Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR IMPROVING DETECTION OF B CELL IMMUNOGLOBULIN
GENE RECOMBINATION
10011 This application claims the benefit of priority of United States
Provisional Patent Application number 61/318,417, filed March 29, 2010.
Field of the Invention
10021 The present invention relates to methods for identifying and
quantifying B-cell immunoglobulin gene recombination. More specifically,
the invention relates to methods for designing primers for increasing the
number of PCR-amplified products from immunoglobulin cDNA and/or RNA.
Background of the Invention
10031 Once considered a "backwater" of scientific study, immunology
has become an integral part of the study of the body and its status in both
health and disease. Scientists are continually finding links between the
body's natural defense system and diseases once thought to have nothing to
do with immunity. Cells of the immune system provide the most significant
part of the inflammatory response, and inflammation may be associated with
diseases as diverse as cardiovascular disease, kidney disease, diabetes,
arthritis, and cancer. The inflammatory response must be carefully
balanced, and when that balance is not maintained it can result in diseases
caused by immune deficiency or, at the other end of the spectrum,
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" autoimmune diseases" such as Systemic Lupus Erythematosus (SLE),
Rheumatoid Arthritis (RA), and Sarcoidosis.
(0041 Dr. Anthony Fauci, M.D., Director of the United States National
Institute of Allergy and Infectious Disease (NIAID), has been quoted as
saving that "Fdlefininq the status of the human immune system in health
and disease is a maior goal of human immunology research" (NIH News,
March 8, 2010, http://www.nih.oov.news/health/mar2010/niaid-08a.htm).
Scientists at NIAID recognized that "Fclurrent methods that examine gene
expression differences in mixtures of immune cells in blood do not take into
account that, even among healthy individuals, there is a wide range of
variation in the proportion of each cell type" (Dr. Mark Davis, NIH News,
March 8, 2010). Their team's approach to the problem is a new
mathematical approach to analyze molecular data obtained through the use
of microarrav technology - cell specific significance analysis of microarravs
(csSAM).
(0051 The antibody response provided by B Cells produces a
significant degree of diversity in the response of the immune system to
challenge. Challenged by antigen, B cells migrate into B cell follicles and
establish germinal centers (GCs). Rearranged immunoglobulin genes,
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themselves a significant source of diversity, are further modified by class-
switch recombination of the constant regions and somatic hypermutation of
the hypervariable regions. The mutation rate in these regions has been
estimated to be about 106 higher than that of spontaneous genetic
mutations.
10061 What are needed are more sensitive methods for detection of B
cell diversity for providing a better understanding of the status of the
immune system in health and disease.
Summary of the Invention
10071 The invention relates to a method for improving PCR
amplification of immunoglobulin recombination regions and increasing the
number of detectable recombinant molecules, the method comprising adding
from about 3 to about 5 randomly generated nucleotides to the 3' end, the
5' end, or both the 3' and 5' ends, of at least one primer for PCR
amplification of one or more immunoglobulin variable regions.
Brief Description of the Drawings
10081 Figure 1 illustrates the results of PCR amplification of
immunoglobulin variable regions from a population of B cells from a human
patient. Lanes 1-3 illustrate the relative yields of PCR products using
control
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primers (without randomly-generated nucleotide sequence at the 3' end),
and 4-6 illustrate the relative numbers of amplified sequences using
experimental primers (with randomly-generated nucleotide sequence at the
3'end). Lane 1 represents amplification products from RNA of a normal
individual, Lane 2 from a CLL patient, and Lane 3 is a blank as negative
control. Lane 4 represents amplification products from RNA isolated from a
normal individual, lane 5 from a CLL patient, and Lane 6 is a negative
control. Amplification conditions were the same. The addition of the 3,
randomly-generated nucleotides to the primers, as shown by the difference
in intensity between the bands in lanes 1, 2, 4, and 5, produced significantly
more amplification products.
(0091 Figure 2 illustrates the results of sequencing of multiple targets
for detection of rearrangements from different individuals. Detection of
these rearrangements and their relative frequencies, the absence of certain
sequences, etc., may provide valuable information about the status of the
immune system and its role in health and disease.
Detailed Description
(0101 The inventor has developed a new method for improving PCR
amplification of immunoglobulin recombination regions and increasing the
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number of detectable recombinant molecules from a sample of a B cell
population of a human and/or animal. The invention comprises adding from
about 3 to about 5 randomly generated nucleotides to the 3' end, the 5' end,
or both the 3' and 5' ends, of at least one primer for PCR amplification of
one or more immunocilobulin variable regions. The method provides
increased numbers of amplification products as compared with amplification
reactions which utilize primers without randomly-generated nucleotides at
one or both ends.
