scholarly journals Detection of Fetal Aneuploidies by QF-PCR in Transcervical Cell Samples

ISRN Genetics ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-5
Author(s):  
Riccardo Cioni ◽  
Cecilia Bussani ◽  
Mariarosaria Di Tommaso
Keyword(s):  
Tandem Repeats ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
First Trimester ◽  
Pcr Analysis ◽  
Trophoblastic Cells ◽  
Standard Karyotype ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction ◽  
Fetal Aneuploidies

Objective. To evaluate the accuracy in the diagnosis of aneuploidies of a quantitative fluorescent polymerase chain reaction (QF-PCR) assay on trophoblastic cells recovered from transcervical cells samples (TCCs) collected by intrauterine lavage (IUL). Study Design. DNA analysis was performed on cells of seemingly trophoblastic origin isolated from IUL samples collected prior to first trimester termination of pregnancy. The analysis was performed by multiplex QF-PCR, using a panel of 29 polymorphic short tandem repeats (STRs) for the chromosomes X, Y, 21, 13, and 18. Results. The QF-PCR analysis on placental samples revealed that among the three cases studied there were two cases of trisomy 21 and one case of monosomy X; the comparison of peak profiles obtained from IUL, placental, and maternal samples confirmed the diagnosis of aneuploidy in all three cases. Conclusion. This study suggests that the detection of chromosomal aneuploidies in micromanipulated TCC samples can be achieved by QF-PCR amplification of selected highly polymorphic and chromosome specific markers. With respect to standard karyotype, QF-PCR analysis has the limitation that only numerical abnormalities of selected chromosomes can be detected but retains the advantages of being quicker, less expensive, and less lab demanding.

Rapid Aneuploidy Detection of Chromosomes 13, 18, 21, X and Y Using Quantitative Fluorescent Polymerase Chain Reaction with Few Microdissected Fetal Cells

2015 ◽  
Vol 38 (1) ◽  
pp. 65-76 ◽  
Author(s):  
Ahmed Emad ◽  
Josée Lamoureux ◽  
Annie Ouellet ◽  
Régen Drouin
Keyword(s):  
Polymerase Chain Reaction ◽  
Tandem Repeats ◽  
Single Cells ◽  
Pcr Analysis ◽  
Limiting Factor ◽  
Chain Reaction ◽  
Fetal Cells ◽  
Allele Dosage ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction

Objectives: Analysis of DNA from small numbers of cells, such as fetal cells in maternal blood, is a major limiting factor for their use in clinical applications. Traditional methods of single-cells whole genome amplification (SCs-WGA) and accurate analysis have been challenging to date. Our purpose was to assess the feasibility of using a few fetal cells to determine fetal sex and major chromosomal abnormalities by quantitative fluorescent polymerase chain reaction (QF-PCR). Methods: Cultured cells from 26 amniotic fluid samples were used for standard DNA extraction and recovery of 5 fetal cells by laser-capture microdissection. SCs-WGA was performed using the DNA from the microdissected cells. PCR amplification of short tandem repeats specific for chromosomes 13, 18, 21, X and Y was performed on extracted and amplified DNA. Allele dosage and sexing were quantitatively analyzed following separation by capillary electrophoresis. Results: Microsatellite QF-PCR analysis showed high concordance in chromosomal copy number between extracted and amplified DNA when 5 or more cells were used. Results were in concordance with that of conventional cytogenetic analysis. Conclusion: Satisfactory genomic coverage can be obtained from SCs-WGA. Clinically, SCs-WGA coupled with QF-PCR can provide a reliable, accurate, rapid and cost-effective method for detection of major fetal chromosome abnormalities.

scholarly journals Distribution of diandric and digynic triploidy depending on gestational age

2021 ◽  
Author(s):  
Diana Massalska ◽  
Katarzyna Ozdarska ◽  
Tomasz Roszkowski ◽  
Julia Bijok ◽  
Anna Kucińska-Chahwan ◽  
...  
Keyword(s):  
Gestational Age ◽  
Pregnancy Loss ◽  
First Trimester ◽  
Parental Origin ◽  
Second Trimester ◽  
Chain Reaction ◽  
Spontaneous Pregnancy ◽  
First Trimester Of Pregnancy ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction

Abstract Purpose To establish the distribution of diandric and digynic triploidy depending on gestational age. Methods 107 triploid samples tested prospectively in a single genetic department during a four-year period were analyzed for parental origin of triploidy by Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) (n=95) with the use of matching parental samples or by MS-MLPA (n=12), when parental samples were unavailable. Tested pregnancies were divided into three subgroups with regard to the gestational age at spontaneous pregnancy loss: <11 gestational weeks, 11–14 gestational weeks, and >14 gestational weeks. Results Diandric triploidy constituted overall 44.9% (46.5% in samples miscarried <11 gestational weeks, 64.3% in samples miscarried between 11 and 14 gestational weeks, and 27.8% in pregnancies which survived >14 gestational weeks). Conclusions The distribution of diandric and digynic triploidy depends on gestational age. The majority of diandric triploid pregnancies is lost in the first trimester of pregnancy. In the second trimester, diandric cases are at least twice less frequent than digynic ones.

scholarly journals Suitability of different tissue fixatives for subsequent PCR analysis of Cysticercus ovis

2012 ◽  
Vol 49 (2) ◽  
pp. 67-70 ◽  
Author(s):  
M. Kolesárová ◽  
R. Herich ◽  
M. Levkut ◽  
J. Čurlík ◽  
M. Levkut
Keyword(s):  
Retrospective Studies ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Fresh Tissue ◽  
Carnoy’S Solution ◽  
Negative Results ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Cysticercus Ovis

AbstractPCR amplification of specific DNA regions is a powerful tool for retrospective studies, but not all preservation or fixation methods render DNA that is suitable for subsequent amplification. Several factors affect sensitivity of polymerase chain reaction (PCR) amplification. There were reported the effects of commonly used fixation solutions — 10 % neutral buffered formalin, 20 % neutral buffered formalin and Carnoy’s solution and the efficiency of PCR amplification in fresh tissue and paraffin (or wax) embedded samples of Cysticercus ovis. DNA from samples was isolated and PCR product of 1300 bp was amplified. Results indicated that the samples fixed in Carnoy’s solution produced reliable amplification of desired fragments. The samples that were fixed in 10 % and 20 % neutral buffered formalin brought negative results.

Microbial characteristics of a methanogenic phenol-degrading sludge

2005 ◽  
Vol 52 (1-2) ◽  
pp. 73-78 ◽  
Author(s):  
T. Zhang ◽  
S. Z. Ke ◽  
Y. Liu ◽  
H.P. Fang
Keyword(s):  
Dna Analysis ◽  
Pcr Amplification ◽  
Upflow Anaerobic Sludge Blanket ◽  
Anaerobic Sludge ◽  
Chain Reaction ◽  
Microbial Characteristics ◽  
Polymerase Chain ◽  
Anaerobic Sludge Blanket

Microbial properties of a methanogenic granular phenol-degrading sludge were characterized using the 16S rRNA/DNA-based techniques, including polymerase chain reaction (PCR) amplification, cloning, DNA sequencing, and fluorescence in situ hybridization (FISH). The sludge was sampled from an upflow anaerobic sludge blanket reactor, which removed 98% of phenol (up to 1260 mg/l) in wastewater at 26°C with 12 hours of hydraulic retention. Based on DNA analysis, the Eubacteria in the sludge was composed of 13 operational taxonomy units (OTUs). Two OTUs, one resembling Clostridium and the other remotely resembling Desulfotomaculum, were likely responsible for the conversion of phenol to benzoate, which was further degraded by five Syntrophus-resembling OTUs to acetate and H2/CO2; methanogens lastly converted acetate and H2/CO2 into methane. The role of six remaining OTUs remains unclear. Overall, the sludge was composed of 26±6% Eubacteria and 74±9% methanogens, of which 54±6% were acetotrophic Methanosaetaceae, 14±3% and 3±2% were hydrogenotrophic Methanomicrobiales and Methanobacteriaceae, respectively.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

scholarly journals Development of a Variable Number of Tandem Repeats Typing Scheme for the Bacterial Rice Pathogen Xanthomonas oryzae pv. oryzicola

2012 ◽  
Vol 102 (10) ◽  
pp. 948-956 ◽  
Author(s):  
Shuai Zhao ◽  
Lucie Poulin ◽  
Luis M. Rodriguez-R ◽  
Natalia Forero Serna ◽  
Shu-Yan Liu ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Bacterial Pathogen ◽  
Pcr Amplification ◽  
Cost Effective ◽  
Variable Number ◽  
Xanthomonas Oryzae ◽  
Chinese Strain ◽  
Epidemiological Monitoring ◽  
Population Structures ◽  
Polymerase Chain

