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Lecture 14

MCDB 2150 Lecture 14: Class 14- Genetic Analysis- DNA sequencing

Molecular Cell & Developmental Biology
Course Code
MCDB 2150
Jennifer Knight

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Unit 2 study guide
Class 14: Genetic Analysis: DNA sequencing
Next generation sequencing: sanger sequencing, pyrosequencing, sequencing by synthesis, sequencing by
Sanger Sequencing
o Utilizes DNA polymerase to generate a complementary copy to a single stranded DNA template
o In each reaction, a primer complementary to the template initiates a DNA synthesis reaction from the 3’
end adding nucleotides
o Contains a mixture of four di-deoxyucleotides, one for each base
Lack 3’ OH group required for further DNA extension resulting in terminated nucleotide
Each has a fluorescent dye allowing for automatic detection of the DNA sequence
o As a result, many copies of different sized DNA fragments are generated in each reaction, terminated at
all the nucleotide positions of the template molecule by ddNTPs
o The reaction mixtures are loaded on the sequencing machine into capillary electrophoresis tubes
o Electrophoresis is used to separate the molecules by size, the smaller fragments travel farther
o Read through the fluorescent emission using a laser and detector
DNA sequencing is the process of determining the precise order of nucleotides with a DNA molecule
o We can only completely characterize an organism at the molecular level when the sequence is known
o Enhances understanding of the genome structure, function, and mechanisms of regulation
Sanger sequencing:
o 1977 Fredrick Sanger developed the method of
DNA sequencing known as the “Sanger Method”
Di-deoxynucleotide chain termination
o Di-deoxynucleotide triphosphates (ddNTPs)
terminate the sequencing reaction at different
positions in the growing DNA strands because
they lack a hydroxyl (-OH) group at their 3’ end
o Components: DNA template, DNA primer, DNA
polymerase, dNTPS (A, T, G, C), ddNTPs
o Reading the gel: sequencing gels are read from
bottom to top (5’ to 3’)
Upgraded Sanger sequencing: how it works
o 1. Reaction mixture:
Primer and DNA template
DNA polymerase
ddNTPs with fluorochromes
o 2. Primer elongation and chain termination
o 3. Capillary gel electrophoresis separation of DNA fragments
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