinations of the foregoing. FIG. 1 gives an overview on one
embodiment of the invention. Lymphoid cells (100) each have a
distinct identifying nucleic acid (102), which in the figure is
represented as a unique binary number. In one aspect, the
identifying nucleic acids are the clonotypes of the lymphocytes. In
addition, each cell has and/or expresses various nucleic acids of
interest (104), or target nucleic acids, represented by the letters
"a", "b", "c" and "w", which may be genomic DNA, expressed genes,
or the like. Cells (100) are disposed (106) in single cell reactors
(110), which in this example are illustrated as micelles of a
water-in-oil emulsion (108), although a variety of single cell
reactors may be used, including but not limited to, plates with
arrays of nanoliter-volume wells, microfluidic devices, and the
like, as described more fully below. In one aspect, single-cell
emulsion (126) is generated using a microfluidic emulsion
generator, such as disclosed by Zeng et al, Anal. Chem., 82:
3183-3190 (2010), or the like.
[0016] Reactors (110) contain a PCA reaction mixture that, for
example, may comprise a nucleic acid polymerase, outer primers and
linking primers (described more fully below), nucleoside
triphosphates, a buffer solution, and the like. In some
embodiments, a PCA reaction mixture may also include one or more
cell lysing reagents, to access of such reagents to target nucleic
acids. For each reactor (110) containing a cell, PCA reaction (112)
generates fusion products (114) that may comprise one or more pairs
of sequences, such that one member of the pair is the identifying
nucleic acid of the cell in the reactor and the other member is a
nucleic acid of interest, such as an expressed gene. In other
embodiments, fusion products may comprise triplets of sequences, or
higher order concatenations. In the method of the invention, a
single kind of fusion product may be generated for each cell (or
per reactor) or a plurality of different kinds of fusion products
may be generated for each cell (or per reactor). Such plurality may
be in the range of from 2 to 500, or from 2 to 200, or from 2 to
100, or from 2 to 20. In one embodiment, such plurality may be in
the range of from 2 to 10.
[0017] After completion of PCA reaction (112), emulsion (126) is
broken and fusion products (114) are isolated (116). Fusion
products (114) are represented in FIG. 1 as conjugates (118) of
identifying nucleic acids (102) and target nucleic acids (128). A
variety of conventional methods may be used to isolate fusion
products (114), including, but not limited to, column
chromatography, ethanol precipitation, affinity purification after
use of biotinylated primers, gel electrophoresis, or the like. As
part of PCA reaction (112) or after isolation (116), additional
sequences may be added to fusion products (114) as necessary for
sequencing (120). Sequencing may be carried out using a
conventional high-throughput instrument (122), e.g. Genome Analyzer
IIx (Illumina, Inc., San Diego), or the like. Data from instrument
(122) may be organized and displayed (124) in a variety of ways. In
particular, where target nucleic acids are selected gene expression
products, e.g. mRNAs, plots may be constructed that display
per-cell expression levels of selected gene for an entire
population or subpopulation, in a manner similar to that for flow
cytometry data, as illustrated by plot (130). Each cell is
associated with a unique clonotype that is linked via the PCA
reaction to genes expressed in the cell in a proportion related to
their cellular abundance. Thus, by counting the number of expressed
gene sequences linked to a specific clonotype sequence, one obtains
a measure of expression for such gene in the cell associated with
the specific clonotype. As illustrated in plot (130), three
subpopulations of cells are indicated by the presence of separate
clusters (132, 134, and 136) based on expression levels of gene w
and gene a. In one aspect, whenever gene expression levels are
monitored, at least one gene is selected as an internal standard
for normalizing the expression measurements of other genes.
Polymerase Cycling Assembly (PCA) Reaction Formats
[0018] Polymerase cycling assembly (PCA) reactions permit a
plurality of nucleic acid fragments to be fused together to form a
single fusion product in one or more cycles of fragment annealing
and polymerase extension, e.g. Xiong et al, FEBS Micro biol. Rev.,
32: 522-540 (2008). PCA reactions come in many formats. In one
format of interest, PCA follows a plurality of polymerase chain
reactions (PCRs) taking place in a common reaction volume, wherein
each component PCR includes at least one linking primer that
permits strands from the resulting amplicon to anneal to strands
from another amplicon in the reaction and to be extended to form a
fusion product or a precursor of a fusion product. PCA in its
various formats (and under various alternative names) is a
well-known method for fragment assembly and gene synthesis, several
forms of which are disclosed in the following references: Yon et
al, Nucleic Acids Research, 17: 4895 (1989); Chen et al, J. Am.
Chem. Soc., 116: 8799-8800 (1994); Stemmer et al, Gene, 164: 49-53
(1995); Hoover et al, Nucleic Acids Research, 30: e43 (2002); Xiong
et al, Biotechnology Advances, 26: 121-134 (2008): Xiong et al,
FEBS Microbiol. Rev., 32: 522-540 (2008); and the like.
[0019] Some PCA formats useful in the present invention are
described in FIGS. 2A-2C, 3A-3C, 4A-4C, 5A-5D, and 6A-6E. FIGS.
