The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
Bai Xiao - One of the best experts on this subject based on the ideXlab platform.
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a rapid detection for α thalassemia by pcr combined with Dissociation Curve analysis
Experimental and Molecular Pathology, 2011Co-Authors: Xingyuan Jia, Lirong Wang, Jingzhong Liu, Limei Yao, Ning Tang, Ren Cai, Bai XiaoAbstract:Deletion mutations of 3.7 kb and 4.2 kb of α-globin gene are the most common causes of α-thalassemia (-α(3.7)/, -α(4.2)/). A simple, rapid assay by using a single-tube PCR to detect the two deletions has been needed. In this study, a pair of shared primers was designed for α2 and α1 gene but with length-different amplicons (159 bp and 409 bp). On the Dissociation Curve analysis profile after PCR, there shows two obvious peaks which represent the two different amplicons. Relative copy number of α2 and α1 gene can be deduced from the ratio of the two peaks. A comprehensive diagnosis for α-thalassemia 10 genotypes of deletions can be achieved when combined with a single-tube duplex PCR for detecting --SEA and non-deletional alleles of αα or α(T)α. Besides, a single-tube multiplex PCR, which is a cost-effective version of dual-priming-oligonucleotide based system, was designed for two common mutations of α-thalassemia in China (Hb Constant Spring and Hb Quong Sze), and these two mutations can be identified in samples by use of Dissociation Curve analysis. In all, using above three PCRs followed by Dissociation Curve analysis, three deletions and two mutations of α-thalassemia in the populations of southern China and Southeast Asia can be detected for molecular diagnosis or prenatal diagnosis. A blinded study of 163 samples was performed using this new assay and it was concordant with the original methods. This comprehensive molecular assay is simple, rapid, automatic and cost-effective, and can be used to diagnose α-thalassemia in this geographical area.
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rapid diagnosis of the α thalassemia 1 southeast asian type deletion using a single tube real time sybr polymerase chain reaction combined with Dissociation Curve analysis
Hemoglobin, 2009Co-Authors: Jingzhong Liu, Bai Xiao, Qingtao Wang, Lirong WangAbstract:Dear Sir,A single tube polymerase chain reaction (PCR) with three primers and SYBR GREEN1 combined with Dissociation Curve analysis was set up that can clearly differentiate between Hb Bart's hydrops fetalis, normal subjects and − −SEA heterozygotes. This method seems to be simpler than that using a two-tube real-time SYBR-PCR with two different primer sets followed by analyses of ΔCT and CT ratio.
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improvement in the detection of alpha0 and deletional alpha thalassemia by real time pcr combined with Dissociation Curve analysis
Acta Haematologica, 2009Co-Authors: Jingzhong Liu, Lirong Wang, Ning Tang, Ren Cai, Quanzhang Liu, Han Han, Bai XiaoAbstract:The prevailing cause of alpha-thalassemia in Southeast Asia is the presence of 3 deletion mutations in the alpha-globin genes (-SEA, -alpha(3.7) and -alpha(4.2)). Current detection methods include gap polymerase chain reaction (PCR), multiplex PCR and real-time PCR with SYBR Green 1 combined with Dissociation Curve analysis. To improve and simplify a previously published method that requires 4 separate reactions, a duplex PCR assay was designed to detect both the nondeletional and the -SEA alleles. This duplex PCR can successfully identify the nondeletional allele and both the -SEA carrier and homozygous genotypes. The combination of the duplex PCR and 2 gap PCRs (for detection of -alpha(3.7) and -alpha(4.2)) can diagnose all types of deletional alpha-thalassemia. Our method was validated by analysis of 195 DNA samples, the results of which were consistent with prior diagnoses. The developed assay can reliably diagnose alpha0-thalassemia and all types of deletional alpha-thalassemia. The diagnostic method is simple, rapid, accurate, automated, inexpensive and has a high throughput.
