Reliability of detection by polymerase chain reaction of the sickle cell-containing region of the β-globin gene in single human blastomeres

1992 ◽  
Vol 7 (5) ◽  
pp. 630-636 ◽  
Author(s):  
Susan J. Pickering ◽  
Josie M. McConnell ◽  
Martin H. Johnson ◽  
Peter R. Braude
Keyword(s):  
Polymerase Chain Reaction ◽  
Sickle Cell ◽  
Globin Gene ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Critical analysis: use of polymerase chain reaction to diagnose leprosy

2016 ◽  
Vol 52 (1) ◽  
pp. 163-169 ◽  
Author(s):  
Flaviane Granero Maltempe ◽  
Vanessa Pietrowski Baldin ◽  
Mariana Aparecida Lopes ◽  
Vera Lúcia Dias Siqueira ◽  
Regiane Bertin de Lima Scodro ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Public Health Problem ◽  
Reference Laboratory ◽  
Chain Reaction ◽  
Molecular Biology Technique ◽  
Pcr Inhibitor ◽  
Significant Difference ◽  
Polymerase Chain ◽  
Pcr Techniques

ABSTRACT Leprosy is a neglected tropical disease and an important public health problem, especially in developing countries. It is a chronic infectious disease that is caused by Mycobacterium leprae, which has a predilection for the skin and peripheral nerves. Although it has low sensitivity, slit-skin smear (SSS) remains the conventional auxiliary laboratory technique for the clinical diagnosis of leprosy. Polymerase chain reaction (PCR) is a molecular biology technique that holds promise as a simple and sensitive diagnostic tool. In the present study, the performance of two PCR methods, using different targets, PCR-LP and PCR-P, were compared with SSS with regard to leprosy diagnosis in a reference laboratory. M. leprae DNA was extracted from 106 lymph samples of 40 patients who had clinical suspicion of leprosy. The samples were subjected to both PCR techniques and SSS. Amplification of the human b-globin gene was used as PCR inhibitor control. The specificity of both PCR techniques was 100%, and sensitivity was 0.007 and 0.015 µg/ml for PCR-LP and PCR-P, respectively. No significant difference was found between either the PCR-LP or PCR-P results and SSS results (p > 0.05). Although PCR is not yet a replacement for SSS in the diagnosis of leprosy, this technique may be used as an efficient auxiliary tool for early detection of the disease, especially in endemic regions. This strategy may also be useful in cases in which SSS results are negative (e.g., in paucibacillary patients) and cases in which skin biopsy cannot be performed.

scholarly journals Diagnosis of thalassemia using cDNA amplification of circulating erythroid cell mRNA with the polymerase chain reaction [published erratum appears in Blood 1992 Jun 15;79(12):3397]

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2433-2437 ◽  
Author(s):  
SZ Huang ◽  
GP Rodgers ◽  
FY Zeng ◽  
YT Zeng ◽  
AN Schechter
Keyword(s):  
Polymerase Chain Reaction ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Polymerase Chain ◽  
Alpha Beta

Abstract We have developed a technique to diagnose the alpha- and beta- thalassemia (thal) syndromes using the polymerase chain reaction to amplify cDNA copies of circulating erythroid cell messenger RNA (mRNA) so as to quantitate the relative amounts of alpha-, beta-, and gamma- globin mRNA contained therein. Quantitation, performed by scintillation counting of 32P-dCTP incorporated into specific globin cDNA bands, showed ratios of alpha/beta-globin mRNA greater than 10-fold and greater than fivefold increased in patients with beta 0- and beta (+)- thal, respectively, as well as a relative increase in gamma-globin mRNA levels. Conversely, patients with alpha-thalassemia showed a decreased ratio of alpha/beta-globin mRNA proportional to the number of alpha- globin genes deleted. This methodology of ascertaining ratios of globin mRNA species provides a new, simplified approach toward the diagnosis of thalassemia syndromes, and may be of value in other studies of globin gene expression at the transcription level.