2A-2C illustrate an exemplary PCA scheme ("Scheme 1") for joining
two separate fragments A' (208) and B' (210) into a single fusion
product (222). Fragment A' (208) is amplified with primers (204))
and (202) and fragment B' (210) is amplified with primers (206) and
(204) in the same PCR mixture. Primers (200) and (206) are "outer"
primers of the PCA reaction and primers (202) and (204) are the
"inner" primers of the PCA reaction. Inner primers (202) and (204)
each have a tail (203 and 205, respectively) that are not
complementary to A' or B' (or adjacent sequences if A' and B' are
segments imbedded in a longer sequence). Tails (203) and (205) are
complementary to one another. Generally, such inner primer tails
are selected for selective hybridization to its corresponding inner
primer (and not elsewhere); but otherwise such tails may vary
widely in length and sequence. In one aspect, such tails have a
length in the range of from 8 to 30 nucleotides; or a length in the
range of from 14 to 24 nucleotides. As the PCRs progress (212),
product fragments A (215) and B (217) are produced that incorporate
tails (203) and (205) into end regions (214) and (216),
respectively. During the PCRs product fragments A (215) and B (217)
will denature and some of the "upper" strands (215a) of A anneal
(218) to lower strands (217b) of B and the 3' ends are extended
(219) to form (220) fusion product A-B (222). Fusion product A-B
(222) may be further amplified by an excess of outer primers (200)
and (206). In some embodiments, the region of fusion product (222)
formed from tails (203) and (205) may include one or more primer
binding sites for use in later analysis, such as high-throughput
sequencing. Typically, in PDA reactions the concentrations of outer
primers are greater than the concentrations of inner primers so
that amplification of the fusion product continues after initial
formation. For example, in one embodiment for fusing two target
nucleic acids outer primer concentration may be from about 10 to
100 times that of the inner primers, e.g. 1 .mu.M for outer primers
and 0.01 .mu.M for inner primers. Otherwise, a PCA reaction may
comprise the components of a PCR.
[0020] A variation of Scheme 1 is illustrated in FIGS. 3A-3C as
Scheme 1(a). As above, fragment A (300) is amplified using primers
(304) and (306) and fragment B' (302) is amplified using primers
(308) and (312) in PCRs carried out in a common reaction mixture.
Outer primers (304) and (312) are employed as above, and inner
primer (308) has tail (310); however, instead of tail (310) being
complementary to a corresponding tail on primer (306), it is
complementary to a segment on the end of fragment A, namely, the
same segment that primer (306) is complementary to. The PCRs
produce (315) fragments A and B, where B is identical to B' (302)
with the addition of segment (316) created by tail (310) of primer
(308). As above, as temperature cycling continues (particularly as
inner primers become exhausted), the upper fragments of fragment A
anneal (318) to the lower fragment of fragment B and are extended
to produce fusion product A-B (320), which may be further amplified
using primers (304) and (312).
[0021] Another embodiment of a PCA that may be used with the
invention ("Scheme 2") is illustrated in FIGS. 4A-4C. The
embodiment is similar to that of FIGS. 2A-2C, except that outer
primers (404) and (414) have tails (408) and (418), respectively,
which permit further amplification of a fusion product with
predetermined primers. As discussed more fully below, this
embodiment is well-suited for multiplexed amplifications. Fragment
A' (400) is amplified with primers (404) and (406), having tails
(408) and (410), respectively, to produce fragment A, and fragment
B' (402) is amplified with primers (412) and (414), having tails
(416) and (418), respectively, to produce (420) fragment B. Tails
(410 and 416) of inner primers (406 and 412) are selected to
complementary (415) to one another. Ends of fragments A and B are
augmented by segments (422, 424, 426 and 428) generated by tails
(408, 410, 416 and 418, respectively). As with previously described
embodiments, upper strands of fragment A anneal (430) to lower
strands of fragment B and are extended (432) to form (434) fusion
product A-B (436) that may be further amplified (437) using primers
(438 and 440) that are the same as primers (404 and 414), but
without tails.
[0022] As mentioned above, the embodiment of FIGS. 4A-4C, may be
used in a multiplex PCA reaction, which is illustrated in FIGS.
5A-5D. There fragments A' (501), B' (502), C' (503), and D' (504)
are amplified in PCRs in a common reaction mixture using primer
sets (506 and 508) for fragment A', (514 and 516) for fragment B',
(522 and 524) for C', and (530 and 532) for D'. All primers have
tails: outer primers (506, 516, 522 and 532) each have tails (512,
520, 526 and 536, respectively) that permit both fragment
amplification and subsequent fusion product amplification.
Sequences of tails (512) and (520) may be the same or different
from the sequences of tails (526) and (536), respectively. In one
embodiment, the sequences of tails (512, 520, 526 and 536) are the
same. Tails of inner primers (518 and 510) are complementary (511)
to one another, likewise, tails of inner primers (528 and 534) are
complementary (513) to one another. The above PCRs generate
fragments A (541), B (542), C (543) and D (544), which further
anneal (546) to one another to form complexes (548 and 550) which
are extended to form fusion products A-B (552) and C-D (554),
respectively.
[0023] FIGS. 5E and 5F illustrate a generalization of the above
embodiment in which multiple different target nucleic acids (560),
A.sub.1', A.sub.2', . . . A.sub.K', are linked to the same target
nucleic acid, X' (562) to form (564) multiple fusion products
X-A.sub.1, X-A.sub.2, . . . X-A.sub.K (566). This embodiment is of
particular interest when target nucleic acid, X, is a segment of
recombined sequence of a lymphocyte, which can be used as a tag for
the lymphocyte that it originates from. In one aspect, X is a
clonotype, such as a segment of a V(D)J region of either a B cell
or T cell. In one embodiment, a plurality of target nucleic acids,
A.sub.1, A.sub.2, . . . A.sub.K, are fused to the clonotype of its
cell of origin. In another embodiment, such plurality is between 2
and 1000; and in another embodiment, it is between 2 and 100; and
in another embodiment, it is between 2 and 10. In PCA reactions of
these embodiments, the concentration of inner primer (568) may be
greater than those of inner primers of the various A; nucleic acids
so that there is adequate quantities of the X amplicon to anneal
with the many stands of the A, amplicons. In accordance with a
method of the invention, the fusion products (566) are extracted
from the reaction mixture (e.g. via conventional double stranded
DNA purification techniques, such as available from Qiagen, or the
like) and sequenced. The sequences of the outer primers may be
selected to permit direct use for cluster formation without further
manipulation for sequencing systems such as a Genome Analyzer
(Illumina, San Diego, Calif.). In one aspect, X may be a clonotype
and A.sub.1, A.sub.2, . . . A.sub.K may be particular genes or
transcripts of interest. After sequencing fusion products, per cell
gene expression levels may be tabulated and/or plotted as shown in
FIG. 1.
