Properties of Samples With Segregating Polymerase Chain Reaction (PCR) Dropout Mutations Within a Species

In polymerase chain reaction (PCR)-based DNA sequencing studies, there is the possibility that mutations at the binding sites of primers result in no primer binding and therefore no amplification. In this article, we call such mutations PCR dropouts and present a coalescent-based theory of the distribution of segregating PCR dropout mutations within a species. We show that dropout mutations typically occur along branch sections that are at or near the base of a coalescent tree, if at all. Given that a dropout mutation occurs along a branch section near the base of a tree, there is a good chance that it causes the alleles of a large fraction of a species to go unamplified, which distorts the tree shape. Expected coalescence times and distributions of pairwise sequence differences in the presence of PCR dropout mutations are derived under the assumptions of both neutrality and background selection. These expectations differ from when PCR dropout mutations are absent and may form the basis of inferential approaches to detect the presence of dropout mutations, as well as the development of unbiased estimators of statistics associated with population-level genetic variation.

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[31] Analysis of DNA sequence variation at population level by polymerase chain reaction and denaturing gradient gel electrophoresis

Methods in Enzymology - Molecular Evolution: Producing the Biochemical Data ◽

10.1016/0076-6879(93)24032-p ◽

1993 ◽

pp. 419-428 ◽

Cited By ~ 2

Author(s):

Enrique P. Lessa

Keyword(s):

Polymerase Chain Reaction ◽

Dna Sequence ◽

Gel Electrophoresis ◽

Sequence Variation ◽

Denaturing Gradient Gel Electrophoresis ◽

Population Level ◽

Chain Reaction ◽

Dna Sequence Variation ◽

Gradient Gel Electrophoresis ◽

Polymerase Chain

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Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis

Journal of Chromatography A ◽

10.1016/j.chroma.2013.02.062 ◽

2013 ◽

Vol 1286 ◽

pp. 229-234 ◽

Cited By ~ 8

Author(s):

Márta Kerékgyártó ◽

Nóra Németh ◽

Tamás Kerekes ◽

Zsolt Rónai ◽

András Guttman

Keyword(s):

Polymerase Chain Reaction ◽

Binding Sites ◽

Gel Electrophoresis ◽

Multiplex Polymerase Chain Reaction ◽

Chain Reaction ◽

Capillary Gel Electrophoresis ◽

Polymerase Chain ◽

Wfs1 Gene

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Thyroid hormone receptor β mRNA expression in Sertoli cells isolated from prepubertal testis

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0140131 ◽

1995 ◽

Vol 14 (1) ◽

pp. 131-134 ◽

Cited By ~ 25

Author(s):

S Palmero ◽

P De Marco ◽

E Fugassa

Keyword(s):

Polymerase Chain Reaction ◽

Thyroid Hormone ◽

Sertoli Cell ◽

Sertoli Cells ◽

Binding Sites ◽

Hormone Receptor ◽

Thyroid Hormone Receptor ◽

Chain Reaction ◽

Polymerase Chain ◽

Thyroid Hormone Receptor Β

ABSTRACT A polymerase chain reaction (PCR)-based assay was used to evaluate the expression of thyroid hormone receptor β mRNA in Sertoli cells isolated from both prepubertal rat and piglet testes. The expression of an mRNA coding for the functional thyroid hormone receptor β isoform, as established by the PCR assay, agrees with the presence of specific tri-iodothyronine (T3)-binding sites in the Sertoli cell nuclei of both species, as previously evaluated by displacement analysis. The results ratify the existence of a functional T3 receptor in the prepubertal testis and confirm the Sertoli cell as a specific target for thyroid hormone action on the developing testis. On the other hand, in both peripubertal rat (Palmero et al. 1988; Jannini et al. 1990) and piglet (Palmero et al. 1992) testes, high-affinity, low-capacity T3 binding sites have been specifically localised at the Sertoli cell level and TRal mRNA expression has been detected very recently in immature Sertoli cells (Jannini et al. 1994). The aim of the present work was to test if, in prepubertal Sertoli cells isolated from both immature rat and piglet testes, the expression of an erbAβ mRNA specifically coding for the TR protein could be detected employing an highly sensitive polymerase chain reaction (PCR)-based assay.

