DNA Sequencing

Reasons for DNA Sequencing

(philosophical, pioneering)
Sequencing of DNA „
„
Deciphering “code of life”
Understanding organisms,
physiology, evolution, disease,
cellular behaviour, etc.
Key to Genomics
R. Saicharan
Dept. of Biotechnology,
SNIST, Hyderabad Reasons for DNA Sequencing
(practical day-to-day)
„ checking mutations
„ checking constructs in cloning
„ constructing phylogenies
„ finding genes

Why and What Why and What?

‡ Sequencing” means finding the order of
nucleotides on a piece of DNA .
‡ Nucleotide order determines Amino acid order,
and by extension, protein structure and
function (proteomics)
‡ An alteration in a DNA sequence can lead to
an altered or non functional protein, and hence
to a harmful effect in a plant or animal.
‡ Understanding a particular DNA sequence can
shed light on a genetic condition and offer hope
for the eventual development of treatment
‡ DNA technology is also extended to
environmental, agricultural and forensic
applications.

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 Methods of sequencing Founders of Sequence Tech
‡ Historically there are two main methods of
DNA sequencing:
‡ Maxam & Gilbert, using chemical sequencing
‡ Sanger, using dideoxynucleotides.
‡ Modern sequencing equipment uses the
principles of the Sanger technique
Fred Sanger Wally Gilbert
MRC-Cambridge Harvard

Chemical or Maxam-Gilbert Sequencing

How it Works
Methods
‡ The fundamental idea behind both
methods is the same. ‡ Around 1977, Wally Gilbert (Harvard) and
‡ One needs a known starting point on
his technician Allan Maxam developed
the DNA and then a method to detect chemical sequencing. In the late 70s, it
where each base is positioned on the was the method of choice.
DNA strand. ‡ Uses a chemical reaction specific for each
‡ Both methods terminate a DNA strand
nucleotide.
at a given nucleotide by either
synthesizing to the base or by
chemically breaking the DNA.

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 Maxam and Gilbert Sequencing Reading the Sequence
- One st r a nd o f D N A i s l abe l ed on t he end w i th r ad i oac ti v it y
- Ca r ry ou t 5 s e par a t e ch e m ic a l r eac ti on s wh i ch a tt ack spec ifi c group s on
ba s es
G , G+ A , C+ T , C, and A>C .

32 P

‡ In the 70's the chemical method of sequencing was widely ‡ Also, the price of enzymes dropped and became
used because it was more reproducible and available than more available, making the enzymatic method of
the enzymatic method.
sequencing easier and more reliable.
‡ The enzymatic method required single stranded DNA-
templates, oligonucleotide primers and pure enzymes which ‡ The chemical method is now mostly used to
were not readily available at the time. sequence regions of DNA that are troublesome by
‡ However once M13 and phagemid vectors were developed the other method, or to assay for proteins that
to produce single stranded DNA templates, oligonucleotide bind to specific sites on the DNA.
synthesis procedures were worked out to make inexpensive
DNA primers.

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The Sanger Technique Because they lack the -OH (which allows nucleotides
to join a growing DNA strand), replication stops.

‡ Uses dideoxynucleotides (dideoxyadenine, dideoxyguanine,

etc)
Normally, this would
‡ These are molecules that resemble normal nucleotides but be where another
lack the normal -OH group. phosphate
Is attached, but with no -
OH
group, a bond can not form
and replication stops

