CN103154015A

CN103154015A - Method for improving detection of b cell immunoglobulin gene recombination

Info

Publication number: CN103154015A
Application number: CN2011800244208A
Authority: CN
Inventors: 韩建
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-03-29
Filing date: 2011-03-29
Publication date: 2013-06-12
Also published as: CA2794800A1; AU2011235281A1; WO2011123473A1; EP2558479A4; JP2013523129A; EP2558479A1

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

Be used for improving the method that detects the gene recombination of B cell immunoglobulin

The application requires the right of priority of the U.S. Provisional Patent Application submitted on March 29th, 2010 number 61/318,417.

Invention field

The present invention relates to the method for identification and the gene recombination of quantification B cell immunoglobulin.More specifically, the present invention relates to be designed for increase from the method for the primer of the PCR-amplified production quantity of immunoglobulin (Ig) cDNA and/or RNA.

Background of invention

When considering " the stagnation area " of scientific research, immunology has become the major portion of the research of health and the state when healthy and disease thereof.Scientist constantly finds the natural defending system of health and the contact between disease, and this once was considered to irrelevant with immunology.Immune cell provides the highlight of inflammatory response, and inflammation can with various disease, as cardiovascular diseases, ephrosis, diabetes, sacroiliitis and related to cancer.Inflammatory response is balance carefully, and when this balance can not get keeping, the disease that caused by immune deficiency be can cause, perhaps " autoimmune disease " such as systemic lupus erythematous (SLE), rheumatic arthritis (RA) and sarcoidosis caused at the type spectrum the other end.

Anthony doctor Fauci, M.D., American National transformation reactions and transmissible disease (the UnitedStates National Institute of Allergy and Infectious Disease of institute, NIAID) president, owing to proposing " the human immune system state that definition is in healthy and disease is the major objective of people's immunology research " (NIH news (NIH News), on March 8th, 2010 Http:// www.nih.gov.news/health/mar2010/niaid-08a.htm) and be cited.NIAID scientist recognize " not considering variation of the ratio of cell type separately of broad range in healthy individuals even be used at present checking the method for gene expression difference in the immunocyte mixture that is in blood " (Mark doctor Davis, NIH news (NIH News), on March 8th, 2010).Mathematical method---the significance analysis (csSAM) of microarray cell-specific that their team is a kind of analysis by the molecular data that uses microarray technology and obtain about the method for this problem

The antibody response that is provided by the B cell in immunity system for the diversity of replying middle generation significance degree of attacking.By the attack of antigen, the B cell transfer is to B cell folliculus and build germinal center (GCs).The immunoglobulin gene of resetting, itself is as significant diversity source, further by constant region type-conversion is recombinated and the somatic hypermutation of hypervariable region is modified.Mutation rate in these zones has been estimated high by approximately 10 than spontaneous genetic mutation ⁶Doubly.

Need the sensitiveer multifarious method of detection B cell, so that the better understanding to the immunity system state that is in healthy and disease to be provided.

Summary of the invention

The present invention relates to for improving immunoglobulin (Ig) recombination zone pcr amplification and increasing the method that can detect recombinant molecule quantity, described method comprises and will approximately 3 be added into 3 ' end at least one primer of the one or more immune globulin variable regions of pcr amplification to about 5 random Nucleotide that produce, 5 ' end, or 3 ' and 5 ' end is both.

The accompanying drawing summary

Fig. 1 diagram is from the pcr amplification result of the immune globulin variable region of people patient's B cell mass.Swimming lane 1-3 diagram is used the fractional yield of the PCR product of contrast primer (at the nucleotide sequence of 3 ' end without random generation), and the 4-6 diagram is used the relative populations of the extension increasing sequence of experiment primer (nucleotide sequence that has random generation at 3 ' end).Swimming lane 1 expression is from the amplified production of normal individual RNA.Swimming lane 2 is from CLL patient, and swimming lane 3 is blank, as negative control.Swimming lane 4 expressions come the amplified production of the RNA of free normal individual separation, and swimming lane 5 is from CLL patient, and swimming lane 6 is negative controls.Amplification condition is identical.Add 3 ' the random Nucleotide that produces to primer, as by as shown in the intensity difference between band in

swimming lane

1,2,4 and 5, produce significantly more amplified production.

Fig. 2 illustrates the sequencing result of a plurality of targets, and described target is for detection of the rearrangement from Different Individual.Detect these rearrangements and relative frequency thereof, lack some sequence etc. the valuable information that acts on about the immunity system state and in health and disease can be provided.

