Preparing Gene of interest for GateWay cloning (2 step PCR process) v2

2022 ◽  
Author(s):  
Johannes Wolfram JWD Debler
Keyword(s):  
Gateway Cloning ◽  
Specific Pcr ◽  
Attachment Sites ◽  
Pcr Product ◽  
Primer Sequence

GateWay recombination cloning is achieved by flanking your gene of interest with GateWay attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers with 5' extensions. Since those primes create 31 bp and 30 bp 5' primer extensions respectively, plus about 20 bp of actual binding primer sequence it becomes expensive fast if you need 2 x ~50 bp primers for every GOI. We therefore use a 2 step PCR process to attach GateWay attB1 and attB2 sites. We first run a gene specific PCR with primers carrying short 5' extesions, and then a second PCR utilizing universal GateWay primers which bind to the short extension of the first PCR product to create the full attB1 and attB2 sites. This protocol has been adapted from: 2-STEP GATEWAY PCR EXPERIMENTS

scholarly journals The polymorphism of ETR1 gene in autochthonous apple cultivars

Genetika ◽  
2007 ◽  
Vol 39 (3) ◽  
pp. 387-394 ◽  
Author(s):  
Sladjana Maric ◽  
Radovan Boskovic ◽  
Milan Lukic
Keyword(s):  
Plant Hormone ◽  
Allelic Polymorphism ◽  
Ethylene Receptor ◽  
Gene Encoding ◽  
Fruit Storage ◽  
Specific Pcr ◽  
Pcr Product ◽  
Apple Cultivars ◽  
Storage Ability

Ethylene is a plant hormone, which plays an important role in the ripening of climacteric fruits such as the apple. We studied allelic polymorphism of the ETR1 gene, encoding ethylene receptor, in 23 autochthonous apple cultivars. The polymorphism was revealed by combining the gene specific PCR and restriction of PCR product. Four alleles of the ETR1 gene (a, b, c and d) were detected, and their possible association with the fruit storage ability examined.

scholarly journals Rapid Detection Method of Salmonella-specific PCR Product by DNA-immobilized Quartz Crystal Microbalance.

1999 ◽  
Vol 16 (1) ◽  
pp. 57-63
Author(s):  
Takahisa MIYAMOTO ◽  
Sudsai TREVANICH ◽  
Takashi OKABE ◽  
Satosi TOMODA ◽  
Ken-ichi HONJOH ◽  
...  
Keyword(s):  
Quartz Crystal Microbalance ◽  
Rapid Detection ◽  
Quartz Crystal ◽  
Detection Method ◽  
Specific Pcr ◽  
Pcr Product

Prognosis of glioblastoma with faint MGMT methylation-specific PCR product

2015 ◽  
Vol 122 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Chih-Yi Hsu ◽  
Hsiang-Ling Ho ◽  
Shih-Chieh Lin ◽  
Yi-Chun Chang-Chien ◽  
Ming-Hsiung Chen ◽  
...  
Keyword(s):  
Mgmt Methylation ◽  
Methylation Specific Pcr ◽  
Specific Pcr ◽  
Pcr Product

scholarly journals Multiplex-Polymerase Chain Reaction Assay for the Authentication of the Mackerel Scomber colias in Commercial Canned Products

2006 ◽  
Vol 89 (3) ◽  
pp. 708-711 ◽  
Author(s):  
Carlos Infante ◽  
Manuel Manchado
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Nontranscribed Spacer ◽  
Specific Pcr ◽  
Scomber Colias ◽  
Pcr Product ◽  
Polymerase Chain ◽  
Specific Fragment

Abstract A multiplex-polymerase chain reaction (PCR) system was developed for the authentication of the mackerel Scomber colias in commercial canned products. This novel method consists of an S. colias-specific fragment [159 base pairs (bp)] located in the nontranscribed spacer (NTS) sequence, and a Scomber genus-specific PCR product in the 5S rRNA gene (196201 bp) as a positive amplification control. The system was assayed using 18 different canned products labeled as S. colias. A positive identification was made in all but one sample, revealing this methodology as a potential molecular tool for direct application in the authentication of S. colias canned products.

