Single Molecule Real Time Sequencing
Encyclopedia
Single molecule real time sequencing (also known as SMRT) is a parallelized single molecule DNA sequencing
by synthesis technology developed by Pacific Biosciences
. Single molecule real time sequencing utilizes the zero-mode waveguide
(ZMW), developed in the laboratories of Harold G. Craighead and Watt W. Webb
at Cornell University. A single DNA polymerase
enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA (also known as a base) being incorporated by DNA polymerase
. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
. The fluorescent dye molecule is attached to the phosphate chain of the nucleotide. When the nucleotide is incorporated by the DNA polymerase, the fluorescent dye is cleaved off with the phosphate chain as a part of a natural DNA synthesis
process during which a phosphodiester bond
is created to elongate the DNA chain. The cleaved fluorescent dye molecule then diffuses out of the detection volume so that the fluorescent signal is no longer detected.
confinement structure that consists of a circular hole in an aluminum cladding film deposited on a clear silica substrate. The ZMW holes are ~70 nm in diameter and ~100 nm in depth. Due to the behavior of light when it travels through a small aperture, the optical field decays exponentially inside the chamber. The observation volume within an illuminated ZMW is ~20 zeptoliters (20 X 10−21 liters). Within this volume, the activity of DNA polymerase incorporating a single nucleotide can be readily detected.
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
by synthesis technology developed by Pacific Biosciences
Pacific Biosciences
Pacific Biosciences is a biotechnology company founded in 2004 that develops and manufactures systems for gene sequencing and some novel real time biological observation. They describe their platform as single molecule real time sequencing , based on the properties of zero-mode waveguides...
. Single molecule real time sequencing utilizes the zero-mode waveguide
Zero-mode waveguide
A zero-mode waveguide is an optical waveguide that guides light energy into a volume that is small in all dimensions compared to the wavelength of the light....
(ZMW), developed in the laboratories of Harold G. Craighead and Watt W. Webb
Watt W. Webb
Watt W. Webb is known for his co-invention of Multiphoton microscopy in 1990.- Biography :Professor Watt W...
at Cornell University. A single DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA (also known as a base) being incorporated by DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
Technology
The DNA sequencing is done on a chip that contains many ZMWs. Inside each ZMW, a single active DNA polymerase with a single molecule of single stranded DNA template is immobilized to the bottom through which light can penetrate and create a visualization chamber that allows monitoring of the activity of the DNA polymerase at a single molecule level. The signal from a phospho-linked nucleotide incorporated by the DNA polymerase is detected as the DNA synthesis proceeds which results in the DNA sequencing in real time.Phospholinked nucleotide
For each of the nucleotide bases, there are four corresponding fluorescent dye molecules that enable the detector to identify the base being incorporated by the DNA polymerase as it performs the DNA synthesisDNA synthesis
DNA synthesis commonly refers to:*DNA replication - DNA biosynthesis *Polymerase chain reaction - enzymatic DNA synthesis *Oligonucleotide synthesis - chemical synthesis of nucleic acids...
. The fluorescent dye molecule is attached to the phosphate chain of the nucleotide. When the nucleotide is incorporated by the DNA polymerase, the fluorescent dye is cleaved off with the phosphate chain as a part of a natural DNA synthesis
DNA synthesis
DNA synthesis commonly refers to:*DNA replication - DNA biosynthesis *Polymerase chain reaction - enzymatic DNA synthesis *Oligonucleotide synthesis - chemical synthesis of nucleic acids...
process during which a phosphodiester bond
Phosphodiester bond
A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two 5-carbon ring carbohydrates over two ester bonds. Phosphodiester bonds are central to all known life, as they make up the backbone of each helical strand of DNA...
is created to elongate the DNA chain. The cleaved fluorescent dye molecule then diffuses out of the detection volume so that the fluorescent signal is no longer detected.
Zero-mode waveguide
The zero-mode waveguide (ZMW) is a nanophotonicPhotonics
The science of photonics includes the generation, emission, transmission, modulation, signal processing, switching, amplification, detection and sensing of light. The term photonics thereby emphasizes that photons are neither particles nor waves — they are different in that they have both particle...
confinement structure that consists of a circular hole in an aluminum cladding film deposited on a clear silica substrate. The ZMW holes are ~70 nm in diameter and ~100 nm in depth. Due to the behavior of light when it travels through a small aperture, the optical field decays exponentially inside the chamber. The observation volume within an illuminated ZMW is ~20 zeptoliters (20 X 10−21 liters). Within this volume, the activity of DNA polymerase incorporating a single nucleotide can be readily detected.
Sequencing performance
Pacific Biosciences expects to commercialize SMRT sequencing in 2010 or 2011. The prototype of the SMRT chip contains ~3000 ZMW holes that allow parallelized DNA sequencing. Each of the ZMW holes produces approximately 1,500 bp (base pair) read lengths at a speed of 10 bp per second.Application
Single molecule real time sequencing will be applicable for a broad range of genomics research, namely:- De novo genome sequencing: The read length from the single molecule real time sequencing is currently comparable to that from the Sanger sequencing method based on dideoxynucleotideDideoxynucleotidesDideoxynucleotides, or ddNTPs, are nucleotides lacking a 3'-hydroxyl group on their deoxyribose sugar. Since deoxyribose already lacks a 2'-OH, dideoxyribose lacks hydroxyl groups at both its 2' and 3' carbons...
chain termination. The longer read length allows de novo genome sequencing and easier genome assemblies. - Individual whole genome sequencing: Individual genome sequencing may utilize the single molecule real time sequencing method for personalized medicinePersonalized medicinePersonalized medicine is a medical model emphasizing in general the customization of healthcare, with all decisions and practices being tailored to individual patients in whatever ways possible...
. - Resequencing: A same DNA molecule can be resequenced independently by creating the circular DNA template and utilizing a strand displacing enzyme that separates the newly synthesized DNA strand from the template.
- Methylation detection: the dynamics of polymerase can indicate whether a base is methylated.