Sequencing methods

Gene Sequencing

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

Automated Gene Sequencing

DNA is the main carrier of genetic information in living organisms. DNA molecules are extremely long, large, and consist of repeating nucleotides. Nucleotides are the bases of DNA and consist of adenine (A), thymine (T), guanine (G), and cytosine (C). The structure of a DNA molecule is double stranded, consisting of two DNA strands wound around each other to form a double helix. The nucleotides of the two strands are complementary to each other such that adenine cross-links with thymine (A-T), and guanine cross-links with cytosine (G-C). The goal of DNA sequencing is to determine the order of bases for a specific piece of DNA.

DNA sequencing is achieved by utilizing labeled nucleotides for incorporation into a copy of a piece of DNA. The DNA sequence can then be derived by the positions of the labeled nucleotides. First, the DNA segment to be copied, called the template DNA, is separated into two strands by heating. An enzyme is used to make complementary copies of the individual strands with the labeled nucleotides. The DNA segments are then separated according to length by electrophoresis in a polyacrylamide gel. Electrophoresis is the movement of electrically charged particles through a porous substance (polyacrylamide gel) under the influence of an electric field provided by a high voltage unit. Once the DNA is separated, several different techniques are available to allow for analysis of the DNA sequence.

Traditional methods of manual DNA sequencing utilize radioactive isotopes such as phosphorous-32, sulfur-35, and phosphorous-33, incorporated into specific nucleotides (A,C,T,G). Radioactive labeled nucleotides allow for reading the sequence by a technique known as autoradiography. The gel that contains the separated DNA segments is exposed to X-ray film for a period of time. The radiation causes dark spots on the film to indicate its location. Four individual lanes are required for manual sequencing in order to determine the full DNA sequence. An individual must interpret the results of this process and typically the results are entered into a computer for storage and linking to other results.

seq

Automated DNA sequencing utilizes a fluorescent dye to label the nucleotides instead of a radioactive isotope. The fluorescent dye is not an environmentally hazardous chemical and has no special handling or disposal requirements. Instead of using X-ray film to read the sequence, a laser is used to stimulate the fluorescent dye. The Perkin-Elmer Applied Biosystems (ABI) DNA sequencers are designed to discriminate all four fluorescent dye wavelengths simultaneously, which allows for complete DNA sequencing in one lane on the gel.Varying degrees of automation are also available.

For full automation, all that is required is to load a sample tray with template DNA; the equipment performs the labeling and analysis. The other option is to perform the labeling reactions with fluorescent dyes, load the samples onto a gel, and place the gel into the DNA sequencer. The equipment performs the separation and analysis. The system automatically identifies the nucleotide sequence and saves the information on the computer. Thus, only a review of the data is necessary to ensure no anomalies were misidentified by the computer.

SEQ

 

SEQ-1

 

Pyrosequencing

Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the “sequencing by synthesis” principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides. The technique was developed by Mostafa Ronaghi and Pål Nyrén at the Royal Institute of Technology in Stockholm in 1996.

 

PYRO

Procedure

“Sequencing by synthesis” involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemiluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template.

ssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5´ phosphosulfate (APS) and luciferin.

  1. The addition of one of the four deoxynucleoside triphosphates (dNTPs) (dATPαS, which is not a substrate for a luciferase, is added instead of dATP to avoid noise) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation releases pyrophosphate (PPi) stoichiometrically.
  2. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP acts as a substrate for the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a camera and analyzed in a pyrogram.
  3. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.

Pyrosequencing is most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available.

The templates for pyrosequencing can be made both by solid phase template preparation (streptavidin-coated magnetic beads) and enzymatic template preparation (apyrase+exonuclease). So Pyrosequencing can be differentiated into two types, namely Solid Phase Pyrosequencing and Liquid Phase Pyrosequencing.pyroseq

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