10111 In the germinal centers, B cells modify rearranged
immunoglobulin genes by somatic hypermutation. This hypermutation
provides additional diversity and antigen-binding specificity. It also
introduces mutations in and around the V-region of the immunoglobulin
heavy and light chain genes. Primer design has recently improved because
computer programs have been developed to assist in the design of
degenerate primers that will more readily bind to these sequences.
However, to detect the recombinations that represent a real population of
antibody molecular recombinations, the inventor has discovered that the
number of detectable molecules can be increased by the use of primers that
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bind at the function of the hypervariable region and take into account the
highly variable nature of the sequence in this region.
10121 The invention provides a method for amplifying RNA and/or
cDNA from a human or animal blood sample. Samples may, however, also
be taken from bone marrow or other B-cell sources in the human or animal
body. "Recombined immunoglobulin sequences" represent the various
genetic rearrangements that have occurred within the body, resulting in a
diverse variety of antibodies.
10131 Amplification may be performed by a variety of methods known
to those of skill in the art, and this may be made easier by the use of
commercially available kits. Methods for amplifying multiple targets from a
single sample have been described, for example, in U.S Patent Application
Publication Number 20070141575, which describes a method known as
TEM-PCR, and U.S. Patent Application Publication Number 20090253183,
which describes a method known as ARM-PCR.
10141 In the first step of the ARM-PCR method, for example, high-
concentration, target-specific, nested primers are used to perform a target-
specific first amplification procedure. Primers are selected based upon their
potential for binding to known immunoglobulin heavy chain variable region
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sequences (IcHV). As mentioned previously, a number of computer
programs are available for aiding in the selection of primers, and to those of
skill in the art primer selection is made easier by certain principles that
are
known in the art. Target-specific primers may be used to amplify one or
more (and preferably multiple) target nucleic acids. Nested primer
concentration may generally be between 5-50 pmol. Selected primers are
" tapped" with additional nucleotides to provide an additional sequence that
is not specific for the target nucleic acid(s) so that amplification of the
target
nucleic acid with such a primer will also incorporate into the resulting
amplicon a binding site for a common primer that, unlike a target-specific
primer, may be used to further amplify unrelated target nucleic acid
amplicons. Amplification is performed for approximately 10-15 cycles, the
reaction is terminated, and the resulting amplicons are rescued from the
reaction mix for use in a second, target-independent amplification
procedure, comprising a polymerase chain reaction primed by common
primers which will, in a relatively indiscriminate manner, provide
amplification of unrelated nucleotide sequences represented by the variety of
amplicons rescued from the target-specific reaction.
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(0151 Amplicon rescue is performed to minimize or eliminate the
primers of the first reaction, while providing amplicons for use in the second
amplification using common primers. Amplicon rescue may be performed in
a variety of ways. For example, a small sampling from the completed first
amplification reaction may be taken to provide amplicons for the second
amplification. When a small sample is taken, it provides sufficient numbers
of amplicons for the second amplification, while significantly decreasing
(i.e.., diluting) the remaining numbers of primers of the first amplification.
Amplicon rescue may also be performed by removing a significant portion of
the contents of the reaction system of the first amplification and adding to
the remaining contents the common primer(s) with the necessary
enzyme(s), nucleotides, buffer(s), and/or other reagents to perform a
second amplification utilizing the common primer(s) to amplify the rescued
amplicons in a second reaction system. Separation techniques may also be
utilized to rescue amplicons. Such techniques may rely on size differences
between the primers and amplicons, on taps that have been attached to the
amplicons, the primers, or both, or other methods known to those of skill in
the art. Once separated, all of the rescued amplicons or a part of the
rescued amplicons may be used in the second amplification.
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(0161 In ARM-PCR, the second amplification is performed using fresh
buffer, nucleotides, and common primer(s). Common primers are chosen to
provide efficient amplification of the rescued amplicons to provide
significant
numbers of copies of those amplicons at the end of the second amplification.
