JP2013523129A - Method for improving detection of B cell immunoglobulin genetic recombination - Google Patents

Abstract

Disclosed are methods for generating primers that increase the production of PCR products representing various genetic recombination events and antibodies present in human or animal derived samples.

Description

  This application claims the benefit of priority of US Provisional Patent Application No. 61 / 318,417, filed March 29, 2010.

The present invention relates to methods for identifying and quantifying B cell immunoglobulin genetic recombination. More specifically, the present invention relates to a method for designing primers for increasing the number of PCR amplification products from immunoglobulin cDNA and / or RNA.

BACKGROUND OF THE INVENTION Immunology, once considered a “stagnation field” in scientific research, has become an integral part of the study of conditions in both the body and its health and disease. Scientists are continually discovering an association between the body's natural defense system and disease, once thought to have nothing to do with immunity. Cells of the immune system provide the most important part of the inflammatory response, and inflammation can be associated with a variety of diseases such as cardiovascular disease, kidney disease, diabetes, arthritis, and cancer. The inflammatory response must be carefully balanced, and if that balance is not maintained, the disease caused by immune deficiency, or its opposite systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and It can lead to “autoimmune diseases” such as sarcoidosis.

  Dr. Anthony Fauci, MD, director of the National Institute of Allergy and Infectious Disease (NIAID), said, “Understanding the state of the human immune system in health and disease Is a major goal of immunological research ”(NIH News, March 8, 2010, http://www.nih.gov.news/health/mar2010/niaid-08a.htm Patent Document 1)). NIAID scientists said, “Current methods of examining gene expression differences in a mixture of immune cells in the blood take into account the wide variation in the proportion of each cell type, even among healthy individuals. (Dr. Mark Davis, NIH News, March 8, 2010 (Non-Patent Document 2)). Their team approach to the challenge is a new mathematical approach to analyze molecular data obtained through the use of microarray technology-cell specific microarray significance analysis (csSAM).

The antibody response provided by B cells creates a considerable degree of diversity relative to the immune system's response to antigenic stimulation. When stimulated by an antigen, B cells migrate into the B cell follicle and build the germinal center (GC). Rearranged immunoglobulin genes, which are themselves an important source of diversity, are further modified by constant region class switch recombination and hypervariable region somatic hypermutation. The mutation rate in these regions is estimated to be about 10 ⁶ higher than spontaneous gene mutations.

  What is needed is a more sensitive method for detecting B cell diversity to provide a better understanding of the state of the immune system in health and disease.

Dr. Anthony Fauci, M.D., NIH News, March 8, 2010, http://www.nih.gov.news/health/mar2010/niaid-08a.htm Dr. Mark Davis, NIH News, March 8, 2010

  The present invention is a method for improving PCR amplification of immunoglobulin recombination regions and increasing the number of detectable recombinant molecules, comprising at least for PCR amplification of one or more immunoglobulin variable regions. It relates to a method comprising adding about 3 to about 5 randomly generated nucleotides to the 3 ′ end, 5 ′ end, or both 3 ′ and 5 ′ ends of one primer.

FIG. 6 illustrates the results of PCR amplification of immunoglobulin variable regions from a B cell population derived from a human patient. Lanes 1-3 illustrate the relative production of PCR products by using control primers (which do not have a randomly generated nucleotide sequence at the 3 'end), lanes 4-6 are tested Figure 2 illustrates the relative number of sequences amplified by using primers (having a randomly generated nucleotide sequence at the 3 'end). Lane 1 represents the amplification product from normal individual RNA, lane 2 represents from a CLL patient, and lane 3 is a blank as a negative control. Lane 4 represents the amplification product from RNA isolated from normal individuals, lane 5 represents from a CLL patient, and lane 6 is a negative control. Amplification conditions were the same. Significantly more amplification product is produced by adding a randomly generated nucleotide 3 'to the primer, as shown by the intensity difference between bands in lanes 1, 2, 4, and 5. It was. FIG. 4 illustrates the results of multiple target sequencing for the detection of rearrangements in different individuals. Detection of these rearrangements and their relative frequency, lack of certain sequences, etc. can provide valuable information about the state of the immune system and its role in health and disease.

DETAILED DESCRIPTION The inventors have developed a new method for improving the PCR amplification of immunoglobulin recombination regions and increasing the number of detectable recombinant molecules from samples of human and / or animal B cell populations. developed. The present invention provides about 3 to about 5 at the 3 ′ end, 5 ′ end, or both 3 ′ and 5 ′ ends of at least one primer for PCR amplification of one or more immunoglobulin variable regions. Adding randomly generated nucleotides. The method provides an increase in the number of amplification products as compared to amplification reactions that utilize primers that do not have randomly generated nucleotides at one or both ends.

