DNA Sequencing

1
DNA Sequencing
From Extraction to Information
2
The Process

• Step 1 DNA Extraction
• Genomic DNA extraction from the organism
  (bacteria)

• Step 2 PCR Amplification
• Amplification of the DNA segment of interest (16S
  gene)

• Step 3 DNA Sequencing
• Sequencing of the PCR product (amplified 16S gene)

3
Step 1 DNA Extraction
DNA Extraction
DNA
Cells with DNA
4
Step 2 PCR Amplification
PCR Product (Amplified Target Gene)
Target Gene
Target Gene
PCR Amplification
DNA
5
Step 3 DNA Sequencing
PCR product (Amplified Target Gene)
Sequence of Target Gene
DNA Sequencing
AGCTGCTAAGCTTG AGCTTGCACAAGCT TAGCTTGCAAGCTT AGCTT
GCAAGCTTG CAAGCTTGCAAGCT TGCAAGCTTGCAAG CTTGCAACGT
TGCA AGCTTGCAAGCTTG AAGCTTGCAAGCTA
6
Chapter 1 DNA Extraction
7
The Cell and its Components
DNA (1)
phospholipids (2)
30 chemicals
polysaccharides (2)
Ions, small molecules (4)
RNA (6)
70 H2O
proteins (15)
8
Three basic steps of DNA extraction

• Disruption of cell and lysis
• Removal of proteins and other biochemicals
• Recovery of DNA

9
Disruption of cell and lysis

• Cells are broken down into components using a
  Lysis Buffer containing
• EDTA disrupts cell membrane and inhibits DNases
• SDS denatures proteins and solubilizes cell
  membranes
• Proteinase K breaks down proteins
• RNase A breaks down RNA
• Solution is incubated at 55ºC 1-3 hours (or
  overnight)

10
Disruption of cell and lysis
DNA
RNA
ions
lipids
proteins




EDTA
RNase A
SDS
Proteinase K
EDTA
11
Disruption of cell and lysis

• After lysis, cell extract contains DNA, proteins,
  and other chemicals/biochemicals

cell extract
12
Removal of proteins and biochemicals

• Solid phase binding (silica membrane)
• Cell extract is applied to a silica membrane
  column
• DNA binds to membrane
• all other molecules flow through and are removed

DNA bound to membrane
Silica membrane
centrifugation
cell extract
flow-thru
13
Recovery of DNA

• Elution of silica membrane
• a low-salt buffer or water is added to the
  membrane
• bound DNA falls off of the membrane

elution
add water (or buffer)
DNA bound to membrane
centrifugation
DNA in solution
14
Review Step 1 DNA Extraction
DNA Extraction
DNA
Cells with DNA
15
Chapter 2 PCR Amplification
16
Step 2 PCR Amplification
PCR Product (Amplified Target Gene)
Target Gene
Target Gene
PCR Amplification
DNA
17
Part I DNA Polymerization
18
DNA Building Block
base
5
P
P
P
OCH2
O
sugar
4
1
H
H
phoshpate groups
3
2
OH
H
deoxyribose nucleotide triphosphate (dNTP)
19
DNA Polymerization Basics
A
Existing DNA Strand
O
G
O
P
O
C
O
P
O
T
O
P
Phosphodiester bond
O
H
dNTP
O
T
P
P
P
O
H
A
O
P
P
P
O
H
O
C
P
P
P
O
H
20
DNA Polymerization

• The synthesis of DNA requires
• DNA template
• Primer short oligonucleotide necessary for DNA
  polymerase to start
• DNA polymerase enzyme that constructs the DNA
  chain
• deoxyribonucleotide triphosphates (dNTPs)
  building blocks of DNA

A
C
C
G
G
G
A
A
G
C
C
C
C
G
G
A
T
G
A
DNA polymerase
C
A
T
G
DNA polymerase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
21
DNA Replication Review

• Step 1 Denaturation separation of the two
  strands of the DNA duplex
• Gyrase pulls apart the strands creating a
  replication bubble
• Helicase travels down DNA molecule, breaking the
  hydrogen bonds that hold the two strands together

gyrase
helicase
helicase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
gyrase
22
DNA Replication Review

• Step 2 Annealing of primers to the DNA template
  strand
• Primase synthesizes small complementary strands
  of RNA (primers) to the single strands of the
  DNA template

primase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
G
C
C
C
C
G
G
A
T
G
A
G
primase
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
23
DNA Replication Review

