Techniques in Biotechnology - DNA Sequencing

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13 Terms

1
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What is DNA Sequencing?

DNA sequencing: 

  • Determines order of nucleotide bases 

2
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What happens during DNA Sequencing?

DNA Sequencing 

  • Phosphate + sugar + base = nucleotide 

  • During DNA sequencing, DNA polymerase assist in adding nucleotides to a template strand 

  • As each nucleotide is added, it bonds to the next at the hydroxyl group in the sugar/phosphate backbone. 

3
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Describe the process of DNA Sequencing

DNA Sequencing: Sanger Technique 

  1. DNA is heated so it denatures into single strands (DNA turns into template strand and complementary strand, template strand will be used: 

 

  1. Primer is added to the template strand, to provide a starting point for later nucleotide addition 

 

  1. 4 Reaction mixtures are set up. One will be used for each base type. 

 

  1. Template strand that will be sequenced, with the annealed primer, is added to each reaction mixture. Many copies are added to each mixture. 

 

  1. DNA polymerase is added. It will allow free nucleotides to be added to the template strand later. 

 

  1. Free nucleotides (dNTP's) of all types added to each reaction mixture and some radioactively labelled for later identification. 

 

dNTP = deoxy nucleoside triphosphate 

dATP = adenine 

dTTP = thymine 

dCTP = cytosine 

dGTP = guanine 

 

  1. Modified nucleotides (ddNTP's – dideoxy nucleotide triphosphate) added 

  • Only one type is added per reaction mixture 

  • These lack the hydroxyl group, so no more nucleotides can attach 

  • This terminates the chain when it is added to the template strand by the DNA polymerase 

  • This means that in the flask with the T added, the template strands will always terminate with a T. They will be different lengths depending on the point at which the ddTTp gets added. So the flask with the T added will end up with lots of different lengths of copied strand, each starting with the primer and ending with a T. 

 

  1. Gel electrophoresis used to separate bits of DNA according to size 

  • One reaction mixture per gel lane 

  • A current is run through the gel mixture and the negatively charged DNA strands move towards the positive terminal. 

  • The shortest strands move the fastest 

  • The strands show up as bands on the gel 

  • Can then determine sequence as we know which modified nucleotide terminates each chain 

4
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What is the 1st step in the Sanger Technique?

  1. DNA is heated so it denatures into single strands (DNA turns into template strand and complementary strand, template strand will be used: 

5
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What is the 2nd step in the Sanger Technique?

  1. Primer is added to the template strand, to provide a starting point for later nucleotide addition 

6
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What is the 3rd step in the Sanger Technique?

  1. 4 Reaction mixtures are set up. One will be used for each base type. 

7
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What is the 4th step in the Sanger Technique?

  1. Template strand that will be sequenced, with the annealed primer, is added to each reaction mixture. Many copies are added to each mixture. 

8
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What is the 5th step in the Sanger Technique?

  1. DNA polymerase is added. It will allow free nucleotides to be added to the template strand later. 

9
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What is the 6th step in the Sanger Technique?

  1. Free nucleotides (dNTP's) of all types added to each reaction mixture and some radioactively labelled for later identification. 

10
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What is the 7th step in the Sanger Technique?

  1. Modified nucleotides (ddNTP's – dideoxy nucleotide triphosphate) added 

  • Only one type is added per reaction mixture 

  • These lack the hydroxyl group, so no more nucleotides can attach 

  • This terminates the chain when it is added to the template strand by the DNA polymerase 

  • This means that in the flask with the T added, the template strands will always terminate with a T. They will be different lengths depending on the point at which the ddTTp gets added. So the flask with the T added will end up with lots of different lengths of copied strand, each starting with the primer and ending with a T. 

11
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What is the last step in the Sanger Technique?

  1. Gel electrophoresis used to separate bits of DNA according to size 

  • One reaction mixture per gel lane 

  • A current is run through the gel mixture and the negatively charged DNA strands move towards the positive terminal. 

  • The shortest strands move the fastest 

  • The strands show up as bands on the gel 

  • Can then determine sequence as we know which modified nucleotide terminates each chain 

12
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What are the uses of DNA Sequencing?

  • DNA Sequencing can be used to: 

  • Compare DNA sequences to detect changed alleles 

  • Readily identify point mutations, small insertions, and deletions by comparing. 

  • Track sequences over time to show evolutionary change 

  • Diseases that can be determined by DNA sequencing 

    • Sickle-cell anaemia 

    • Cystic fibrosis 

    • Some hereditary forms of cancer 

  • Also used for paternity tests and forensic identification 

13
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What are some DNA profiling techniques?

DNA Profiling Techniques 

  • DNA can be used as a means of identification 

  • Specific restriction enzymes cut DNA at specific base sequences, leaving various lengths 

  • Lengths vary between individuals as base sequences differ between individuals