scholarly journals Prenatal diagnosis of pyruvate kinase deficiency

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2354-2356 ◽  
Author(s):  
L Baronciani ◽  
E Beutler
Keyword(s):  
Pyruvate Kinase ◽  
Genomic Dna ◽  
Direct Method ◽  
Pcr Amplification ◽  
Prenatal Testing ◽  
Restriction Endonuclease Analysis ◽  
Diagnostic Methods ◽  
Pyruvate Kinase Deficiency ◽  
A Direct Method ◽  
Endonuclease Analysis

Abstract Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a direct method of PCR amplification and restriction endonuclease analysis, detected mutations in fetus genomic DNA. The second method, using two polymorphic sites linked to the PKRL gene, enabled us to establish which chromosome had been inherited from each parent.

scholarly journals Prenatal diagnosis of pyruvate kinase deficiency

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2354-2356 ◽  
Author(s):  
L Baronciani ◽  
E Beutler
Keyword(s):  
Pyruvate Kinase ◽  
Genomic Dna ◽  
Direct Method ◽  
Pcr Amplification ◽  
Prenatal Testing ◽  
Restriction Endonuclease Analysis ◽  
Diagnostic Methods ◽  
Pyruvate Kinase Deficiency ◽  
A Direct Method ◽  
Endonuclease Analysis

Prenatal testing for pyruvate kinase deficiency is often requested by parents who already have an affected child. However, before the development of molecular biologic techniques there were no suitable diagnostic methods. We present here two cases in which the diagnosis was established, one using amniotic fluid cells, the other cord blood. Two different approaches were used. The first, using a direct method of PCR amplification and restriction endonuclease analysis, detected mutations in fetus genomic DNA. The second method, using two polymorphic sites linked to the PKRL gene, enabled us to establish which chromosome had been inherited from each parent.

scholarly journals Comparison of Genomic Methods for Differentiating Strains of Enterococcus faecium: Assessment Using Clinical Epidemiologic Data

1998 ◽  
Vol 36 (11) ◽  
pp. 3327-3331 ◽  
Author(s):  
Connie Savor ◽  
Michael A. Pfaller ◽  
Julie A. Kruszynski ◽  
Richard J. Hollis ◽  
Gary A. Noskin ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Gel Electrophoresis ◽  
Genomic Dna ◽  
Restriction Endonuclease Analysis ◽  
Pulsed Field Gel Electrophoresis ◽  
Agarose Gel Electrophoresis ◽  
Subspecies Level ◽  
Vancomycin Resistant ◽  
Ideal Method ◽  
Endonuclease Analysis

Genomic DNA extracted from 45 vancomycin-resistantEnterococcus faecium (VRE) isolates was cleaved withHindIII and HaeIII and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates, HindIII found six were the same type and two were unique types. HaeIII found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single “ideal” method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection.

scholarly journals Pyruvate kinase deficiency: epidemiology, molecular analyses and modern diagnostic approaches (literature review)

2020 ◽  
Vol 7 (2) ◽  
pp. 86-93
Author(s):  
A. V. Bankole ◽  
E. A. Chernyak
Keyword(s):  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Molecular Genetic ◽  
Clinical Manifestations ◽  
Diagnostic Methods ◽  
Compound Heterozygous ◽  
Red Cell ◽  
Molecular Genetic Study ◽  
Pyruvate Kinase Deficiency ◽  
Diagnostic Approaches

Red cell pyruvate kinase deficiency is the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Pyruvate kinase is the enzyme involved in the last step of glycolysis – the transfer of a phosphate group from phosphoenolpyruvate producing the enolate of pyruvate and ATP (50 % of total energy ATP of erythrocytes). ATP deficiency directly shortened red cell lifespan. Affected red blood cells are destroyed in the splenic capillaries, leading to the development of chronic hemolytic anemia. It is an autosomal recessive disease, caused by homozygous and compound heterozygous mutations in the PKLR gene. There are no exact data on the incidence of pyruvate kinase deficiency, but the estimated frequency varies from 3: 1,000,000 to 1:20,000. The clinical features of the disease and the severity are highly variable. Diagnosis of pyruvate kinase deficiency is based on the determination of pyruvate kinase activity and molecular genetic study of the PKLR gene. The variety of clinical manifestations, possible complications, as well as the inaccessibility of diagnostic methods complicate the diagnosis.

scholarly journals Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms.

2016 ◽  
Vol 63 (1) ◽  
Author(s):  
Beata Krawczyk ◽  
Józef Kur ◽  
Karolina Stojowska-Swędrzyńska ◽  
Marta Śpibida
Keyword(s):  
Pcr Amplification ◽  
Dna Amplification ◽  
Restriction Endonuclease Analysis ◽  
Site Amplification ◽  
Genomic Fingerprinting ◽  
Restriction Sites ◽  
Double Digestion ◽  
Pcr Method ◽  
Alternative Techniques ◽  
Endonuclease Analysis

A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal.

scholarly journals P34.8 (GP37) is not essential for baculovirus replication

2001 ◽  
Vol 82 (2) ◽  
pp. 299-305 ◽  
Author(s):  
Xiao-Wen Cheng ◽  
Peter J. Krell ◽  
Basil M. Arif
Keyword(s):  
Northern Blot Analysis ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Restriction Endonuclease Analysis ◽  
Gene Encoding ◽  
Conserved Domains ◽  
Reading Frame ◽  
Viral Plaque ◽  
Green Fluorescent ◽  
Endonuclease Analysis

Previous reports have indicated that p34.8 (gp37) may be essential for the replication of Autographa californica nucleopolyhedrovirus (AcMNPV) because no virus with inactivated p34.8 was isolated. We have ascertained the requirement for this gene by attempting to inactivate it with a large insertion [the gene encoding GFP (green fluorescent protein)] or by deleting all the conserved domains from the open reading frame (ORF). The gene encoding GFP was inserted into the NotI site of the p34.8 ORF and a viral plaque containing the insertion was propagated in SF-21 cells. Similarly, 531 bp (NotI–XbaI) containing all conserved domains were deleted from the ORF. All mutants were authenticated by PCR amplification, restriction endonuclease analysis, DNA sequencing, and Southern and Northern blot analysis. It was found that inactivation of p34.8 of AcUW1-LacZ (AcMNPV containing a lacZ gene in the p10 locus) had no effect on the biological property of virus, such as virulence and kinetics. These two independent methods showed that p34.8 is not essential for replication and that this locus could provide another site for the engineering of baculoviruses.

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