MULTIPLEX REAL TIME PCR SYSTEM FOR CHIMERISM MONITORING AFTER ALLOGENEIC HEMATOPOIETIC STEM CELLS TRANSPLANTATION
(Abstract release date: 05/19/16)
EHA Library. Fevraleva I. 06/09/16; 135052; PB2152
Dr. Irina Fevraleva
Contributions
Contributions
Abstract
Abstract: PB2152
Type: Publication Only
Background
Modern chimerism techniques require high sensitivity and capacity to predict relapse after allogeneic hematopoietic stem cells transplantation (allo-HSCT) several months prior its clinical presentation. These techniques have to be simple in use, time saving and inexpensive permitting repetitive measure in the course of treatment. Here we propose multiplex real time polymerase chain reaction technique targeted to unique polymorphic sites in recipient/donor alleles conforming these requirements.
Aims
To develop high sensitive and versatile test-system based on multiplex real time PCR technique for donor/recipient chimerism analysis after allo-HSCT.
Methods
We have used the panel of primers and molecular probes, proposed by M. Alizadeh et al., targeted to biallelic polymorphisms sites (insertions/deletions) in the human genome. All probes were modified by one of four fluorescent dyes: FAM, R&G, ROX and Cy5, and appropriate quenchers: BQH1 or BQH2. Genomic DNAs of donors and patients were isolated from the bone marrow or peripheral blood. Informative markers for each donor/recipient pair were selected by initial screening. For special cases (sibling transplantation) with no informative markers found among Alizadeh set we extend analyzed panel with additional primers and Taqman probes for other genome SNPs (for example rs1801131, rs1801133, rs587776796). Chimerism was detected also with STR-PCR using COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci ( 'GORDIZ'Ltd., Moscow, Russia).
Results
We have combined twenty pairs of primers and twelve Taqman fluorescent probes into seven parallel real time PCR multiplex reactions. Each of seven simultaneous PCR was analyzed by four channel, detecting FAM, R&G, ROX and Cy5 fluorescence. Alleles present only in donor or recipient DNA were considered informative and used for chimerism monitoring. Sensitivity of the method was estimated in control experiments with serial donor/recipient DNA sample dilutions. The same sample dilutions were analyzed with classic method, based on PCR of short tandem repeats (STR-PCR) followed by fragment analysis of the PCR-products. Multiplex method proposed detects as low as 0.03% recipient DNA admixture to donor DNA, while the standard STR-PCR method accuracy is limited to 1%.
Conclusion
We have developed fast multiplex Taqman real time PCR technique for evaluating polymorphic alleles informative for donor/recipient chimerism analysis after allo-HSCT. Method is convenient, simple, time saving, more sensitive and could replace classic STR-PCR approach.
Session topic: E-poster
Keyword(s): Allogeneic bone marrow transplant, Chimerism quantification, Real time PCR
Type: Publication Only
Background
Modern chimerism techniques require high sensitivity and capacity to predict relapse after allogeneic hematopoietic stem cells transplantation (allo-HSCT) several months prior its clinical presentation. These techniques have to be simple in use, time saving and inexpensive permitting repetitive measure in the course of treatment. Here we propose multiplex real time polymerase chain reaction technique targeted to unique polymorphic sites in recipient/donor alleles conforming these requirements.
Aims
To develop high sensitive and versatile test-system based on multiplex real time PCR technique for donor/recipient chimerism analysis after allo-HSCT.
Methods
We have used the panel of primers and molecular probes, proposed by M. Alizadeh et al., targeted to biallelic polymorphisms sites (insertions/deletions) in the human genome. All probes were modified by one of four fluorescent dyes: FAM, R&G, ROX and Cy5, and appropriate quenchers: BQH1 or BQH2. Genomic DNAs of donors and patients were isolated from the bone marrow or peripheral blood. Informative markers for each donor/recipient pair were selected by initial screening. For special cases (sibling transplantation) with no informative markers found among Alizadeh set we extend analyzed panel with additional primers and Taqman probes for other genome SNPs (for example rs1801131, rs1801133, rs587776796). Chimerism was detected also with STR-PCR using COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci ( 'GORDIZ'Ltd., Moscow, Russia).
Results
We have combined twenty pairs of primers and twelve Taqman fluorescent probes into seven parallel real time PCR multiplex reactions. Each of seven simultaneous PCR was analyzed by four channel, detecting FAM, R&G, ROX and Cy5 fluorescence. Alleles present only in donor or recipient DNA were considered informative and used for chimerism monitoring. Sensitivity of the method was estimated in control experiments with serial donor/recipient DNA sample dilutions. The same sample dilutions were analyzed with classic method, based on PCR of short tandem repeats (STR-PCR) followed by fragment analysis of the PCR-products. Multiplex method proposed detects as low as 0.03% recipient DNA admixture to donor DNA, while the standard STR-PCR method accuracy is limited to 1%.
Conclusion
We have developed fast multiplex Taqman real time PCR technique for evaluating polymorphic alleles informative for donor/recipient chimerism analysis after allo-HSCT. Method is convenient, simple, time saving, more sensitive and could replace classic STR-PCR approach.
