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Article
Effect of Dimethylsulfoxide and Betaine on duplex polymerase chain reaction of human beta-globin gene amplification

Author: Zuhair Mohammad Ali Jeddoa
Journal: journal of kerbala university مجلة جامعة كربلاء ISSN: 18130410 Year: 2012 Volume: 10 Issue: 2 Pages: 171-177
Publisher: Kerbala University جامعة كربلاء

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Abstract

A variety of additives and enhancing agents can be included in PCR amplifications to increase yield, specificity and consistency of PCR products. Blood samples were collected from (35) apparently healthy individual for DNA extraction and duplex PCR amplification according to the four different PCR component set up include: 1- standard set up (without PCR additives), 2- standard PCR set up plus 5% DMSO, 3- standard PCR set up plus 1M betaine, and 4- standard PCR set up plus 5% DMSO and 1M betaine. The results revealed that the standard optimization condition of duplex PCR amplification is enough to reveal a good PCR amplification of human beta-globin gene, and the studied PCR additives are useful in improvement of amplification in combination of 5 % DMSO and 1 M betaine.

يمكن استعمال العديد من الأضافات والمواد التي تزيد من انتاج سلاسل الحامض النووي منقوص الأوكسجين وزيادة خصوصية تضخيمها في تفاعل السلسلة المتبلمرة (PCR) , , جمعت عينات الدم من 35 شخص سليم مظهريا لغرض اجراء استخلاص الحامض النووي منقوص الأوكسجين ومن ثم تضخيم المنطقة من الجين المشمول بالدراسة باستخدام تفاعل السلسلة المتبلمرة طبقا لأربعة نماذج مختلفة من المواد المستخدمة بالتفاعل والتي شملت: 1-التضخيم باستعمال المواد القياسية ( بدون اضافة محسنات التفاعل), 2- بأضافة 5% من ثنائي ميثيل سلفوكسايد , 3- بأضافة 1 مولاري من مادة البيتايين , و4- بأضافة 5% من ثنائي ميثيل سلفوكسايد مع 1 مولاري من مادة البيتايين. أظهرت النتائج ان استعمال المواد القياسية في تضخيم المناطق المدروسة من جين الكلوبين – بيتا البشري تكون كافية لأعطاء نتائج جيدة في عملية التضخيم , وان اضافة المحسنات المشمولة بالدراسة ساعدت على زيادة كمية الحامض النووي منقوص الأوكسجين في المناطق المضخمة وخصوصا عند استعمال أضافة 5% من ثنائي ميثيل سلفوكسايد مع 1 مولاري من مادة البيتايين .


Article
Evaluation of (HPLC) Patterns of Sickle Cell Anaemia Patients in Comparison with Apparently Healthy Individuals

Author: Zuhair Mohammad Ali Jeddoa
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2011 Volume: 4 no 1 Issue: 9 Pages: 980-987
Publisher: Kerbala University جامعة كربلاء

