Riddhi Patel

English 202 C, 025

October 28, 2016

Analyzing the mechanisms behind a particular type

of DNA Sequencing: Pyrosequencing
What is DNA?
DNA, or deoxyribonucleic acid, is a type of nucleic acid that holds the genetic
information in humans and almost all other organisms. DNA drives gene
expression and serves as an instruction set to code for proteins needed to carry
out the various functions in our body. Most DNA is located in the nucleus of a
cell, but a small amount of DNA can also be found in the mitochondria.
The building blocks of a nucleic
acid, nucleotides1, comprise of a
sugar (six-carbon, deoxyribose), a
phosphate group and a nitrogenous
base (adenine (A), guanine (G),
cytosine (C), and thymine (T)). DNA
bases pair up with each other, A
with T and C with G, to form units
called base pairs via hydrogen
bonding2 as shown in Figure 1 to
the left. This results in the doublehelix conformation of a double
stranded DNA molecule.
Figure 1: DNA double-helix molecule.
Base pairs formed via hydrogen bonding
between the nitrogenous bases: A and T, and
G and C.

Human DNA consists of about 3

billion bases; the order, or sequence,
of these bases determines the
information available for building and
maintaining an organism.

Riddhi Patel
English 202 C, 025
October 28, 2016

What is DNA Sequencing?

DNA sequencing is a laboratory technique used to determine the exact sequence
of nucleotides (nitrogenous bases: A, C, G, and T) in a DNA molecule. It includes
a variety of methods/technology that is used to determine the order of the four
nitrogenous bases in a given strand of DNA. This process is based on
quantifiable data. The DNA base sequence carries the information a cell needs to
assemble protein and RNA molecules. DNA sequence information is important
to scientists investigating the functions of genes. The technology of DNA
sequencing was made faster and less expensive as a part of the Human Genome
Project.

Sanger Sequencing:
First and most conventional form of DNA sequencing method introduced
by Frederick Sanger in the 1970s is the Chain-Termination method or
Sanger Sequencing. Sanger sequencing is a method of DNA sequencing
based on the selective incorporation of chain-terminating
dideoxynucleotides3 by DNA polymerase4. This method is based on the
natural process of DNA Replication, and the short fragments created are
aligned based on size/length to sequence the data. Whereas the latest,
most effective and efficient technology is Pyrosequencing which relies on
a series of biochemical reactions and consequent light production to
sequence the nucleotides.

What is Pyrosequencing?
Pyrosequencing is a method of DNA sequencing that relies on light changes and
detection of pyrophosphate release upon nucleotide incorporation, as opposed
the chain termination method with the addition of dideoxynucleotides. Its the
latest sequence-based detection technology that enables rapid and accurate
quantification of sequence variation as a result of streamlined protocols, analysis
flexibility, and elegant output. Figure 2 depicts the overview and the biochemical
mechanisms behind the process of Pyrosequencing.

Patel 3

https://www.researchgate.net/figure/24180702_fig1_Fig-1-Pyrosequencing-chemistry-biochemical-reactionsand-enzymes-involved-in-the

Figure 2: An overview of the pyrosequencing reaction process. A polymerase

catalyzes incorporation of nucleotide(s) into a nucleic acid chain. As a result of
the incorporation, a pyrophosphate5 (PPi) molecule(s) is released and
subsequently converted to ATP6, by ATP sulfurylase7. Light is produced in the
consequent luciferase reaction during which a luciferin molecule is oxidized.
This light is detected by a CCD camera and the peak data are recorded using a
program called Pyrogram. After each nucleotide addition, a washing step is
performed to allow iterative addition.

What are the steps behind the mechanism of the process?

Step 1:
DNA template is immobilized on a bead. A DNA segment is amplified and the
strand to serve as the Pyrosequencing template is biotinylated. After
denaturation, the biotinylated8 single-stranded PCR9 amplicon is isolated.

Patel 4
Step 2:
A primer10 and DNA polymerase are added to the DNA template. The amplicon is then
allowed to hybridize with a sequencing primer. Hybridized primer and singlestranded template are incubated with the enzymes DNA polymerase, ATP
sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5'
phosphosulfate (APS) and luciferin.
Step 3:
A dNTP substrate is introduced. If DNA polymerase adds that nucleotide to the new
DNA strand, pyrophosphate is released. The first deoxy ribonucleotide triphosphate
(dNTP) is added to the reaction. DNA polymerase catalyzes addition of the
dNTP to the sequencing primer, if it is complementary to the base in the template
strand. Each incorporation event is accompanied by the release of pyrophosphate
(PPi) in a quantity equimolar to the amount of incorporated nucleotide.
Step 4:
The release of PPi triggers a chemical reaction involving the firefly enzyme luciferase,
which generates a flash of light. ATP sulfurylase converts the released PPi to ATP in
the presence of adenosine 5' phosphosulfate (APS). This ATP drives the
luciferase-mediated conversion of luciferin to oxyluciferin11 that generates visible
light in amounts that are proportional to the amount of ATP.
Step 5:
A charge coupled device camera detects the light and Pyrogram records the height of the
peak. Solutions of each of the four dNTPs are successively washed across the
immobilized DNA template, and a detector records whether light is produced in
the presence of each particular dNTP. The light produced in the luciferasecatalyzed reaction is detected by a charge coupled device (CCD) camera and seen
as a peak in the raw data output known as Pyrogram12. The height of each peak
(light signal) is proportional to the number of nucleotides incorporated.
Step 6:
Continuous degradation of free dNTPs by apyrase allows the process to restart. Apyrase,
a nucleotide-degrading enzyme, continuously degrades unincorporated

