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Article
DETECTION OF A, A- MUTATIONS OF G6PD GENE ON MOLECULAR LEVEL IN IRAQI POPULATION
الكشف عن طفرات A, A- المصاحبة لأنزيم نازع هيدروجين الكلوكوز-6- فوسفات (G6PD) على المستوى الجزيئي بإستعمال تقنية PCR/RFLP في عينات محلية

Author: Rana A. Al-Temmemy
Journal: Iraqi Journal of Biotechnology المجلة العراقية للتقانات الحياتية ISSN: 18154794 Year: 2012 Volume: 11 Issue: 2 Pages: 357-368
Publisher: Baghdad University جامعة بغداد

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Abstract

The study involved the extraction of deoxyribonucleic acid (DNA) from 71 samples of random G6PD patients and 85 samples apparently healthy individuals from different Iraqi populations respectively, which was then amplified by polymerase chain reaction (PCR) and later subjected to digestion by restriction enzymes (Nla III and Fok I) to create restriction fragment length polymorphism (RFLP) to enable the detection of mutation that caused G6PD deficiency namely A and A-. The results of the current study showed that Iraqis were affected by G6PD deficiency in a percentage 7.2% and showed that the affected cases were attributed to A mutation in 4.2% while 0% was recorded for A- mutation.

تضمنت الدراسة إستخلاص الدنا من 71 عينة مصابة سرسرياً بنقص نازع هيدروجين الكلوكوز-6-فوسفات (G6PD) و 85 حالة من الاصحاء ظاهرياً ثم أخضعت هذه العينات لتفاعل التضاعف التسلسلي ((PCR بعدها تم إستعمال احد الطرق المعتمدة (RFLP) وذلك باستعمال انزيمات قاطعة (Nla III and Fok I ) للكشف عن الطفرتين قيد الدراسة. بينت نتائج الدراسة الحالية إن نسبة الإصابة بمرض نقص نازع هيدروجين الكلوكوز-6-فوسفات في العراق هي 7.2% وان الطفرة نوع A قد تسببت ب 4.2% من مجمل حالات الإصابة في حين لم تسجل أي إصابة بالطفرة من نوع A-.

Keywords

G6pd Detection --- Mutations --- Rflp


Article
GENOTYPING OF THE ABO BLOOD GROUP SYSTEM IN IRAQ POPULATION USING PCR-RFLP
التنميط الوراثي لمجموعة الدم ABO للمجتمع االعراقي بإستعمال PCR-RFLP

Author: Mohammed I. Nader
Journal: Iraqi Journal of Biotechnology المجلة العراقية للتقانات الحياتية ISSN: 18154794 Year: 2012 Volume: 11 Issue: 2 Pages: 464-474
Publisher: Baghdad University جامعة بغداد

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Abstract

Due to a large number of alleles that could give similar phenotypes but differ in genomic structure. This study study was conducted to determine the ABO genotyping by use PCR-RFLP method. Blood samples withdrawn from 30 unrelated individuals who appear to be healthy. ABO system phenotype grouping was detected by two immunoagglutination tests. Slide and ELISA were done for all samples. Homozygous allele was selected by chosen special family which gives known offspring according to Mendel laws. The DNA was extracted by using two methods, the manual method by using extra gene kit gives relatively high yield concentration (5-10µg/ml) but slightly low purity (0.9-3.3) and the automated BioRobot EZ1 gives slightly lower yield of DNA(4-8 µg/ml) but high purity (1.6–2.2) in comparison with manual method . Two separate segments of ABO gene the glycosyltransferases gene (216pb) in exon 6 and (703pb) in exon 7 were amplified using two sets of specific primers. Restriction digisten of the two amplification PCR productes was used for discrimination of AB and O allells.