Describe in detail

The inventor has developed and has been used for improving immunoglobulin (Ig) recombination zone pcr amplification and the novel method of increase from the detected recombinant molecule quantity of the B cell mass sample of people and/or animal.The present invention includes and will approximately 3 be added into 3 ' end at least one primer of the one or more immune globulin variable regions of pcr amplification to about 5 random Nucleotide that produce, 5 ' end, or 3 ' and 5 ' end is both.Described method is compared without the amplified reaction of the primer of the random Nucleotide that produces at an end or two ends with utilization, and the amplified production that increases quantity is provided.

In germinal center, the B cell is modified the immunoglobulin gene of rearrangement by somatic hypermutation.This height sudden change provides other diversity and antigen-binding specificity.It also introduces sudden change near the V of heavy chain immunoglobulin and light chain gene district's neutralization.Improve in the recent period design of primers, assisted to design easier degenerated primer of being combined with these sequences because developed computer program.Yet in order to detect the restructuring of true group of expression antibody molecule restructuring, the inventor has been found that detectable molecular amounts can increase by using following primer, the high change nature of described primer sequence in the combination of hypervariable region joint and in considering this district.

The invention provides for the method for amplification from RNA and/or the cDNA of human or animal's blood sample.Yet sample can also be available from marrow or other B cell sources of human or animal body.The different heredity that occured in " recombination immunoglobulin sequence " expression body are reset, and it causes different diversity antibody.

Amplification can be undertaken by the known several different methods of those skilled in the art, and it can be purchased test kit by use and simplifies.Being used for amplification records and narrates from the method for a plurality of targets of single sample, for example, in U.S. Patent Application Publication No. 20070141575, it has described the method that is called TEM-PCR, with U.S. Patent Application Publication No. 20090253183, it has described the method that is called ARM-PCR.

For example, in the first step of ARM-PCR method, use high density, target specificity, nested primers to carry out target specificity the first amplification program.Primer is selected in conjunction with the potentiality of known immunoglobulin heavy chain variable region sequence (IgHV) based on them.As previously mentioned, exist many computer programs to assist to select primer, and for those skilled in the art, Primer selection is because some principle as known in the art becomes simple.Can be with target Auele Specific Primer one or more (and preferably a plurality of) target nucleic acids that increase.Nested primers concentration can be generally 5-50pmol.Selected primer is by other Nucleotide " mark ", thereby provide the other sequence that is not specific to target nucleic acid, so that use the target nucleic acid of such primer to increase also to generating thus the binding site of introducing in amplicon about universal primer, described universal primer, different from the target Auele Specific Primer, can be used for the further irrelevant target nucleic acid amplicon of amplification.Approximately 10-15 circulation carried out in amplification, termination reaction, and by the consequent amplicon of rescue in reaction mixture, not use in the dependency amplification program at the second target, described program comprises by universal primer and starts polymerase chain reaction, described universal primer provides the amplification of the irrelevant nucleotide sequence that is represented by the multiple amplicon of saving in the target specific reaction in relatively indiscriminate mode.

Carry out the amplicon rescue, thereby minimize or eliminate the primer of the first reaction, be provided at simultaneously the amplicon that uses in the second amplification of using universal primer.The amplicon rescue can be carried out by different way.For example, can carry out the small-scale sampling from the first amplified reaction of completing, to be provided for the amplicon of the second amplification.When carrying out small-scale when taking a sample, the amplicon that is used for the second amplification that it provides sufficient amount significantly reduces the volume residual of (that is, dilution) the first amplimer simultaneously.Amplicon rescue can also be undertaken by following steps: remove the major portion of the first amplified reaction system for content thing and add universal primer and for utilizing described universal primer to carry out the second necessary enzyme of amplification, Nucleotide, damping fluid and/or other reagent to remaining content, thereby in the second reactive system the amplicon of amplification rescue.Can also utilize isolation technique from the rescue amplicon.Such technology can depend on the difference in size between primer and amplicon, has been connected in amplicon, primer or label both, or the known additive method of those skilled in the art.One section separation, all the amplicon of the amplicon of rescue or part rescue can be used for the second amplification.

In ARM-PCR, the second amplification uses fresh buffer, Nucleotide and universal primer to carry out.Select universal primer, with effective amplification of amplicon that rescue is provided, thereby provide the copy of those amplicons of remarkable quantity when the second amplification finishes.

By the not amplification that drives of dependency universal primer of the amplification and second that reaction is divided into first, the target Auele Specific Primer drives, target, described method is by using the target Auele Specific Primer that specificity is provided, thereby kind and quantity and the sensitivity by using nested primers, high density target Auele Specific Primer and using universal primer to obtain of the nucleic acid that represented by particular target of only increasing, thereby so that non-specific (target is dependency not) amplification to be provided than high copy number.In addition, use high density primer in the first amplification, and the rescue of subsequently amplicon---particularly when separating a part the first amplification by following steps and carries out the amplicon rescue: by remove described part and be placed in new reactive system or by remove the first major portion that increases and to the essential reagent of its interpolation be formed for second, target the second reactive system of dependent amplification not---so that it is suitable for automatic operation.Not only these steps can be carried out in the reactive system of the limit pollution possibility of relative closure, and the combination results of the first amplification, amplicon rescue and the second amplification that are provided by described method a kind of carry out in during less than 2 hours about specificity, Sensitive Detection method from a plurality of targets of a plurality of samples.