  At the germinal center, B cells modify the 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 immunoglobulin heavy and light chain genes. In recent years, the design of primers has been improved as computer programs have been developed to help design degenerate primers that bind more easily to these sequences. However, to detect recombination representing the actual recombination population of antibody molecules, use primers that bind at the junction of the hypervariable region and take into account the highly variable nature of the sequence in this region. By doing so, the inventors have discovered that the number of detectable molecules can be increased.

  The present invention provides a method for amplifying RNA and / or cDNA from human or animal blood samples. However, the sample may be taken from bone marrow or other B cell sources in the human or animal body. “Recombinant immunoglobulin sequence” refers to the various genetic rearrangements that occur in the body, resulting in various diversity of antibodies.

  Amplification may be performed by various methods known to those skilled in the art, which may be made easier by use of a commercially available kit. Methods for amplifying multiple targets from a sample include, for example, US Patent Publication No. 20070141575 describing a method known as TEM-PCR, and US describing a method known as ARM-PCR. This is described in Japanese Patent Application Publication No. 20090253183.

  For example, in the first step of the ARM-PCR method, a target-specific first amplification procedure is performed using a high concentration of target-specific nested primers. Primers are selected based on their likelihood of binding to a known immunoglobulin heavy chain variable region sequence (IgHV). As mentioned above, numerous computer programs are available to assist in the selection of primers, and for those skilled in the art, primer selection is made easier by certain principles known in the art. . One or more (preferably multiple) target nucleic acids may be amplified using target-specific primers. The concentration of the nested primer may be generally between 5 and 50 pmol. The selected primer is “tagged” with additional nucleotides to provide additional sequences that are not specific for the target nucleic acid, so that amplification of the target nucleic acid with such a primer results in a target-specific primer. Unlike, the common primer binding site that can be used to further amplify an irrelevant target nucleic acid amplicon is also incorporated into the resulting amplicon. Amplification is performed for approximately 10-15 cycles to terminate the reaction, and the resulting amplicon is rescued from the reaction mixture, and these are used for irrelevant nucleotide sequences corresponding to the various amplicons rescued from the target specific reaction. Used in a second target-independent amplification procedure involving the polymerase chain reaction primed with a common primer that provides amplification in a relatively indiscriminate manner.

  Amplicon rescue is performed to reduce or eliminate the first reaction primers as much as possible while providing an amplicon for use in a second amplification using a common primer. Amplicon rescue can be performed in various ways. For example, an amplicon for the second amplification can be prepared by collecting a small sample from the completed first amplification reaction. When a small sample is taken, it provides a sufficient number of amplicons for the second amplification, while at the same time the remaining number of primers for the first amplification is greatly reduced (ie diluted). Amplicon rescue removes most of the contents of the first amplification reaction and utilizes a common primer to amplify the rescued amplicon in the second reaction to the remaining contents. This may be done by adding a common primer along with the enzymes, nucleotides, buffers, and / or other reagents necessary to perform the second amplification. Amplicons may be rescued using separation techniques. Such techniques may depend on the size difference between the primer and the amplicon, the tag attached to the amplicon, the primer, or both, or other methods known to those skilled in the art. Once separated, all or a portion of the rescued amplicon may be used for the second amplification.

  In ARM-PCR, a second amplification is performed using new buffers, nucleotides, and common primers. The common primers are selected to provide efficient amplification of the rescued amplicons and to provide a substantial number of copies of those amplicons at the end of the second amplification.

  By separating the reaction into a first amplification driven by a target-specific primer and a second amplification driven by a common primer independent of the target, the method allows the type of nucleic acid present in a particular target. Provides specificity by using target specific primers that amplify only and numbers, and higher use of nested primers and high concentrations of target specific primers, as well as non-specific (target independent) amplification Provides the sensitivity achieved by the use of a common primer provided in copy number. In addition, the use of a high concentration of primer in the first amplification followed by rescue of the amplicon (especially by removing a portion of the first amplification and placing it in a new reaction system, or One of the first amplifications by either removing the majority of the amplification and adding to it the reagents necessary to form a second reaction system for the second amplification independent of the target. Amplicon rescue by isolating parts) is suitable for automation. Not only can these steps be performed in a relatively closed reaction system that limits the possibility of contamination, but also the first amplification, amplicon rescue, and second amplification provided by the method. The combination provides a specific and sensitive detection method in less than 2 hours for multiple targets from multiple samples.