هدفت الدراسة الى الكشف الجزيئي لمجموعة الدم ABO ومطابقتها مع الكشوف المختبرية المعتمدة على النمط الظاهري وإيجاد أنسب الظروف لعمل الكشف الجزيئي. توجد العديد من السمات التي تتشابة بالنمط الظاهري لكنها تختلف بالتركيب الوراثي ومن أبرز الأمثلة عليها جين مجموعة الدم ABO. جمعت عينات الدم من 30 شخص بدو من الفحص الظاهري أنهم أصحاء, أستعملت طريقتين للتحري عن النمط الظاهري لفصائل الدم لكل النماذج. الطريقة الاولى التلازن المباشر على الشريحة, الطريقة الثانية هي اليلايزا، إذ تطابقت النتائج لطريقتان علماً ان الطريقتان لم تميزا نوع الاليل. تم إنتخاب أفراد من أبوين معلومين لصنف الدمAB) ) حيث يكون نسل هذه العوائل متجانس الزيجة Homozygous وذلك لقلة صفة التجانس الوراثي في المجتمعات مقارنةً بالطرز الهجين . Heterozygous عزلت المادة الوراثية بطريقتين, الطريقة اليدوية بإستعمال عدة العزل وكانت ذات تركيز عالي نسبياً (10 – 5 ميكروغرام) لكنها بنقاوة دون المثالية (3.3 – 0.9). كانت نقاوة الطريقة الميكانكية بإستعمال جهاز عزل المادة الوراثية الالي (EZI )أفضل (2.2- 1.6) لكنها أقل تركيزاً (8 – 4 ميكروغرام) مقارنهً بالطريقة اليدوية. ضبط تركيز المادة الوراثية مع مايلائم طريقة تفاعل التضاعف التسلسلي (PCR)وأستعملت تسلسلات من بادىء البلمرة( Primers ) مخصصة لمضاعفة قطعتان تمثل جينABO بجميع اليلاته. تم مضاعفة قطعتان من جين ABO, القطعة الاولى في المحور 6 تتضمن النيوكليوتيد 261, والقطعة الثانية في المحور7 والمتضمنة النيوكليوتيد 703(جين ABO يتكون من 7 اكسونات و 6 انترونات) نتجَ من مضاعفة القطعة الاولى 200 زوج قاعدة بينما نتج عن القطعة الثانية 128 زوج قاعدة. فصلت نواتج بإستعمال النيوكليوتيدات 261 و 703 لتميز اليلات مجاميع الدم A,B,O بواسطة أنزيمات التقييد.

Keywords

Abo Blood --- PCR-RFLP --- Genotyping


Article
Molecular Basis of G6PD Deficiency in Babylon : Iraq

Authors: Munaf S. Dauod --- William M. Frankool --- Fadhil J. Al-Touma
Journal: Iraqi National Journal Of Chemistry المجلة العراقية الوطنية لعلوم الكيمياء ISSN: 22236686 Year: 2010 Issue: 39 Pages: 571-588
Publisher: Babylon University جامعة بابل