ARM-PCR and TEM-PCR method both all are used for increasing a plurality of target sequences by the inventor, and when with Combination of Methods of the present invention, are used for the primer that generation increase immunoglobulin (Ig) RNA and/or cDNA sequence sample can detect target quantity.As an example, add 3 random Nucleotide that produce by 3 ' end at primer sequence, and make ARM-PCR and TEM-PCR method both more effectively increase from a plurality of targets of B cell sample.This provides at least 64 kinds of different possibilities for detecting the other target that can have high sudden change, if use the primer without the random end that produces, described target can not detect, because the primer mispairing will cause lacking in conjunction with reducing and increasing of target and described mispairing.

Methods of other a plurality of targets that are used for increasing also can be used together with method of the present invention, and provide ARM-PCR and TEM-PCR method as the inventor's be successfully used to realize to increase example of required method of a plurality of targets.

Be primer dimer interference amplification and be very difficult to remove with the common subject matter that exists of high-flux sequence.By forward and the reverse primer that uses 40 base pair length, dimer can have 80 base pairs so long.The product that is produced by ordinary method can have the 150-250 base pair so short.The new design of primers of using inventive method to provide, and with the combination of the described primer of outside movement with amplification and the longer inserting step of order-checking, the PCR product surpasses 350bp, this makes and easier product and dimer is separated.

Method of the present invention is effective to generate the primer of the antibody sequence that is difficult to detect before more effectively amplification.This makes the multiple sequence that exists in its sample available from any single individuality of more easily increasing, thereby determines that more easily which kind of rearrangement can exist with upper frequency, and which kind of can not exist etc. fully.In the past, this was more difficult, the fact that it is difficult to detect owing to some clone, and this is owing to the mispairing pairing by the caused primer of height sudden change that causes those clones to produce.

The random Nucleotide that produces can be at 5 ' end, and 3 ' end, or two ends are added into primer sequence, although use in the experience of Ig molecule and ARM-PCR/TEM-PCR the inventor, they are the most effective when being added into 3 ' end.The approximately 2-that the random Nucleotide that produces can also be included in polynucleotide primers 3 ' or 5 ' end is 5 Nucleotide approximately.Provide outstanding result in amplification with in detecting recombination immunoglobulin sequence from human blood about the result of 3 of 3 ' the end random Nucleotide that produce, during the annealing temperature of the higher range of particularly having used in having used the PCR reaction.

The present invention can further describe by following examples:

Embodiment

Blood sample is available from Conversant Healthcare Systems, Inc., and Huntsville, the Alabama is to be used for the amplification shown in Fig. 1.In order to use the first amplified reaction of Qiagen single step RT-PCR test kit (Qiagen, Carlsbad, California), utilize the Qiagen test kit to extract mRNA.Add ThermoScript II in the sample: 50 ℃, 40 minutes (30 minutes, minimum RT), thus activate 15 minutes at 95 ℃ of initial PCR.Carry out the enrichment circulation: 94 ℃, 30 seconds → 63 ℃, 2 minutes → 72 ℃, 30 seconds, carry out 15 circulations.94 ℃, 30 seconds → 72 ℃, the 2-step of 2 minutes looped 15 circulations, 72 ℃ of final prolongations 10 minutes.In the second amplified reaction that uses Qiagen Multiplex PCR test kit, initial PCR activation was carried out 15 minutes at 95 ℃, followed to go on foot circulation by 3-: 94 ℃, 30 seconds → 55 ℃, 30 seconds → 72 ℃, 30 seconds, carry out 40 circulations, 72 ℃ of final prolongations 5 minutes.Also add the restructuring from Promega

Ribonuclease inhibitor.

Claims

1. for increasing the method for amplified production quantity, described amplified production is from the amplification of the primer generation of the recombination immunoglobulin sequence of human or animal's sample, described method comprises at least one primer of the amplification that is used for described primer generation, 3 ' end at described primer sequence, 5 ' end, or 3 ' end and 5 ' end both locate, introduce approximately 2 to about 5 random Nucleotide that produce.

2. the process of claim 1 wherein that the Nucleotide of described random generation is introduced in 3 ' end of described primer sequence.

3. the process of claim 1 wherein 3 random Nucleotide that produce at 3 ' end of described primer sequence, 5 ' end, or 3 ' end and 5 ' end are both located to be introduced in described primer sequence.

4. the process of claim 1 wherein that 3 random Nucleotide that produce are introduced in 3 ' end of described primer sequence.