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Related Concept Videos

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.
Sanger Sequencing01:57

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
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...
Lagging Strand Synthesis01:59

Lagging Strand Synthesis

During replication, the complementary strands in double-stranded DNA are synthesized at different rates. Replication first begins on the leading strand. Replication starts later, occurs more slowly, and proceeds discontinuously on the lagging strand.
There are several major differences between synthesis of the leading strand and synthesis of the lagging strand. 1) Leading strand synthesis happens in the direction of replication fork opening, whereas lagging strand synthesis happens in the...
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...

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Updated: Jun 11, 2026

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
07:38

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

Published on: October 6, 2017

Real-time DNA sequencing from single polymerase molecules.

Jonas Korlach1, Keith P Bjornson, Bidhan P Chaudhuri

  • 1Pacific Biosciences, Menlo Park, California, USA.

Methods in Enzymology
|June 29, 2010
PubMed
Summary
This summary is machine-generated.

Pacific Biosciences developed single-molecule, real-time (SMRT) DNA sequencing. This method detects DNA polymerase activity and provides kinetic parameters, enabling detailed analysis of DNA synthesis.

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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • DNA sequencing technologies are crucial for genomic research.
  • Previous methods had limitations in read length and real-time analysis.

Purpose of the Study:

  • To describe the single-molecule, real-time (SMRT) DNA sequencing method.
  • To illustrate the accessibility of DNA polymerization dynamics.

Main Methods:

  • Real-time sequencing of single DNA molecules using fluorescently labeled nucleotides.
  • Detection of sequential base additions by DNA polymerase.
  • High-throughput, parallel operational mode.

Main Results:

  • Achieved sequencing rates of several bases per second with kilobase read lengths.
  • Demonstrated direct measurement of base-specific kinetic parameters.
  • Characterized heterogeneities in DNA synthesis rates from single molecules.

Conclusions:

  • SMRT DNA sequencing provides sequence information and polymerization dynamics.
  • This method offers new insights into enzyme kinetics and DNA synthesis processes.
  • Enables detailed analysis of DNA polymerase activity at the single-molecule level.