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Abstract

AbstractObjective: The objective of this study was to investigate the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) genes in hyperbilirubinemic neonates in Babylon province of Iraq by using molecular methods (genomic DNA extraction, PCR and RFLP analysis) and then to investigate the type of G6PD variant predominantly present. Methods: The study included a total of 236 full-term male neonates, 183 of them were associated with severe hyperbilirubinemia which were admitted in Teaching Hospital of Pediatric and Maternity / Babylon during 1st , Oct., 2007 to 14th , July, 2008 with age ranged between 1 – 28 days, their TSB levels ≥ 15 mg/dl , while another 53 neonates were used as control group. The blood sample taken from each neonate was divided into two aliquots: the first aliquot was used for hemoglobin (Hb), total and conjugated bilirubin (TSB and SCB), G6PD activity. The second aliquot was used for molecular analysis including genomic DNA extraction and then application of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) protocols. Results and Discussion: Severe hyperbilirubinemic neonates were screened for erythrocyte G6PD enzyme activity measurements, severe G6PD deficiency was detected in 22 of the total 183 hyperbilirubinemic neonates included and their activity levels was significantly decreased (P < 0.05) to 0.34 ± 0.17 U/g Hb as compared with control value 10.02 ± 1.17 U/g Hb. The incidence of severe G6PD deficiency in neonatal hyperbilirubinemic neonates identified was 12.02 %. TSB levels were markedly elevated to (23.01 ± 5.0 mg/dl), whereas the control value was 0.75 ± 0.23 mg/dl. The mean ± SD values of each of SCB and Hb were significantly lower than that found in controls (P < 0.05) and reached to 0.063 ± 0.036 mg/dl ; 13.32 ± 0.94 g/dl as compared with that found in control neonates 0.19 ± 0.11 mg/dl ; 15.91 ± 1.62 g/dl respectively. Conjugated bilirubin was undetectable in sera of 10 of 22 neonates (45.5%) with severe G6PD deficiency which imply a partial defect of bilirubin conjugation. The molecular part of the study involved the extraction of genomic DNA from hyperbilirubinemic neonates with severe G6PD deficiency which detected by agarose electrophoresis and then amplified by PCR and finally was subjected to digestion by endonuclease restriction enzymes to create RFLP to enable the detection of mutation that caused G6PD deficiency. The overall majority of affected severe G6PD-deficient neonates with hyperbilirubinemia in Babylon province : Iraq were due to G6PD Med variant (C563T, Ser 188 Phe) in which 19 out of 22 (86.4%) have this type of gene mutation, and only one G6PD A- variant (4.55%) (G202A ; A376G mutations) was diagnosed, whereas 2 of 22 (9.1%) remain with unknown G6PD variants. Conclusion: The predominant G6PD gene detected in hyperbilirubinemic neonate with severe G6PD deficiency in Babylon province was G6PD Med.