• Step 3 Extension of newly constructed
  complementary DNA molecules
• DNA polymerase adds bases to the ends of the
  primers, constructing an exact copy of the
  template

DNA polymerase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
G
C
C
C
C
G
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
DNA polymerase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
24
DNA Replication Review

• Another DNA Polymerase replaces the RNA primer
  with dNTPs
• The final result two copies of replicated DNA

DNA polymerase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
G
C
C
C
C
G
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
DNA polymerase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
G
A
T
G
A
G
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
25
Polymerization
1) DNA Template
Mg2
Mg2
Mg2
Mg2
Mg2
Mg2
Mg2
2) Primer
3) DNA Polymerase
Mg2 ions
4) dNTPs
dNTPs
5) Mg2 ions
DNA Polymerase
DNA Polymerase
Primer
Phosphodiester bond
DNA Template
26
Part II PCR
27
The Polymerase Chain Reaction

• Polymerase Chain Reaction cycling process
  consisting of the same 3 steps of DNA
  replication, with some differences
• temperature cycling removes the need for other
  enzymes (gyrase/helicase, or primase)
• PCR uses pre-made oligonucleotide DNA primers

gyrase
DNA polymerase
primase
helicase
28
The Polymerase Chain Reaction

• During PCR, a thermocycler brings the reaction
  mix to 3 different temperatures analagous to the
  3 steps of DNA replication
• Denaturation (94C) of the DNA template by heat
• Annealing (37-70C) of the primers to the
  template
• Extension (72C) of the DNA strand by DNA
  polymerase
• These steps are repeated for 25 to 30 cycles

94C
65C
72C
denaturation
annealing
extension
29
Thermocycler Program

• Initial Denaturation 94C 2 min
• Start Cycle
• Denaturation 94C 30 sec
• Annealing 65C 30 sec
• Extension 72C 30 sec
• Repeat Cycle 29 times (total 30 cycles)
• Final Extension 72C 7 min
• Hold 4C 8

30
Denaturation

• Denaturation occurs at 94C
• The high temperature is used to break down the
  hydrogen bonds that hold the two strands together

94C
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
31
Annealing

• Annealing occurs at 37-70C
• Oligonuclotide DNA primers anneal to their
  complementary sequences on the template strands
• Annealing temperature depends on the melting
  temperature (Tm) of the primer (dependent on base
  composition)

65C
94C
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
32
Extension

• Extension occurs at 72C
• DNA polymerase attaches to the primers and
  extends the new DNA strand
• The 3 steps (denaturation, annealing, and
  extension) are repeated for another 24 to 29
  cycles

72C
65C
DNA polymerase
G
A
T
G
A
G
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
T
A
G
T
A
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
DNA polymerase
T
C
A
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
G
G
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
33
Target Sequence

• A desired target sequence is identified
• To isolate the target sequence, primers that
  flank the region must be constructed
• The DNA segment that is then amplified contains
  the region of interest

Template DNA
Forward Primer
Reverse Primer
Target Sequence of interest
PCR Product
34
PCR Cycle 1
Denaturation
Extension
Annealing
DNA Copies
4
Target Copies
0
Target Sequence of interest
35
PCR Cycle 2
Denaturation
Extension
Annealing
DNA Copies
8
Target Copies
2
36
PCR Cycle 3
Denaturation
Extension
Annealing
DNA Copies
16
Target Copies
8
37
PCR Cycle 4
Denaturation
Extension
Annealing
DNA Copies
32
Target Copies
22
38
PCR Cycle 5
Denaturation
Extension
Annealing
DNA Copies
64
Target Copies
52
39
PCR Amplification First 10 cycles
40
PCR Amplification First 15 cycles
41
PCR Amplification After 30 cycles
42
PCR Amplification After 30 cycles
Cycle Target Copies Cycle Target Copies
1 0   16 65,504
2 0   17 131,038
3 2   18 262,108
4 8   19 524,250
5 22   20 1,048,536
6 52   21 2,097,110
7 114   22 4,194,260
8 240   23 8,388,562
9 494   24 16,777,168
10 1,004   25 33,554,382
11 2,026   26 67,108,812
12 4,072   27 134,217,674
13 8,166   28 268,435,400
14 16,356   29 536,870,854
15 32,738   30 1,073,741,764
43
Review Step 1 DNA Extraction
DNA Extraction
DNA
Cells with DNA
44
Review Step 2 PCR Amplification
PCR Product (Amplified Target Gene)
Target Gene
Target Gene
PCR Amplification
DNA
45
Chapter 3DNA Sequencing
46
Nucleotides
BASE
BASE
OCH2
P
P
P
OCH2
P
P
P
O
O
H
H
H
H
OH
H
OH
OH
deoxyribose NTP (dNTP) (Makes up DNA)
ribose NTP (NTP) (Makes up RNA)
BASE
OCH2
P
P
P
O
H
H
H
H
dideoxyribose NTP (ddNTP)
47
DNA Sequencing