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detection of alpha thalassemia using real time pcr and Dissociation Curve analysis
Chinese journal of medical genetics, 2007Co-Authors: Mei Yan, Lirong Wang, Zhanyong Wang, Yan Zhou, Yan Liang, Bai Xiao, Jingzhong LiuAbstract:Objective To establish an automatic, high throughput, quick detection method of α-thalassemia. Methods The genotypes of -α^4.2 and -α^3.7 were detected by two real-time fluorescence PCRs using SYBR-Greenl （SYBR-PCR） with the analysis of Dissociation Curve （DC） and melting temperature （Tm）. The PCR products were recombined into T-vector and the correct cloning was selected as positive control. Sensitivities were gained from a serious dilution of the recombinant as SYBR-PCR template, from which detection deadlines for two kinds of alleles could be determined. Totally 110 samples were detected by this technique. Results The length of product of - α^4.2 and -α^3.7 were 1.65 kb and 1.9 kb respectively, and the Tm were （81.5 ± 0.5） ℃ and （82.5 ± 0.5） ℃ respectively. The detection deadline were 9 × 10^2 copies and 4.3 × 10^2 copies respectively. The sensitivity of the technique was much higher than that of regular PCR plus gel electrophoresis method, and the detection results of the technique were the same as that of multiplex PCR. Conclusion The genotypes of -α^4.2, - α^3.7, non-deletion αα/and --^SEA can be sensitively, exactly diagnosed by the real-time PCR with SYBR-Greenl combined with the Dissociation Curve analysis. The assay is automatic, low-cost, high throughput, and easy for quality control without fluorescence probe. Key words: real-time polymerase chain reaction; SYBR-Green 1; Dissociation Curve; α-thalassemia
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molecular diagnosis of α thalassemia by combining real time pcr with sybr green1 and Dissociation Curve analysis
Translational Research, 2006Co-Authors: Jingzhong Liu, Mei Yan, Lirong Wang, Yan Zhou, Zhangyong Wang, Bai XiaoAbstract:The aim of the study was to set up an automatic molecular diagnostic method for deletional α-thalassemia without gel electrophoresis and TaqMan probe. Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by Dissociation Curve (DC) analysis were used to detect the −− SEA , − α 3.7 , −α 4.2 , and non-deletion-type alleles (α α or α T α), respectively. Positive results of the SYBR-PCRs were defined by the special shapes of the Dissociation Curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon ≥ cutoff values. Molecular diagnosis of α-thalassemia was determined by combining all four SYBR-PCR results. The specific Tms for the SYBR-PCR1-4, which was used to detect the −− SEA , − α 3.7 , −α 4.2 , and non-deletion-type alleles were 82.5 ± 1°C, 82.8 ± 1°C, 81.5 ± 1°C, and 83.0 ± 1°C, respectively. The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70. The C T VS log copies of a recombinant plasmid DNA showed a good linear relationship between 10 5 ∼ 10 0 . Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method. Diagnostic outcomes of the 120 α-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods. The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of α-thalassemia.
Penny K Riggs - One of the best experts on this subject based on the ideXlab platform.
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application of Dissociation Curve analysis to radiation hybrid panel marker scoring generation of a map of river buffalo b bubalis chromosome 20
BMC Genomics, 2008Co-Authors: Kelli J Kochan, Elisabete M J Amaral, Richa Agarwala, Alejandro A Schaffer, Penny K RiggsAbstract:Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification Dissociation Curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and Dissociation Curve analysis for use as a novel scoring method. As proof of principle for this Dissociation Curve method, we generated new maps of river buffalo (Bubalus bubalis) chromosome 20 by both Dissociation Curve analysis and conventional marker scoring. We also obtained sequence data to augment Dissociation Curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46%) were suitable as cross-species markers for Dissociation Curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly. Dissociation Curve analysis is reliable and efficient for radiation hybrid panel scoring, and is more sensitive and robust than conventional gel-based typing methods. Several markers could be scored only by the new method, and ambiguous scores were reduced. PCR-based Dissociation Curve analysis decreases both time and resources needed for construction of radiation hybrid panel marker maps and represents a significant improvement over gel-based methods in any species.
