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Wize University Biology Textbook > Lab Techniques for Biology

Whole Genome Sequencing

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DNA Sequencing

So you want to sequence a genome… where do you start?
  • Purification – isolating DNA from an organism or cells.
  • Fragmentation – breaking up the genome into smaller fragments to be sequenced.
  • Amplification – make more copies of our fragments.
  • Sequence fragments – assigning nucleotide bases to our fragments.
  • Re-assembly of fragments – put all the sequence fragments back together to create a continuous sequence.

Types of Sequencing

  1. Sanger Sequencing
  • Also known as the dideoxy method = dideoxynucleotides have an H on the 3’ carbon of the sugar-phosphate backbone instead of OH attached to a fluorescent tag.
  • How it works:
  • Amplified fragments are replicated again in the presence of the ddNTPs.
  • Replication enzyme uses normal nucleotide bases and then randomly inserts a ddNTP that stops replication.
  • Fluorescently labeled sequencing fragments are run through electrophoresis to separate them by size.
  • The fluorescent tags are all different colors for their respective nucleotide base, need to subject gel to fluorescent filter.
  • Order of sequence = shortest to longest (fragment that travelled most to fragment that travelled least).
  • Most useful in sequencing single genes.
Photo by Tdpaustian / CC BY

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  1. Whole Genome Shotgun Sequencing
  • Useful in sequencing the entire genome.
  • How it works:
  • Isolate genome DNA and break up into overlapping fragments.
  • Do this by using 2 DNA samples that have been digested by 2 different restriction enzymes.
  • Clone each fragment into a plasmid vector.
  • Sequence the genomic DNA fragment in each clone.
  • Use computer programs to re-align sequences based on areas of overlapping.
Adapted from photo by Commins, J., Toft, C., Fares, M. A. / CC BY

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  1. Next Generation Sequencing
Allows one sequencing instrument to carry out billions of sequencing reactions at the same time.
  • Useful for multiple gene sequencing of whole genome sequencing.
  • How it works:
  • DNA fragments prepared by adding double stranded linkers to both ends of the fragment.
  • Fragments are amplified using PCR primers that are complementary to linkers.
  • Primers are then used to covalently bond the DNA fragments to a solid surface in a tight cluster.
  • The fragments are analyzed using a special microscope that detects which fluorescent labeled base is added to the DNA template by polymerase.
Photo by DMLapato / CC BY
Extra Practice
PCR is a method which has been used to...
You are studying a particular mutant fly which has misshapen wings. You believe you know which gene has been mutated to cause the misshapen wing phenotype but you do not know exactly what the mutation is (i.e. point mutation? Deletion? Insertion?) What is the best experimental protocol to determine the sequence of the mutated gene?
Given the Sanger sequencing results, what is the correct template DNA sequence - Practice Question
Given the following Sanger sequencing results, which of the following is the correct sequence of the template DNA?
Given the Sanger sequencing results, what is the correct template DNA sequence - Practice Question
Given the following Sanger sequencing results, which of the following is the correct sequence of the template DNA?
Practice: DNA Techniques
You are studying a particular mutant fly which has misshapen wings. You believe you know which gene has been mutated to cause the misshapen wing phenotype but you do not know exactly what the mutation is (i.e. point mutation? Deletion? Insertion?) What is the best experimental protocol to determine the sequence of the mutated gene?
Which is the forward and reverse primer of a PCR reaction given the DNA sequence - Practice Question
You have 2 DNA primers that are supposed to be able to amplify the below DNA fragment. You forgot to write down the 5' to 3' direction on the primer tubes, so you are unsure which is the forward and which is the backward primer. Given the primer sequences shown from 5' to 3', which is the forward and which is the reverse?
Given the Sanger sequencing results, what is the correct template DNA sequence - Practice Question
Given the following Sanger sequencing results, which of the following is the correct sequence of the template DNA?
Given the following banding pattern from a Sanger sequencing experiment, what would be the first 10 bases of the sequence of the template DNA being sequenced?
As a famous molecular biologist, you are asked to sequence a novel gene found in the DNA of a new species of insect. Which of the following things is NOT required for you to successfully sequence this gene using Sanger sequencing?
Which of the following statements about Sanger DNA sequencing is correct?
PCR is a method which has been used to...
You are studying a particular mutant fly which has misshapen wings. You believe you know which gene has been mutated to cause the misshapen wing phenotype but you do not know exactly what the mutation is (i.e. point mutation? Deletion? Insertion?) What is the best experimental protocol to determine the sequence of the mutated gene?
_____________________ is a method by which billions of DNA template fragments are bound to a solid surface via linkers and then recorded as DNA polymerase adds fluorescently tagged dNTPs to the template strand.
You suspect the presence of Simple Sequence Length Polymorphism in a piece of genetic material, which of the following techniques is most suitable to confirm and count the number of repeats?