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Inclusive and semi-quantitative amplification is the key for immune repertoire analysis.

 

Compared to normal individuals, patients with different diseases may have their immune repertoire changed quantitatively or qualitatively. Quantitative changes may be apparent as increases and decreases in repertoire diversity. Qualitative changes may present as increased sharing of disease-specific CDR3s in T or B cells.

 

A good amplification technology is critical for quantitatively and qualitatively studying the immune repertoire. A good method should be inclusive and semi-quantitative. Inclusive means 100,000-500,000 unique CDR3s can be amplified from one sample, with one experiment. Semi-quantitative means that biases should not be introduced during the amplification.

 

At iRepertoire, we have developed a multiplex PCR method that allows for the immune repertoire to be amplified inclusively and semi-quantitatively.

 

No one can imagine biotechnology without PCR. There are many variations of the basic PCR technology, just visit Wikipedia to find more than 20 different PCR based methods. We have been focused on developing mPCR (multiplex PCR) technologies for the past 10 years. First, we invented the patented tem-PCR technology (for target enriched multiplex PCR technology). In tem-PCR (see figure below), nested primers were designed for every target needing to be co-amplified (Fo, Fi, Ro and Ri) in the multiplex assay. The inside primers for all the targets share the same tag sequence which will bind to a pair of “Superprimers” (FS and RS). All these nested primers and the superprimers are in the same reaction system together, though the nested gene specific primers are used at an extremely low concentration. Only the superprimers are at high concentration for the exponential amplification of the targets. The tem-PCR technology worked because it solved the primer (loci) incompatibility problem. We used the tem-PCR technology successfully to develop several multiplex PCR assays for infectious diseases diagnoses (references 1-12).

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Depiction of the tem-PCR technology which  solved the loci incompatibility problem in multiplex PCR.

In 2009, we developed the next generation mPCR technology, the arm-PCR (amplicon rescued multiplex PCR, patent 7,999,092). For arm-PCR (the animated graphic at the top of the page), we are using a high concentration of the nested primers in the initial PCR round. Then, we rescue the amplicon and perform a second round of PCR by adding fresh communal primers (recognizing the shared tag sequence already introduced during the first round of amplification) and enzymes. In mPCR, since the exponential phase of the amplification is carried out by one pair of primers for all the potential targets, all CDR3s are amplified in a semi-quantitative manner. 

 

Amplify the immune repertoire of your samples contamination free and automatically.

 

Preparation of immune repertoire libraries have also been automated using the iCubate cassette. The two rounds of reactions are set up automatically in the iCubate cassette so that we do not need to worry about contaminating the laboratory environment with a high concentration of primers. In addtion, primer-dimers are automatically removed and the resulting library is ready for QC and sequencing. For more information about iRepertoire's immune repertoire on the iCubate cassette, contact us.

References:

 

  1. Jian Han,  David C. Swan, Sharon J. Smith, Shanjuan H. Lum, Susan E. Sefers, Elizabeth R. Unger, and Yiwei Tang. (2006) Simultaneous Amplification and Identification of 25 Human Papillomavirus Types with Templex Technology. Journal of Clinical Microbiology. 44(11). 4157-4162.

  2. Han J. Molecular differential diagnoses of infectious diseases: is the future now? In: Stratton C. and Tang YW. Eds. Advanced Technologies in Diagnostic Microbiology. New York. NY. Springer Publishing Company. (2006)

  3. John Brunstein, and Eva Thomas. (2006) Direct Screening of Clinical Specimens for Multiple Respiratory Pathogens Using the Genaco Respiratory Panels 1 and 2. Diagn Mol Pathol. 15(3). 169-173.

  4. Haijing Li, Melinda A. McCormac, R. Wray Estes, Susan E. Sefers, Ryan K. Dare, James D. Chapell, Dean D. Erdman, Peter F. Wright, and Yi-Wei Tang. (2007) Simultaneous Detection and High-Throughput Identification of a panel of RNA viruses Causing Respiratory Tract Infections. Journal of Clinical Microbiology, July. 45(7) 2105-2109.

