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DNA Sequencing - Sangers Method

The document summarizes DNA sequencing methods, including Sanger sequencing and next-generation sequencing. It describes how Sanger sequencing works by making copies of DNA fragments of varying lengths terminated by fluorescent chain terminators. Next-generation sequencing involves massively parallel, smaller scale reactions that have made sequencing faster and cheaper.

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kavya nainita
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213 views10 pages

DNA Sequencing - Sangers Method

The document summarizes DNA sequencing methods, including Sanger sequencing and next-generation sequencing. It describes how Sanger sequencing works by making copies of DNA fragments of varying lengths terminated by fluorescent chain terminators. Next-generation sequencing involves massively parallel, smaller scale reactions that have made sequencing faster and cheaper.

Uploaded by

kavya nainita
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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DNA sequencing

How the sequence of nucleo de bases (As, Ts, Cs, and Gs)
in a piece of DNA is determined.

Key points:
DNA sequencing is the process of
determining the sequence of nucleo des
(As, Ts, Cs, and Gs) in a piece of DNA.

In Sanger sequencing, the target DNA is


copied many mes, making fragments of
different lengths. Fluorescent “chain
terminator” nucleo des mark the ends of the
fragments and allow the sequence to be
determined.

Next-genera on sequencing techniques are


new, large-scale approaches that increase
the speed and reduce the cost of DNA
sequencing.

What is sequencing?
You may have heard of genomes being
sequenced. For instance, the human genome
was completed in 2003, a er a many-year,
interna onal effort. But what does it mean to
sequence a genome, or even a small fragment of
DNA?

DNA sequencing is the process of determining


the sequence of nucleo de bases (As, Ts, Cs, and
Gs) in a piece of DNA. Today, with the right
equipment and materials, sequencing a short
piece of DNA is rela vely straigh orward.

Sequencing an en re genome (all of an


organism’s DNA) remains a complex task. It
requires breaking the DNA of the genome into
many smaller pieces, sequencing the pieces, and
assembling the sequences into a single long
"consensus." However, thanks to new methods
that have been developed over the past two
decades, genome sequencing is now much faster
and less expensive than it was during the Human
Genome Project1 .

In this ar cle, we’ll take a look at methods used


for DNA sequencing. We'll focus on one well-
established method, Sanger sequencing, but we'll
also discuss new ("next-genera on") methods
that have reduced the cost and accelerated the
speed of large-scale sequencing.

Sanger sequencing: The chain


termina on method
Regions of DNA up to about 900 base pairs in
length are rou nely sequenced using a method
called Sanger sequencing or the chain
termina on method. Sanger sequencing was
developed by the Bri sh biochemist Fred Sanger
and his colleagues in 1977.

In the Human Genome Project, Sanger


sequencing was used to determine the
sequences of many rela vely small fragments of
human DNA. (These fragments weren't
necessarily 900 bp or less, but researchers were
able to "walk" along each fragment using
mul ple rounds of Sanger sequencing.) The
fragments were aligned based on overlapping
por ons to assemble the sequences of larger
regions of DNA and, eventually, en re
chromosomes.

Although genomes are now typically sequenced


using other methods that are faster and less
expensive, Sanger sequencing is s ll in wide use
for the sequencing of individual pieces of DNA,
such as fragments used in DNA cloning or
generated through polymerase chain reac on
(PCR).

Ingredients for Sanger


sequencing
Sanger sequencing involves making many copies
of a target DNA region. Its ingredients are similar
to those needed for DNA replica on in an
organism, or for polymerase chain reac on
(PCR), which copies DNA in vitro. They include:

A DNA polymerase enzyme

A primer, which is a short piece of single-


stranded DNA that binds to the template
DNA and acts as a "starter" for the
polymerase

The four DNA nucleo des (dATP, dTTP,


dCTP, dGTP)

The template DNA to be sequenced


However, a Sanger sequencing reac on also
contains a unique ingredient:

Dideoxy, or chain-termina ng, versions of all


four nucleo des (ddATP, ddTTP, ddCTP,
ddGTP), each labeled with a different color
of dye

_Image credit: "Whole-genome sequencing: Figure 1," by OpenStax


College, Biology (CC BY 4.0)._

Dideoxy nucleo des are similar to regular, or


deoxy, nucleo des, but with one key difference:
they lack a hydroxyl group on the 3’ carbon of
the sugar ring. In a regular nucleo de, the 3’
hydroxyl group acts as a “hook," allowing a new
nucleo de to be added to an exis ng chain.

