DNA sequencing techniques

1. What is DNA Sequencing?
• “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.

3. importance
• 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.

4. DNA Sequence variation can change the Protein
produced by a particular gene
•Simple point mutations such as this can cause altered protein
shape and function.
•Diseases such as Sickle Cell Anaemia and Cystic Fibrosis
are caused by point mutations
Gene A
from
person 1
Codon results
in particular
Amino Acid (AA)
sequence
GCA AGA GAT AAT TGT
Ala Arg Asp Asn Cys
Gene A
from
person 2
Codon change
makes no
difference in
AA
(redundant
code)
GCG AGA GAT AAT TGT
Ala Arg Asp Asn Cys
Gene A
from
person 3
Codon change
results in
different AA
sequence
GCA AAA GAT AAT TGT
Ala Lys Asp Asn Cys

8. DNA Sequencing—General Principle
DNA sequencing actually involves synthesizing DNA
sub-fragments of all possible lengths and separating
them on a gel.
Therefore, if the template were 200 base pairs in
length, there would be 200 different sub-fragments
ranging from one base pair to 200 base pairs

9. Methods in DNA
Sequencing

10. DNA Sequencing methods
• Basic DNA sequencing
1. Sanger Sequencing method (using dideoxynucleotides, chain termination method)
(99.9% accurate but costly, $ 1000/ 1 million bp)
1. Maxam & Gilbert Sequencing method (using chemical sequencing, chemical
termination method)
• Advanced DNA Sequencing
1. Shot gun sequencing method
• Next Generation DNA sequencing
1. Solid sequencing (99.9% accurate but cost effective, $ 0.50-0.20/1 million bp)
2. Ilumina sequencing (98 % accurate, $ 0.05-0.15/ 1 million bp)
3. Pyro sequencing

12. Frederick Sanger
• Discovered DNA sequencing by chain termination method
• Nobel Prize 1 (1958)
• Complete amino acid
sequence of insulin
• Nobel Prize 2 (1980)
• For DNA sequencing

14. Let’s illustrate this using the eight-base sequence
ACGATTAG as an example

15. In practice, the template, primers, radioactive dNTPs
for all four bases, and DNA polymerase are all mixed
together. This mixture is then distributed among four
tubes, each with a different dideoxynucleotide.
Since the largest DNA sub-fragments generated by
sequencing reactions are usually only 200–300 base
pairs in length, the fragments are too small to be
resolved by the large pores of agarose and must be
separated using a polyacrylamide gel.

17. Dideoxy nucleotides
• Incorporation of a dideoxynucleotide to growing
DNA strand terminates its further extension
• Are added in small proportion
• dATP ddATP
• dGTP ddGTP
• dCTP ddCTP
• dTTP ddTTP

18. 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)

19. • 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.

20. • 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

27. DNA fragments were
originally detected by
radioactive labeling,
although nowadays
fluorescent dyes are
normally used as labels.

29. Sample: Chain Termination Output

30. Dye Sequencing
• Four different labels
• Each of the four nucleotide chains has a different dye
• Individual dyes fluoresce at unique wavelengths
• Vast majority of sequencing projects
• easier
• cheaper

31. Sample: Dye Sequencing Output

33. DNA Sequence Files

34. A 'Scan' of one gel lane:
• Nowadays, we don't even have to 'read' the
sequence from the gel - the computer does
that for us. This is an example of what the
sequencer's computer shows for one sample.
• This is a plot of the colors detected in one
'lane' of a gel (one sample), scanned from
smallest fragments to largest. The computer
even interprets the colors by printing the
nucleotide sequence across the top of the plot.

35. Maxam & Gilbert Chemical Sequencing
• The chemical sequencing is based on the ability of base-specific
chemical reagents.
• Four sets of deoxyoligonucleotides are generated by subjecting a
purified 3′- or 5′-end-labeled deoxyoligonucleotide to a base-specific
chemical reagent
• That randomly cleaves DNA at one or two specific nucleotides.
• Because only end-labeled fragments are observed following
autoradiography of the sequencing gel, four DNA ladders are
observed as shown in Figure below….

36. Chemical sequencing method of Maxam and
Gilbert• Base-specific chemical reagents:
Hydrazine.
Dimethyl sulfate (DMS).
or Formic acid to specifically modify bases
within the DNA molecule.
• Then Piperidine is added to catalyze strand
breakage at these modified nucleotides.
• In the first reaction the specificity resides with
hydrazine, DMS, or formic acid, which reacts
with only few of the bases.
• The second reaction, there must be
quantitative cleavage of piperidine strand.

37. Chemical sequencing

38. The chemical mechanisms of the reactions are as
follows
• The shaded base at the 3′end of the fragments to the right of
the gel indicates bases that have been chemically modified and
• Then fragments of these bases can be displaced by piperidine
after each cleavage.
• For example, after a limited reaction with dimethyl sulfate
(DMS), which is specific for G′s, followed by quantitative release
of the modified G residues by piperidine,
• A set of oligonucleotides are generated that terminate at the
base immediately 5′of each G in the sequence.

39. Conti…..
• Formic acid is specific for purines (G′s and A′s), a fragment that
terminates in G or A will produce a band in the G A lane.
• Hydrazine in the absence of NaCl cleaves T′s and C′s resulting
in a band in the T C lane.
• Hydrazine in the presence of NaCl cleaves only C′s; thus, a
band is observed in the C lane.

41. Assembling Small Genomes by Shotgun
Sequencing
• In shotgun sequencing the genome is broken randomly
into short fragments (1 to 2 kbp long) suitable for
sequencing.
• The fragments are ligated into a suitable vector and
then partially sequenced.
• Around 400–500 bp of sequence can be generated
from each fragment in a single sequencing run.
• Computerized searching for overlaps between
individual sequences then assembles the complete
sequence.
• Overlapping sequences are assembled to generate
contigs.
• The term contig refers to a known DNA sequence that
is contiguous and lacks gaps.

42. • No genetic map or prior information is needed about
the organism whose genome is to be sequenced
• The original limitation to shotgun sequencing was
the massive data handling that is required. The
development of faster computers has overcome this
problem.
• Nowadays, a more important issue is that repetitive
sequences create ambiguities.

44. ShotGun Sequencing

45. Sequencing a Cloned DNA Fragment by
Primer Walking

46. NEXT GENERATION SEQUENCING

47. Ion torrent sequencing machine

51. Barcode: flurecently tagged molecule with radio isotope
Marking the genome fragment