Differential display: a novel pcr-based method for gene isolation and cloning 1
01 Jan 1998-
TL;DR: DDRT-PCR has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, reamplification and cloning of the differentially expressed PCR products; and finally cDNA library screening to isolate the genes of interest.
Abstract: Differential Display (DDRT-PCR) is a powerful technique for analyzing differences in gene expression. DDRT-PCR has the following essential steps: total RNA isolation and purification, cDNA synthesis from mRNAs, PCR amplification of cDNAs, visualization of PCR products, reamplification and cloning of the differentially expressed PCR products, confirmation by northern blot, sequencing the confirmed clones, and finally cDNA library screening to isolate the genes of interest. After its invention in 1992, a number of modifications have been introduced to optimize the technique and specifically to reduce the major problem of "false positives". Since understanding of specific gene expression patterns that regulate developmental and stress responses is a major concern of molecular biology, DDRT-PCR has become a very popular molecular technique during the past decade. Additional index terms: flooding, gene expression, mRNA differential display.
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TL;DR: Focusing on defense-related processes in plants, a brief review and examples of each of these methodologies and their advantages and limitations regarding the study of plant gene expression are presented.
Abstract: The plant genes involved in cellular signaling and
metabolism have not been fully identified, while the function(s) of many of
those which have are as yet incompletely characterized. Gene expression
analysis allows the identification of genes and the study of their relationship
with cellular processes. There are several options available for studying gene
expression, including the use of cDNA and microarray libraries and techniques
such as suppression subtractive hybridization (SSH), differential display (DD),
RNA fingerprinting by arbitrary primed PCR (RAP), expressed sequence tags
(EST), serial analysis of gene expression (SAGE), representational difference
analysis (RDA), cDNA-amplified fragment length polymorphism (cDNA-AFLP) and RNA
sequencing (RNA-Seq). Focusing on defense-related processes in plants, we
present a brief review and examples of each of these methodologies and their
advantages and limitations regarding the study of plant gene expression.
21 citations
Cites background from "Differential display: a novel pcr-b..."
...The differential expression of such sequences can be confirmed by the real time polymerase chain reaction (RT-PCR) or microarray analysis [43,44]....
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TL;DR: Induction of these anti-fungal genes appears to enable tolerant cultivars to withstand fungal infestation compared to susceptible cultivars, and rapid expression of chitinase and stilbene synthase genes may be partly responsible for anthracnose-tolerance in Florida hybrid bunch grapes.
Abstract: Florida hybrid bunch grape (Vitis spp.) cultivars with commercial value are susceptible to anthracnose disease caused by the fungi, Elsinoe ampelina. The Florida grape industry is keen on improving anthracnose tolerance characteristics of these grape cultivars as the cultivation of European grapes is limited in Southeastern United States due to Pierce’s disease incidence. In this connection, a molecular approach was taken to identify and characterize the gene/s differentially expressed upon infestation with E. ampelina in selected Florida hybrid bunch grape cultivars differing in their disease tolerance level. Variation in gene expressions was monitored by employing Differential Display RT-PCR. The results showed significant up-regulation of transcripts ranging between 200 and 800 bp in anthracnose-tolerant (cvs. Blue Lake and Lake Emerald) grape cultivars compared to anthracnose-susceptible (cvs. Suwannee and Blanc du Bois) cultivars. The transcripts that were uniquely induced in tolerant cultivars were identified as chitinase, stilbene synthase, protein/sugar kinases and transcriptional factor. Expression of chitinase and stilbene synthase genes was validated using real time PCR analysis. The results showed that these genes were more rapidly expressed in tolerant cultivars within a day after inoculating with E. ampelina and peaking by day 3, compared to susceptible cultivars. Induction of these anti-fungal genes appears to enable tolerant cultivars to withstand fungal infestation compared to susceptible cultivars. Rapid expression of chitinase and stilbene synthase genes along with others genes may be partly responsible for anthracnose-tolerance in Florida hybrid bunch grapes. Further analysis may shed light on their specific role in anthracnose tolerance.
12 citations
Cites background from "Differential display: a novel pcr-b..."
...DDRTPCR is a powerful technique that detects specific gene expression patterns of two or more samples that are separated on adjacent lanes of sequencing gels (Alves et al. 1998)....
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Journal Article•
TL;DR: The present review emphasized the multidimensional applications of DDRTPCR in studying the differentially expressed gene under biotic and abiotic stress especially the salinity stress.