Once a dideoxy nucleo de has been added to


the chain, there is no hydroxyl available and no
further nucleo des can be added. The chain
ends with the dideoxy nucleo de, which is
marked with a par cular color of dye depending
on the base (A, T, C or G) that it carries.

[Where is the dye a ached?]

Method of Sanger sequencing


The DNA sample to be sequenced is combined
in a tube with primer, DNA polymerase, and
DNA nucleo des (dATP, dTTP, dGTP, and dCTP).
The four dye-labeled, chain-termina ng dideoxy
nucleo des are added as well, but in much
smaller amounts than the ordinary nucleo des.

The mixture is first heated to denature the


template DNA (separate the strands), then
cooled so that the primer can bind to the single-
stranded template. Once the primer has bound,
the temperature is raised again, allowing DNA
polymerase to synthesize new DNA star ng
from the primer. DNA polymerase will con nue
adding nucleo des to the chain un l it happens
to add a dideoxy nucleo de instead of a normal
one. At that point, no further nucleo des can be
added, so the strand will end with the dideoxy
nucleo de.

This process is repeated in a number of cycles.


By the me the cycling is complete, it’s virtually
guaranteed that a dideoxy nucleo de will have
been incorporated at every single posi on of the
target DNA in at least one reac on. That is, the
tube will contain fragments of different lengths,
ending at each of the nucleo de posi ons in the
original DNA (see figure below). The ends of the
fragments will be labeled with dyes that indicate
their final nucleo de.

[Will all the fragments be labeled?]

Image modified from "Sanger sequencing," by Estevezj (CC BY-SA 3.0). The
modified image is licensed under a (CC BY-SA 3.0) license.

A er the reac on is done, the fragments are run


through a long, thin tube containing a gel matrix
in a process called capillary gel electrophoresis.
Short fragments move quickly through the pores
of the gel, while long fragments move more
slowly. As each fragment crosses the “finish line”
at the end of the tube, it’s illuminated by a laser,
allowing the a ached dye to be detected.
The smallest fragment (ending just one
nucleo de a er the primer) crosses the finish
line first, followed by the next-smallest fragment
(ending two nucleo des a er the primer), and so
forth. Thus, from the colors of dyes registered
one a er another on the detector, the sequence
of the original piece of DNA can be built up one
nucleo de at a me. The data recorded by the
detector consist of a series of peaks in
fluorescence intensity, as shown in the
chromatogram above. The DNA sequence is
read from the peaks in the chromatogram.

Uses and limita ons


Sanger sequencing gives high-quality sequence
for rela vely long stretches of DNA (up to about
900 base pairs). It's typically used to sequence
individual pieces of DNA, such as bacterial
plasmids or DNA copied in PCR.

However, Sanger sequencing is expensive and


inefficient for larger-scale projects, such as the
sequencing of an en re genome or metagenome
(the “collec ve genome” of a microbial
community). For tasks such as these, new, large-
scale sequencing techniques are faster and less
expensive.

Next-genera on sequencing
The name may sound like Star Trek, but that’s
really what it’s called! The most recent set of
DNA sequencing technologies are collec vely
referred to as next-genera on sequencing.

There are a variety of next-genera on


sequencing techniques that use different
technologies. However, most share a common
set of features that dis nguish them from Sanger
sequencing:

Highly parallel: many sequencing reac ons


take place at the same me

Micro scale: reac ons are ny and many can


be done at once on a chip

Fast: because reac ons are done in parallel,


results are ready much faster

Low-cost: sequencing a genome is cheaper


than with Sanger sequencing

Shorter length: reads typically range from 50


-700 nucleo des in length

Conceptually, next-genera on sequencing is


kind of like running a very large number of ny
Sanger sequencing reac ons in parallel. Thanks
to this paralleliza on and small scale, large
quan es of DNA can be sequenced much more
quickly and cheaply with next-genera on
methods than with Sanger sequencing. For
example, in 2001, the cost of sequencing a
human genome was almost $100 million. In
2015, it was just $12452 !

Why does fast and inexpensive sequencing


ma er? The ability to rou nely sequence
genomes opens new possibili es for biology
research and biomedical applica ons. For
example, low-cost sequencing is a step towards
personalized medicine – that is, medical
treatment tailored to an individual's needs, based
on the gene variants in his or her genome.

[A ribu on and references]

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Benni S 2 years ago

How were the first primers chosen if the


sequences were unknown?
3 (7
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2
years
Amma's Daughter Anupama ago

The MIT synthe c chemist


Gobind Khorana won the 1968

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