scholarly journals Allele-Specific PCR for the Detection of Azoxystrobin Resistance in Didymella bryoniae

Plant Disease ◽  
2014 ◽  
Vol 98 (12) ◽  
pp. 1681-1684 ◽  
Author(s):  
Mavis J. Finger ◽  
Venkatesan Parkunan ◽  
Pingsheng Ji ◽  
Katherine L. Stevenson
Keyword(s):  
Dna Sequences ◽  
Cyt B ◽  
Didymella Bryoniae ◽  
Specific Pcr ◽  
Pcr Product ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Sensitive Allele ◽  
Cyt B Gene ◽  
Allele Specific Pcr

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is considered the most widespread and destructive disease of watermelon in the southeastern United States. The quinone outside-inhibiting (QoI) fungicide azoxystrobin (AZO), which inhibits mitochondrial respiration by binding to the outer, quinone-oxidizing pocket of the cytochrome bc1 (cyt b) enzyme complex, was initially very effective in controlling GSB. However, resistance to AZO has been observed in D. bryoniae in many watermelon-producing regions. In this study, the DNA sequences of partial cyt b genes of four AZO-resistant (AZO-R) and four AZO-sensitive (AZO-S) isolates of D. bryoniae confirmed the amino acid substitution of glycine by alanine at the 143 codon (G143A) in the AZO-R isolates tested. Allele-specific primers were designed to detect the resistant or sensitive allele at codon 143 of the cyt b gene, which amplified a 165-bp polymerase chain reaction (PCR) product from genomic DNA of nine AZO-R and nine AZO-S isolates of D. bryoniae, respectively. The primer pairs did not amplify DNA from other pathogens tested in the study. The results indicated that the PCR assays developed in the study were specific in differentiating AZO-R and AZO-S isolates and could facilitate AZO resistance detection in D. bryoniae.

PCR Identification of Ruminant Tissue in Raw and Heat-Treated Meat Meals

2006 ◽  
Vol 69 (9) ◽  
pp. 2241-2247 ◽  
Author(s):  
JEONG CHUL HA ◽  
WAN TAE JUNG ◽  
YONG SUK NAM ◽  
TAE WHA MOON
Keyword(s):  
Dna Sequence ◽  
12S Rrna ◽  
Spongiform Encephalopathy ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Product ◽  
Heat Treated ◽  
Mitochondrial 12S Rrna ◽  
Animal Feedstuffs ◽  
Species Specific

To control the spread of bovine spongiform encephalopathy in cattle through contaminated animal feedstuffs, screening of feed products is essential. We designed five pairs of primers to identify specifically raw and heat-treated tissue from cattle, sheep, goat, deer, and ruminants in general. A forward common primer was designed based on a conserved DNA sequence in the mitochondrial 12S rRNA–tRNAval–16S rRNA gene, and reverse primers were designed to hybridize with a species-specific DNA sequence for each species considered. All primers were developed to create a specific PCR product small enough (less than 200 bp) to be suitable for heat-treated material. To evaluate the effect of heat treatment, a severe sterilization condition (133°C at 300 kPa for 20 min) was chosen. Species-specific amplicons were obtained from all types of heat-treated meat meals. Analysis of laboratory-contaminated vegetable meals revealed that the detection limit of the assay was 0.05% for each species analyzed. This PCR-based analysis can be used as a routine method for detecting banned animal-derived ingredients in raw and heat-treated feedstuffs.

scholarly journals Establishment of Methylation-Specific PCR for the Mouse p53 Gene

2011 ◽  
Vol 2011 ◽  
pp. 1-4 ◽  
Author(s):  
Ryuji Okazaki ◽  
Akira Ootsuyama ◽  
Yasuhiro Yoshida ◽  
Toshiyuki Norimura
Keyword(s):  
Cpg Islands ◽  
P53 Gene ◽  
Pcr Primers ◽  
Young Mouse ◽  
Methylation Specific Pcr ◽  
Specific Pcr ◽  
Pcr Product ◽  
Induced Tumors ◽  
Radiation Induced ◽  
Radiation Induced Tumors