[0024] In addition to multiplexed PCA reactions in a parallel sense
to simultaneously generate multiple binary fusion products, as
illustrated in FIGS. 6A-6E, PCA reactions may be multiplexed in a
serial sense to assemble multi-subunit fusion products. As shown in
FIG. 6A, fragments A' (601), B' (602) and C' (603) are amplified in
a common PCR mixture with primer sets (606 and 608) for A', (610
and 612) for B' and (614 and 616) for C'. All primers have tails:
(i) tails (620 and 630) of outer primers (606 and 616) are selected
for amplification of outer fragments A' and C' and further
amplification of three-way fusion product A-B-C (662) shown in FIG.
6E; (ii) tails (622 and 624) of inner primers (608 and 610) are
complementary to one another and (iii) tails (628 and 626) of inner
primers (614 and 612) are complementary to one another. The PCRs
generate (632) fragments A (641), B (642) and C (643), which in the
reaction form (644) complexes (646 and 648) comprising segments LS1
and LS2, respectively, which in turn are extended to form (650)
fusion products A-B (652) and B-C (654). These fusion products are
denatured and some cross anneal (658) to one another by way of the
common B fragment (656) to form a complex which is extended (660)
to form fusion product A-B-C (662). Exemplary, PCA reaction
conditions for the above reaction may be as follows: 39.4 .mu.L
distilled water combined with 10 .mu.L of 10.times. buffer (IX) mM
Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, and 0.01% gelatin), 2
.mu.L of a 10 mM solution of each of the dNTPs, 0.5 .mu.L of Taq
polymerase (5 units/.mu.L), 1 .mu.L of each outer primer (from a
100 .mu.M stock solution) and 10 .mu.L of each inner primer (from a
0.1 .mu.M stock solution).
Single Cell Analysis
[0025] As mentioned above, in one aspect of the invention, cells
from a population are disposed in reactors each containing a single
cell. This may be accomplished by a variety of large-scale
single-cell reactor platforms known in the art, e.g. Clarke et at,
U.S. patent publication 2010/0255471; Mathies et al, U.S. patent
publication 2010/0285975; Edd et al, U.S. patent publication
2010/0021984; Colston et al, U.S. patent publication 2010/0173394;
Love et al, International patent publication WO2009/145925;
Muraguchi et al, U.S. patent publication 2009/0181859; Novak et al,
Angew. Chem. Int. Ed., 50: 390-395 (2011); and the like, which are
incorporated herein by reference. In one aspect, cells are disposed
in wells of a microwell array where reactions, such as PCA
reactions, take place; in another aspect, cells are disposed in
micelles of a water-in-oil emulsion, where micelles serve as
reactors. Micelle reactors generated by microfluidics devices, e.g.
Mathies et al (cited above) or Edd et al (cited above), are of
particular interest because uniform-sized micelles may be generated
and cells encounter lower shear and stress than in bulk
emulsification processes.
[0026] Cells of a sample may be suspended in a PCA reaction mixture
prior to disposition into reactors. In one aspect, a PCA reaction
mixture is substantially the same as a PCR reaction mixture with
inner at least one pair of inner primers and at least one pair of
outer primers. Optionally, a PCA reaction mixture may comprise a
lysing agent to facilitate access of the PCA reagents to target
nucleic acids of isolated cells. Lysing conditions of a PCA
reaction may vary widely and may be based on the action of heat,
detergent, protease, alkaline, or combinations of such factors. The
following references provide guidance for selection of single-cell
lysing conditions where a polymerase-based amplification, such as
PCA, is employed: Thronhill et al, Prenatal Diagnosis, 21; 490-497
(2001); Kim et al. Fertility and Sterility, 92: 814-818 (2009); and
the like. Exemplary lysis conditions for use with PCA reactions are
as follows: 1) cells in H.sub.2O at 96.degree. C. for 15 min,
followed by 15 min at 10.degree. C.; 2) 200 mM KOH, 50 mM
dithiotheitol, heat to 65.degree. C. for 10 min; 3) for 4 .mu.L
protease-based lysis buffer; 1 .mu.L of 17 .mu.M SDS combined with
3 .mu.L of 125 .mu.g/mL proteinase K, followed by incubation at
37.degree. C. for 60 min, then 95.degree. C. for 15 min (to
inactivate the proteinase K); 4) for 10 .mu.L of a detergent-based
lysis buffer; 2 .mu.L H.sub.2O, 2 .mu.L 250 ng/.mu.L polyA, 2
.mu.l, 10 mM EDTA, 2 .mu.L 250 mM dithiothreitol, 2 .mu.L 0.5%
N-laurylsarcosin salt solution. Single-cell analysis platforms,
incubation times, lysis buffer and/or PCA reaction other
components, their concentrations, reactions volumes and the like,
are design choices that are optimized for particular applications
by one of ordinary skill in the art.