  5. Shumei Zou, Jian Han, Leying Wen, Yan Liu, Kassi Cronin, Shanjuan H. Lum, Lu Gao, Jie Dong, Ye Zhang, Yuanji Guo and Yuelong Shu. (2007) Human Influenza A Virus (H5N1) Detection by a Novel Multiplex PCR Typing Method. Journal of Clinical Microbiology. 45(6). 1889-1892.

  6. Yi-Wei Tang, Abdullah Killic, Qunying Yang, Sigrid K. McAllister, Haijing Li, RebeccaS. Miller, Melinda McCormac, Karen D. Tracy, Charles W. Stratton, Jian Han, and Brandi Limbago. (2007) StaphPlex System for Rapid and Simultaneous Identification of Antibiotic Resistance Determinants and Panton-Valentine Leukocidin Detection of Staphylococci from Positive Blood Cultures. Journal of Clinical Microbiology. 45(6). 1867-1873.

  7. Kenneth L. Muldrew., Safedin H. Beqaj, Jian Han, Shanjuan H. Lum, Vicki Clinard, Stephen J. Schultenover, and Yiwei Tang. (2007)  Evaluation of a Digene-Recommended Algorithm for Human Papillomavirus Low-Positive Results Present in a “Retest Zone”. American Journal of Clinical Pathology. 127: 97-102.

  8. Medea Gegia, Nino Mdivani, Rodrigo E. Mendes, Haijing Li, Maka Akhalaia, Archil Salakaia, Jian Han, George Khechinashvili, and Yi-Wei Tang. Prevalence of and Molecular Basis for Tuberculosis Drug Resistance in the Republic of Georgia: Validation of a QIAplex System for Detection of Drug Resistance-Related Mutations. Antimicrobial Agents and Chemotherapy, Feb. 2008, p 725-729

  9. John Brunstein, Christy L. Cline, Steven McKinney, and Eva Thomas. Evidence from Multiplex Molecular Assay for Complex Multi-pathogen Interactions in Acute Respiratory Infections. Journal of Clinical Microbiology, Jan. 2008, p. 97-102.

  10. Robert Benson, Maria L. Tondella, Julu Bhatnagar, Maria da Gloria S. Carvalho, Jacquelyn S. Sampson, Deborah F. Talkington, Anne Whitney, Elizabeth mothershed, Lesley McGee, George Carlone, Vondguraus McClee, Jeannette Guarner, Sherif Zaki, Surang Dejsiri, Kassi Cronin, Jian Han and Barry Fields. Development and Evaluation of a Novel Multiplex PCR Technology for Molecular Differential Detection of Bacterial Respiratory Disease Pathogens. Journal of Clinical Microbiology, June 2008, p.2074-2077

  11. Edward H. Eiland III, Nicholas Beyda, Jian Han, William Lindgren, Randy Ward, Thomas MacAndrew English, Ali Hassoun, and Kathi Hathcock. The Utility of Rapid Micorbiological and Molecular Techniques in Optimizing Antimicrobial Therapy. SRX Pharmacology 2010, Article ID 395215.

  12. Chunlin Wang, Catherine M. Sanders, Qunying Yang, Harry W. Schroeder, Jr., Elijah Wang, Farbod Babrzadeh, Baback Gharizadeh, Richard M. Myers, James R. Hudson, Jr., Ronald W. Davis, and Jian Han. High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets. PNAS Jan 26, 2010 vol. 107(4) 1518-1523.

  13. Kassi Koon, Catherine M. Sanders, Jessica Green, Leslie Molone, Holly White, Delineliz Zayas, Rebecca Miller, Stanley Lu and Jian Han. Co-detection of Pandemic (H1N1) 2009 Virus and Other Respiratory Pathogens. Emerging Infectious Diseases.  2011 vol. 16 (12) 1976-1978