WO2011123473A1

WO2011123473A1 - Method for improving detection of b cell immunoglobulin gene recombination

Info

Publication number: WO2011123473A1
Application number: PCT/US2011/030398
Authority: WO
Inventors: Jian Han
Original assignee: Jian Han
Priority date: 2010-03-29
Filing date: 2011-03-29
Publication date: 2011-10-06
Also published as: JP2013523129A; AU2011235281A1; CN103154015A; EP2558479A1; CA2794800A1; EP2558479A4

Abstract

Disclosed is a method for generating primers to increase the yield of PCR products that represent the various genetic recombination events and antibodies that exist in a sample from a human or animal.

Description

METHOD FOR IMPROVING DETECTION OF B CELL IMMUNOGLOBULIN

GENE RECOMBINATION

Γ0011 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

Γ0021 The present invention relates to methods for identifying and guantifving 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

Γ0031 Once considered a "backwater" of scientific studv, immunology has become an integral part of the studv of the bodv and its status in both health and disease. Scientists are continually finding links between the bodv's natural defense system and diseases once thought to have nothing to do with immunity. Cells of the immune svstem provide the most significant part of the inflammatorv response, and inflammation mav be associated with diseases as diverse as cardiovascular disease, kidney disease, diabetes, arthritis, and cancer. The inflammatorv response must be carefullv 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, "autoimmune diseases" such as Systemic Lupus Erythematosus (SLE^"), Rheumatoid Arthritis (RA), and Sarcoidosis.

Γ0041 Dr. Anthony Fauci, M .D., Director of the United States National Institute of Allergy and Infectious Disease (NIAID^"), has been quoted as saving that "Tdlefininq the status of the human immune system in health and disease is a maior qoal of human immunoloqv research" (NIH News, March 8, 2010, http: //www.nih.aov.news/health/mar2010/niaid-08a .htmy Scientists at NIAID recoanized that "Tclurrent methods that examine aene expression differences in mixtures of immune cells in blood do not take into account that, even amona healthy individuals, there is a wide ranae of variation in the proportion of each cell tvpe" (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 throuah the use of microarrav technoloav ^~ cell specific sianificance analysis of microarravs (csSAM^").

Γ0051 The antibody response provided by B Cells produces a sianificant dearee of diversity in the response of the immune svstem to challenae. Challenaed by antiaen, B cells miarate into B cell follicles and establish aerminal centers (GCs). Rearranaed immunoalobulin aenes, themselves a significant source of diversity, are further modified bv class- switch recombination of the constant regions and somatic hvpermutation of the hvpervariable regions. The mutation rate in these regions has been estimated to be about 10⁶ higher than that of spontaneous genetic mutations.

Γ0061 What are needed are more sensitive methods for detection of B cell diversity for providing a better understanding of the status of the immune svstem in health and disease.

Summary of the Invention

Γ0071 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

Γ0081 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 vields of PCR products using control primers (without randomlv-qenerated nucleotide sequence at the 3' end^"), and 4-6 illustrate the relative numbers of amplified sequences usinq experimental primers (with randomlv-qenerated 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 neqative control. Lane 4 represents amplification products from RNA isolated from a normal individual, lane 5 from a CLL patient, and Lane 6 is a neqative control. Amplification conditions were the same. The addition of the 3' randomlv-qenerated nucleotides to the primers, as shown bv the difference in intensity between the bands in lanes 1, 2, 4, and 5, produced siqnificantlv more amplification products.

Γ0091 Fiqure 2 illustrates the results of sequencinq of multiple tarqets for detection of rearranqements from different individuals. Detection of these rearranqements and their relative frequencies, the absence of certain sequences, etc., mav provide valuable information about the status of the immune svstem and its role in health and disease.

Detailed Description

Γ0101 The inventor has developed a new method for improvinq PCR amplification of immunoqlobulin recombination reqions and increasinq the 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 immunoglobulin variable regions. The method provides increased numbers of amplification products as compared with amplification reactions which utilize primers without randomlv-generated nucleotides at one or both ends.

Γ0111 In the germinal centers, B cells modify rearranged

immunoglobulin genes bv somatic hvpermutation. 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 readilv bind to these seguences.

However, to detect the recombinations that represent a real population of antibodv molecular recombinations, the inventor has discovered that the number of detectable molecules can be increased bv the use of primers that bind at the junction of the hypervariable region and take into account the highly variable nature of the seguence in this region.

Γ0121 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 seguences" represent the various genetic rearrangements that have occurred within the bodv, resulting in a diverse variety of antibodies.

Γ0131 Amplification mav be performed by a variety of methods known to those of skill in the art, and this mav be made easier bv 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.