Nucleic Acid Sequencing Techniques
[0027] Any high-throughput technique for sequencing nucleic acids
can be used in the method of the invention. DNA sequencing
techniques include dideoxy sequencing reactions (Sanger method)
using labeled terminators or primers and gel separation in slab or
capillary, sequencing by synthesis using reversibly terminated
labeled nucleotides, pyrosequencing, 454 sequencing, sequencing by
synthesis using allele specific hybridization to a library of
labeled clones that is followed by ligation, real time monitoring
of the incorporation of labeled nucleotides during a polymerization
step, polony sequencing, SOLiD sequencing, and the like. These
sequencing approaches can thus be used to sequence fusion products
of target nucleic acids of interest and clonotypes based on T-cell
receptors (TCRs) and/or B-cell receptors (BCRs). In one aspect of
the invention, high-throughput methods of sequencing are employed
that comprise a step of spatially isolating individual molecules on
a solid surface where they are sequenced in parallel. Such solid
surfaces may include nonporous surfaces (such as in Solexa
sequencing, e.g. Bentley et al, Nature, 456: 53-59 (2008) or
Complete Genomics sequencing, e.g. Drmanac et al. Science, 327:
78-81 (2010)), arrays of wells, which may include bead- or
particle-bound templates (such as with 454, e.g. Margulies et al,
Nature, 437: 376-380 (2005) or Ion Torrent sequencing, U.S. patent
publication 2010/0137143 or 2010/0304982), micromachined membranes
(such as with SMRT sequencing, e.g. Eid et al, Science, 323:
133-138 (2009)), or bead arrays (as with SOLiD sequencing or polony
sequencing, e.g. Kim et at, Science, 316: 1481-1414 (2007)). In
another aspect, such methods comprise amplifying the isolated
molecules either before or after they are spatially isolated on a
solid surface. Prior amplification may comprise emulsion-based
amplification, such as emulsion PCR, or rolling circle
amplification. Of particular interest is Solexa-based sequencing
where individual template molecules are spatially isolated on a
solid surface, after which they are amplified in parallel by bridge
PCR to form separate clonal populations, or clusters, and then
sequenced, as described in Bendey et al (cited above) and in
manufacturer's instructions (e.g. TruSeq.TM. Sample Preparation Kit
and Data Sheet, Illumina, Inc., San Diego, Calif., 2010); and
further in the following references: U.S. Pat. Nos. 6,090,592;
6,300,070; 7,115,400; and EP0972081B1; which are incorporated by
reference. In one embodiment, individual molecules disposed and
amplified on a solid surface form clusters in a density of at least
10.sup.5 clusters per cm.sup.2; or in a density of at least
5.times.10.sup.5 per cm; or in a density of at least 10.sup.6
clusters per cm.sup.2. In one embodiment, sequencing chemistries
are employed having relatively high error rates. In such
embodiments, the average quality scores produced by such
chemistries are monotonically declining functions of sequence read
lengths. In one embodiment, such decline corresponds to 0.5 percent
of sequence reads have at least one error in positions 1-75; 1
percent of sequence reads have at least one error in positions
76-100; and 2 percent of sequence reads have at least one error in
positions 101-125.
[0028] In one aspect of the invention, multiplex PCR is used to
amplify members of a mixture of nucleic acids, particularly
mixtures comprising recombined immune molecules such as T cell
receptors, B cell receptors, or portions thereof. Guidance for
carrying out multiplex PCRs of such immune molecules is found in
the following references, which are incorporated by reference:
Morley, U.S. Pat. No. 5,296,351: Gorski, U.S. Pat. No. 5,837,447;
Dau, U.S. Pat. No. 6,087,096: Von Dongen et al, U.S. patent
publication 2006/0234234: European patent publication EP 1544308B1;
Faham et al, U.S. patent publication 2010/0151471; Han, U.S. patent
publication 2010/0021896; Robins et al, U.S. patent publication
2010/033057; and the like. Such amplification techniques are
readily modified by those of ordinary skill in the art to supply
outer primers and linking primers of the invention.
Cancer-Related Applications
[0029] Detecting cross-lineage rearrangements. Some types of
otherwise uncommon rearrangements are common in some cancers and
thus can be used to associate them with tumor. For example, cross
lineage rearrangements, like T cell receptor (.alpha., .beta.,
.gamma. and/or .delta.) in B cells or B cell receptor (IgH, IgK,
and/or IgL) in T cells are common, especially in ALL. The presence
of cross lineage rearrangements is likely to support a malignant
origin of the clonotype. Demonstrating cross lineage rearrangement
can be done by performing linked PCR on a cell by cell basis.
Linked PCR amplifies two distinct targets (for example IgH and
TCR.beta.) and create a linked molecule between the two amplified
targets. These targets from all the amplified cells can be then
pooled and sequenced without losing the information as to whether
the two targets are expressed in the same or different cells. In
order to get amplification even in the absence of the other
rearrangement, another competing product may be used. For example,
for B cells, IgH amplification will always occur while the cross
lineage TCR.beta. may or may not occur. Two competing set of
primers can be used for the amplification of TCR.beta.: one that
amplifies the rearranged sequences and the other the germ line
sequences. Optionally, the two competing sets can be used at
different concentrations allowing the rearranged sequence to
compete more efficiently when present in the cell. All the cells
would have their IgH and TCR.beta. products amplified and linked,
and sequencing would be used to identify those cells with cross
lineage rearrangement. Methods for achieving linked PCR are
disclosed above. One method to detect cells that do not have
functional sequences uses the linked PCR technique mentioned above.