scholarly journals Coexpression of gamma and beta globin mRNA in cells containing a single human beta globin locus: results from studies using single-cell reverse transcription polymerase chain reaction [published erratum appears in Blood 1994 Aug 15;84(4):1357]

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1412-1419 ◽  
Author(s):  
T Furukawa ◽  
G Zitnik ◽  
K Leppig ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Single Cells ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Beta Globin ◽  
Chain Reaction ◽  
Globin Mrna ◽  
Polymerase Chain ◽  
In Cells

Abstract We developed a method detecting globin gene expression in single cells using reverse transcription polymerase chain reaction. epsilon and gamma globin cDNAs are coamplified by an epsilon gamma primer set whereas gamma and beta globin cDNAs are coamplified by a gamma beta primer set and the individual globin cDNAs are distinguished by restriction enzyme digestion. Analysis of RNA preparations from human fetal liver, neonatal red blood cells (RBCs), or adult RBCs showed the expected mRNA species for each stage of human development. Analysis of single cells from a human erythroleukemia line coexpressing gamma and beta globin chains showed heterogeneity in gamma and beta mRNA contents. The method was subsequently used to test whether only one or more than one globin genes are expressed in cells that contain a single human beta globin locus. We found that about 50% of single cells from MEL x fetal erythroid cell hybrids containing a single human beta globin locus coexpressed gamma and beta globin mRNA. This finding is best explained by assuming that both gamma and beta genes are simultaneously transcribed from the same beta globin locus implying that the LCR can simultaneously interact with more than one globin gene promoter.

scholarly journals Interaction of rare illegitimate recombination event and a poly A addition site mutation resulting in a severe form of alpha thalassemia

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3356-3362 ◽  
Author(s):  
P Fortina ◽  
T Parrella ◽  
M Sartore ◽  
E Gottardi ◽  
V Gabutti ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Sequence Analysis ◽  
Recombination Event ◽  
Globin Gene ◽  
Severe Form ◽  
Illegitimate Recombination ◽  
Chain Reaction ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Polymerase Chain

Abstract The clinical diversity of thalassemia depends on interaction of diverse genetic defects. We have characterized a severe form of alpha thalassemia caused by coinheritance of a rare alpha-globin gene deletion and a nondeletional defect in a southern Italian family. The proband, a 7-year-old girl, exhibited an abnormal hemoglobin electrophoresis pattern with hemoglobin H and hemoglobin Barts, indicating inheritance of H and hemoglobin Barts, indicating inheritance of a severe form of alpha thalassemia. Southern blot analysis of DNA showed normal as well as aberrant alpha-globin gene fragments indicating heterozygosity for a deletional form of alpha thalassemia in the proband and her mother. The coinheritance of a nondeletional form of alpha thalassemia (alpha alpha T) was suspected because of the severity of the proband's phenotype and the presence of normal alpha-globin gene fragments in the father. Selective polymerase chain reaction of the paternal alpha 1- and alpha 2-globin genes in the proband followed by DNA sequence analysis showed an AATAAA to AATGAA mutation in the polyadenylation signal sequence of the alpha 2-globin gene. Genomic DNA mapping and sequence analysis of a unique polymerase chain reaction product generated across the deletion breakpoint of the maternal allele showed a 5,201-bp deletion extending from 870 nucleotides 5′ of the alpha 2-globin gene to nucleotide +519 in the alpha 1-globin gene. This deletion is similar to that previously suggested by blotting studies in a Greek family (Pressley et al, Nucleic Acids Res 8:4889, 1980) and removes the entire alpha 2-globin gene and a portion of the 5′ end of the alpha 1-globin gene. Sequence characterization of the resultant aberrant truncated alpha 1-globin gene from the proband showed a 27 nucleotide duplication corresponding to the 3′ end of the alpha-globin gene IVS-2 region separated by the insertion of a tetranucleotide (GGTT), suggesting that this deletion is caused by an illegitimate recombination event.