الخلاصة يعتبر مرض عوز نازعة هيدروجين الكلوكوز- 6- فوسفات (G6PD deficiency) من الأمراض المنتشرة في مناطق مختلفة من دول العالم حيث أن عدد المصابين يتجاوز اﻠـــ 400 مليون من اﻠﺬكور والإناث ومن حديثي الولادة والأعمار الأخرى و يعتبر من الأسباب المؤدية إلى حدوث مرض اليرقان الولادي اﻠﺬي يسبب مضاعفات سريرية خطيرة تؤدي إلى تلف الدماغ ومن ثم الموت. وقد تم تشخيص أكثر من 442 نوع من أنماط الإنزيم (G6PD Variants) باستعمال عدد كبير من التقنيات الحيوية ومنها التقنيات الجزيئية Molecular analytical methods والتي تحدد الطفرات الوراثية التي تحدث في الجينات المسئولة عن التصنيع الحيوي للأنماط المختلفة من الإنزيم . إن أحد أهم أهداف ﻫﺬه الدراسة هو تحديد نسبة انتشار المرض لدى حديثي الولادة من اﻠﺬكور ﺬو النمو الجنيني المتكامل والمصابين بمرض اليرقان الولادي في محافظة بابل وتحديد الطفرات الوراثية في تتابع القواعد النتروجينية للجينات المسئولة عن تصنيع الأنماط الإنزيمية المختلفة (G6PD Variants) والتي تسبب حدوث مرض اليرقان الولادي الحاد باستعمال طرق جزيئية وباستعمال236 عينة من الذكور حديثي الولادة للفترة من 1/ 10 2007/ولغاية 12/ 7/ 2008 ولأعمار تراوحت ما بين 1 – 28 يوما حيث تم توزيعهم إلى مجموعتين اعتمادا على تركيز البيليروبين الكلي TSB وكما يلي:- - المجموعة الأولى وهي مجموعة السيطرة والتي شملت 53 (22.46%) حديث الولادة وكان تركيزالبيليروبين الكلي طبيعيا (1 mg/dl (TSB <. - المجموعة الثانية والتي شملت 183 (77.54%) حديث الولادة مصابين باليرقان الولادي وكان تركيز البيليروبين قد ارتفع وبشدة ≥15 mg /dl) TSB). وتضمنت ﻫﺬه الدراسة متابعة تأثير العوز الحاد للإنزيم على تركيز كل من الهيموكلوبين , البيليروبين الكلي TSB والبيليروبين المقترن SCB في مصل الدم. ولقد وجد أن نشـــــــــــــاط الإنزيم قد انخفض معنويا بشكل حاد في 22 حالة من حالات اليرقان الولادي ووصل إلى IU/g Hb 0.34 ± 0.17 مقارنة مع المعدل الطبيعي وان معدل الـ TSB قد ارتفع إلى 23.01 ± 5.0 mg/dl . وان نسبة انتشار النقص الحاد للإنزيم في محافظة بابل كانت 12.02%) ). وقد اقترن ﺬلك بانخفاض معنوي في تركيز البيليروبين المقترن SCB وﻫﺬا يوضح عدم حدوث عملية الاقتران للبيليروبين في خلايا الكبد لغــــــــرض التخلص منه بسبب عدم نضج ميكانيكية الاقتران إضافة إلى وجود نقص في بعض الإنزيمات المسئولة عن ﺬلك ومنها إنزيم اﻠـــ UGT1A1 . وقد وجد أن هناك ارتباط معنوي(P<0.05) سالب مابين نشاط إنزيم الــ G6PD المنخفض بشدة وتركيز البيليروبين الكلي TSB اﻠﺬي ارتفع إلى أكثر من .15 mg/dl وقد تضمن المحور الجزيئي التحري عن الطفرات الوراثية للقواعد النتروجينية التي تحدث في الجينات المسئولة عن التصنيع الحيوي لإنزيم اﻠـــ G6PD باستعمال التقنيات الجزيئية Molecular Analysis والتي تعتمد على استخلاص جزيئة اﻠـحامض النووي منقوص الأوكسجين Genomic DNA التي تم استخلاصها من دم الذكور حديثي الولادة والمصابين بالنقص الحاد في نشاط إنزيم اﻠـــ G6PD باستعمال طقم خاص تم استيراده من شركة Roche الألمانية ومن ثم متابعة دراسة التحليل الجيني للطفرات الوراثية حيث أخضعت العينات المرضية مع مجموعة السيطرة إلى تفاعل التضاعف التسلسلي Polymerase Chain Reaction للحامض النووي بعدها تم استعمال طريقة الهضم بالأنزيمات المعتمدة (RFLP) للكشف عن الطفرات في الجينات المسئولة عن التصنيع الحيوي لنمطي الإنزيم G6PD Med and G6PD A- . وقد بينت الدراسة أن الطفرات التي تم تشخيصها والتي تحدث في الجينات المسئولة عن التصنيع الحيوي للإنزيم والتي تسبب نقصان حاد في نشاطه الحيوي في مصل الدم باستعمال الطرق الجزيئية ومن ثم إحداث اليرقان الولادي في حديثي الولادة بمحافظة بابل كانت 19 حالة من النمط المتوسطي للإنزيم G6PD Med(C563T) ونسبته% (86.4 ) بينما تم تشخيص حالة مرضية واحدة من النمط الأفريقي G6PD A- (G202A ; A376G) وكانت نسبته هي ( %4.55) ولكن بقيت حالتين (% 9.1) لم يتم تشخيص نوع الطفرة الوراثية في الجين المسئول عن النمط الإنزيمي لهما .


Article
Molecular Basis of G6PD Deficiency in Hyperbilirubinemic Neonates in Middle Euphrates Province : Iraq

Authors: William M. Frankool --- Fadhil Jawad Al-Tu'ma
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2010 Volume: 3 no.3, 4 Issue: 7 Pages: 867-881
Publisher: Kerbala University جامعة كربلاء