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Abstract

background: Sickle cell haemoglobin (HbS) results from an autosomal recessively inherited mutation in which the amino-acid glutamine is replaced by valine at position 6 in the beta globin chain of haemoglobin (Hb). Sickle cells have a reduced deformability and are easily destroyed, causing occlusion of the microcirculation and a chronic haemolytic anaemia with a median Hb concentration level of about 9 g/dl. Routine electrophoresis methods and High performance liquid chromatography (HPLC) were used to screen normal and variant Hb, and allowed the verification of the Hb observed with electrophoresis and precise quantification of their proportion.Objectives: 1- This study aimed to evaluate the chromatographic pattern of Hb types (HbA, HbF, HbA2 and HbS) of sickle cell anemia patients in comparison with the apparently healthy individuals.2- To study the Hb chromatographic patterns according to the gender, age and blood groups.3- To evaluate the efficiency of variant Hb testing system in detection of HbS type of sickle cell anaemia patients.Methods: A total of eighty four sickle cell anemia patients who were attending to theIbn Al-Baladi pediatrics hospital (in Baghdad) and Kerbala teaching pediatricshospital, the samples were (39) males and (45) females , from April of 2006 toFebruary of 2007 . And thirty seven of case controls with matched age and sex wererandomly selected from apparently healthy individuals. High performance liquidchromatography (HPLC) was adopted to determine the different types of Hb forpatients and control groups using variant Hb testing system which depend upon theseparation and quantification of Hb types by high performance liquid chromatographytechnique.Results: The study of Hb chromatographic patterns of samples revealed that there were no significant differences (p ≥ 0.05) between the values of HbA2 for the patients and control groups and there were significant differences (P≤ 0.05) for HbF and highly significant differences (P≤ 0.01) for HbA and HbS percentages of patients in comparison with the control group. The results of Hb chromatographic patterns of samples according to the gender revealed that there were no significant differences at (p≥ 0.05) between males and females within patient and control groups. The results of Hb chromatographic patterns of Hb types for patients and control groups according to the age groups revealed heterogeneous results with highest HbF, HbA, and HbA2 percentages of patients were (10.83±3.32, 57.6±7.33, and 4.22 ±1.88) respectively in age group less than five years old, while it was (54.33±8.9) for HbS type in age group (6-10) years old. As for control group, the highest HbF percentages was (8.2±4.09) in age group less than five years old, for HbA was (87.22±5.86) in age group (6-10) years old, for HbA2 was (3.6±0.23) in age group (11-15) years old, and for HbS was (0.3±0.05) in age group less than five years old. Finally, the results also showed that there were no significant differences at (P ≥ 0.05) for Hb chromatographic patterns of different Hb types percentages according to the blood groups of studied samples within group (patients or controls).Conclusion:1- The study of Hb chromatographic patterns is useful for the diagnosis of sickle cell anaemia.2- There are no significant effects of gender and blood groups on the chromatographic patterns of different Hb types of sickle cell anaemia patients in comparison to the apparently healthy individuals.3- HPLC is an excellent, powerful diagnostic tool for the direct identification of HbS.


Article
Molecular characterization of Beta- thalassemia Mutations in Holy Karbala
الكشف الجزيئي عن الطفرات المسببة لفقر الدم البحري (بيتا) في كربلاء المقدسة

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Abstract

AbstractBeta- thalassemia is autosomal recessive disease that spread inhigh percentages around the world. the importance of the presentstudy was in evaluating the disease in holy Kerbala، Iraq using moleculartechniques.A total of 36 of blood samples were collected from beta- thalassemiamajor patients، 20 samples intermedia thalassemic patients fromthalassemia center- AL-Hussain medical city in holy Kerbalagovernorate، and 20 samples from apparently healthy individuals as acontrol group.The DNA molecules were extracted from all samples for molecularcharacterization of four β- thalassemia mutations are [IVS1-110(G>C)،CD8/9(+G)،-87(C>G)،CD41/42(-TCTT)] using PCR basedtechnique called Amplification Refractory Mutation System (ARMS).The results revealed that IVS1-110 is the common mutation70%) in the studied samples، the CD8/9(+G) mutation was ) 14diagnosed for first time in holy Kerbala 2(10%)، the -87(C>G) wasdiagnosed for the first time in Iraq in percentage 4(20%) and finally theCD 41/42 was not diagnosed in the studied samples.Geographical distribution of diagnosed mutations were studied thedistribution of mutations within Kerbala regions، the results showedthat the city center was more affected with diagnosed mutations.Finally، results also revealed that heterozygotes were the moregenotypic patterns 20(74.07%) of mutations in comparison withhomozygotes with were 5(18.52%)، and the compound cases were2(7.41%) of studied mutations.Key words: Thalassemia، ARMS-PCR، Mutations، IVS1-110(G>C)،CD8/9(+G)،-87(C>G)،CD41/42(-TCTT)، Kerbala.

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