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Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas

Blood ◽

10.1182/blood.v73.7.1759.bloodjournal7371759 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1759-1762 ◽

Cited By ~ 1

Author(s):

BY Ngan ◽

J Nourse ◽

ML Cleary

Keyword(s):

Polymerase Chain Reaction ◽

Large Fraction ◽

Dna Amplification ◽

Nucleotide Sequencing ◽

Chromosome 18 ◽

Chain Reaction ◽

Specific Primers ◽

Cluster Region ◽

Polymerase Chain ◽

Follicular Lymphomas

A majority of t(14;18) translocations have been shown to cluster at one of two sites on chromosome 18, called the major breakpoint region (mbr) or the minor cluster region, (mcr), which map within or flanking the bcl-2 proto-oncogene, respectively. We have determined the nucleotide sequence for a portion of the mcr, and constructed oligonucleotides that were used to perform the polymerase chain reaction (PCR) in conjunction with universal immunoglobulin primers to specifically amplify t(14;18) breakpoints in DNA obtained from follicular lymphomas. Eight of ten breakpoints that were detectable on Southern blots using DNA probes for the mcr could be detected due to specific amplification by the PCR technique using an mcr-specific primer. Direct nucleotide sequencing of the enzymatically amplified DNAs showed that the breakpoints clustered within a 500 nucleotide region, and five occurred within three nucleotides of each other. These data show a remarkable clustering of some t(14;18) breakpoints at a site on chromosome 18, at least a 30-kb distance from the bcl-2 gene. Our findings also indicate that mcr-specific primers may be used in conjunction with previously described mbr-specific primers in a highly sensitive DNA amplification technique to detect a large fraction of t(14;18) breakpoints.

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Detection of chromosomal translocation t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas

Blood ◽

10.1182/blood.v73.7.1759.1759 ◽

1989 ◽

Vol 73 (7) ◽

pp. 1759-1762 ◽

Cited By ~ 154

Author(s):

BY Ngan ◽

J Nourse ◽

ML Cleary

Keyword(s):

Polymerase Chain Reaction ◽

Large Fraction ◽

Dna Amplification ◽

Nucleotide Sequencing ◽

Chromosome 18 ◽

Chain Reaction ◽

Specific Primers ◽

Cluster Region ◽

Polymerase Chain ◽

Follicular Lymphomas

Abstract A majority of t(14;18) translocations have been shown to cluster at one of two sites on chromosome 18, called the major breakpoint region (mbr) or the minor cluster region, (mcr), which map within or flanking the bcl-2 proto-oncogene, respectively. We have determined the nucleotide sequence for a portion of the mcr, and constructed oligonucleotides that were used to perform the polymerase chain reaction (PCR) in conjunction with universal immunoglobulin primers to specifically amplify t(14;18) breakpoints in DNA obtained from follicular lymphomas. Eight of ten breakpoints that were detectable on Southern blots using DNA probes for the mcr could be detected due to specific amplification by the PCR technique using an mcr-specific primer. Direct nucleotide sequencing of the enzymatically amplified DNAs showed that the breakpoints clustered within a 500 nucleotide region, and five occurred within three nucleotides of each other. These data show a remarkable clustering of some t(14;18) breakpoints at a site on chromosome 18, at least a 30-kb distance from the bcl-2 gene. Our findings also indicate that mcr-specific primers may be used in conjunction with previously described mbr-specific primers in a highly sensitive DNA amplification technique to detect a large fraction of t(14;18) breakpoints.

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Polymorphic locus rs1061624 of the ТNFR2 gene is associated with the development of arterial hypertension in males

Kardiologiia ◽

10.18087/cardio.2020.8.n996 ◽

2020 ◽

Vol 60 (8) ◽

pp. 78-83

Author(s):