Γ0141 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 sequences (IgHV^"). 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 bv certain principles that are known in the art. Tarqet-specific primers mav be used to amplify one or more (and preferably multiple^") target nucleic acids. Nested primer concentration mav generally be between 5-50 pmol. Selected primers are "tagged" with additional nucleotides to provide an additional seguence that is not specific for the target nucleic acidfs^") 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, mav 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 bv common primers which will, in a relatively indiscriminate manner, provide

amplification of unrelated nucleotide seguences represented bv the variety of amplicons rescued from the target-specific reaction. Γ0151 Amplicon rescue is performed to minimize or eliminate the primers of the first reaction, while providinq amplicons for use in the second amplification usinq common primers. Amplicon rescue mav be performed in a variety of wavs. For example, a small samplinq from the completed first amplification reaction mav 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 siqnificantlv decreasinq (i.e.., dilutinq^") the remaininq numbers of primers of the first amplification. Amplicon rescue mav also be performed bv removinq a siqnificant portion of the contents of the reaction svstem of the first amplification and addinq to the remaininq contents the common primer(s^") with the necessarv

enzvme(s^"), nucleotides, buffer(s^"), and/or other reaqents to perform a second amplification utilizinq the common primer(s^") to amplify the rescued amplicons in a second reaction svstem. Separation techniques mav also be utilized to rescue amplicons. Such techniques mav relv on size differences between the primers and amplicons, on taqs 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 mav be used in the second amplification. Γ0161 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.

Γ0171 Bv 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 onlv the kinds and numbers of nucleic acids present from a particular target, and sensitivity achieved bv 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 copv numbers. Furthermore, the use of high- concentration primers in a first amplification, followed bv amplicon rescue— particularly when amplicon rescue is performed bv isolating a portion of the first amplification bv either removing that portion and placing it into a new reaction svstem or bv removing a significant portion of the first amplification and adding to that the necessarv reagents to form a second reaction svstem for a second, target-independent amplification— lends itself to automation. Not onlv can these steps be performed within a relatively closed reaction system, which limits the possibility of contamination, but the combination of first amplification, amplicon rescue, and second amplification provided bv the method produces a specific, sensitive detection method for multiple targets from multiple samples within a period of less than 2 hours.

Γ0181 Both the ARM-PCR and the TEM-PCR methods have been used bv the inventor to amplify multiple target seguences, 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 seguences. As an example, both the ARM-PCR and TEM- PCR methods were made more efficient for amplifying multiple targets from a B-cell sample bv the addition of 3 randomlv-generated nucleotides at the 3' end of the primer seguences. This provides at least 64 different possibilities for detection of additional targets which mav have

hvpermutations 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.

Γ0191 Additional methods for amplification of multiple targets mav also be used with the method of the invention, and the ARM-PCR and TEM- PCR methods are provided as examples of methods that have successfully been used bv the inventor for accomplishinq the desired method of amplification of multiple targets.

Γ0201 One maior problem that exists with hiqh throughput sequencing is that primer di-mers interfere with amplification and are verv difficult to remove. Bv usinq forward-in and reverse-in primers that are 40 base pairs lonq, a di-mer could be as lonq as 80 base pairs. Products produced bv conventional methods mav be as short as 150-250 base pairs. With the new primer desiqn provided bv the method of the invention, combined with the step of movinq the primers outwards to amplify and sequence a lonqer insert, the PCR products are over 350bp, makinq it much easier to separate the products from di-mers.

Γ0211 The method of the invention is useful for producinq 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 sinqle individual so that it will be easier to ascertain which rearranqements mav be present at a qreater frequency, which mav be absent entirely, etc. Previously, this was made more difficult bv the fact that certain clones were difficult to detect due to the mismatched pairing of primers due to hvpermutations leading to the production of those clones.

Γ0221 Randomlv-generated nucleotides mav be added to the primer seguence at the 5' end, the 3' end, or at both ends, although in the inventor's experience with Ig molecules and ARM-PCR/TEM-PCR, thev have been most useful when added to the 3' end. Randomlv-generated nucleotides mav 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 randomlv-generated nucleotides at the 3' end have provided outstanding results in the amplification and detection of recombinant immunoglobulin seguences from human blood, particularly when annealing temperatures in the higher ranges used in PCR reactions have been used.

Γ0231 The invention mav be further described bv means of the following examples:

Examples

Γ0241 Blood samples were obtained from Conversant Healthcare Systems, Inc., Huntsville, Alabama, for the amplifications shown in Fig. 1. For a first amplification reaction using the Oiagen OneStep RT-PCR Kit (Qiagen, Carlsbad, California^"), mRNA was extracted using a Oiagen kit. To the sample was added Reverse Transcriptase: 50° C, for 40 min (30 min minimum RJ) 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 cvcles. A 2-step cvclinq of 94°C, 30 sec→72°C, 2 min was performed for 15 cvcles, with a final extension at 72°C for 10 min. In a second amplification reaction usinq the Oiaqen 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 cvcles, with final extension at 72°C for 5 min . Recombinant RNasin® Ribonuclease Inhibitor from Promeqa was also added .

Claims

What is claimed is:

1. A method for increasinq the number of amplification products from a primer-generated amplification of recombined immunoglobulin sequences from a sampie from a human or animal, the method comprising incorporating into at least one primer used for the primer- generated amplification from about 2 to about 5 randomly generated nucleotides at the 3' end, the 5' end, or at both the 3' end and the 5' end of the primer sequence.

2. The method of claim 1 wherein the randomly generated nucleotides are incorporated into the 3' end of the primer sequence,

3. The method of claim 1 wherein 3 randomly generated nucleotides are incorporated into the primer sequence at the 3' end, the 5' end, or at both the 3' end and the 5' end of the primer sequence,

4. The method of claim 1 wherein 3 randomly generated nucleotides are incorporate into the 3' end of the primer sequence,