Xanthomonas oryzae pv. oryzicola is an important bacterial pathogen responsible for outbreaks of bacterial leaf streak (BLS) on rice, mostly occurring in Asia and parts of Africa. To better monitor epidemics and assess population structures, efficient tools that allow the precise identification and diagnosis of pathogenic populations are needed. In this study, we explored variable numbers of tandem repeats (VNTR) as a fast, reliable, and cost-effective molecular typing tool. Screening of three X. oryzae pv. oryzicola genome sequences (Philippine strain BLS256, Chinese strain GX01, and Malian strain MAI10) predicted 28 candidate VNTR loci. Primer pairs for polymerase chain reaction (PCR) amplification of all 28 loci were designed and applied to a panel of 20 X. oryzae pv. oryzicola strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci that are shared among Asian and African X. oryzae pv. oryzicola strains. A dendrogram constructed from 25 VNTR loci indicated that most Asian strains are clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali is related to Asian strains, pointing to a possible introduction of Asian strains to the African continent. The new VNTR-based tool described here is useful for studies of population structures and epidemiological monitoring of X. oryzae pv. oryzicola.

scholarly journals Molecular cloning of RhD cDNA derived from a gene present in RhD- positive, but not RhD-negative individuals

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 651-655 ◽  
Author(s):  
MA Arce ◽  
ES Thompson ◽  
S Wagner ◽  
KE Coyne ◽  
BA Ferdman ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Molecular Genetic ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Blood Group System ◽  
Pcr Cloning ◽  
Antigenic Protein ◽  
Genomic Libraries ◽  
Polymerase Chain ◽  
Rh Blood Group

The Rh blood group system plays a major role in immune and nonimmune hemolytic states. Although an Rh cDNA has been previously cloned, there is no information on which Rh antigenic protein it encodes. Using polymerase chain reaction (PCR) amplification, we have identified this original Rh clone, here designated Rh21, and an additional Rh cDNA clone, Rh13, that is 96% nucleotide- and 92% amino acid-identical to Rh21, with the substitutions scattered throughout the sequence. A molecular genetic approach was used to match this Rh clone with an Rh specificity. The mRNA transcript for Rh13 was present in reticulocytes from RhD-positive individuals, but was absent from the reticulocytes of RhD-negative individuals. Using conventional screening of genomic libraries, as well as PCR cloning, partial genomic clones for these two Rh cDNAs were obtained. Based on PCR analysis and Southern blots, the Rh21 gene was present in all individuals, but an intact Rh13 gene was only present in RhD-positive and not RhD-negative individuals. Thus, by correlating the presence of Rh mRNA and gene sequences with individual Rh phenotypes, we were able to establish that the new Rh13 cDNA clone represents the RhD protein.

scholarly journals Fingernail, as an Alternative Source of DNA for Forensic Investigations and Medical Diagnostics A new technique to obtain template DNA in critical situations

1996 ◽  
Vol 45 (1-2) ◽  
pp. 301-302
Author(s):  
U. Ricci ◽  
M.L. Giovannucci Uzielli
Keyword(s):  
Tandem Repeats ◽  
Dna Analysis ◽  
Dna Typing ◽  
Pcr Amplification ◽  
Medical Diagnostics ◽  
Personal Identification ◽  
Forensic Identification ◽  
Alternative Source ◽  
Chelex 100

The use of PCR (polymerase chain reaction) is having a profound impact on forensic science and medical diagnostics.Limited amounts of biological material and/or degraded low molecular weight DNA can be anticipated for forensic identification and often also for diagnostic investigations in fetuses, stillbirths and children with birth defects.In vitro amplification of DNA, via PCR, represents an important tool for overcoming some of the limitations. Nevertheless, the problem of availability of biological samples as DNA source is often critic.In order to obtain a useful and rapid procedure of DNA analysis in such difficult situations, we have recently developed a simple DNA extraction method using Chelex 100 and a PCR based amplification technique, and using fingernail as an alternative source of DNA.Chelex 100 procedures result in denaturated DNA samples, not suitable for RFLPS analysis. Nevertheless we demonstrated that no differences are detectable between the genotypes obtained by PCR amplification using the conventional phenol-chlorophorm or saline extraction and the Chelex based procedure.A DNA sample of 3-5 micrograms weight was easily obtained from fingernail clippings, and enabled us to perform several tests by using DNA typing methodologies, both for personal identification (in various forensic, medical and social situations) and in disputed parentage.Our laboratory uses as polymorphic markers a set of several VNTRs (variable number of tandem repeats) and, more recently, a series of unlinked STRs (short tandem repeats). The use of DNA typing with STR loci provides an accurate, highly sensitive and rapid assay for parentage testing, forensic identification and medical applications.

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