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application of Dissociation Curve analysis to radiation hybrid panel marker scoring generation of a map of river buffalo b bubalis chromosome 20
BMC Genomics, 2008Co-Authors: Kelli J Kochan, Elisabete M J Amaral, Richa Agarwala, Alejandro A Schaffer, Penny K RiggsAbstract:Background Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification Dissociation Curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and Dissociation Curve analysis for use as a novel scoring method.
Jingzhong Liu - One of the best experts on this subject based on the ideXlab platform.
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a rapid detection for α thalassemia by pcr combined with Dissociation Curve analysis
Experimental and Molecular Pathology, 2011Co-Authors: Xingyuan Jia, Lirong Wang, Jingzhong Liu, Limei Yao, Ning Tang, Ren Cai, Bai XiaoAbstract:Deletion mutations of 3.7 kb and 4.2 kb of α-globin gene are the most common causes of α-thalassemia (-α(3.7)/, -α(4.2)/). A simple, rapid assay by using a single-tube PCR to detect the two deletions has been needed. In this study, a pair of shared primers was designed for α2 and α1 gene but with length-different amplicons (159 bp and 409 bp). On the Dissociation Curve analysis profile after PCR, there shows two obvious peaks which represent the two different amplicons. Relative copy number of α2 and α1 gene can be deduced from the ratio of the two peaks. A comprehensive diagnosis for α-thalassemia 10 genotypes of deletions can be achieved when combined with a single-tube duplex PCR for detecting --SEA and non-deletional alleles of αα or α(T)α. Besides, a single-tube multiplex PCR, which is a cost-effective version of dual-priming-oligonucleotide based system, was designed for two common mutations of α-thalassemia in China (Hb Constant Spring and Hb Quong Sze), and these two mutations can be identified in samples by use of Dissociation Curve analysis. In all, using above three PCRs followed by Dissociation Curve analysis, three deletions and two mutations of α-thalassemia in the populations of southern China and Southeast Asia can be detected for molecular diagnosis or prenatal diagnosis. A blinded study of 163 samples was performed using this new assay and it was concordant with the original methods. This comprehensive molecular assay is simple, rapid, automatic and cost-effective, and can be used to diagnose α-thalassemia in this geographical area.
-
rapid diagnosis of the α thalassemia 1 southeast asian type deletion using a single tube real time sybr polymerase chain reaction combined with Dissociation Curve analysis
Hemoglobin, 2009Co-Authors: Jingzhong Liu, Bai Xiao, Qingtao Wang, Lirong WangAbstract:Dear Sir,A single tube polymerase chain reaction (PCR) with three primers and SYBR GREEN1 combined with Dissociation Curve analysis was set up that can clearly differentiate between Hb Bart's hydrops fetalis, normal subjects and − −SEA heterozygotes. This method seems to be simpler than that using a two-tube real-time SYBR-PCR with two different primer sets followed by analyses of ΔCT and CT ratio.
-
improvement in the detection of alpha0 and deletional alpha thalassemia by real time pcr combined with Dissociation Curve analysis
Acta Haematologica, 2009Co-Authors: Jingzhong Liu, Lirong Wang, Ning Tang, Ren Cai, Quanzhang Liu, Han Han, Bai XiaoAbstract:The prevailing cause of alpha-thalassemia in Southeast Asia is the presence of 3 deletion mutations in the alpha-globin genes (-SEA, -alpha(3.7) and -alpha(4.2)). Current detection methods include gap polymerase chain reaction (PCR), multiplex PCR and real-time PCR with SYBR Green 1 combined with Dissociation Curve analysis. To improve and simplify a previously published method that requires 4 separate reactions, a duplex PCR assay was designed to detect both the nondeletional and the -SEA alleles. This duplex PCR can successfully identify the nondeletional allele and both the -SEA carrier and homozygous genotypes. The combination of the duplex PCR and 2 gap PCRs (for detection of -alpha(3.7) and -alpha(4.2)) can diagnose all types of deletional alpha-thalassemia. Our method was validated by analysis of 195 DNA samples, the results of which were consistent with prior diagnoses. The developed assay can reliably diagnose alpha0-thalassemia and all types of deletional alpha-thalassemia. The diagnostic method is simple, rapid, accurate, automated, inexpensive and has a high throughput.
