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Related Concept Videos

DNA as a Genetic Template02:05

DNA as a Genetic Template

Two structural features of the DNA molecule provide a basis for the mechanisms of heredity: the four nucleotide bases and its double-stranded nature. The Watson-Crick model of double-helical DNA structure, proposed in 1952, drew heavily upon the X-ray crystallography work of researchers Rosalind Franklin and Maurice Wilkins. Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine for their work in 1962. Franklin was, controversially, excluded from the prize for...
DNA as a Genetic Template02:05

DNA as a Genetic Template

Two structural features of the DNA molecule provide a basis for the mechanisms of heredity: the four nucleotide bases and its double-stranded nature. The Watson-Crick model of double-helical DNA structure, proposed in 1952, drew heavily upon the X-ray crystallography work of researchers Rosalind Franklin and Maurice Wilkins. Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine for their work in 1962. Franklin was, controversially, excluded from the prize for...
The Replisome03:01

The Replisome

DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...
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.
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

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Related Experiment Video

Updated: Jun 6, 2026

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
10:32

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Published on: December 6, 2017

A shRNA library constructed through the generation of loop-stem-loop DNA.

Yuji Nishikawa1, Tomoyasu Sugiyama

  • 1Graduate School of Bionics, Tokyo University of Technology, Hachioji, Tokyo, Japan.

The Journal of Gene Medicine
|November 25, 2010
PubMed
Summary

This study presents an efficient four-step method for generating short hairpin RNA (shRNA) libraries from randomized oligonucleotides, simplifying genetic screening. The novel shRNA maintains RNA interference (RNAi) specificity, offering a valuable tool for functional genomics research.

More Related Videos

Design and Synthesis of a Reconfigurable DNA Accordion Rack
07:44

Design and Synthesis of a Reconfigurable DNA Accordion Rack

Published on: August 15, 2018

Related Experiment Videos

Last Updated: Jun 6, 2026

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source
10:32

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Published on: December 6, 2017

Design and Synthesis of a Reconfigurable DNA Accordion Rack
07:44

Design and Synthesis of a Reconfigurable DNA Accordion Rack

Published on: August 15, 2018

Area of Science:

  • Molecular Biology
  • Genetics
  • Functional Genomics

Background:

  • Short hairpin RNA (shRNA) libraries are crucial for genetic screening.
  • Existing library construction methods involve 4-9 complex reaction steps.

Purpose of the Study:

  • To develop an efficient, streamlined method for generating shRNA-coding DNA.
  • To reduce the number of reaction steps in shRNA library construction.

Main Methods:

  • Synthesized blunt-end stem-loop DNA from randomized oligonucleotides using Klenow fragment.
  • Created loop-stem-loop DNA by ligating stem-loop DNA with a hairpin linker.
  • Generated inverted repeat-containing stem-loop DNA and cloned it into an expression vector.
  • Evaluated shRNA specificity and efficiency via luciferase activity assays.

Main Results:

  • Successfully generated shRNA-coding DNA from randomized oligonucleotides with an average GC content of 46.6%.
  • Each synthesized sequence was unique.
  • shRNA produced by this method suppressed luciferase activity more effectively than typical shRNAs at lower doses.

Conclusions:

  • Developed a four-step shRNA library construction method without polymerase chain reaction.
  • The generated shRNA demonstrated retained RNA interference (RNAi) specificity.
  • This simplified method is valuable for functional genomics and genetic screening.