In this case, the linking has to occur for the two alleles of the
same target. For this purpose, 3 stage PCR can be performed. The
first PCR of an immune cell genomic rearrangement is done from one
cell with a set of primers (primer A and B) that allow the
rearrangement of both alleles to be amplified. Primers A and B are
then removed (e.g. by dilution) and a portion of these PCR products
can be reamplified with a second set of primers (C and D) which
also allow the same product to be amplified. Primers C and B can
additionally be designed to include a sequence homology at their 5'
termini that allows these 2 PCR products (from A/B and from C/D) to
anneal to each other and extend to produce a linked product. After
removing primers from this reaction, the two PCR products can be
mixed and reamplified by PCR using primers A and D. The result is a
linking of the two products, and in 50% of molecules they will
carry both alleles. Sequencing would identify high frequency linked
non-functional sequences. Specific high frequency non-functional
sequences that are consistently linked to a second non-functional
sequence are indicative of the potential cancer cell
[0030] In addition to serving as a marker of cells that have become
cancerous IgH is often one of the two pathological translocation
partners in lymphoid neoplasms. One example is the t(11:14) that
puts the J segment of IgH in close proximity to the cycline D1
(CCND1) gene resulting in its overexpression. This rearrangement
which is referred to as BCL1-IgH occurs in as many as 60-70% of
mantle cell lymphoma as well as other lymphoid neoplasms (e.g, 20%
of multiple myeloma). Another example is t(14:18) that puts the J
segment of IgH in close proximity to BCL2 resulting in its over
expression. This rearrangement occurs in up to 90% of follicular
lymphoma and 20% of large B cell lymphoma. These rearrangements are
typically identified by cytogenetics, Southern blotting, or FISH.
PCR has the potential to identify rearrangement at very high
sensitivity and specificity as shown by BCR-ABL for the detection
of Philadelphia chromosome. Different PCR techniques have been used
to the assessment of translocations relevant to lymphoma, with the
recently introduced real time PCR (e.g, for BCL2-IgH) being
probably the most advanced. There are a few features of BCL1-IgH
and BCL2-IgH that make their detection less sensitive and specific
than that of BCR-ABL. First, in contrast to BCR-ABL, BCL1-IgH and
BCL2-IgH do not generate a fusion protein, and there is no splicing
event that generates predictable molecular structure. Instead the
breakpoints may span a large region. There are common breakpoints
that allow the detection of up to 88% of BCL2-IgH using a
combination of primers and .about.40% of the BCL1-IgH. This results
in missing some patients that have the translocation. Second, these
rearrangements may be present in normal individuals that would
never get cancer. For example, BLC2-IgH translocation has been
found at the level of .about.10.sup.5 in a large fraction of the
normal individuals with over .about.4% carrying BCL2-IgH at a
frequency of > 1/25K. The frequency of BCL2-IgH gets higher with
increasing age. It is also hypothesized that different people may
have distinct levels of "background" translocation. Presumably the
presence of this translocation in normal sample is due to the fact
that tumorgenesis is a multi-step process and the BCL2-IgH is not
sufficient for tumors to emerge. The presence of this low level
background puts a limit on the sensitivity of detection.
[0031] Amplification of with a pool of the J primers complementary
to all the J segments and primers complementary to the regions
upstream of the BCL1 or BCL2 translocation breakpoints can be
sequenced. This can generate a method for sensitive detection of
these translocations and the cancer cells they appear in. First,
deep sequencing of individual isolated molecules (e.g, 100K or 1
million reads) can allow the detection of the appropriate sequences
from a small number of cells in a background of amplifications of
other loci. In addition, the problem of the background
translocations in normal individuals may ameliorate the problem
that real time PCR suffer from. There is evidence that, at least in
some cases, the background translocations are not clonal but rather
appear repeatedly in the same patient. Using sequencing one can
distinguish the different translocation events to obtain frequency
of the independent translocation events. Since the breakpoint of
different translocations is likely to be distinct translocation
events can be distinguished from each other. Alternatively or
additionally, a linking PCR using the translocation with a B or T
cell receptor gene can be done to provide a unique barcode for the
translocation. The linking can also be done statistically using a
set of dilution samples as described above.
[0032] Similarly additional data relating to the status of the cell
containing the cancer-related clonotype can be used to predict
likelihood of recurrence. For example, the presence of certain
markers (surface or non-surface) can be an indication of the
functional status of the cell and hence the likelihood of
recurrence. Sequencing before and after the capture of cells with
the relevant markers can determine the fraction of cells with the
cancer clonotype that carry the relevant markers. Similarly some
markers relevant to the likelihood of recurrence (e.g., expression
of some gene relating to cell growth) can be assessed at the RNA
level. This can be done by several methods including linking PCR as
described above. Finally, it is possible that the level of immune
receptor specific RNA in the tumor cell can have functional
consequence and association with the likelihood of recurrence. This
level can be assessed by doing linking PCR between a control gene 1
that can link to either the immune receptor rearrangement or
control gene 2. The relative fraction of the two products can be
indicative of the relative amount of the RNA in the cell. Another
method involves comparing the RNA level to the DNA level of the
immune receptor rearrangement. The frequency of the cancer-specific
clonotype in the DNA identifies the relative level of the
cancer-specific clonotype. The frequency of the same clonotype can
then be assessed from RNA, and the relative frequency in RNA and in
DNA can be followed. A change in this relative frequency can be
indicative of a change in the likelihood of recurrence.
[0033] While the present invention has been described with
reference to several particular example embodiments, those skilled
in the art will recognize that many changes may be made thereto
without departing from the spirit and scope of the present
invention. The present invention is applicable to a variety of
sensor implementations and other subject matter, in addition to
those discussed above.
DEFINITIONS
[0034] Unless otherwise specifically defined herein, terms and
symbols of nucleic acid chemistry, biochemistry, genetics, and
molecular biology used herein follow those of standard treatises
and texts in the field, e.g. Kornberg and Baker, DNA Replication,
Second Edition (W. H. Freeman, New York, 1992); Lehninger,
Biochemistry, Second Edition (Worth Publishers, New York, 1975);
Strachan and Read. Human Molecular Genetics. Second Edition
(Wiley-Liss, New York, 1999); Abbas et al, Cellular and Molecular
Immunology, 6.sup.th edition (Saunders, 2007).
