DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain react

1
DNA sequencing with Thermus aquaticus DNA
polymeraseand direct sequencing of polymerase
chain reaction-amplified DNAMICHAEL A.
INNIS, KENNETH B. MYAMBO, DAVID H. GELFAND, AND
MARY ANN D. BROW, Cetus Corporation,
Emeryville, CA PNAS, 1988, 85 9436-9440,

• Presented by
• Charlotte Girondel Bunnee Goldberg

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Thermus aquaticus
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Thermus aquaticus
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Thermus aquaticus
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Historical Perspective

• Mid 1960s Microbiologist, Professor Thomas
  Brock, discovers Thermus aquaticus.
• 1971 - Nobel Prize laureate H. Gobind Khorana and
  Kjell Kleppe, describes PCR process using DNA
  polymerase from E. coli.
• 1976 Alice Chien colleagues isolates DNA
  polymerase from T. aquaticus.
• 1983 Kary Mullis, Cetus Corp, optimizes PCR
  methods using TAQ polymerase
• 1993 - Shares Nobel prize in Chemistry with
  Michael Smith for refining PCR methods.

6
Historical Perspective

• Mid 1960s Microbiologist, Professor Thomas
  Brock, discovers Thermus aquaticus.
• 1971 - Nobel Prize laureate H. Gobind Khorana and
  Kjell Kleppe, describes PCR process using DNA
  polymerase from E. coli.
• 1976 Alice Chien colleagues isolates DNA
  polymerase from T. aquaticus.
• 1983 Kary Mullis, Cetus Corp, optimizes PCR
  methods using TAQ polymerase
• 1993 - Shares Nobel prize in Chemistry with
  Michael Smith for refining PCR methods.

7
Historical Perspective

• Mid 1960s Microbiologist, Professor Thomas
  Brock, discovers Thermus aquaticus.
• 1971 - Nobel Prize laureate H. Gobind Khorana and
  Kjell Kleppe, describes PCR process using DNA
  polymerase from E. coli.
• 1976 Alice Chien colleagues isolates DNA
  polymerase from T. aquaticus.
• 1983 Kary Mullis, Cetus Corp, optimizes PCR
  methods using TAQ polymerase
• 1993 - Shares Nobel prize in Chemistry with
  Michael Smith for refining PCR methods.

8
Historical Perspective

• Mid 1960s Microbiologist, Professor Thomas
  Brock, discovers Thermus aquaticus.
• 1971 - Nobel Prize laureate H. Gobind Khorana and
  Kjell Kleppe, describes PCR process using DNA
  polymerase from E. coli.
• 1976 Alice Chien colleagues isolates DNA
  polymerase from T. aquaticus.
• 1983 Kary Mullis, Cetus Corp, optimizes PCR
  methods using TAQ polymerase
• 1993 - Shares Nobel prize in Chemistry with
  Michael Smith for refining PCR methods.

9
Purpose

• To optimize PCR methods using an M13 DNA strand
  along with Taq DNA polymerase and direct
  sequencing using the Sanger sequencing method.
• Automating the preparation of DNA templates and
  performing the sequencing reactions.

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What is PCR?

• PCR In vitro technique using DNA polymerase to
  exponentially amplify dsDNA fragment.
• Asymmetric PCR Used to amplify one strand more
  than the other.

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Sequencing Methods
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.

• 1. Template Preparation
• 2. Sequencing Rxns
• a. Annealing
• b. Labeling
• c. Extension/
• Termination
• Sequencing PCR Products

• Single-stranded M13mp10 DNA prepared.

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Sequencing Methods
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.

• 1. Template Preparation
• 2. Sequencing Rxns
• a. Annealing
• b. Labeling
• c. Extension/
• Termination
• Sequencing PCR Products

• Consist in hybridizing an oligonucleotide primer
  to the prepared template.
• Material Primer, template, Taq sequencing
  buffer.
• Performed for each set of four sequencing
  reactions.

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Sequencing Methods
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.

