DNA Sequencing Scramble

1
DNA Sequencing Scramble

• Class instructions

2
In this lesson

•  
• DNA sequencing is a powerful method for analyzing
  DNA
• Ratio of chain terminators (ddNTPs) is important
• Importance of complementary base pairing
• DNA polymerase only works in the 5- 3 direction
• Electrophoresis separates DNA fragments by size
• Chain terminators stop the reaction visualize
  the result
• Why fluorescent labels are used more than
  radioisotopes
• Software (bioinformatics) is used to line up
  sequences

3
The devil is in the detail!

• The 5 prime and 3 prime ends of the bases must
  be round the right way!

IMPORTANT Do not take bases apart!!!
4
Correct base pairing is critical!

• Green (Guanine) pairs with yellow (Cytosine)

• Blue (Adenine) pairs with orange (Thymine)

5
Bases with white sugars are CHAIN TERMINATORS
or dideoxynucleotides (ddNTP)
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This is the DNA we are going to sequence
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This is the primer that will begin the reaction
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Annealed primer
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Automated sequencing

• A whole class activity
• There are four pots, one for each nucleotide,
  each with 15 normal and 5 ddNTPs
• Produce a copy of our template using the labeled
  nucleotides
• Align resulting products in one column to read
  sequence

10
The activity

• Each student takes it in turns to synthesise a
  new strand
• Randomly select a nucleotide from each pot
• Add them in the 5 to 3 direction
• If you pull out a ddNTP, your chain is finished,
  it is the next students turn

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The result - electrophoresis

• Line up all the fragments from longest to
  smallest in one column
• Read the sequence from the smallest fragment
  upwards

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Automated Sequencing Results
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Electrophoresis gel is read by a laser - printout
produced
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Sanger Sequencing

• Original method of sequencing
• Chain terminating nucleotides are radioactively
  labeled
• Sequencing takes place in four separate tubes,
  each with one ddNTP
• All four results are then run in four separate
  lanes on a gel, separated by size

15
The activity

• As before put the template at front of the class
• Denature the two strands and anneal the primer
• Then line up the automated sequencing products in
  size order using one lane per base.

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Sanger Sequencing Result - Electrophoresis

• Line up products in 4 columns one for each
  base

Read sequence in this direction
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How an electrophoresis gel would look
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The difference between two methods

• Original Sanger method requires four lanes
  because all fragments are the same colour.
• Radioactive labels hazardous
• Automated sequencing can be run in one lane
  because each fragment is coloured differently.
• Coloured labels safe