[0035] "Amplicon" means the product of a polynucleotide
amplification reaction; that is, a clonal population of
polynucleotides, which may be single stranded or double stranded,
which are replicated from one or more starting sequences. The one
or more starting sequences may be one or more copies of the same
sequence, or they may be a mixture of different sequences.
Preferably, amplicons are formed by the amplification of a single
starting sequence. Amplicons may be produced by a variety of
amplification reactions whose products comprise replicates of the
one or more starting, or target, nucleic acids. In one aspect,
amplification reactions producing amplicons are "template-driven"
in that base pairing of reactants, either nucleotides or
oligonucleotides, have complements in a template polynucleotide
that are required for the creation of reaction products. In one
aspect, template-driven reactions are primer extensions with a
nucleic acid polymerase or oligonucleotide ligations with a nucleic
acid ligase. Such reactions include, but are not limited to,
polymerase chain reactions (PCRs), linear polymerase reactions,
nucleic acid sequence-based amplification (NASBAs), rolling circle
amplifications, and the like, disclosed in the following references
that are incorporated herein by reference: Mullis et al, U.S. Pat.
Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et
al, U.S. Pat. No. 5,210,015 (real-time PCR with "taqman" probes);
Wittwer et al, U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No.
5,399,491 ("NASBA"); Lizardi, U.S. Pat. No. 5,854,033; Aono et al,
Japanese patent publ. JP 4-262799 (rolling circle amplification);
and the like. In one aspect, amplicons of the invention are
produced by PCRs. An amplification reaction may be a "real-time"
amplification if a detection chemistry is available that permits a
reaction product to be measured as the amplification reaction
progresses, e.g. "real-time PCR" described below, or "real-time
NASBA" as described in Leone et al, Nucleic Acids Research, 26:
2150-2155 (1998), and like references. As used herein, the term
"amplifying" means performing an amplification reaction. A
"reaction mixture" means a solution containing all the necessary
reactants for performing a reaction, which may include, but not be
limited to, buffering agents to maintain pH at a selected level
during a reaction, salts, co-factors, scavengers, and the like.
[0036] "Clonotype" means a recombined nucleotide sequence of a T
cell or B cell encoding a T cell receptor (TCR) or B cell receptor
(BCR), or a portion thereof. In one aspect, a collection of all the
distinct clonotypes of a population of lymphocytes of an individual
is a repertoire of such population, e.g. Arstila et al, Science,
286: 958-961 (1999); Yassai et al, Immunogenetics, 61: 493-502
(2009); Kedzierska et al, Mol. Immunol., 45(3): 607-618 (2008); and
the like. A "clonotype profile," or "repertoire profile," is a
tabulation or representation of clonotypes of a population of T
cells and/or B cells (such as a peripheral blood sample containing
such cells) that includes substantially all of the repertoire's
clonotypes and their relative abundances. As used herein,
"clonotype profile," "repertoire profile," and "repertoire" are
used interchangeably. (That is, the term "repertoire," as discussed
more fully below, means a repertoire measured from a sample of
lymphocytes). In one aspect of the invention, clonotypes comprise
portions of an immunoglobulin heavy chain (IgH) or a TCR .beta.
chain. In other aspects of the invention, clonotypes may be based
on other recombined molecules, such as immunoglobulin light chains
or TCR.alpha. chains, or portions thereof.
[0037] "Complementarity determining regions" (CDRs) mean regions of
an immunoglobulin (i.e., antibody) or T cell receptor where the
molecule complements an antigen's conformation, thereby determining
the molecule's specificity and contact with a specific antigen. T
cell receptors and immunoglobulins each have three CDRs: CDR1 and
CDR2 are found in the variable (V) domain, and CDR3 includes some
of V, all of diverse (D) (heavy chains only) and joint (J), and
some of the constant (C) domains.
[0038] "Kit" refers to any delivery system for delivering materials
or reagents for carrying out a method of the invention. In the
context of reaction assays, such delivery systems include systems
that allow for the storage, transport, or delivery of reaction
reagents (e.g., primers, enzymes, etc. in the appropriate
containers) and/or supporting materials (e.g., buffers, written
instructions for performing the assay etc.) from one location to
another. For example, kits include one or more enclosures (e.g.,
boxes) containing the relevant reaction reagents and/or supporting
materials. Such contents may be delivered to the intended recipient
together or separately. For example, a first container may contain
an enzyme for use in an assay, while a second container contains
primers.
[0039] "Microfluidics device" means an integrated system of one or
more chambers, ports, and channels that are interconnected and in
fluid communication and designed for carrying out an analytical
reaction or process, either alone or in cooperation with an
appliance or instrument that provides support functions, such as
sample introduction, fluid and/or reagent driving means,
temperature control, detection systems, data collection and/or
integration systems, and the like. Microfluidics devices may
further include valves, pumps, and specialized functional coatings
on interior walls, e.g. to prevent adsorption of sample components
or reactants, facilitate reagent movement by electroosmosis, or the
like. Such devices are usually fabricated in or as a solid
substrate, which may be glass, plastic, or other solid polymeric
materials, and typically have a planar format for ease of detecting
and monitoring sample and reagent movement, especially via optical
or electrochemical methods. Features of a microfluidic device
usually have cross-sectional dimensions of less than a few hundred
square micrometers and passages typically have capillary
dimensions, e.g. having maximal cross-sectional dimensions of from
about 500 .mu.m to about 0.1 .mu.m. Microfluidics devices typically
have volume capacities in the range of from 1 .mu.L to a few nL,
e.g. 10-100 nL. The fabrication and operation of microfluidics
devices are well-known in the art as exemplified by the following
references that are incorporated by reference: Ramsey, U.S. Pat.