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Abstract


Background: Neonates G6PD deficiency screening has been recognized as an essential component of public health care in most developed and some Mediterranean countries. However, such screening is yet to be widely embraced in Iraq. More than 442 variants of G6PD have been identified by various molecular methods. The aim of the present study was to determine the normal values of G6PD and deficiency prevalence of this enzyme in male neonates and then determination of the type molecular variant of G6PD prevalence in Middle Euphrates Province of Iraq.
Objective: The objective of this study was to investigate the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in hyperbilirubinemic neonates in Middle Euphrates province of Iraq. Molecular methods (genomic DNA extraction, polymerase chain reaction and restriction fragment length polymorphism analysis) and then investigate the type of G6PD variant predominantly present have been performed.
Methods: The study included a total of 917 full-term male neonates which were divided into two groups:
The first group which include 704 neonates (76.8%) associated with severe hyperbilirubinemia were admitted in Middle Euphrates Province Teaching Hospitals of Maternity and Pediatrics during 1st Oct., 2007 to 12th July, 2008 with age ranged between 1 – 28 days, their total serum protein , TSB levels ≥ 15 mg/dl.
The second group which include 213 neonates (23.2%) with the same age ranged were used as control group, their TSB levels ˂ 1 mg/dl. The blood sample taken from each neonate was divided into two aliquots: the first aliquot was used for the determination of total and serum conjugated bilirubin (TSB and SCB), and G6PD activity. The second aliquot was used for molecular analyses including genomic DNA extraction and then application of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) protocols.
Results and Discussion: Severe hyperbilirubinemic neonates were screened for erythrocyte G6PD enzyme activity, severe G6PD deficiency was detected in 75 of 704 hyperbilirubinemic neonates included and their activity levels was significantly decreased (P < 0.05) to less than 10% of that found in control group. Therefore, the incidence of severe G6PD deficiency identified in Middle Euphrates Province of Iraq was 10.65%. TSB levels were markedly elevated to ( ≥ 15 mg/dl), whereas the mean ± SD values of SCB were significantly lower than that found in controls (P < 0.05) , SCB was undetectable in 32 of 75 (42.67%) of hyperbilirubinemic neonates with severe G6PD deficiency which imply a partial defect of bilirubin conjugation. The molecular part of the study involved the extraction of genomic DNA from hyperbilirubinemic neonates with severe G6PD deficiency which was detected by agarose gel electrophoresis and then amplified by PCR and finally was subjected to digestion by endonuclease restriction enzymes to create RFLP and to enable the detection of mutation that caused G6PD deficiency. The majority of affected severe G6PD deficient neonates with hyperbilirubinemia in Middle Euphrates province – Iraq, were due to G6PD Med variant (C563T, Ser 188 Phe) , of such 67 of 75 neonates (89.3%) have this type of mutation, and 5 of 75 (6.67%) have G6PD A- variant (G202A ; A376G mutations), whereas only 3 of 75 (5.3%) remain unknown G6PD variants which require future molecular studies.
Conclusion: The predominant G6PD gene detected in hyperbilirubinemic neonate with severe G6PD deficiency in Middle Euphrates province was G6PD Med.variant.
Keywords : Hyperbilirubinemia, G6PD gene, Polymerase Chain Reaction , RFLP.


Article
Molecular Characterization of Severe G6PD Deficiency in Hyperbilirubinemic Neonates in Karbala : Iraq……

Authors: Fadhil J. Al-Touma --- William M. Frankool
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2009 Volume: 2 no.8, 9 Issue: 5 Pages: 663-680
Publisher: Kerbala University جامعة كربلاء

