Sanger Sequencing

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Last updated 3:35 PM on 10/15/26
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8 Terms

1
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Why was Maxam-Gilbert sequencing used before sanger sequencing but not anymore?

  • Would cleave DNA after modifying it and then run a gel. So didnt need DNA polymerase or primers


  • Hazardous chemicals

  • Complex and labour intensive

  • difficult to scale up


<ul><li><p>Would cleave DNA after modifying it and then run a gel. So didnt need DNA polymerase or primers</p></li></ul><p></p><ul><li><p>Hazardous chemicals</p></li><li><p>Complex and labour intensive</p></li><li><p>difficult to scale up</p></li></ul><p></p>
2
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Why was sanger sequencing preferred after it came out?

Easier to automate and produced longer more accurate reads

<p>Easier to automate and produced longer more accurate reads</p>
3
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Whats the difference between a dNTP and a ddNTP?

A dNTP has an OH group on the sugar so chain can continue building

a ddNTP has a H instead so stops the chain from elongating. Also fluorescently labelled so nucleotide can be detected

<p>A dNTP has an OH group on the sugar so chain can continue building</p><p>a ddNTP has a H instead so stops the chain from elongating. Also fluorescently labelled so nucleotide can be detected</p>
4
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What do you need to add into a sanger sequencing reaction

  • ONE primer

  • dNTPs

  • ddNTPs

  • DNA template

  • DNA polymerase

  • buffer


<ul><li><p>ONE primer</p></li><li><p>dNTPs</p></li><li><p>ddNTPs</p></li><li><p>DNA template</p></li><li><p>DNA polymerase</p></li><li><p>buffer</p></li></ul><p></p>
5
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How does sanger sequencing work?

Similar to PCR, polymerase will elongate complementary strand with dNTPs until ddNTPs is incorporated, over and over again, produces variable length frgaments that can be separated by capillary electrophoresis

<p>Similar to PCR, polymerase will elongate complementary strand with dNTPs until ddNTPs is incorporated, over and over again, produces variable length frgaments that can be separated by capillary electrophoresis</p>
6
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how did people use to detect the ddNTPs

run 4 separate reactions and do 4 separate gels and detect radioactive ddNTPs

<p>run 4 separate reactions and do 4 separate gels and detect radioactive ddNTPs</p>
7
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What are the strengths of sanger sequencing?

  • VERY accurate

  • Long-ish base reads

  • Very easy to use


<ul><li><p>VERY accurate</p></li><li><p>Long-ish base reads</p></li><li><p>Very easy to use</p></li></ul><p></p>
8
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What are the weaknesses of sanger sequencing?

  • Low throughput

  • Costly

  • Not good with microsatellites


<ul><li><p>Low throughput</p></li><li><p>Costly</p></li><li><p>Not good with microsatellites</p></li></ul><p></p>