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Related Concept Videos

Transfer RNA Synthesis02:36

Transfer RNA Synthesis

One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Transfer RNA Synthesis02:36

Transfer RNA Synthesis

One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
Synthetic Biology02:55

Synthetic Biology

Synthetic biology is an interdisciplinary science that involves using principles from disciplines such as engineering, molecular biology, cell biology, and systems biology. It involves remodeling existing organisms from nature or constructing completely new synthetic organisms for applications such as protein or enzyme production, bioremediation, value-added macromolecule production, and the addition of desirable traits to crops, to name a few.
Golden rice
Golden rice is a genetically modified...
RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...

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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

Published on: December 29, 2021

TopDown real-time gene synthesis.

Mo Chao Huang1, Wai Chye Cheong, Hongye Ye

  • 1Institute of Bioengineering and Nanotechnology, The Nanos, Singapore.

Methods in Molecular Biology (Clifton, N.J.)
|February 14, 2012
PubMed
Summary
This summary is machine-generated.

A novel TopDown one-step gene synthesis method simplifies long DNA assembly. This cost-effective approach optimizes oligonucleotide assembly and amplification, improving efficiency and kinetics for DNA synthesis.

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Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems
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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biotechnology

Background:

  • Conventional gene synthesis methods can be complex and costly.
  • Efficient assembly of long DNA sequences remains a challenge in molecular biology.

Purpose of the Study:

  • To introduce a simple, cost-effective, one-step gene synthesis method for long DNA sequences.
  • To improve the efficiency and control of gene synthesis through optimized primer and annealing temperature strategies.

Main Methods:

  • Developed a TopDown one-step gene synthesis method.
  • Utilized differential melting temperatures for outer primers and assembly oligonucleotides.
  • Employed distinct annealing temperatures for assembly and amplification steps.
  • Integrated LCGreen I DNA fluorescence dye for real-time monitoring of PCR-based gene synthesis.

Main Results:

  • The TopDown method effectively assembles long DNA sequences.
  • Eliminated interference between polymerase chain reaction (PCR) assembly and amplification.
  • Real-time monitoring provided insights into stepwise efficiency and kinetics.
  • Optimized gene synthesis conditions by comparing fluorescence data with gel electrophoresis.

Conclusions:

  • The TopDown one-step gene synthesis method offers a simplified and efficient approach for constructing long DNA molecules.
  • Real-time monitoring enhances the understanding and optimization of PCR-based gene synthesis processes.