-
detection of alpha thalassemia using real time pcr and Dissociation Curve analysis
Chinese journal of medical genetics, 2007Co-Authors: Mei Yan, Lirong Wang, Zhanyong Wang, Yan Zhou, Yan Liang, Bai Xiao, Jingzhong LiuAbstract:Objective To establish an automatic, high throughput, quick detection method of α-thalassemia. Methods The genotypes of -α^4.2 and -α^3.7 were detected by two real-time fluorescence PCRs using SYBR-Greenl （SYBR-PCR） with the analysis of Dissociation Curve （DC） and melting temperature （Tm）. The PCR products were recombined into T-vector and the correct cloning was selected as positive control. Sensitivities were gained from a serious dilution of the recombinant as SYBR-PCR template, from which detection deadlines for two kinds of alleles could be determined. Totally 110 samples were detected by this technique. Results The length of product of - α^4.2 and -α^3.7 were 1.65 kb and 1.9 kb respectively, and the Tm were （81.5 ± 0.5） ℃ and （82.5 ± 0.5） ℃ respectively. The detection deadline were 9 × 10^2 copies and 4.3 × 10^2 copies respectively. The sensitivity of the technique was much higher than that of regular PCR plus gel electrophoresis method, and the detection results of the technique were the same as that of multiplex PCR. Conclusion The genotypes of -α^4.2, - α^3.7, non-deletion αα/and --^SEA can be sensitively, exactly diagnosed by the real-time PCR with SYBR-Greenl combined with the Dissociation Curve analysis. The assay is automatic, low-cost, high throughput, and easy for quality control without fluorescence probe. Key words: real-time polymerase chain reaction; SYBR-Green 1; Dissociation Curve; α-thalassemia
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molecular diagnosis of α thalassemia by combining real time pcr with sybr green1 and Dissociation Curve analysis
Translational Research, 2006Co-Authors: Jingzhong Liu, Mei Yan, Lirong Wang, Yan Zhou, Zhangyong Wang, Bai XiaoAbstract:The aim of the study was to set up an automatic molecular diagnostic method for deletional α-thalassemia without gel electrophoresis and TaqMan probe. Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by Dissociation Curve (DC) analysis were used to detect the −− SEA , − α 3.7 , −α 4.2 , and non-deletion-type alleles (α α or α T α), respectively. Positive results of the SYBR-PCRs were defined by the special shapes of the Dissociation Curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon ≥ cutoff values. Molecular diagnosis of α-thalassemia was determined by combining all four SYBR-PCR results. The specific Tms for the SYBR-PCR1-4, which was used to detect the −− SEA , − α 3.7 , −α 4.2 , and non-deletion-type alleles were 82.5 ± 1°C, 82.8 ± 1°C, 81.5 ± 1°C, and 83.0 ± 1°C, respectively. The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70. The C T VS log copies of a recombinant plasmid DNA showed a good linear relationship between 10 5 ∼ 10 0 . Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method. Diagnostic outcomes of the 120 α-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods. The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of α-thalassemia.
Lirong Wang - One of the best experts on this subject based on the ideXlab platform.