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The Sanger method requires
‡ Multiple copies of single stranded template DNA
‡ A suitable primer (a small piece of DNA that can pair
with the template DNA to act as a starting point for
replication)
‡ DNA polymerase (an enzyme that copies DNA,
adding new nucleotides to the 3’ end of the template
‡ A ‘pool’ of normal nucleotides
‡ A small proportion of dideoxynucleotides labeled in
some way ( radioactively or with fluorescent dyes)
‡ The template DNA pieces are replicated,
incorporating normal nucleotides, but occasionally
and at random dideoxy (DD) nucleotides are taken
up.
‡ This stops replication on that piece of DNA
‡ The result is a mix of DNA lengths, each ending with
a particular labeled DDnucleotide.
‡ Because the different lengths ‘travel’ at different rates
during electrophoresis, their order can be
determined.
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 Sequencing Hardware
‡ Originally four separate sets of DNA, primer and
a single different DD nucleotide were produced
and run on a gel.
‡ Modern technology allows all the DNA, primers,
etc to be mixed and the fluorescent labeled
DDnucleotide ‘ends’ of different lengths can be
‘read’ by a laser.
‡ Additionally, the gel slab has been replaced by
polymer filled capillary tubes in modern
equipment.

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Step 1- Before submission for sequencing DNA A Nanodrop readout of known concentration
purity & concentration is checked with the to be run as a control
‘Nanodrop’

Cost?
‡ Cost is dependant on a number of factors but
typically in 2003:
Step 2 -Samples are
‡ Each tube of sample DNA costs $27 to run.
received and stored
‡ An entire set of 96 tubes from one source (the
in the refrigerator
capacity of the present equipment) costs
and a request filed
$960.
‡ The methods used will readily analyze DNA
fragments of 500-1000 bases in length,
depending on the quality of DNA used
‡ Note – the dye alone to run 5000 reactions
costs $61,000

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Samples arrive in Eppendorf tubes

Step 3 - paperwork. Each request is assigned a

‘well’ in the sample tray and volumes of
primers, water, dye, etc are calculated. A typical
‘run’ has samples from a number of researchers

Step 5 - Reagents, etc

‡ Each reaction requires several reagents:
‡ Specific primers for the DNA in question
‡ Fluorescent Dye attached to DD nucleotides
(Big Dye)
‡ Deionised water
‡ DNA polymerase
‡ Additionally, a ‘control’ sample of a known DNA
Step 4- Samples are agitated then centrifuged in is prepared so it can run at the same time as the
an Ultracentrifuge to be sure they are in the experimental DNA.
bottom of their Eppendorf tubes.

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 Step 6 - Preparing the wells
Micropipettors come ‡ The Sample wells are loaded with DNA
in a range of sizes. to be sequenced.
They have
‡ Great care needs to be taken to ensure
disposable tips that
that each sample goes into its assigned
hold tiny amounts of
required reagents.
well.
‡ Reagents are added (water, dye,
primers) in required amounts
‡ The sample wells are ‘spun’ to ensure
that the DNA and reagents are mixed
and at the bottom of the sample wells.

Step 7 - The
samples are run
through a cycle
sequencing
process to get the
fluorescent dyes
incorporated by the
DNA.
The DNA and
reagents are
alternately heated
and cooled over a
Sample tray and micropipettor. Each tray holds
96 samples 2 1/2 hour period.

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Step 8 - Sample purification to get rid of
extra dye and salts
‡ Unincorporated dye and salts can interfere with DNA
analysis and need to be removed
‡ Samples are centrifuged, precipitated with 95% ethanol,
centrifuged again, and drained
‡ The process is repeated with 70% ethanol
‡ Dry samples are either analyzed immediately or stored in
the dark (light degrades the fluorescent dyes used)
‡ Just before sequencing formamide is added to ensure
that the DNA remains linear

Entering data from the record sheet into the

Sequencer software programme

Capillary tubes

Reagents

Sample tray
goes here

Step 10- The sequencer is warmed up,

reagents are refreshed and the sample tray is
inserted Inside the sequencer

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The Sequencer Apparatus

‡ Each sample tray has 96 wells (1 per sample), and

the analyzer (3100 model) has the capacity to
analyze 16 wells at a time
‡ Robotic apparatus moves the sample tray so each of
the 16 wells is in contact with a separate capillary
tube filled with a polymer - this replaces a lane on an
electrophoresis gel
‡ Labeled DNA from that well moves up the capillary
tube, with smaller labeled fragments moving more
quickly than longer ones

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