(0171 By separating the reactions into a first, target-specific primer-
driven amplification and a second, target-independent common primer-
driven amplification, the method provides specificity through the use of
target-specific primers to amplify only the kinds and numbers of nucleic
acids present from a particular target, and sensitivity achieved by the use of
nested primers, the high concentration of target-specific primers, and the
use of the common primer(s) to provide non-specific (target-independent)
amplification at higher copy numbers. Furthermore, the use of high-
concentration primers in a first amplification, followed by amplicon rescue-
particularly when amplicon rescue is performed by isolating a portion of the
first amplification by either removing that portion and placing it into a new
reaction system or by removing a significant portion of the first
amplification
and adding to that the necessary reagents to form a second reaction system
for a second, target-independent amplification-lends itself to automation.
Not only can these steps be performed within a relatively closed reaction
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system, which limits the possibility of contamination, but the combination of
first amplification, amplicon rescue, and second amplification provided by the
method produces a specific, sensitive detection method for multiple targets
from multiple samples within a period of less than 2 hours.
10181 Both the ARM-PCR and the TEM-PCR methods have been used
by the inventor to amplify multiple target sequences, and when combined
with the method of the present invention for generating primers which
increase the number of detectable targets in a sample of immunoglobulin
RNA and/or cDNA sequences. As an example, both the ARM-PCR and TEM-
PCR methods were made more efficient for amplifying multiple targets from
a B-cell sample by the addition of 3 randomly-generated nucleotides at the
3' end of the primer sequences. This provides at least 64 different
possibilities for detection of additional targets which may have
hypermutations that would, if primers without the randomly-generated ends
were used, not be detectable because the primer mismatch would result in
decreased binding and lack of amplification of a target with such a
mismatch.
10191 Additional methods for amplification of multiple targets may
also be used with the method of the invention, and the ARM-PCR and TEM-
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PCR methods are provided as examples of methods that have successfully
been used by the inventor for accomplishing the desired method of
amplification of multiple targets.
10201 One maior problem that exists with high throughput
sequencing is that primer di-mers interfere with amplification and are very
difficult to remove. By using forward-in and reverse-in primers that are 40
base pairs long, a di-mer could be as long as 80 base pairs. Products
produced by conventional methods may be as short as 150-250 base pairs.
With the new primer design provided by the method of the invention,
combined with the step of moving the primers outwards to amplify and
sequence a longer insert, the PCR products are over 350bp, making it much
easier to separate the products from di-mers.
10211 The method of the invention is useful for producing primers
that will more efficiently amplify antibody sequences that were previously
difficult to detect. This will make it easier to amplify the variety of
sequences that are present in a sample taken from any single individual so
that it will be easier to ascertain which rearrangements may be present at a
greater frequency, which may be absent entirely, etc. Previously, this was
made more difficult by the fact that certain clones were difficult to detect
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due to the mismatched pairing of primers due to hypermutations leading to
the production of those clones.
(0221 Randomly-generated nucleotides may be added to the primer
sequence at the 5' end, the 3' end, or at both ends, although in the
inventor's experience with Iq molecules and ARM-PCR/TEM-PCR, they have
been most useful when added to the 3' end. Randomly-generated
nucleotides may also comprise from about 2 to about 5 of the nucleotides at
either the 3' or the 5' end of the polynucleotide primer. Results with 3
randomly-generated nucleotides at the 3' end have provided outstanding
results in the amplification and detection of recombinant immunoglobulin
sequences from human blood, particularly when annealing temperatures in
the higher ranges used in PCR reactions have been used.
(0231 The invention may be further described by means of the
following examples:
Examples
(0241 Blood samples were obtained from Conversant Healthcare
Systems, Inc., Huntsville, Alabama, for the amplifications shown in Fiq. 1.
For a first amplification reaction using the Qiagen OneStep RT-PCR Kit
(Qiagen, Carlsbad, California), mRNA was extracted using a Qiagen kit. To
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the sample was added Reverse Transcriptase: 50 C, for 40 min (30 min
minimum RT) for an initial PCR activation at 95 C for 15
minutes. Enrichment cvclinq was performed at 94 C, 30 sec--->63 C, 2
min--->72 C, 30 sec for 15 cycles. A 2-step cvclinq of 94 C, 30 sec--->72 C, 2
min was performed for 15 cycles, with a final extension at 72 C for 10 min.
In a second amplification reaction using the Qiagen Multiplex PCR Kit, the
initial PCR activation was performed at 95 C for 15 min, followed 3-step
cvclinq: 94 C, 30 sec--->55 C, 30 sec--->72 C, 30 sec for 40 cycles, with
final
extension at 72 C for 5 min. Recombinant RNasin`'-) Ribonuclease Inhibitor
from Promega was also added.
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