  Both ARM-PCR and TEM-PCR methods have been used by the inventor to amplify multiple target sequences and increase the number of detectable targets in samples of immunoglobulin RNA and / or cDNA sequences It is combined with the method of the present invention to produce a primer to be made. As an example, by adding three randomly generated nucleotides to the 3 'end of the primer sequence, both ARM-PCR and TEM-PCR methods are more efficient for amplifying multiple targets from B cell samples. It became. This cannot be detected when using primers that do not have randomly generated ends because primer mismatches are thought to result in reduced binding and lack of amplification of targets with such mismatches. Provides at least 64 different possibilities for the detection of additional targets that may have hypermutations.

  Additional methods for amplification of multiple targets may be used with the methods of the present invention, and examples of methods that have been successfully used by the present inventors to achieve desirable methods of amplifying multiple targets include: PCR and TEM-PCR methods are provided.

  One major challenge that exists with high-throughput sequencing is that primer dimers interfere with amplification and are very difficult to remove. By using forward-in and reverse-in primers that are 40 base pairs long, the dimer can be as long as 80 base pairs. Products produced by conventional methods can be as short as 150-250 base pairs. By combining the new primer design provided by the method of the present invention with the steps of transferring the primer out and amplifying and sequencing the longer insert, it is much more likely that the PCR product exceeds 350 bp and the product is separated from the dimer. To be easier.

  The methods of the invention are useful for producing primers that more efficiently amplify antibody sequences that were previously difficult to detect. This makes it easier to amplify the diverse sequences present in a sample taken from any one individual, and as a result, rearrangements can exist more frequently or not at all. It becomes easier to confirm. This has previously been made more difficult by the fact that certain clones are difficult to detect due to primer mismatch pairing due to hypermutation leading to the production of those clones.

  Randomly generated nucleotides are most useful when added to the 3 ′ end in our experience with Ig molecules and ARM-PCR / TEM-PCR, but the 5 ′ end, 3 ′ end Or may be added to the primer sequence at both ends. The randomly generated nucleotides may comprise from about 2 to about 5 nucleotides at either the 3 ′ or 5 ′ end of the polynucleotide primer. Results using 3 randomly generated nucleotides at the 3 'end show amplification and detection of recombinant immunoglobulin sequences from human blood, especially when using the higher range of annealing temperatures used in PCR reactions Provides impressive results.

  The invention can be further illustrated by the following examples.

  Blood samples were obtained from Conversant Healthcare Systems, Inc., Huntsville, Alabama for the amplification shown in FIG. For the first amplification reaction using the Qiagen OneStep RT-PCR kit (Qiagen, Carlsbad, California), mRNA was extracted using the Qiagen kit. Reverse transcriptase was added to the sample: 50 ° C for 40 minutes (minimum 30 minutes RT), 95 ° C for 15 minutes for initial PCR activation. The concentration cycle was performed in 15 cycles of 94 ° C. for 30 seconds → 63 ° C. for 2 minutes → 72 ° C. for 30 seconds. Fifteen cycles of 94 ° C for 30 seconds → 72 ° C for 2 minutes were performed for 15 cycles, and the final extension at 72 ° C for 10 minutes was performed. In the second amplification reaction using the Qiagen Multiplex PCR kit, the first PCR activation is performed at 95 ° C for 15 minutes, followed by 40 steps of 3 step cycles of 94 ° C for 30 seconds → 55 ° C for 30 seconds → 72 ° C for 30 seconds. Cycled, followed by a final extension at 72 ° C for 5 minutes. Recombinant RNasin® Ribonuclease Inhibitor from Promega was also added.

Claims (4)

  A method for increasing the number of amplification products by amplification provided by a primer of a recombinant immunoglobulin sequence from a sample of human or animal origin, wherein the method is for at least one primer used for amplification provided by the primer. Incorporating about 2 to about 5 randomly generated nucleotides at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the primer sequence.   The method of claim 1, wherein the randomly generated nucleotide is incorporated at the 3 ′ end of the primer sequence.   The method of claim 1, wherein for the primer sequence, 3 randomly generated nucleotides are incorporated at the 3 ′ end, 5 ′ end, or both the 3 ′ end and the 5 ′ end of the primer sequence.   The method of claim 1, wherein three randomly generated nucleotides are incorporated at the 3 ′ end of the primer sequence.

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