Eric Tran

English 363- M/W @ 10 AM

2/26/18

Sanger Method usage of high-powered technology. With the

The Sanger Method, also known as rapid progression of science, the

Dideoxy Sequencing, is a laboratory process understanding of Sanger Method has served

in which dideoxynucleotides (ddNTPs) are as the basis of genetic analysis and opened

used to map the sequence of a select DNA the paths to newer methods that incorporate

genome. Developed in the 1970s, the Sanger modern technology (Figure 1). Better known

method was one of the first DNA as Next Generation Sequencing, processes

sequencing techniques that became the such as Illumina Sequencing and

forefront of modern molecular genetics Pyrosequencing were made possible through

(Pierce, 2017). This type of DNA the understanding of Sanger Method (Pierce,

sequencing method is an effective 2017). Next Generation Sequencing is

combination of laboratory techniques such highly accurate, reducing the probability of

as polymerase chain reaction (PCR), micro- committing errors in comparison to the

pipetting, and gel electrophoresis. execution of Sanger method. This expansion

of the capabilities in upgraded DNA

History. Frederick Sanger was a
sequencing methods allows thousands of
British biochemist who won the 1980 Nobel
DNA samples to be sequenced at once in the
Peace Prize for the creation of the Sanger
same time it takes the limited Sanger
Method in 1977 (Sanger-Facts). Sanger and
method to sequence one DNA sample.
his colleagues were one of the first to

establish a standard procedure of sequencing

DNA (Sanger-Facts). Sanger method

became a simple and cost-effective way to

determine the DNA sequence without the

 Eric Tran
English 363- M/W @ 10 AM
2/26/18

makes it difficult for free nucleotides to

attach and continue the replication chain in

DNA replication processes (Brunstein,

2017). Because of their chemical structure,

the ddNTP variations of A, C, T and G are

picked up by DNA polymerase in DNA

synthesis and “terminate” DNA sequences at

specific sites (Brunstein, 2017). The

Figure 1: A flowchart overview of the several types of Next-
generation DNA sequencing that result from the basis of Sanger terminations caused by the ddNTPs create
method. (Sánchez, 2011).
Key Components. Nucleotides varying fragment lengths of newly
(dNTPs), classified as monomers, are the synthesized DNA at specific nucleotide
basic building blocks of DNA. There are sites.
four distinct types of nucleotides that appear

in a DNA sequence: A (Adenine), T

(Thymine), C (Cytosine), and G (Guanine).

The purpose of Sanger method is to identify

the specific order of the nucleotides that an

original DNA template is composed of. The

dideoxynucleotides used in Sanger method

are special types of nucleotides that lack Figure 2: The differences in the chemical structure between a
dideoxynucleotide (ddNTP) and a deoxynucleotide (dNTP).
hydroxyl groups (OH-) in their chemical (OpenStax College, 2013).

structure (Pierce, 2017). Depicted in Figure

2, the lack of hydroxyl groups in ddNTPs

 Eric Tran
English 363- M/W @ 10 AM
2/26/18

Procedure. In Sanger method, the attachments. Starting at the bottom of the

polymerase chain reaction (PCR) is the first autoradiogram, the first band is the shortest

laboratory technique used to create length because short fragments travel the

numerous copies of template DNA strands fastest through the gel. The specific ddNTP

for ddNTP termination. Four polymerase type of the shortest band represents the first

chain reactions with distinct types of nucleotide of the complementary DNA

ddNTPs are conducted in separate vials. sequence (Pierce, 2017). The bands of the

The resulting products yield large sample gel are read like a musical score and

sizes of distinguishable DNA fragments, sequenced in order from bottom to top for

shortened with ddATP, ddTTP, ddCTP, and complementary DNA strand construction

ddGTP (Brunstein, 2017). The next lab (Pierce, 2017). Once the complementary

technique, gel electrophoresis, separates the sequence of DNA is obtained, the

fragments of each type of ddNTP reaction in corresponding nucleotides can be placed

four wells of agarose gel (Brunstein, 2017). together to establish the sequence of the

