scholarly journals Preparation of DNA Ladder Based on Multiplex PCR Technique

2010 ◽  
Vol 2010 ◽  
pp. 1-3 ◽  
Author(s):  
Tian-Yun Wang ◽  
Li Guo ◽  
Jun-he Zhang
Keyword(s):  
Molecular Biology ◽  
Dna Marker ◽  
Dna Fragments ◽  
Dna Ladder ◽  
Hot Start ◽  
Target Dna ◽  
Touchdown Pcr ◽  
Lambda Dna ◽  
Molecular Weight Standard ◽  
Multiple Pcr

DNA molecular weight standard control, also called DNA marker (ladder), has been widely used in the experiments of molecular biology. In the paper, we report a method by which DNA marker was prepared based on multiple PCR technique. 100–1000 bp DNA fragments were amplified using the primers designed according to the 6631 ~ 7630 position of lambda DNA. Target DNA fragments were amplified using Touchdown PCR combined with hot start PCR, respectively, followed extracted by phenol/chloroform, precipitated with ethanol and mixed thoroughly. The results showed that the 100–1000 bp DNA fragments were successfully obtained in one PCR reaction, the bands of prepared DNA marker were clear, the size was right and could be used as control in the molecular biology experiment. This method could save time and be more inexpensive, rapid, simple when compared with the current DNA Ladder prepared means.

scholarly journals Construction of DNA ladder for determination of small size DNA fragments

2021 ◽  
Vol 19 (3) ◽  
pp. 539-545
Author(s):  
Vo Thi Thuong Lan ◽  
Le Thi Thanh
Keyword(s):  
Molecular Biology ◽  
Restriction Enzyme ◽  
Dna Markers ◽  
Dna Marker ◽  
Dna Fragments ◽  
Dna Ladder ◽  
Time Saving ◽  
Pcr Product ◽  
Dna Fragment

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.

scholarly journals Straightforward Procedure for Laboratory Production of DNA Ladder

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Vo Thi Thuong Lan ◽  
Pham Thi Thanh Loan ◽  
Pham Anh Thuy Duong ◽  
Le Thi Thanh ◽  
Ngo Thi Ha ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Tandem Repeat ◽  
Restriction Site ◽  
Restriction Enzymes ◽  
Dna Fragments ◽  
Repeated Dna ◽  
Dna Ladder ◽  
Time Saving ◽  
Partial Digestion ◽  
Unique Restriction

DNA ladder is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare a DNA ladder that consists of 10 fragments from 100 to 1000 bp. This protocol is a combination of routinely employed methods: cloning, PCR, and partial digestion with restriction enzymes. DNA fragments of 100 bp with unique restriction site at both ends were self-ligated to create a tandem repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR and partially digested by restriction enzymes to produce a ladder containing multimers of the repeated DNA fragments. Our procedure for production of DNA ladder could be simple, time saving, and inexpensive in comparison with current ones widely used in most laboratories.

scholarly journals Specificity-Enhanced Hot-Start PCR: Addition of Double-Stranded DNA Fragments Adapted to the Annealing Temperature

BioTechniques ◽  
2000 ◽  
Vol 28 (2) ◽  
pp. 278-282 ◽  
Author(s):  
Peter Kainz ◽  
Angela Schmiedlechner ◽  
Hans Bernd Strack
Keyword(s):  
Annealing Temperature ◽  
Dna Fragments ◽  
Double Stranded Dna ◽  
Hot Start

Advances in visualizing transcription factor-DNA interactions

Genome ◽  
2020 ◽  
Author(s):  
Rachel M. Price ◽  
Marek A Budzynski ◽  
Shivani Kundra ◽  
Sheila S. Teves
Keyword(s):  
Molecular Biology ◽  
Dna Sequences ◽  
Imaging Techniques ◽  
Specific Interaction ◽  
Dna Interactions ◽  
Dynamic Information ◽  
Transcription Process ◽  
Target Dna ◽  
Biology Research

At the heart of the transcription process is the specific interaction between transcription factors (TFs) and their target DNA sequences. Decades of molecular biology research have led to unprecedented insights into how TFs access the genome to regulate transcription. In the last 20 years, advances in microscopy have enabled scientists to add imaging as a powerful tool in probing two specific aspects of TF-DNA interactions: structure and dynamics. In this review, we examine how applications of diverse imaging technologies can provide structural and dynamic information that complements insights gained from molecular biology assays. As a case study, we discuss how applications of advanced imaging techniques have reshaped our understanding of TF behavior across the cell cycle, leading to a rethinking in the field of mitotic bookmarking.

scholarly journals High-Frequency Genetic Recombination and Reactivation of Orthopoxviruses from DNA Fragments Transfected into Leporipoxvirus-Infected Cells