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a rapid detection for α thalassemia by pcr combined with Dissociation Curve analysis
Experimental and Molecular Pathology, 2011Co-Authors: Xingyuan Jia, Lirong Wang, Jingzhong Liu, Limei Yao, Ning Tang, Ren Cai, Bai XiaoAbstract:Deletion mutations of 3.7 kb and 4.2 kb of α-globin gene are the most common causes of α-thalassemia (-α(3.7)/, -α(4.2)/). A simple, rapid assay by using a single-tube PCR to detect the two deletions has been needed. In this study, a pair of shared primers was designed for α2 and α1 gene but with length-different amplicons (159 bp and 409 bp). On the Dissociation Curve analysis profile after PCR, there shows two obvious peaks which represent the two different amplicons. Relative copy number of α2 and α1 gene can be deduced from the ratio of the two peaks. A comprehensive diagnosis for α-thalassemia 10 genotypes of deletions can be achieved when combined with a single-tube duplex PCR for detecting --SEA and non-deletional alleles of αα or α(T)α. Besides, a single-tube multiplex PCR, which is a cost-effective version of dual-priming-oligonucleotide based system, was designed for two common mutations of α-thalassemia in China (Hb Constant Spring and Hb Quong Sze), and these two mutations can be identified in samples by use of Dissociation Curve analysis. In all, using above three PCRs followed by Dissociation Curve analysis, three deletions and two mutations of α-thalassemia in the populations of southern China and Southeast Asia can be detected for molecular diagnosis or prenatal diagnosis. A blinded study of 163 samples was performed using this new assay and it was concordant with the original methods. This comprehensive molecular assay is simple, rapid, automatic and cost-effective, and can be used to diagnose α-thalassemia in this geographical area.
-
rapid diagnosis of the α thalassemia 1 southeast asian type deletion using a single tube real time sybr polymerase chain reaction combined with Dissociation Curve analysis
Hemoglobin, 2009Co-Authors: Jingzhong Liu, Bai Xiao, Qingtao Wang, Lirong WangAbstract:Dear Sir,A single tube polymerase chain reaction (PCR) with three primers and SYBR GREEN1 combined with Dissociation Curve analysis was set up that can clearly differentiate between Hb Bart's hydrops fetalis, normal subjects and − −SEA heterozygotes. This method seems to be simpler than that using a two-tube real-time SYBR-PCR with two different primer sets followed by analyses of ΔCT and CT ratio.
-
improvement in the detection of alpha0 and deletional alpha thalassemia by real time pcr combined with Dissociation Curve analysis
Acta Haematologica, 2009Co-Authors: Jingzhong Liu, Lirong Wang, Ning Tang, Ren Cai, Quanzhang Liu, Han Han, Bai XiaoAbstract:The prevailing cause of alpha-thalassemia in Southeast Asia is the presence of 3 deletion mutations in the alpha-globin genes (-SEA, -alpha(3.7) and -alpha(4.2)). Current detection methods include gap polymerase chain reaction (PCR), multiplex PCR and real-time PCR with SYBR Green 1 combined with Dissociation Curve analysis. To improve and simplify a previously published method that requires 4 separate reactions, a duplex PCR assay was designed to detect both the nondeletional and the -SEA alleles. This duplex PCR can successfully identify the nondeletional allele and both the -SEA carrier and homozygous genotypes. The combination of the duplex PCR and 2 gap PCRs (for detection of -alpha(3.7) and -alpha(4.2)) can diagnose all types of deletional alpha-thalassemia. Our method was validated by analysis of 195 DNA samples, the results of which were consistent with prior diagnoses. The developed assay can reliably diagnose alpha0-thalassemia and all types of deletional alpha-thalassemia. The diagnostic method is simple, rapid, accurate, automated, inexpensive and has a high throughput.