An electric current stimulates the gel, desired DNA strand.

forcing the negatively charged DNA to

move towards the positive end. An

autoradiogram of the agarose gel is created

using X-ray film to visualize the DNA, as

shown in Figure 3 (Pierce, 2017). The

agarose gel displays four columns of bands

that represent ranges of DNA fragment

Figure 3: An example of an autoradiogram of gel electrophoresis
lengths established by the dideoxynucleotide used to obtain the complementary DNA sequence. (Kimmel, 2016)
 Eric Tran
English 363- M/W @ 10 AM
2/26/18

Experimental Usage. Sanger measures such as medicine and vaccines can

method is not limited to the construction of be rapidly progressed further.

only human DNA genomes. For example, a

Future Applications. Methods of
research team from the Peter Doherty
DNA sequencing open opportunities to
Institute for Infection and Immunity
advance our understanding of science,
performed Sanger sequencing to map out
technology, and humanity. The theory of
multiple genomes of human influenza A
Sanger method inspired many scientists to
viruses (Deng, 2015). The experimentation
uncover the mysteries of the human genome.
of six subtypes of the influenza viruses was
The Human Genome Project has been an
conducted to further enhance the traditional
ongoing mission to analyze and compare
usage of Sanger method for genomic
human DNA to that of other organisms with
mapping (Deng, 2015). It has been
DNA sequencing methods (Pierce, 2017).
concluded through experimentation that
These relationships can be used to identify
Sanger sequencing utilizing a minimum set
model organisms with similarities to human
of primers can be a practical method to
DNA for experimentation. This process,
create genomes of viruses in a shorter
through Sanger sequencing, provides ethical
amount of time (Deng, 2015). Through this
and financial solutions to experimentation.
method, comparisons between healthy and
DNA sequencing enables the possibility of
infected human DNA can be drawn to
personalized medicine, where the analysis of
determine the causal factors of the sickness.
DNA leads to predictions and preventions of
By determining the genome of viruses, our
inheritable diseases.
understanding to create preventative
 Eric Tran
English 363- M/W @ 10 AM
2/26/18

References

Brunstein, J. (2017). Back to Basics: Sanger sequencing and its applications. MLO: Medical
Laboratory Observer, 49(7), 58-60.

Deng, Spirason, Iannello, Jelley, Lau, & Barr. (2015). A simplified Sanger sequencing method
for full genome sequencing of multiple subtypes of human influenza A viruses. Journal
of Clinical Virology, 68, 43-48.

Frederick Sanger - Facts. (n.d.). Retrieved February 20, 2018, from

https://www.nobelprize.org/nobel_prizes/chemistry/laureates/1958/sanger-facts.html

Kimmel, E. (2016) Sanger Method of DNA Sequencing: Figure 2. [JPEG]. Retrieved from
http://www.execulink.com/~ekimmel/sanger.htm

OpenStax College, Biology (2013) Whole-genome sequencing: Figure 1. [JPEG]. Retrieved

from https://cnx.org/contents/GFy_h8cu@10.7:5l844Z38@7/Whole-Genome-
Sequencing.

Pierce, B. A. (2017). Genetics: A Conceptual Approach (6th ed.). Plymouth, MI: Macmillan
Learning Curriculum Solutions.

Sánchez, A. (2011). Next Generation DNA Sequencing. [JPEG]. Retrieved from

https://www.slideshare.net/ueb52/introduction-to-next-generation-sequencing-v2

Post-write
In my definition, I employed the usage of the examples, graphics, and principle of operation
Markel definition strategies. Examples are utilized in the applications argument where
experiments sampled from sources are used to highlight the benefits of Sanger Method. Figures
1-3 represent the graphics portion that supplement the explanations of the complicated process.
The principle of operation strategy is described through the procedure paragraph where the
Sanger Method process is outlined in a systematic manner.