2003 ◽  
Vol 77 (13) ◽  
pp. 7281-7290 ◽  
Author(s):  
Xiao-Dan Yao ◽  
David H. Evans
Keyword(s):  
Vaccinia Virus ◽  
Genetic Recombination ◽  
Virus Genome ◽  
Dna Fragments ◽  
Gene Encoding ◽  
Plaque Purification ◽  
Infected Cells ◽  
Clone And Express ◽  
Recombination Reactions ◽  
Multiple Pcr

ABSTRACT Poxvirus DNA is not infectious because establishing an infection requires the activities of enzymes packaged in the virion. This barrier can be overcome by transfecting virus DNA into cells previously infected with another poxvirus, since the resident virus can provide the trans-acting systems needed to reactivate transfected DNA. In this study we show that cells infected with a leporipoxvirus, Shope fibroma virus (SFV), can reactivate vaccinia virus DNA. Similar heterologous packaging systems which used fowlpox-infected cells to reactivate vaccinia virus have been described, but SFV-infected cells promoted a far more efficient reaction that can produce virus titers exceeding 106 PFU/μg of transfected DNA. SFV-promoted reactions also exploit the hyperrecombinogenic systems previously characterized in SFV-infected cells, and these coupled recombination and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green protein without the need for plaque purification or selectable markers. The ability of SFV-infected cells to reactivate fragments of vaccinia virus was ultimately limited by the number of recombinational exchanges required and one cannot reconstruct vaccinia virus from multiple PCR fragments spanning essential portions of the genome. These observations suggest that recombination is an integral part of poxvirus reactivation reactions and provide a useful new technique for altering the structure of poxvirus genomes.

Polymerase Chain Reaction–Mediated Characterization of Molds Belonging to the Aspergillus flavus Group and Detection of Aspergillus parasiticus in Peanut Kernels by a Multiplex Polymerase Chain Reaction

2002 ◽  
Vol 65 (5) ◽  
pp. 840-844 ◽  
Author(s):  
RUEY-SHYANG CHEN ◽  
JWU-GUH TSAY ◽  
YU-FEN HUANG ◽  
ROBIN Y.-Y. CHIOU
Keyword(s):  
Polymerase Chain Reaction ◽  
Aspergillus Flavus ◽  
Aspergillus Parasiticus ◽  
Regulatory Protein ◽  
Aflatoxin Production ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Target Dna ◽  
Polymerase Chain ◽  
Aspergillus Flavus Group

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxi-genicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.

6. Genomics and the microbial world

2018 ◽  
pp. 88-108
Author(s):  
John Archibald
Keyword(s):  
Molecular Biology ◽  
Single Cell ◽  
Human Microbiome ◽  
Gene Sequencing ◽  
Dna Fragments ◽  
Microbial World ◽  
Single Cell Genomics ◽  
Genomic Encyclopedia

Together, molecular biology and genomics have made it possible to explore the diversity and ecology of the 99 per cent of microbes that cannot be cultured in laboratories. ‘Genomics and the microbial world’ considers environmental gene sequencing; metagenomics, the isolation and analysis of large DNA fragments taken directly from the environment; and single-cell genomics and transcriptomics. It also examines the human microbiome; it is now clear that a complex array of factors contribute to the establishment and maintenance of the human microbiome over the course of a lifetime. Bioprospecting in the metagenomics era is also discussed along with the ambitious Genomic Encyclopedia of Bacteria and Archaea project.

Comparison of DNA fragments as donor DNAs upon sequence conversion of cleaved target DNA

2017 ◽  
Vol 36 (6) ◽  
pp. 428-434 ◽  
Author(s):  
Tetsuya Suzuki ◽  
Takashi Imada ◽  
Yasuo Komatsu ◽  
Hiroyuki Kamiya
Keyword(s):  
Dna Fragments ◽  
Target Dna

Influence of Sieving Polymer Concentration and Applied Voltage on Separation Accuracy of the Capillary Gel Electrophoresis for Small DNA Fragments

2020 ◽  
Vol 990 ◽  
pp. 101-105
Author(s):  
Tomoka Nakazumi ◽  
Yusuke Hara
Keyword(s):  
Applied Voltage ◽  
Gel Electrophoresis ◽  
Polymer Concentration ◽  
Dna Fragments ◽  
Dna Ladder ◽  
Capillary Gel Electrophoresis ◽  
Two Factors ◽  
Sufficient Degree ◽  
Separation Conditions

In this study, measurement conditions of Capillary gel electrophoresis (CGE) were optimized in order to increase the separation accuracy for small DNA fragments. We adopted a 20-bp DNA Ladder including 13 small double-stranded fragments (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, and 500-bp). The applied voltage and sieving polymer concentration were assessed because these two factors significantly affect the separation accuracy of DNA fragments. As a result, we succeeded in optimizing CGE separation conditions for small DNA fragments with a sufficient degree of accuracy.

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