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[Rapid detection of three common deletional alpha thalassemias in Chinese by single-tube multiplex PCR].
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005 Apr; 22(2):180-4.ZY

Abstract

OBJECTIVE

To develop a simple, rapid, accurate, and cost-effective single-0tube multiplex polymerase chain reaction (PCR) assay, which could be used for molecular screening and prenatal diagnosis, for detection of three commonest deletional alpha-thalassemias (-- (SEA), -alpha (3.7) and -alpha (4.2)) in Chinese population.

METHODS

Four groups of primers were designed on the basis of gap-PCR, and the PCR reaction condition was optimized systematically with the purpose of amplifying effectively specific DNA fragments that are indicative of the respective genotypes of these three deletional alpha thalassemias. In addition, a pair of primers was designed to amplify LIS1 3' untranslated region (UTR) fragment for use as a separate control for amplification running. A total of 72 blood and prenatal archival DNA samples with various known alpha thalassemia genes or normal alpha globin gene sequence that had been confirmed by Southern blotting analysis or DNA sequencing were collected to test the specificity of this assay by blind analysis. In addition, DNA samples from nine couples at high risk of alpha thalassemia were also analyzed to evaluate the reliability of this technique in prenatal implementation.

RESULTS

Homozygote, heterozygote and double heterozygote of the three commonest deletional alpha thalassemias were well detected simultaneously by this established method. For normal allele, a 2.4 kb amplified band as a systematic control and an alpha (2) gene-specific amplicon of 1.8 kb were produced. Besides the two amplified fragments of normal allele, it was found that a 1.3 kb, a 2.0 kb or a 1.6 kb amplified band could be simultaneously shown for representing --(SEA), -alpha (3.7) and -alpha (4.2) alleles, respectively, in the heterozygous states. In a blind test, this technique accurately detected 100% of the DNA samples previously characterized by Southern blotting or DNA sequencing, and it was successfully applied to prenatal diagnosis of alpha thalassemia in nine at-risk families.

CONCLUSION

The single-tube multiplex PCR protocol presented in this study is easy-to-handle, rapid, reliable and is cost-effective for detecting --(SEA), -alpha (3.7) and -alpha (4.2) chromosomes, and it is suitable for large-scale population screening and for rapid molecular genotyping in clinics.

Authors+Show Affiliations

Zhuhai Institute of Medical Genetics, Zhuhai Municipal Maternal and Child Healthcare Hospital, ZhuhaiìGuangdong, 519001 P. R. China. geneticsman@21cn.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

15793780

Citation

Zhou, Yuqiu, et al. "[Rapid Detection of Three Common Deletional Alpha Thalassemias in Chinese By Single-tube Multiplex PCR]." Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, vol. 22, no. 2, 2005, pp. 180-4.
Zhou Y, Zhang Y, Li L, et al. [Rapid detection of three common deletional alpha thalassemias in Chinese by single-tube multiplex PCR]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005;22(2):180-4.
Zhou, Y., Zhang, Y., Li, L., Li, W., Mo, Q., Zheng, Q., & Xu, X. (2005). [Rapid detection of three common deletional alpha thalassemias in Chinese by single-tube multiplex PCR]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics, 22(2), 180-4.
Zhou Y, et al. [Rapid Detection of Three Common Deletional Alpha Thalassemias in Chinese By Single-tube Multiplex PCR]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005;22(2):180-4. PubMed PMID: 15793780.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Rapid detection of three common deletional alpha thalassemias in Chinese by single-tube multiplex PCR]. AU - Zhou,Yuqiu, AU - Zhang,Yongliang, AU - Li,Liyan, AU - Li,Wendian, AU - Mo,Qiuhua, AU - Zheng,Qing, AU - Xu,Xiangmin, PY - 2005/3/29/pubmed PY - 2009/4/3/medline PY - 2005/3/29/entrez SP - 180 EP - 4 JF - Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics JO - Zhonghua Yi Xue Yi Chuan Xue Za Zhi VL - 22 IS - 2 N2 - OBJECTIVE: To develop a simple, rapid, accurate, and cost-effective single-0tube multiplex polymerase chain reaction (PCR) assay, which could be used for molecular screening and prenatal diagnosis, for detection of three commonest deletional alpha-thalassemias (-- (SEA), -alpha (3.7) and -alpha (4.2)) in Chinese population. METHODS: Four groups of primers were designed on the basis of gap-PCR, and the PCR reaction condition was optimized systematically with the purpose of amplifying effectively specific DNA fragments that are indicative of the respective genotypes of these three deletional alpha thalassemias. In addition, a pair of primers was designed to amplify LIS1 3' untranslated region (UTR) fragment for use as a separate control for amplification running. A total of 72 blood and prenatal archival DNA samples with various known alpha thalassemia genes or normal alpha globin gene sequence that had been confirmed by Southern blotting analysis or DNA sequencing were collected to test the specificity of this assay by blind analysis. In addition, DNA samples from nine couples at high risk of alpha thalassemia were also analyzed to evaluate the reliability of this technique in prenatal implementation. RESULTS: Homozygote, heterozygote and double heterozygote of the three commonest deletional alpha thalassemias were well detected simultaneously by this established method. For normal allele, a 2.4 kb amplified band as a systematic control and an alpha (2) gene-specific amplicon of 1.8 kb were produced. Besides the two amplified fragments of normal allele, it was found that a 1.3 kb, a 2.0 kb or a 1.6 kb amplified band could be simultaneously shown for representing --(SEA), -alpha (3.7) and -alpha (4.2) alleles, respectively, in the heterozygous states. In a blind test, this technique accurately detected 100% of the DNA samples previously characterized by Southern blotting or DNA sequencing, and it was successfully applied to prenatal diagnosis of alpha thalassemia in nine at-risk families. CONCLUSION: The single-tube multiplex PCR protocol presented in this study is easy-to-handle, rapid, reliable and is cost-effective for detecting --(SEA), -alpha (3.7) and -alpha (4.2) chromosomes, and it is suitable for large-scale population screening and for rapid molecular genotyping in clinics. SN - 1003-9406 UR - https://www.unboundmedicine.com/medline/citation/15793780/[Rapid_detection_of_three_common_deletional_alpha_thalassemias_in_Chinese_by_single_tube_multiplex_PCR]_ DB - PRIME DP - Unbound Medicine ER -