• Dideoxy method of DNA sequencing (Sanger Method)
• Single-stranded DNA to be sequenced serves as a
  template strand for DNA synthesis
• single primer is used for DNA synthesis
  initiation
• use of dNTPs along with labeled ddNTPs

BASE
BASE
OCH2
OCH2
P
P
P
P
P
P
O
O
H
H
H
H
OH
H
H
H
dNTP
ddNTP
48
DNA Polymerization using ddNTPs
A
A
O
O
G
O
P
G
O
P
O
O
C
O
P
C
O
P
O
O
T
O
P
C
O
P
O
H
O
H
T
O
P
P
P
T
O
P
P
P
O
H
H
A
O
P
P
P
A
O
P
P
P
O
H
O
H
C
O
P
P
P
Chain Termination
O
H
49
Sequence Reaction

• BigDye Terminator v3.1 Sequencing
• a Dye Terminator Cycle Sequencing Master Mix is
  used for sequencing reaction.
• Components include
• DNA polymerase I, Mg2, buffer
• dNTPs in ample quantities
• (dATP, dTTP, dCTP, dGTP)
• ddNTPs in limited quantities, each labeled with a
  tag that fluoresces a different color
• (ddATP, ddTTP, ddCTP, ddGTP)

50
The Polymerase Chain Reaction

• PCR makes use of a thermocycler to bring the
  reaction mix to three different temperatures
• Denaturation (94C) of the DNA template by heat
• Annealing (37-70C) of the primers to the
  template
• Extension (72C) of the DNA strand by DNA
  polymerase
• These steps are repeated for 25 to 30 cycles

94C
65C
72C
denaturation
annealing
extension
51
Sequencing Reaction

• Sequencing reaction is a cycled reaction using a
  thermocycler (as in the Polymerase Chain
  Reaction)
• Like PCR, it consists of 3 steps
• Denaturation, Annealing, Extension
• these 3 steps are repeated for 30 cycles
• Unlike PCR, it involves a single primer and
  labeled ddNTPs
• extension proceeds normally until, by chance, DNA
  polymerase inserts a ddNTP, terminating the chain

52
Sequencing Reaction
DNA polymerase
G
A
A
C
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
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T
A
C
T
C
A
A
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C
A
C
A
G
G
C
A
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G
T
T
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A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
53
Sequencing Reaction
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
DNA polymerase
G
A
A
C
T
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
54
Sequencing Reaction
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
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G
G
C
C
C
T
T
C
G
T
G
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C
C
G
T
A
C
A
A
C
T
G
G
C
G
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A
A
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A
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G
C
C
C
T
T
G
T
G
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C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
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C
A
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G
G
C
C
C
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G
T
G
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C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
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C
A
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G
G
C
C
C
G
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G
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C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
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C
A
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G
G
C
C
G
T
G
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C
C
G
T
A
C
A
A
C
T
G
G
C
G
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A
A
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C
A
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G
C
G
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G
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C
C
G
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A
C
A
A
C
T
G
G
C
G
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A
A
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C
A
T
G
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
DNA polymerase
G
A
A
C
T
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
55
Applied Biosystems 3130xl Genetic Analyzer

• 16-channel capillary electrophoresis capable of
  various genomic analysis functions

56
Capillary Electrophoresis

• the sequencing reactions are loaded into the
  ABI3130xl
• Samples are taken up by capillaries containing
  polyacrylamide gel (Performance Optimized Polymer
  (POP-7)
• Fragments are separated by length from shortest
  to longest by electrophoresis

Detector
-

Capillary array
Laser
Samples
57
Electrophoresis

• Electrophoresis is a technique used to separate
  DNA or protein molecules on the basis of size and
  charge
• Typical method used for analyzing, identifying
  and purifying DNA fragments