• 1. Template Preparation
• 2. Sequencing Rxns
• a. Annealing
• b. Labeling
• c. Extension/
• Termination
• Sequencing PCR Products

• Extending primer with DNA polymerase in 4
  separate reaction mixtures
• Material a labeled dNTP (dATPa-35S) in
  limiting concentrations, mixture of unlabeled
  dNTPs (C-G-T)

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Sequencing Methods
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.

• 1. Template Preparation
• 2. Sequencing Rxns
• a. Annealing
• b. Labeling
• c. Extension/
• Termination
• Sequencing PCR Products

• Performed 4 separate extension/termination by
  adding aliquots of the labeling mix to each below
  
• "G-mix" (dNTP, ddGTP, MgCI2)
• "A-mix (dNTP, ddATP, MgCl2)
• "T-mix" (dNTP, ddTTP, MgCl2)
• "C-mix" (dNTP, ddCTP, MgCl2).
• loaded onto buffer gradient sequencing gel

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Sequencing Methods
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.
ss M13mp10 DNA with a 400-base insert in the
EcoRI site of the polylinker. 20-mer primers
from the 5 end RG05 (5'-AGGGTTTTCCCAGTCACGAC-3')
Reverse primer RG02(5'-GTGTGGAATTGTGAGCGGAT3'),
respectively. PCR Cycle 35 cycles of
denaturation Each cycle at 93C for 30 sec,
primer annealing at 50C for 1 min, and
extension at 72C for 1 min.

• 1. Template Preparation
• 2. Sequencing Rxns
• a. Annealing
• b. Labeling
• c. Extension/
• Termination
• Sequencing PCR Products

• Asymmetric PCR to prepare the templates
• Sequencing The primer is labeled, labeling step
  omitted, directly chain-termination reaction.
• gel electrophoresis

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Steps in PCR
Supply
1. DNA template
2. Primers
3. dNTP
4. TAQ Pol
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Sanger Sequencing Method
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Results . Figure 1A
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Results . Figure 1A cont.
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Results . 1B
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Problems to resolve

• Deal with misincorporation of dNTPs and ddNTPs
• Sequence through GC-rich DNA
• Resolve gel compression
• Facilitate automation for large-scale sequencing
  projects

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Results . Figure 2
c. Chase with concentrated dNTP
A. Standard ddNTP termination lanes
B. Lanes limited by 1 of the dNTPs
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Results . Figure 3
The effects of temperature on the labeling
reaction
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Results . Figure 3
The effect of incubation time on the extension
reaction

• High dNTP concentrations to ensure maximum
  processivity and fidelity

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Results . Figure 4

• Sequencing through GC-Rich DNA and eliminating
  band compression
• Base analogs
• c7GTP
• dITP

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Results . Figure 5
Coupling DNA Sequencing to the PCR
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Discussion

• Taq DNA polymerase has high processivity
• Add NT at a rate of 60 nt/ sec
• No 3 exonuclease activity
• Band compressions resolved using
• Coupling asymmetric PCR with direct sequencing
• GTP base analog, C7 GTP
• higher percentage gel (7 acrylamide at 21 hrs)

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PCR Today

• All sequencing is done with dideoxy Sanger method
  now.
• Automated DNA sequence analysis (fluorescent
  ddNTPs).
• Capillaries sequencers much faster.
• PCR with taq polymerase
• New ways to prepare templates

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Further Reading

• Brock, Thomas D. (Oct 11, 1985). "Life at high
  temperatures." Science. 230132.
• Chien A, Edgar DB, Trela JM. Deoxyribonucleic
  Acid Polymerase from the Extreme Thermophile
  Thermus aquaticus. J of Bacteriology. 1976, p.
  1550-1557
• Kary Mullis, Dancing Naked in the Mind Field
  (Pantheon Books, 1998).
• Kleppe K, Ohtsuka E, Kleppe R, Molineux I,
  Khorana HG.J Mol Biol. 1971 Mar
  1456(2)341-61.Studies on polynucleotides. XCVI.
  Repair replications of short synthetic DNA's as
  catalyzed by DNA polymerases.