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Abstract

Objective: The objective of this study was to investigate the molecular basis ofglucose-6-phosphate dehydrogenase (G6PD) genes in hyperbilirubinemic neonates inKarbala province of Iraq by using molecular methods (genomic DNA extraction, PCRand RFLP analysis) and then to investigate the type of G6PD variant predominantlypresent.Methods: The study included a total of 253 full-term male neonates, 197 of themassociated with severe hyperbilirubinemia which were admitted in Karbala TeachingHospital of Pediatrics during the period from 1st October 2007 to 14th July 2008 withage ranged between 1 – 28 days, their TSB levels ≥ 15 mg/dl, and another 53 neonateswere used as control group. The blood sample taken from each neonate was dividedinto two aliquots: the first aliquot was used for total and conjugated serum bilirubin(TSB and SCB), G6PD activity. The second aliquot was used for molecular analysisincluding genomic DNA extraction and then application of polymerase chain reaction(PCR) and restriction fragment length polymorphism (RFLP) protocols.Results and Discussion: Severe hyperbilirubinemic neonates were screened forerythrocyte G6PD enzyme activity measurements, severe G6PD deficiency wasdetected in 18 of the total 197 hyperbilirubinemic neonates included and their activitylevels was significantly decreased (P < 0.05) to 0.56 ± 0.32 U/g Hb. The incidence ofsevere G6PD deficiency identified was found 9.14%. TSB levels were markedlyelevated to (20.26 ± 4.96 mg/dl), whereas the mean ± SD values of SCB weresignificantly lower than that found in controls (P < 0.05) and reached to 0.053 ± 0.046mg/dl , and it was undetectable in 5 of 18 neonates (27.78%) with severe G6PDdeficiency which imply a partial defect of bilirubin conjugation. The molecular part ofthe study involved the extraction of genomic DNA from hyperbilirubinemic neonateswith severe G6PD deficiency which detected by agarose electrophoresis and thenamplified by PCR and finally was subjected to digestion by endonuclease restrictionenzymes to create RFLP to enable the detection of mutation that caused G6PDdeficiency. The overall majority of affected severe G6PD neonates withhyperbilirubinemia in Kerbala province : Iraq were due to G6PD Med variant(C563T, Ser 188 Phe) in which 17 out of 18 (94.4%) have this type of mutation, andonly one G6PD A- variant (5.56%) (G202A ; A376G mutations) was diagnosed.Conclusion: The predominant G6PD gene detected in hyperbilirubinemic neonatewith severe G6PD deficiency in Kerbala province was G6PD Med.


Article
Association Between Polycystic Ovary Syndrome and Genetic Polymorphisms of CYP 17 Gene in Iraqi Women

Author: Marwa B. Mohammed1, Salwa J. AL-Awadi2, Mahfoodha A. Omran3
Journal: Iraqi Journal of Biotechnology المجلة العراقية للتقانات الحياتية ISSN: 18154794 Year: 2015 Volume: 14 Issue: 2 Pages: 99-110
Publisher: Baghdad University جامعة بغداد

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Abstract

Polycystic ovary syndrome (PCOS), whose genetic basis is not completely well understood, it is the most common endocrine disorder in women in their reproductive years. Blood samples were collected from two groups. The first group included 61 females with PCOS and the second group included 30 normal females to detect the presence of mutation in the CYP17 gene. The two groups were genotyped and a comparison was done between them. The results showed a significant difference (p<0.05) in FSH levels in patients and normal females and there is no significant difference in the levels of Testosterone, prolactin and LH. Moreover, it showed a significant difference (p<0.05) in the levels of HDL in both patients and normal females and no significant difference in the levels of (LDL, VLDL, Triglyceride and cholesterol). Two comparisons for genotype were done: one between age and genotype and the second between BMI and genotype for each group. The results showed two types of genotype, which were a TT wild type and a heterozygote TC mutant type. Furthermore, the results showed a significant difference (p<0.05) in genotype TT and TC in the group of age less than 25 years old and no significant difference in these genotypes in age groups (25-35) and those with more than 35 years old. It is concluded from this study that this single nucleotide polymorphism in the CYP 17 gene was not associated with PCOS in Iraqi women.

Keywords

PCOS --- BMI --- CYP17 --- SNP --- RFLP --- genotype.


