Cycle Sequencing

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Cycle Sequencing
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Broad and Long Term Objective

• To characterize a single clone from an
  Emiliania huxleyi cDNA library using sequence
  analysis

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Research Plan
Preparation of Competent Cells and Bacterial
Transformation
Growth of Transformant and Plasmid MiniPrep
Cycle Sequencing
Sequence analysis
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Todays Laboratory Objectives

• To Sequence the E. huxleyi cDNA insert in the
  pMAB plasmid
• using cycle sequencing
• To learn how to interpret cycle sequencing data
• To learn how to characterize a DNA sequence using
  various web-based bioinformatics tools

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What is Cycle Sequencing?

• Based on the Sanger Dideoxy chain termination
  method
• DNA synthesis reaction whereby fluorescent
  dideoxynucleotides are incorportated into the
  newly replicated DNA by DNA polymerase in a
  primer extension reaction
• Thermal cycling reaction

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Primer Extension ReactionWhenever a
fluorescently labeled dideoxynucleotide is
incorporated chain termination occurs
DNA Sequencing Dideoxy Chain Termination Method
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DNA Polymerase

• an enzyme used in sequencing to extends the 3'
  end of a primer along a ssDNA template using
  dNTPs and ddNTPs
• DNA pol-1 cannot distinguish a between normal
  dideoxynucleotides (ddNTPs) and a chemically
  modified fluorescent ddNTPs
• Whether the enzyme incorporates by completmentary
  base pairing a dNTP or a fluorescent ddNTP
  depending on the concentration ratio of
  ddNTPs/dNTPs
• 4. Each time the enzyme place a ddNTP the
  sequence will be "terminated", because ddNTPs
  don't have a 3' end.

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Primer Extension Reaction Components

• DNA template
• Primer
• Fluorescently labeled ddNTPs
• dNTPs
• Buffer
• Amplitaq termal stable DNA Polymerase

In this example DNA polymerase will produce 21
terminated sequences
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Cycling Parameters

• Step 1 96 C for 1 minute hot start
• Step 2 96 C for 10 seconds denaturation
• Step 3 50 C for 14 seconds annealing
• Step 4 60 C for 4 minutes primer extension
• Step 5 Cycle back 24 times to Step 2
• Step 6 Hold at 4 until purification

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Capillary or PAGE Electrophoresis

• These 21 sequences are separated in a denaturing
  polyacrylamide electrophoresis. (denaturing, to
  separate the template).
• Each sequence will move in the gel as a function
  of its size, the shortest first.
• They will pass according to their sizes in front
  of a excitatory laser beam and a fluorescence
  detector. As a sequence passes through the
  excitatory laser beam, it fluoresces according to
  its terminal ddNTP. The successsion of the
  fluorescences is recorded.

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Instrumentation
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Reaction Products are Separated on a
Polyacrylamide Gel
Each ddNTP is labeled with a flourescent
molecule Cytosine-blue Guanosine-yellow
Adenine-Green Thymidine-Red Each different
colored bands represents a different sized
fragment of DNA, the last nucleotide of which is
represented by the dye
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Electrophoretogram