Nos. 6,001,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al,
U.S. Pat. Nos. 5,126,022 and 6,054,034; Nelson et al, U.S. Pat. No.
6,613,525: Maher et al, U.S. Pat. No. 6,399,952; Ricco et al,
International patent publication WO 02/24322; Bjornson et al,
International patent publication WO 99/19717; Wilding et al, U.S.
Pat. Nos. 5,587,128; 5,498,392; Sia et al, Electrophoresis, 24:
3563-3576 (2003); Unger et al, Science, 288: 113-116 (2000);
Enzelberger et al, U.S. Pat. No. 6,960,437.
[0040] "Polymerase chain reaction," or "PCR," means a reaction for
the in vitro amplification of specific DNA sequences by the
simultaneous primer extension of complementary strands of DNA. In
other words, PCR is a reaction for making multiple copies or
replicates of a target nucleic acid flanked by primer binding
sites, such reaction comprising one or more repetitions of the
following steps: (i) denaturing the target nucleic acid, (ii)
annealing primers to the primer binding sites, and (iii) extending
the primers by a nucleic acid polymerase in the presence of
nucleoside triphosphates. Usually, the reaction is cycled through
different temperatures optimized for each step in a thermal cycler
instrument. Particular temperatures, durations at each step, and
rates of change between steps depend on many factors well-known to
those of ordinary skill in the art, e.g. exemplified by the
references: McPherson et al, editors, PCR: A Practical Approach and
PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995,
respectively). For example, in a conventional PCR using Taq DNA
polymerase, a double stranded target nucleic acid may be denatured
at a temperature>90.degree. C. primers annealed at a temperature
in the range 50-75.degree. C., and primers extended at a
temperature in the range 72-78.degree. C. The term "PCR"
encompasses derivative forms of the reaction, including but not
limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,
multiplexed PCR, and the like. PCR reaction volumes typically range
from a few hundred nanoliters, e.g. 200 nL, to a few hundred .mu.L,
e.g. 200 .mu.L. "Reverse transcription PCR," or "RT-PCR," means a
PCR that is preceded by a reverse transcription reaction that
converts a target RNA to a complementary single stranded DNA, which
is then amplified, e.g. Tecott et al, U.S. Pat. No. 5,168,038,
which patent is incorporated herein by reference. "Real-time PCR"
means a PCR for which the amount of reaction product, i.e.
amplicon, is monitored as the reaction proceeds. There are many
forms of real-time PCR that differ mainly in the detection
chemistries used for monitoring the reaction product, e.g. Gelfand
et al, U.S. Pat. No. 5,210,015 ("taqman"); Wittwer et al, U.S. Pat.
Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al,
U.S. Pat. No. 5,925,517 (molecular beacons); which patents are
incorporated herein by reference. Detection chemistries for
real-time PCR are reviewed in Mackay et al, Nucleic Acids Research,
30: 1292-1305 (2002), which is also incorporated herein by
reference. "Nested PCR" means a two-stage PCR wherein the amplicon
of a first PCR becomes the sample for a second PCR using a new set
of primers, at least one of which binds to an interior location of
the first amplicon. As used herein, "initial primers" in reference
to a nested amplification reaction mean the primers used to
generate a first amplicon, and "secondary primers" mean the one or
more primers used to generate a second, or nested, amplicon.
"Multiplexed PCR" means a PCR wherein multiple target sequences (or
a single target sequence and one or more reference sequences) are
simultaneously carried out in the same reaction mixture, e.g.
Bernard et al, Anal. Biochem., 273: 221-228 (1999)(two-color
real-time PCR). Usually, distinct sets of primers are employed for
each sequence being amplified. "Quantitative PCR" means a PCR
designed to measure the abundance of one or more specific target
sequences in a sample or specimen. Quantitative PCR includes both
absolute quantitation and relative quantitation of such target
sequences. Quantitative measurements are made using one or more
reference sequences that may be assayed separately or together with
a target sequence. The reference sequence may be endogenous or
exogenous to a sample or specimen, and in the latter case, may
comprise one or more competitor templates. Typical endogenous
reference sequences include segments of transcripts of the
following genes: .beta.-actin, GAPDH, .beta.-microglobulin,
ribosomal RNA, and the like. Techniques for quantitative PCR are
well-known to those of ordinary skill in the art, as exemplified in
the following references that are incorporated by reference:
Freeman et al, Biotechniques, 26: 112-126 (1999); Becker-Andre et
al, Nucleic Acids Research, 17: 9437-9447 (1989); Zimmerman et al,
Biotechniques, 21: 268-279 (1996); Diviacco et al, Gene, 122:
3013-3020 (1992); Becker-Andre et al, Nucleic Acids Research, 17:
9437-9446 (1989); and the like.
[0041] "Polymerase cycling assembly" or "PCA" reaction (also
referred to herein as "linked PCR") means a PCR that comprises at
least one pair of outer primers and at least one pair of inner
primers. An inner primer has a 3' portion that is complementary to
a target nucleic acid (or its complement) and a 5' portion that is
complementary to the 5' portion of another inner primer
corresponding to a different target nucleic acid.
[0042] "Primer" means an oligonucleotide, either natural or
synthetic that is capable, upon forming a duplex with a
polynucleotide template, of acting as a point of initiation of
nucleic acid synthesis and being extended from its 3' end along the
template so that an extended duplex is formed. Extension of a
primer is usually carried out with a nucleic acid polymerase, such
as a DNA or RNA polymerase. The sequence of nucleotides added in
the extension process is determined by the sequence of the template
polynucleotide. Usually primers are extended by a DNA polymerase.