Article
Genotyping of Cryptosporidium Isolates from Clinical Samples

Author: Thanaa Ismael Jawad
Journal: Medical Journal of Babylon مجلة بابل الطبية ISSN: 1812156X 23126760 Year: 2015 Volume: 12 Issue: 3 Pages: 632-637
Publisher: Babylon University جامعة بابل

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Abstract

Cryptosporidium is an intracellular protozoan that infects the gastrointestinal tract of human and other animals. Two main species of this parasite are known to infect human C. hominis and C. parvum with the latter has a zoonotic transmission. This study aimed to genotype the clinical isolates of Cryptosporidium from children with diarrhea.A total of 64 fecal samples were collected from children with diarrhea. Cryptosporidial infection was detected using modified acid fast stain. DNA of the parasite was extracted from oocysts of positive fecal samples and nested PCR method was used for TRAP-C2 gene amplification. Restriction fragment length polymorphism (RFLP) was applied for genotyping.Four fecal samples from 64 (0.625%) gave positive result for Cryptosporidium. RFLP-PCR suggested that 3 samples of these related to C. parvum and 1 sample of mixed infection with both C. parvum and C. hominis. From these results it can be concluded that most infections in children with cryptosporidiosis are of bovine origin.

Keywords

nested-PCR --- RFLP --- TRAP-C2 --- cryptosporidiosis


Article
Analysis of Polymorphism of Melatonin Receptor Type 1A (MTNR1A) Gene, in Iraqi Local Sheep Using PCR-RFLP Technique

Author: Rahawy M. A.1 , AL-Timimi, I. H.2 , Najwa Sh. Ahmad3
Journal: Iraqi Journal of Biotechnology المجلة العراقية للتقانات الحياتية ISSN: 18154794 Year: 2016 Volume: 15 Issue: 3 Pages: 85-95
Publisher: Baghdad University جامعة بغداد

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Abstract

The gene encoding melatonin receptor type 1A (MTNR1A) gene by PCR-RFLP, the aim of the present study of polymorphism MTNR1A gene associated with breeding out of season and correlated with prolactin hormone in Iraqi sheep breed,. Blood samples were randomly collected from 60 ewes from Agriculture Research-Ruminant Researches station breeding station, Baghdad, and genomic DNA was isolated using a Geneius TM Micro gDNA Extraction Kit. A large fragment of exon 2 of MTNR1A gene was amplified by PCR using specific primer pairs and the PCR product was digested with RsaI enzyme which cuts the amplicon to several fragments. There are four cleavage sites (53 bp, 267 bp, 23 bp, 411 bp, 70 bp) for Rsa I within the amplification fragment, Digestion with Rsa1yielded polymorphic fragments of 267 bp when the cleavage site was present (allele R) and a single 290 bp fragment when the cleavage site was (allele r). Three genotypes RR (411 bp/267 bp), Rr (411/290 bp/267 bp) and rr (411 bp/290 bp). Three fragments of 23bp, 53 bp and 70bp were been not related allelic polymorphism .Restricted digestion allowed the determination of two alleles (R, r), Result gene frequencies were 0.40 and 0.60 for the alleles R and r respectively, in local ewes and genotype frequencies of RR (20%), Rr (40%) and rr (40%) in ewes with a significant at (P<0.01), the level of prolactin relation to the genotype differences that the highest hormonal level linked to the RR genotype (234.65±17,48ng/ml), and high significant (P<0.01) with lower values for prolactin hormone that linked to Rr and rr genotype (217. 02 ± 28.12; 182.91 ± 21.35 ng/ml) in local Iraqi ewes and no-significant values between the out and the in season lambing months, however, higher significant variation (P<0.01) of frequencies of R genotype (62.50%) in out-seasonal lambing months than in-seasonal lambing months (37.50%). It was concluded that the alleles frequencies of MTNR1A gene ‘r’ higher than ‘R’ alleles, Analysis of the MTNR1A polymorphism its relationship with prolactin concentration of Moreover show the indirect effect of higher prolactin concentration with genotype RR of the MTNR1A gene polymorphism and can be used a genetic selection marker for spring (out-of-season) breeding in Iraqi local sheep.

Keywords

MTNR1A --- PCR-RFLP --- Polymorphism --- Iraqi sheep.