Polymerase Chain Reaction Facilitates Archival Autopsy Studies of Sickle Cell Disease

1993 ◽  
Vol 13 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Elizabeth A. Manci ◽  
Donald E. Culberson ◽  
Guey-Jen Lee Chen ◽  
Vipul Mankad ◽  
Vijay V. Joshi ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Disease ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Identification of four novel delta-globin gene mutations in Greek Cypriots using polymerase chain reaction and automated fluorescence- based DNA sequence analysis

Blood ◽  
1991 ◽  
Vol 78 (12) ◽  
pp. 3298-3305 ◽  
Author(s):  
P Trifillis ◽  
P Ioannou ◽  
E Schwartz ◽  
S Surrey
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Restriction Enzyme ◽  
Globin Gene ◽  
Pcr Amplification ◽  
Enzyme Digestion ◽  
Acceptor Site ◽  
Restriction Enzyme Digestion ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract The molecular basis of most beta-thalassemia syndromes has been defined, while the spectrum of mutations causing delta-thalassemia is not well characterized. In an attempt to identify such mutations, the region encompassing the delta-globin gene from three Greek Cypriot families suspected of having delta-thalassemia was amplified by polymerase chain reaction (PCR), and DNA sequence determined using an automated fluorescence-based sequencer. Four novel mutations were identified: a G----T change at codon 27 that results in an alanine to serine change; a C----T change at codon 116 converting arginine to cysteine; a T----C change at codon 141 converting leucine to proline; and an AG----GG change at the consensus 3′-acceptor site in IVS-2. While the latter is clearly a thalassemic mutation, the low hemoglobin A2 in the first three may be due to either decreased production or instability of the altered delta-globin chain. All four mutations may be detected by PCR amplification of genomic DNA followed by restriction enzyme digestion. Two mutations abolish restriction sites while two create new cleavage sites. Screening for molecular defects that cause delta-thalassemia or unstable delta-globin by PCR amplification and restriction enzyme digestion will lead to correct diagnosis of beta/delta-thalassemia compound heterozygotes and improved genetic counseling.

Detection of α-Thalassemia of Southeast Asian (--SEA) Deletion by Novel Multiplex PCR

2020 ◽  
Vol 103 (8) ◽  
pp. 741-747
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Control Strategy ◽  
Predictive Value ◽  
Prevention And Control ◽  
Globin Gene ◽  
Southeast Asian ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
And Control

Background: Southeast Asian (--SEA) deletion of α-thalassemia is the most common α-thalassemia-1 mutation in Thailand and neighborhood countries. The absence of α-globin gene production in a homozygous fetus is the most serious and leads to death in utero or soon after birth and life-threatening maternal complications. Therefore, sensitive and accurate assays for detecting α-thalassemia--SEA deletion are crucial for thalassemia diagnosis. Materials and Methods: The present study evaluated multiplex polymerase chain reaction (PCR)--SEA, comparing with the routinely performed gap-PCR for α-thalassemia diagnosis in prenatal thalassemia prevention and control strategy commonly used in Thailand. Four primers were employed to detect the deleted region of α-globin gene family. This, thus, provides high accuracy in discriminating heterozygous and homozygous --SEA deletion. Results: Multiplex PCR--SEA assay showed the results with 100% sensitivity, specificity, positive predictive value, negative predictive value, and efficiency in comparison to the routinely performed gap-PCR used in prenatal thalassemia prevention and control strategy. In addition, the multiplex PCR--SEA assay displayed lower detection limit for heterozygous detection. Conclusion: The novel multiplex PCR--SEA in the present study was proofed to be a good alternative for thalassemia diagnosis in thalassemia prevention and control strategy. The advantage of the assay is the capability to identify three wild types and one deleted fragment comparing with one wild type and one deleted fragment in the routinely performed gap-PCR. This is very useful, especially in the genes where polymorphism is common. Therefore, the multiplex PCR--SEA provides a better protocol for α-thalassemia--SEA diagnosis. Keywords: α-thalassemia, Hemoglobin Bart’s hydrop fetalis, Multiplex PCR, Polymerase chain reaction, Prenatal diagnosis, --SEA deletion

Sign in / Sign up

Export Citation Format

Share Document