M. I. Moskalenko ◽

I. V. Ponomarenko ◽

S. N. Milanova ◽

I. N. Verzilina ◽

O. A. Efremova ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Arterial Hypertension ◽

Binding Sites ◽

Polymorphic Locus ◽

Group Analysis ◽

Control Group ◽

Chain Reaction ◽

Regulatory Potential ◽

Polymerase Chain ◽

Regulatory Significance

Aim To study the involvement of cytokine polymorphous loci in development of arterial hypertension (AH) in men from the Central Black Earth region of Russia.Materials and methods 821 men were evaluated, including 564 patients with AH and 257 individuals of the control group. Analysis of 8 cytokine mononucleotide polymorphisms (MNP) was performed using the real-time polymerase chain reaction with TagMan probes. Statistical analysis was performed with the STATISTICA (v.10.0) and PLINK (v.1.06) software. The regulatory potential of MNP was analyzed with the HaploReg (v.4.1) service (http://archive.broadinstitute.org).Results The rs1061624 ТNFR2 polymorphous locus was associated with development of AH in men in recessive (odd ratio (OR), 0.33; 95 % confidence interval (CI): 0.18–0.61, рperm=0.0004) and additive (OR, 0.50, 95 % CI: 0.34–0.74, рperm=0.0006) genetic models and exerted a protective effect in development of AH. The rs1061624 MNP of the ТNFR2 gene has a regulatory significance; it is located in the DNA sites hypersensitive to the action of DNAase 1 and in binding sites for transcriptional factors and histones that mark enhancers and promoters in different organs and tissues.Conclusion The rs1061624 ТNFR2 gene polymorphism is involved in the development of AH in men of the Central Black Earth region of Russia.

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Transient upregulation of μ opioid receptor mRNA levels in nucleus accumbens during chronic cocaine administration

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y98-023 ◽

1998 ◽

Vol 76 (3) ◽

pp. 278-283 ◽

Cited By ~ 33

Author(s):

Anahit V Azaryan ◽

Barbara J Clock ◽

John G Rosenberger ◽

Brian M Cox

Keyword(s):

Polymerase Chain Reaction ◽

Nucleus Accumbens ◽

Opioid Receptor ◽

Binding Sites ◽

Maximum Effect ◽

Chain Reaction ◽

Cocaine Administration ◽

Polymerase Chain ◽

Baseline Levels ◽

Continuous Cocaine

Chronic continuous cocaine administration for 3 days has been shown to upregulate the level of µ opioid receptor (MOR) mRNA in the nucleus accumbens (n. acc.) of rat brain. Dopamine (DA) antagonists, SCH 23390, eticlopride, and nafadotride, blocked, and DA agonists, SKF 38393, R(+)-6-bromo-APB hydrobromide, and bromocriptine, mimicked the cocaine-induced upregulation of MOR mRNA, suggesting involvement of both subfamilies of DA receptors in the effect of cocaine. In the present study the time course of cocaine-induced and DA agonist induced alterations in the level of MOR mRNA in n. acc. has been determined and compared with the changes in the level of MOR binding sites. Male Sprague-Dawley rats were treated with saline, cocaine (50 mg ·kg-1 ·day-1), or DA agonists for periods between 24 and 336 h. Expression of MOR mRNA in n. acc. was estimated using quantitative competitive polymerase chain reaction assays following reverse transcription. The cocaine-induced upregulation of MOR mRNA in n. acc. was transient, developing 2 days after exposure, and peaking at 3 days with return to baseline levels by 4 days of chronic continuous cocaine treatment. The temporal characteristics of DA agonist induced increase in the levels of MOR mRNA in n. acc. were similar to those of cocaine, with maximum effect after 3 days of treatment. The density of [3H]DAMGO binding sites in n. acc. was 30% higher after 3 days of cocaine administration than in saline-treated control animals, but returned toward baseline levels after 4 days of cocaine treatment. No changes in the binding of [3H]DAMGO were detected after 7 or 14 days exposure to cocaine. The affinity of [3H]DAMGO to n. acc. membranes (~2.0 nM) was unchanged during the cocaine treatment.Key words: cocaine, dopamine, dopamine agonists, µ opioid receptor, nucleus accumbens, polymerase chain reaction.

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High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166944 ◽

1996 ◽

Vol 54 ◽

pp. 896-897

Author(s):

G. W. Hacker ◽

I. Zehbe ◽

J. Hainfeld ◽

A.-H. Graf ◽

C. Hauser-Kronberger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Messenger Rna ◽

High Performance ◽

Detection System ◽

Direct Methods ◽

Genetic Alterations ◽

Chain Reaction ◽

Protein Encoding ◽

Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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