Article
Determination of Different Species of Animal from their Meats by Using PCR-RFLP Technique of Mitochondria Gene COI

Authors: Asaad Y. Ayied --- Zahra R. Al-Mossawi
Journal: Basrah Journal of Agricultural Sciences مجلة البصرة للعلوم الزراعية ISSN: 18175868 Year: 2017 Volume: 30 Issue: 1 Pages: 59-64
Publisher: Basrah University جامعة البصرة

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Abstract

This study was conducted at Dr. Taleb A. Jaayad of Molecular Genetics, Department of Animal Production, College of Agriculture, University of Basrah. Samples of fresh and canned meat of cattle, buffalo, sheep, goats, chicken and turkey were collected randomly from different areas of Basrah province, as well as blood samples of camel. The aim was to determine different animal species from their meat (except camel). DNA was extracted from meat tissue (0.2 gm) and blood by using DNA kit (Invetrogen). DNA purity was estimated by using wavelength (260-280), to be 1.8-2.0 ng. PCR was used to amplify mtco1 gene using a general primer and gave a band of 710 bp for all species used in this study. Different species were determined by using Taq restricted enzyme. Cattle, buffalo, chicken and turkey showed one band of 637 bp. Taq enzyme has recognized sheep and goat, while sheep did not show any band to the fragment 710 bp. However, goat showed a band at 650 bp. Furthermore, camel produced two bands of 303 and 403 bp.

Keywords

Animal --- species --- meat --- mt CO1 --- PCR-RFLP.


Article
Detection of genotypes for Giardia lamblia in Iraqi patients feces by using PCR-RFLP techniques based on GDH gene characterization
التحري عن الطرز الوراثية لطفيلي Giardia lamblia في براز المرضى العراقيين بوساطة تقنية PCR-RFLP واستنادا الى خصائص جين GDH

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Abstract

This study was carried out during the period from March 2016 to January 2017 to detect of genotypes and subgenotypes of Giardia lamblia in human feces by PCR-Restriction fragment length polymorphism based on the detection of glutamate dehydrogenase gene and study the corralation between genotype and prescence of symptoms , a total of 101 stool samples were taken from patients(male and female), aged(2-72) years, who suffering from acute or persistent diarrhea and examined by microscope and molecular techniques , the gdh gene was amplified from 19 cases only by a conventional PCR assay by using a specific oligonucleotide sequence for coding region by gdh gene at size 432bp and typed by RFLP analysis, the results showed the total infection with Giardia lamblia was 84(83.1%) according to the results of microscopic examination, of the 19 positive samples, 2(1.98%) were infected with genotype AI , 3(2.97%) genotype AII , 5(4.95%) genotype BIII and 9(8.91%) infected with genotype BIV, with significance differences between the genotype and precence of symptoms

اجريت هذه الدراسة خلال المدة الواقعة بين اذار 2016 الى كانون الثاني 2017 كان الهدف منها التحري عن الطرز الوراثية لطفيلي Giardia lamblia في البراز البشري بوساطة تقنية تقييد اطوال القطع متعددة الاشكال PCR-RFLP وذلك من خلال الكشف عن جين gdh ، تم خلال هذه المدة جمع 101 عينة براز من مرضى يشكون من اسهال حاد واعراض الإصابة بداء الجيارديات و من عدد من عمال المطاعم وصالونات الحلاقة والفنادق بأعتبارهم مصدر للعدوى بالطفيليات، تم تضخيم جين gdh من 19عينة بوساطة تقنية PCR التقليدية وتم تنميطها جينيا بوساطة تقنية RFLP PCR-وذلك من خلال ادخال نوعين من الانزيمات القاطعة، من مجموع 19 عينة موجبة لفحص PCR وجد %)1.98)2 مصابين بالطراز الوراثي AI ،3(2.97%) مصابين بالطراز الوراثي AII ، 5(4.95%) مصابين بالطراز الوراثي BIII و 9(8.91%) مصابين بالطراز الوراثي BIV ، لاتوجد فروقات حقيقية بين نسبة الإصابة والعمر، الجنس،المهنة، التباين الفصلي،والمنطقة الجغرافية.

Keywords

Giardia lamblia --- GDH gene --- PCR-RFLP

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