Abstract: The current ongoing studies and elucidations of control mechanisms of stress tolerance at molecular level that might facilitate the usage of molecular tools for developing further tolerant floras, is mainly centered on the expression of specific genes related to stress. Differential Display Reverse Transcriptase polymerase chain reaction (DDRT-PCR), a competent, profound and reproducible technology, is more advantageous in numerous ways than other approaches of gene expression analysis. Until 1992, the only and single technique applied for isolation of differentially expressed genes was subtractive hybridization or differential hybridization. In 1992, a different and innovative PCR-based method called Differential Display (DDRT-PCR) was developed by Liang and Pardee. The technological simplicity and wider applicability made this technique very novel. It has successfully utilized in a number of organisms starting from yeast to mammals. This technique was introduced and developed to accelerate the identification of differentially expressed genes to overcome the shortcomings of earlier known methods which were sensitive to error, unresponsive and strenuous. The present review emphasized the multidimensional applications of DDRTPCR in studying the differentially expressed gene under biotic and abiotic stress especially the salinity stress.
2 citations
Cites methods from "Differential display: a novel pcr-b..."
...The only and single technique applied for isolation of differentially expressed genes was subtractive hybridization or differential hybridization until 1992; no other method to designate the aggregate figure of genes answerable to up/ down-regulation in certain situations was known While it is a consistent technique, it is tiresome, time taking and tough to execute, moreover, it involves mRNA in hefty amounts which in many conditions can be limited [10,12]....
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TL;DR: The DD-RTPCR technique can be one more tool to be used in gene expression analysis, including establishing intra and inter-caste relations in insects, as demonstrated in the study of mandibular glands of minina and media workers and soldiers of the ant species A. s.
Abstract: The DD-RTPCR technique is becoming more efficient nowadays to isolate and characterize genes differentially expressed among cells, tissues or individuals, being used here, for the first time in the study of mandibular glands of minina and media workers and soldiers of the ant species Atta sexdens rubropilosa. The expression profile of bands (transcripts), obtained in the present study, permitted the grouping of gene fragments into three distinctive classes. Class I fragments corresponded with those called constitutive genes, which could be associated with the metabolic processes of the mandibular glands. Class II could be the ones that would be codifying more important products in more specific stages than in others, related to the tasks performed in the colony by individuals of different castes. Class III gene fragments (transcripts 1, 2 and 3) must be caste-specific and they were found in the mandibular glands of soldiers (T1), media workers (T2) and minima workers (T3), respectively. Therefore the present study, besides having highlighted the differential gene expression in the mandibular glands of individuals of three castes of A. s. rubropilosa, demonstrated that the DD-RTPCR technique can be one more tool to be used in gene expression analysis, including establishing intra and inter-caste relations in insects.
2 citations
21 Jun 2006
TL;DR: Aplicação das amostras no gel de poliacrilamida e eletroforese em condições desnaturantes e coleta do material para extração de RNA total.......................... 27 3.2.9.1 Dosagem de RNATotal total..................................................... 27 32.8.9 IDENTIFICAÇÃO DOS GENES EXPRESSOS (DDRT-PCR)............. 27
Abstract: ......................................................................................................... 5 1. INTRODUÇÃO................................................................................................ 8 2. OBJETIVOS..................................................................................................... 15 3. MATERIAL E MÉTODOS............................................................................. 18 3.1 MATERIAL................................................................................................... 18 3.2 MÉTODOS.................................................................................................... 18 3.2.1 MORFOLOGIA...................................................................................... 18 3.2.2 HISTOLOGIA......................................................................................... 19 3.2.3 MICROSCOPIA ELETRÔNICA DE VARREDURA........................... 19 3.2.4 MICROSCOPIA ELETRÔNICA DE TRANSMISSÃO........................ 19 3.2.5 HISTOQUÍMICA.................................................................................... 20 3.2.5.1 Detecção de DNA/RNA.................................................................... 20 3.2.5.2 Detecção de Polissacarídeos Ácidos e Neutros................................. 21 3.2.5.3 Detecção de Proteínas Totais............................................................. 21 3.2.5.4 Detecção de Lipídios Ácidos............................................................. 22 3.2.6 DOSAGEM DE PROTEÍNA TOTAL.................................................... 22 3.2.6.1 Preparação do Extrato........................................................................ 22 3.2.6.2 Dosagem Protéica........................................................................... 22 3.2.7 ELETROFORESE DE PROTEÍNAS...................................................... 23 3.2.7.1 Aplicação das amostras no gel de poliacrilamida e eletroforese em condições desnaturantes (SDS-PAGE)................................................................... 24 3.2.8 CITOQUÍMICA ULTRA-ESTRUTURAL............................................. 25 3.2.8.1 Detecção de Proteínas Básicas.......................................................... 25 3.2.8.2 Detecção de Polissacarídeos.............................................................. 25 3.2.8.3 Detecção de Lipídios......................................................................... 26 3.2.9 IDENTIFICAÇÃO DOS GENES EXPRESSOS (DDRT-PCR)............. 27 3.2.9.1 Dosagem de RNA total..................................................... 27 3.2.9.1.1 Coleta do material para extração de RNA total.......................... 27
1 citations
References
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TL;DR: A method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction using a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer.