Primers usually have a length in the range of from 14 to 40
nucleotides, or in the range of from 18 to 36 nucleotides. Primers
are employed in a variety of nucleic amplification reactions, for
example, linear amplification reactions using a single primer, or
polymerase chain reactions, employing two or more primers. Guidance
for selecting the lengths and sequences of primers for particular
applications is well known to those of ordinary skill in the art,
as evidenced by the following references that are incorporated by
reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual,
2.sup.nd Edition (Cold Spring Harbor Press, New York, 2003).
[0043] "Repertoire" means a set of distinct recombined nucleotide
sequences that encode T cell receptors (TCRs) or B cell receptors
(BCRs), or fragments thereof, respectively, in a population of
lymphocytes of an individual, wherein the nucleotide sequences of
the set have a one-to-one correspondence with distinct lymphocytes
or their clonal subpopulations for substantially all of the
lymphocytes of the population. Member nucleotide sequences of a
repertoire are referred to herein as a "clonotype." In one aspect,
a repertoire comprises any segment of nucleic acid common to a T
cell or a B cell population which has undergone somatic
recombination during the development of TCRs or BCRs, including
normal or aberrant (e.g. associated with cancers) precursors
thereof, including, but not limited to, any of the following: an
immunoglobulin heavy chain (IgH) or subsets thereof (e.g. an IgH
variable region, CDR3 region, or the like), an immunoglobulin light
chain or subsets thereof (e.g. a variable region, CDR region, or
the like), T cell receptor .alpha. chain or subsets thereof, T cell
receptor .beta. chain or subsets thereof (e.g. variable region,
CDR3, V(D)J region, or the like), a CDR (including CDR1, CDR2 or
CDR3, of either TCRs or BCRs, or combinations of such CDRs), V(D)J
regions of either TCRs or BCRs, hypermutated regions of IgH
variable regions, or the like. In one aspect, a repertoire is
selected so that its diversity (i.e. the number of distinct nucleic
acid sequences in the set) is large enough so that substantially
every T cell or B cell or clone thereof in an individual carries a
unique nucleic acid sequence of such repertoire. That is, in
accordance with the invention, a practitioner may select for
defining clonotypes a particular segment or region of recombined
nucleic acids that encode TCRs or BCRs that do not reflect the full
diversity of a population of T cells or B cells, however,
preferably, clonotypes are defined so that they do reflect the
diversity of the population of T cells and/or B cells from which
they are derived. That is, preferably each different clone of a
sample has different clonotype. In other aspects of the invention,
the population of lymphocytes corresponding to a repertoire may be
circulating B cells, or may be circulating T cells, or may be
subpopulations of either of the foregoing populations, including
but not limited to, CD4+ T cells, or CD8+ T cells, or other
subpopulations defined by cell surface markers, or the like. In one
embodiment, a repertoire of human TCR (i chains comprises a number
of distinct nucleotide sequences in the range of from
0.3.times.10.sup.6 to 1.8.times.10.sup.6, or in the range of from
0.5.times.10.sup.6 to 1.5.times.10.sup.6, or in the range of from
0.8.times.10.sup.6 to 1.2.times.10.sup.6. Such subpopulations may
be acquired by taking samples from particular tissues, e.g. bone
marrow, or lymph nodes, or the like, or by sorting or enriching
cells from a sample (such as peripheral blood) based on one or more
cell surface markers, size, morphology, or the like. In still other
aspects, the population of lymphocytes corresponding to a
repertoire may be derived from disease tissues, such as a tumor
tissue, an infected tissue, or the like. In a particular
embodiment, a repertoire of the invention comprises a set of
nucleotide sequences encoding substantially all segments of the
V(D)J region of an IgH chain. In one aspect, "substantially all" as
used herein means every segment having a relative abundance of
0.001 percent or higher: or in another aspect, a relative abundance
of 0.0001 percent or higher. In another particular embodiment, a
repertoire of the invention comprises a set of nucleotide sequences
that encodes substantially all segments of the V(D)J region of a
TCR .beta. chain. In another embodiment, a repertoire of the
invention comprises a set of nucleotide sequences having lengths in
the range of from 25-200 nucleotides and including segments of the
V, D, and J regions of a TCR .beta. chain. In another embodiment, a
repertoire of the invention comprises a set of nucleotide sequences
having lengths in the range of from 25-200 nucleotides and
including segments of the V, D, and J regions of an IgH chain. In
another embodiment, a repertoire of the invention comprises a
number of distinct nucleotide sequences that is substantially
equivalent to the number of lymphocytes expressing a distinct IgH
chain. In another embodiment, a repertoire of the invention
comprises a number of distinct nucleotide sequences that is
substantially equivalent to the number of lymphocytes expressing a
distinct TCR .beta. chain. In still another embodiment,
"substantially equivalent" means that with ninety-nine percent
probability a repertoire of nucleotide sequences will include a
nucleotide sequence encoding an IgH or TCR .beta. or portion
thereof carried or expressed by every lymphocyte of a population of
an individual at a frequency of 0.001 percent or greater. In still
another embodiment, "substantially equivalent" means that with
ninety-nine percent probability a repertoire of nucleotide
sequences will include a nucleotide sequence encoding an IgH or TCR
.beta. or portion thereof carried or expressed by every lymphocyte
present at a frequency of 0.0001 percent or greater. The foregoing
sets of clonotypes are sometimes referred to herein as representing
the "full repertoire" of IgH and/or TCR.beta. sequences.
* * * * *