scholarly journals Screening A Trinucleotide Repeat Expansion: How precise PCR can be?

2019 ◽  
Vol 3 (3) ◽  
pp. 34-40
Author(s):  
Ziske Maritska ◽  
Baharudin Baharudin ◽  
Ardy Santosa ◽  
Ching Leng Kee ◽  
Tan Yue Ming ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Trinucleotide Repeat ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Cag Repeats ◽  
Chain Reaction ◽  
Trinucleotide Repeat Expansion ◽  
Pcr Method ◽  
The Mean ◽  
Polymerase Chain

ABSTRACT Background. Trinucleotide Repeat Expansion (TRE) in human DNA could lead to various diseases. An expanded CAG repeat (>31 or 37 repeats, depends on the ethnicity) in Androgen Receptor gene is suggested to be associated with the occurrence of isolated hypospadias. In an effort to identify the exact numbers of repeats, sequencing has been the most favored method to be conducted despite its cost. Objective. This study wished to investigate the possibilities of using Polymerase Chain Reaction (PCR) method to screen expanded repeats in isolated hypospadias, as one of the TRE diseases. Materials and Methods. Numbers of CAG repeat in twelve hypospadias patients and one normal male was first predicted from the visualization of PCR products in 3% agarose gel electrophoreses with 20 bp ladder marker before it was finally sequenced. Results. Two samples gave the same exact result, while the rest showed a range of 1-11 bp differences. Statistically, there was a significant difference between the mean of CAG repeats from PCR method (M=26.1667, SD=6.71272) and the mean of CAG repeats from sequencing (M=23.75, SD=5.70685); t(11)= 4.570, p=0.001. Furthermore, the sensitivity of PCR was 100% and the specificity was 83.33%. Conclusion. It can be concluded that PCR method could be used as a screening method in identifying TRE with large numbers of repeats. However, PCR in TRE disease with small numbers of expanded repeats needs to be followed by sequencing in order to obtain the exact numbers of repeats.   Keywords: Trinucleotide Repeat Expansion, Polymerase Chain Reaction, Sequencing, Isolated Hypospadias

scholarly journals 5′ UTR CGG repeat expansion in GIPC1 is associated with oculopharyngodistal myopathy

Brain ◽  
2020 ◽  
Author(s):  
Jianying Xi ◽  
Xilu Wang ◽  
Dongyue Yue ◽  
Tonghai Dou ◽  
Qunfeng Wu ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Trinucleotide Repeat ◽  
Late Onset ◽  
Age At Onset ◽  
Large Family ◽  
Repeat Expansion ◽  
Chain Reaction ◽  
Cgg Repeat ◽  
Sporadic Cases ◽  
Polymerase Chain

Abstract Oculopharyngodistal myopathy is a late-onset degenerative muscle disorder characterized by ptosis and weakness of the facial, pharyngeal, and distal limb muscles. A recent report suggested a non-coding trinucleotide repeat expansion in LRP12 to be associated with the disease. Here we report a genetic study in a Chinese cohort of 41 patients with the clinical diagnosis of oculopharyngodistal myopathy (21 cases from seven families and 20 sporadic cases). In a large family with 12 affected individuals, combined haplotype and linkage analysis revealed a maximum two-point logarithm of the odds (LOD) score of 3.3 in chromosomal region chr19p13.11-p13.2 and narrowed the candidate region to an interval of 4.5 Mb. Using a comprehensive strategy combining whole-exome sequencing, long-read sequencing, repeat-primed polymerase chain reaction and GC-rich polymerase chain reaction, we identified an abnormal CGG repeat expansion in the 5′ UTR of the GIPC1 gene that co-segregated with disease. Overall, the repeat expansion in GIPC1 was identified in 51.9% independent pedigrees (4/7 families and 10/20 sporadic cases), while the repeat expansion in LRP12 was only identified in one sporadic case (3.7%) in our cohort. The number of CGG repeats was <30 in controls but >60 in affected individuals. There was a slight correlation between repeat size and the age at onset. Both repeat expansion and retraction were observed during transmission but somatic instability was not evident. These results further support that non-coding CGG repeat expansion plays an essential role in the pathogenesis of oculopharyngodistal myopathy.

scholarly journals Diversity ofLeptospiraspp. in Rats and Environment from Urban Areas of Sarawak, Malaysia

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Chai Fung Pui ◽  
Lesley Maurice Bilung ◽  
Kasing Apun ◽  
Lela Su’ut
Keyword(s):  
Polymerase Chain Reaction ◽  
Water Samples ◽  
Urban Areas ◽  
Environmental Samples ◽  
Soil Samples ◽  
Chain Reaction ◽  
Prevalence Studies ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Soil And Water

Various prevalence studies onLeptospirain animals and humans, as well as environmental samples, had been conducted worldwide, including Malaysia. However, limited studies have been documented on the presence of pathogenic, intermediate, and saprophyticLeptospirain selected animals and environments. This study was therefore conducted to detectLeptospiraspp. in rats, soil, and water from urban areas of Sarawak using the polymerase chain reaction (PCR) method. A total of 107 rats, 292 soil samples, and 324 water samples were collected from April 2014 to February 2015. PathogenicLeptospirawas present in 5.6% (6/107) of rats, 11.6% (34/292) of soil samples, and 1.9% (6/324) of water samples. IntermediateLeptospirawas present in 2.7% (8/292) of soil samples and 1.9% (6/324) of water samples. SaprophyticLeptospirawas present in 10.3% (11/107) of rats, 1.4% (4/292) of soil samples, and 0.3% (1/324) of water samples. From this study, 76Leptospiraspp. were isolated. Based on DNA sequencing, the dominantLeptospiraspp. circulating in urban areas of Sarawak are pathogenicLeptospira noguchii, intermediateLeptospira wolffiiserovar Khorat, and saprophyticLeptospira meyeri, respectively. Overall, this study provided important surveillance data on the prevalence ofLeptospiraspp. from rats and the environment, with dominant local serovars in urban areas of Sarawak.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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