Methylation-specific PCR (MSP) of the mouse p53 gene has not yet been reported. We searched the CpG islands, sequenced the bisulfited DNA, and designed PCR primers for methylation and unmethylation sites. DNA from a young mouse produced a strong PCR product with the unmethylated primer and a weaker band with the methylated primer. DNA from an old mouse produced bands of similar intensities with both primers. In radiation-induced tumors, DNA from an old mouse yielded similar bands with both types of primers. We suggest that MSP is a valuable technique for the epigenetic study of the mouse p53 gene.

scholarly journals Studying The Expression of Human P53 Gene in Patients Carrying Breast Cancer.

2017 ◽  
Vol 28 (1) ◽  
pp. 14
Author(s):  
Dawood S. Edan
Keyword(s):  
Breast Cancer ◽  
Healthy Woman ◽  
P53 Gene ◽  
Suppressor Gene ◽  
Women Patients ◽  
Cell Protection ◽  
Specific Pcr ◽  
Pcr Product ◽  
Dna Repair Proteins ◽  
Healthy Sample

The gene P53 can activate DNA repair proteins when DNA has sustained damage. So, it is an important factor in aging. Also, P53 gene can also be modified by mutagens (chemicals, radiation, or viruses), increasing the likelihood for uncontrolled cell division. For this importance, we collected thirty samples from Yarmouk hospital in Baghdad, Iraq. Twenty-nine from women patients carrying breast cancer and the last one was from healthy woman, and the samples that we have taken were embedded in paraffin wax. The extracted RNA from each samples was used to check the expression of a suppressor gene (P53) via Real time PCR. We designed primers of P53 gene on encoded sequence (Exon), to make sure generation a specific PCR product of P53 gene by converting mRNA to cDNA then PCR Product. The expression of each samples was fluctuated between 0.1 fold to 0.2 fold compared with the control (the healthy sample). The highest expression showed was samples 7 (0.2 fold), and the lowest expression showed was samples 27 (0.1 fold) that compared with control expression (sample 19). While the control expression was the highest (0.25 fold) among all samples. The results indicate that tumor may affects on a suppressor gene (P53) and can decrease the gene expression, and eventually can decrease the P53 suppressor protein that can be used it in the cell protection against cancer.

scholarly journals A Multiplex PCR System for the Specific Detection of Cylindrocarpon liriodendri, C. macrodidymum, and C. pauciseptatum from Grapevine

Plant Disease ◽  
2009 ◽  
Vol 93 (8) ◽  
pp. 821-825 ◽  
Author(s):  
Sandra Alaniz ◽  
Josep Armengol ◽  
José García-Jiménez ◽  
Paloma Abad-Campos ◽  
Maela León
Keyword(s):  
Multiplex Pcr ◽  
Extraction Methods ◽  
Specific Pcr ◽  
Pcr Product ◽  
Total Dna ◽  
Polymerase Chain ◽  
Foot Disease ◽  
Species Specific ◽  
Black Foot ◽  
Black Foot Disease

Cylindrocarpon liriodendri and C. macrodidymum are the causal agents of grapevine black foot disease. Recently, a third species, C. pauciseptatum, has been isolated from roots of grapevine showing decline symptoms. Currently, reliable identification of isolates of these species through phenotypical characteristics has not been possible. The polymerase chain reaction (PCR)-based method developed in this study allows a quick and easy detection of Cylindrocarpon spp. associated with grapevine. Three primer pairs annealing to variable, partly species-specific sites of the internal transcribed spacer regions amplified species-specific PCR fragments of different sizes in C. liriodendri, C. macrodidymum, and C. pauciseptatum in a multiplex assay with DNA obtained with both quick and traditional extraction methods. They did not generate any PCR product in other fungal trunk pathogens or contaminants commonly associated with grapevines. When universal fungal ITS primers were used in a nested multiplex PCR, the three primer pairs also detected C. liriodendri, C. macrodidymum, and C. pauciseptatum in total DNA extracted from roots of inoculated grapevines. The designed methods can be used for the diagnosis of these fungi from pure culture or infected grapevines.

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