Patel 5
nucleotides and ATP by removing single phosphates. When degradation is
complete, another nucleotide is added.

Why do we sequence DNA?

More than 99 percent of these base sequences are the same in all people and yet
the minor differences in ones DNA sequence are enough to distinguish an
individual among millions. Hence, DNA acts as a persons unique fingerprint or
an almost infallible form of identification.
Obtaining a sample of someones DNA (hair, saliva, skin cells, etc.) and a sample
from a crime scene or another site, sequencing the two and comparing them can
help identify the individual if the sequences match; it can also help determine
biological relationships. Analyzing the genome through sequencing can
furthermore aid in research when studying different alleles and genes and the
roles they play in certain traits and diseases.
One can get their entire genome sequenced for relatively cheap - about $1,000,
but analyzing the entire genome with traits and genes underlying diseases would
cost 1 million dollars!

Applications

Quantitative DNA methylation analysis

Mutation analysis and genotyping
Allelic quantification
Microbial identification and drug resistance typing
Pharmacogenetics
Clinical research

Conclusion
DNA Sequencing is a very valuable process that helps whether it is in the
context of identifying an individual in a crime scene, a paternity test, or to
determine disease probabilities among many other. Pyrosequencing allows it to
be done in a rapid and accurate manner; Addition of dNTPs is performed
sequentially. As the process continues, the complementary DNA strand is built
up and the nucleotide sequence is determined from the signal peaks in the
Pyrogram trace.

Riddhi Patel
English 202 C, 025
October 28, 2016

Key Terms and definitions:

1. Nucleotides: a compound consisting of a nucleoside linked to a phosphate
group which forms the basic structural unit of nucleic acids such as DNA.
2. Hydrogen Bonding: attractive force between the hydrogen attached to an
electronegative atom of one molecule and an electronegative atom of a
different molecule.
3. Dideoxynucleotides (ddNTP): chain-elongating inhibitors of DNA
polymerase, used in the Sanger method for DNA sequencing.
4. DNA Polymerase: enzymes that create DNA molecules by assembling
nucleotides, the building blocks of DNA.
5. Pyrophosphate (PPi): a phosphorus oxyanion
6. ATP: Adenosine Triphosphate, a high-energy molecule found in every
cell. Its job is to store and supply the cell with needed energy.
7. ATP Sulfurylase: an enzyme that catalyzes a step in the pathway for the
synthesis of active sulfate; the enzyme reacts ATP with sulfate to produce
pyrophosphate and adenosine 5'-phosphosulfate (APS).
8. Biotin: a vitamin of the B complex, found in egg yolk, liver, and yeast;
involved in the synthesis of fatty acids and glucose.
9. Polymerase Chain Reaction (PCR): a technique used in molecular biology
to amplify a single copy or a few copies of a piece of DNA across several
orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
10. Primer: a short strand of RNA or DNA (generally about 18-22 bases) that
serves as a starting point for DNA synthesis.
12. Pyrogram: A graph that displays the signal peaks which help in the
determination of the nucleotide sequence.
11. Luciferin reaction: Luciferin combines with ATP to form luciferyl
adenylate and release PPi. The reaction releases a photon of light as
oxyluciferin (electronically excited state) goes back to the ground state.

Patel 7

References
"DNA Sequencing Fact Sheet." National Human Genome Research Institute
(NHGRI). NIH, 18 Dec. 2015. Web. 03 Nov. 2016.
<https://www.genome.gov/10001177/dna-sequencing-fact-sheet/>.
Qiagen. "Pyrosequencing Technology and Platform Overview." QIAGEN. N.p.,
2013. Web. 03 Nov. 2016.
<https://www.qiagen.com/us/resources/technologies/pyrosequencingresource-center/technology-overview/>.
"What Is DNA? - Genetics Home Reference." U.S National Library of Medicine.
U.S. National Library of Medicine, 1 Nov. 2016. Web. 03 Nov. 2016.
<https://ghr.nlm.nih.gov/primer/basics/dna>.