Abstract: Effective methods are needed to identify and isolate those genes that are differentially expressed in various cells or under altered conditions. This report describes a method to separate and clone individual messenger RNAs (mRNAs) by means of the polymerase chain reaction. The key element is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it anneals at different positions relative to the first primer. The mRNA subpopulations defined by these primer pairs were amplified after reverse transcription and resolved on a DNA sequencing gel. When multiple primer sets were used, reproducible patterns of amplified complementary DNA fragments were obtained that showed strong dependence on sequence specificity of either primer.
5,254 citations
"Differential display: a novel pcr-b..." refers methods in this paper
...RESUMO - Differential Display (DDRT-PCR) é uma poderosa técnica utilizada para análise de diferenças na expressão de genes....
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...DDRT-PCR engloba as seguintes etapas: isolamento e purificação de RNA total, síntese de cDNA, PCR, visualização dos produtos de PCR, reamplificação e clonagem dos fragmentos de interesse (diferencialmente expressos), confirmação por análise de “northern blot”, sequenciamento dos clones confirmados e, finalmente, seleção em bibliotecas de cDNA para isolamento do gene de interesse....
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...COLONNA-ROMANO, S.; LEONE, A. & MARESCA, B. Differentialdisplay reverse transcription-PCR (DDRT-PCR)....
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...Therefore, with properly designed primers and controls, DDRT-PCR could produce results that truly reflect gene expression patterns of different tissues....
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...Após seu desenvolvimento em 1992, DDRT-PCR tem sofrido uma série de modificações com o propósito de melhorar e otimizar a técnica principalmente no que se refere a eliminação ou diminuição de “falsos positivos”....
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TL;DR: Differential display and RNA arbitrary primed polmerase chain reaction are methods recently designed to identify and isolate differentially expressed genes and have been introduced to streamline the techniques.
295 citations
"Differential display: a novel pcr-b..." refers methods in this paper
...…of technical modifications have been introduced to reduce the problem of false positives and to increase the reproducibility of the technique (Liang & Pardee, 1995a; Huang et al., 1996; Jones et al. 1997; Zhao et al. 1995; Doss, 1996; Pfeffer et al. 1995; Averboukh et al., 1996; Chen & Peck,…...
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TL;DR: This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation and found that incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs.
Abstract: In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.
188 citations
"Differential display: a novel pcr-b..." refers methods in this paper
...…have been introduced to reduce the problem of false positives and to increase the reproducibility of the technique (Liang & Pardee, 1995a; Huang et al., 1996; Jones et al. 1997; Zhao et al. 1995; Doss, 1996; Pfeffer et al. 1995; Averboukh et al., 1996; Chen & Peck, 1996; Callard et al.…...
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Journal Article•
TL;DR: Modified long composite primers were developed based on both mRNA differential display and RNA arbitrarily primed PCR fingerprinting methods to increase the reproducibility and sensitivity of the mRNA differentialdisplay while still keeping the characteristics of the original method.
Abstract: To increase the reproducibility and to reduce the false positives in the initial mRNA differential display, modified long composite primers were developed based on both mRNA differential display and RNA arbitrarily primed PCR fingerprinting methods. Ten-base nucleotides were added at the 5' ends of the primers used in the initial mRNA differential display. These included a restriction site to aid cloning. PCR began with one low-stringency cycle (40 degrees C for annealing) followed by 35 high-stringency cycles (60 degrees C for annealing). The modified method significantly improved the reproducibility and sensitivity of the mRNA differential display while still keeping the characteristics of the original method.
154 citations
Additional excerpts
...…have been introduced to reduce the problem of false positives and to increase the reproducibility of the technique (Liang & Pardee, 1995a; Huang et al., 1996; Jones et al. 1997; Zhao et al. 1995; Doss, 1996; Pfeffer et al. 1995; Averboukh et al., 1996; Chen & Peck, 1996; Callard et al. 1994))....
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Journal Article•
TL;DR: It is shown that the purified cDNAs remain contaminated with unrelated cDNA sequences that may lead to the artifactual isolation of false positives in the subsequent steps of the differential display method.
Abstract: The recently described mRNA differential display method provides an attractive tool for the isolation of genes showing regulated expression in a variety of systems. A key step in this technique consists of the isolation of PCR-synthesized radioactive cDNAs corresponding to differentially expressed mRNAs. Here, we show that the purified cDNAs remain contaminated with unrelated cDNA sequences that may lead to the artifactual isolation of false positives in the subsequent steps of the method. A powerful assay for the detection and elimination of this contaminating material, allowing the specific isolation of clones corresponding to the regulated genes identified by the differential display, is provided.
142 citations
Additional excerpts
...…have been introduced to reduce the problem of false positives and to increase the reproducibility of the technique (Liang & Pardee, 1995a; Huang et al., 1996; Jones et al. 1997; Zhao et al. 1995; Doss, 1996; Pfeffer et al. 1995; Averboukh et al., 1996; Chen & Peck, 1996; Callard et al. 1994))....
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