-
detection of alpha thalassemia using real time pcr and Dissociation Curve analysis
Chinese journal of medical genetics, 2007Co-Authors: Mei Yan, Lirong Wang, Zhanyong Wang, Yan Zhou, Yan Liang, Bai Xiao, Jingzhong LiuAbstract:Objective To establish an automatic, high throughput, quick detection method of α-thalassemia. Methods The genotypes of -α^4.2 and -α^3.7 were detected by two real-time fluorescence PCRs using SYBR-Greenl （SYBR-PCR） with the analysis of Dissociation Curve （DC） and melting temperature （Tm）. The PCR products were recombined into T-vector and the correct cloning was selected as positive control. Sensitivities were gained from a serious dilution of the recombinant as SYBR-PCR template, from which detection deadlines for two kinds of alleles could be determined. Totally 110 samples were detected by this technique. Results The length of product of - α^4.2 and -α^3.7 were 1.65 kb and 1.9 kb respectively, and the Tm were （81.5 ± 0.5） ℃ and （82.5 ± 0.5） ℃ respectively. The detection deadline were 9 × 10^2 copies and 4.3 × 10^2 copies respectively. The sensitivity of the technique was much higher than that of regular PCR plus gel electrophoresis method, and the detection results of the technique were the same as that of multiplex PCR. Conclusion The genotypes of -α^4.2, - α^3.7, non-deletion αα/and --^SEA can be sensitively, exactly diagnosed by the real-time PCR with SYBR-Greenl combined with the Dissociation Curve analysis. The assay is automatic, low-cost, high throughput, and easy for quality control without fluorescence probe. Key words: real-time polymerase chain reaction; SYBR-Green 1; Dissociation Curve; α-thalassemia
-
molecular diagnosis of α thalassemia by combining real time pcr with sybr green1 and Dissociation Curve analysis
Translational Research, 2006Co-Authors: Jingzhong Liu, Mei Yan, Lirong Wang, Yan Zhou, Zhangyong Wang, Bai XiaoAbstract:The aim of the study was to set up an automatic molecular diagnostic method for deletional α-thalassemia without gel electrophoresis and TaqMan probe. Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by Dissociation Curve (DC) analysis were used to detect the −− SEA , − α 3.7 , −α 4.2 , and non-deletion-type alleles (α α or α T α), respectively. Positive results of the SYBR-PCRs were defined by the special shapes of the Dissociation Curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon ≥ cutoff values. Molecular diagnosis of α-thalassemia was determined by combining all four SYBR-PCR results. The specific Tms for the SYBR-PCR1-4, which was used to detect the −− SEA , − α 3.7 , −α 4.2 , and non-deletion-type alleles were 82.5 ± 1°C, 82.8 ± 1°C, 81.5 ± 1°C, and 83.0 ± 1°C, respectively. The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70. The C T VS log copies of a recombinant plasmid DNA showed a good linear relationship between 10 5 ∼ 10 0 . Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method. Diagnostic outcomes of the 120 α-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods. The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of α-thalassemia.
Kelli J Kochan - One of the best experts on this subject based on the ideXlab platform.
-
application of Dissociation Curve analysis to radiation hybrid panel marker scoring generation of a map of river buffalo b bubalis chromosome 20
BMC Genomics, 2008Co-Authors: Kelli J Kochan, Elisabete M J Amaral, Richa Agarwala, Alejandro A Schaffer, Penny K RiggsAbstract:Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification Dissociation Curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and Dissociation Curve analysis for use as a novel scoring method. As proof of principle for this Dissociation Curve method, we generated new maps of river buffalo (Bubalus bubalis) chromosome 20 by both Dissociation Curve analysis and conventional marker scoring. We also obtained sequence data to augment Dissociation Curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46%) were suitable as cross-species markers for Dissociation Curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly. Dissociation Curve analysis is reliable and efficient for radiation hybrid panel scoring, and is more sensitive and robust than conventional gel-based typing methods. Several markers could be scored only by the new method, and ambiguous scores were reduced. PCR-based Dissociation Curve analysis decreases both time and resources needed for construction of radiation hybrid panel marker maps and represents a significant improvement over gel-based methods in any species.
-
application of Dissociation Curve analysis to radiation hybrid panel marker scoring generation of a map of river buffalo b bubalis chromosome 20
BMC Genomics, 2008Co-Authors: Kelli J Kochan, Elisabete M J Amaral, Richa Agarwala, Alejandro A Schaffer, Penny K RiggsAbstract:Background Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification Dissociation Curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and Dissociation Curve analysis for use as a novel scoring method.