Session topic: E-poster
Keyword(s): Allogeneic bone marrow transplant, Chimerism quantification, Real time PCR
Abstract: PB2152
Type: Publication Only
Background
Modern chimerism techniques require high sensitivity and capacity to predict relapse after allogeneic hematopoietic stem cells transplantation (allo-HSCT) several months prior its clinical presentation. These techniques have to be simple in use, time saving and inexpensive permitting repetitive measure in the course of treatment. Here we propose multiplex real time polymerase chain reaction technique targeted to unique polymorphic sites in recipient/donor alleles conforming these requirements.
Aims
To develop high sensitive and versatile test-system based on multiplex real time PCR technique for donor/recipient chimerism analysis after allo-HSCT.
Methods
We have used the panel of primers and molecular probes, proposed by M. Alizadeh et al., targeted to biallelic polymorphisms sites (insertions/deletions) in the human genome. All probes were modified by one of four fluorescent dyes: FAM, R&G, ROX and Cy5, and appropriate quenchers: BQH1 or BQH2. Genomic DNAs of donors and patients were isolated from the bone marrow or peripheral blood. Informative markers for each donor/recipient pair were selected by initial screening. For special cases (sibling transplantation) with no informative markers found among Alizadeh set we extend analyzed panel with additional primers and Taqman probes for other genome SNPs (for example rs1801131, rs1801133, rs587776796). Chimerism was detected also with STR-PCR using COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci ( 'GORDIZ'Ltd., Moscow, Russia).
Results
We have combined twenty pairs of primers and twelve Taqman fluorescent probes into seven parallel real time PCR multiplex reactions. Each of seven simultaneous PCR was analyzed by four channel, detecting FAM, R&G, ROX and Cy5 fluorescence. Alleles present only in donor or recipient DNA were considered informative and used for chimerism monitoring. Sensitivity of the method was estimated in control experiments with serial donor/recipient DNA sample dilutions. The same sample dilutions were analyzed with classic method, based on PCR of short tandem repeats (STR-PCR) followed by fragment analysis of the PCR-products. Multiplex method proposed detects as low as 0.03% recipient DNA admixture to donor DNA, while the standard STR-PCR method accuracy is limited to 1%.
Conclusion
We have developed fast multiplex Taqman real time PCR technique for evaluating polymorphic alleles informative for donor/recipient chimerism analysis after allo-HSCT. Method is convenient, simple, time saving, more sensitive and could replace classic STR-PCR approach.
Session topic: E-poster
Keyword(s): Allogeneic bone marrow transplant, Chimerism quantification, Real time PCR
Type: Publication Only
Background
Modern chimerism techniques require high sensitivity and capacity to predict relapse after allogeneic hematopoietic stem cells transplantation (allo-HSCT) several months prior its clinical presentation. These techniques have to be simple in use, time saving and inexpensive permitting repetitive measure in the course of treatment. Here we propose multiplex real time polymerase chain reaction technique targeted to unique polymorphic sites in recipient/donor alleles conforming these requirements.
Aims
To develop high sensitive and versatile test-system based on multiplex real time PCR technique for donor/recipient chimerism analysis after allo-HSCT.
Methods
We have used the panel of primers and molecular probes, proposed by M. Alizadeh et al., targeted to biallelic polymorphisms sites (insertions/deletions) in the human genome. All probes were modified by one of four fluorescent dyes: FAM, R&G, ROX and Cy5, and appropriate quenchers: BQH1 or BQH2. Genomic DNAs of donors and patients were isolated from the bone marrow or peripheral blood. Informative markers for each donor/recipient pair were selected by initial screening. For special cases (sibling transplantation) with no informative markers found among Alizadeh set we extend analyzed panel with additional primers and Taqman probes for other genome SNPs (for example rs1801131, rs1801133, rs587776796). Chimerism was detected also with STR-PCR using COrDIS Plus multiplex kit for amplification of 19 polymorphic STR-markers and amelogenin loci ( 'GORDIZ'Ltd., Moscow, Russia).
Results
We have combined twenty pairs of primers and twelve Taqman fluorescent probes into seven parallel real time PCR multiplex reactions. Each of seven simultaneous PCR was analyzed by four channel, detecting FAM, R&G, ROX and Cy5 fluorescence. Alleles present only in donor or recipient DNA were considered informative and used for chimerism monitoring. Sensitivity of the method was estimated in control experiments with serial donor/recipient DNA sample dilutions. The same sample dilutions were analyzed with classic method, based on PCR of short tandem repeats (STR-PCR) followed by fragment analysis of the PCR-products. Multiplex method proposed detects as low as 0.03% recipient DNA admixture to donor DNA, while the standard STR-PCR method accuracy is limited to 1%.
Conclusion
We have developed fast multiplex Taqman real time PCR technique for evaluating polymorphic alleles informative for donor/recipient chimerism analysis after allo-HSCT. Method is convenient, simple, time saving, more sensitive and could replace classic STR-PCR approach.
Session topic: E-poster
Keyword(s): Allogeneic bone marrow transplant, Chimerism quantification, Real time PCR
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