58
Movement in an Electric Field

• The mobility of molecules in the electrical field
  is also affected by their overall size or
  molecular weight

Lower Molecular weight
Higher Molecular weight

Agarose gel

59
DNA Gel Electrophoresis
These markers are run alongside samples, Helps
determine the length of the PCR sample DNA
fragments of the same length will migrate through
the gel at the same rate
1500bp
1200bp
1100bp
1000bp
1050bp
900bp
800bp
820bp
700bp
650bp
600bp
500bp
400bp
400bp
300bp
280bp
200bp
100bp
60
Electrophoresis

• As the sample travels through the capillaries
• Shorter fragments have less resistance and
  migrate faster
• Longer fragments have more resistance and move
  slower

-

61
Fluorescense detection

• As fragments pass through detector window, the
  fluorescent tag of the ddNTP is excited by a
  laser
• The emission of the tag is picked up by a
  detector and is translated to a colored peak
  unique to the nucleotide

C
T
G
A
62
Sequencing Reaction
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
G
C
C
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
T
G
T
C
C
G
T
A
C
A
A
C
T
G
G
C
G
T
A
A
T
C
A
T
G
DNA polymerase
G
A
A
C
T
C
C
T
A
C
T
C
A
A
G
C
A
C
A
G
G
C
A
T
G
T
T
G
A
C
C
G
C
A
T
T
A
G
T
A
C
C
G
G
G
A
A
G
C
C
C
C
G
63
Capillary Electrophoresis

• As the sample travels through the capillaries
• Shorter fragments have less resistance and
  migrate faster
• Longer fragments have more resistance and move
  slower

-

64
Detection of fluorescent tags
C
G
T
A
A
A
C
A
C
G
G
C
C
C
T
T
C
G
G
G
G
C
65
Final Data

• The final data generated is the complete
  chromatogram and the text version of the DNA
  sequence

ACAACTGGCGTGAATCATGGCCCTTCGGGGCCATTGTTTCTCTGTGGAGG
AGTGCCATGACGAAAGATGAACTGATTGCCCGTCTCCGCTCGCTGGGTGA
ACAACTGAACCGTGATGTCAGCCTGACGGGGACGAAAGAAGAACTGGCGC
TCCGTGTGGCAGAGCTGAAAGAGGAGCTTGATGACACGGATGAAACTGCC
GGTCAGGACACCCCTCTCAGCCGGGAAAATGT
66
Review Step 1 DNA Extraction
DNA Extraction
DNA
Cells with DNA
67
Review Step 2 PCR Amplification
PCR Product (Amplified Target Gene)
Target Gene
Target Gene
PCR Amplification
DNA
68
Review Step 3 DNA Sequencing
PCR product (Amplified Target Gene)
Sequence of Target Gene
DNA Sequencing
AGCTGCTAAGCTTG AGCTTGCACAAGCT TAGCTTGCAAGCTT AGCTT
GCAAGCTTG CAAGCTTGCAAGCT TGCAAGCTTGCAAG CTTGCAACGT
TGCA AGCTTGCAAGCTTG AAGCTTGCAAGCTA
69
BWET.PL4.8F Sequence
70
BWET.PL4.8F Sequence

• GCAGTCGAGCGGAACGAGTTATCTGAACCTTCGGGGAACGATAACGGCGT
  CGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATTGCCCTGATGTGGGGG
  ATAACCATTGGAAACGATGGCTAATACCGCATAATAGCTTCGGCTCAAAG
  AGGGGGACCTTCGGGCCTCTCGCGTCAGGATATGCCCAGGTGGGATTAGC
  TAGTTGGTGAGGTAAGGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGA
  GAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGG
  AGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCAT
  GCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCAGTGA
  GGAAGGTGGTGATGTTAATAGCATCATCATTTGACGTTAGCTGCAGAAGA
  AGCACCGGCTAACTCCGTGCCAGCCG

71
BLAST

• BLAST Basic Local Alignment Search Tool
• www.ncbi.nlm.nih.gov/blast
• an unknown sequence can be matched up to known
  sequences published in GenBank
• Lists all sequences producing significant
  alignments
• Gene identification
• Organism genus/species
• identity alignment/match

72
BLAST Results of BWET.PL4.8F
73
BLAST Results of BWET.PL4.8F

• Summary of best match
• Vibrio sp. BISLTS1 16S ribosomal RNA gene
• Identities 435/436 (99)

74
The End