LIFESCI 7C Lecture Notes - Lecture 8: Shotgun Sequencing, Sequence Assembly, Dideoxynucleotide
Week 8
DNA sequencing makes use of the principles of DNA replication
● Techniques used to sequence DNA follow from our
understanding of DNA replication
● SANGER SEQUENCING METHOD: use of
dideoxynucleotides, which will stop growth of daughter strand →
chain terminator
○ dideoxynucleotide A nucleotide lacking both the 2′
and 3′ hydroxyl groups on the sugar ring.
○ chain terminator A term for a dideoxynucleotide,
which if incorporated into a growing daughter strand
stops strand growth because there is no hydroxyl
group to attack the incoming nucleotide.
○ Mixture of small amount of chain terminators and larger
amounts of the 4 nucleotides, DNA primer, DNA
template, DNA polymerase → can produce series of
interrupted daughter strands → gives us DNA sequence
○ Possible because free 3’ hydroxyl group is essential for
each step in elongation → site of nucleotide attack
● Each of the 4 dideoxynucleotides is chemially labelled w/
different fluorescent dye, so they can be distinguished ina
single reaction
● After DNA synthesis, daughter strands are separated by size w/
gel electrophoresis
○ Smallest migrate quickly → closer to the bottom → in
order of increasing size
○ Fluroescence detector @ bottom of the gell “reads” the
colors of the fragments as they exit the ge0l
○ → we get a trace (graph) of the fluorescent intensities
● Each sequencing rxn can determine sequence of about ~ nucleotide in template DNA
13.1 Genome Sequencing
Complete genome sequences are assembled from smaller pieces
● Even w/ recent advances, DNA sequencing data is obtained in short sequences (less than a few hundred nucs
long)
● How to sequence a long DNA? (like an entire chromosome?)
○ Break up single long DNA into small fragments
■ Fragments sequenced repeatedly to minimize error present in final genome and number/size of
gaps where genome sequence is incomplete
○ When sequences of a sufficient number of short fragments of genome have been obtained → sequence
assembly by computer programs
■ sequence assembly short nucleotide sequences of a long DNA molecule are arranged in the
correct order to generate the complete sequence.
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● Genomic DNA broken into short fragments of a few hundred base pairs → sequenced in
machines → sequences aligned by regions of overlap until complete sequence is known
■ Short sequenes assembled by matching regions of overlap between sequences → shotgun
sequencing
● → shotgun sequencing DNA sequencing method in which the sequenced fragments do
not originate from a particular gene or region, but from sites scattered randomly across
the molecule.
Sequences that are repeated complicate sequence assembly
● Overlaps between fragments must be long enough to both ensure the assembly of sequences is correct and to
determine from which strand of DNA (as DNA is double stranded) the short sequence originated
● repetitive DNA The collective term for repeated sequences of various types in eukaryotic genomes.
○ Dispersed repeats, tandem repeats, simple-sequence repeats
○ COMPLICATE SEQUENCING PROCESS
■ The repeated sequences may appear in many fragments.
■ Fragments from different regions of the chromosomes may appear identical if they contain the
same repeated sequence.
■ Regions of sequence overlap may occur between fragments not actually adjacent in the
genome.
■
○ Long repeated sequence are typically much LONGER than the short fragments sequenced by
automated sequency → may not be detected at all
■ If detected, there is no easy way knowing the # of copies of that repeat or where the repeat
originated
○ Short repeats (e.g. two nucleotides long) also troublesome → any single-stranded fragment w/
alternating AT can fold bakc upon itself to form double-stranded structure pairing A and T (more stable)
→ not easily sequenced
■ Any sequence that contains internal complementarity can form a foldback loop, e.g. 5’-
ATATATA-3’
Case 3: Why sequence your personal genome?
● Genome sequencing becoming increasingly cheap → ability to have personal genome sequenced
● Every person’s genome is unique w/ the exception of identical twins → fusion of unique egg and unique sperm
○ “Human genome” is actually a composite of sequences from different individuals but useless because
most of us share the same genes/regulatory regions, organized the same way on chromosomes
○ Detailed knowledge of own personal genome can be valuable → individual genomes can differ at
millions of nucleotide sites → can account for physical differences, help dredict susceptibility to disease
+ response to meds, etc.
■ → step towards personalized medicine
● personalized medicine An approach in which the treatment is matched to the patient,
not the disease; examination of an individual’s genome sequence, by revealing his or
her disease susceptibilities and drug sensitivities, allows treatments to be tailored to
that individual.
○ → genome info brings benefits but also raises ethical concerns and poses risks
to confidentiality and insurability
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Dna sequencing makes use of the principles of dna replication. Techniques used to sequence dna follow from our understanding of dna replication. Sanger sequencing method: use of dideoxynucleotides, which will stop growth of daughter strand chain terminator. Dideoxynucleotide a nucleotide lacking both the 2 and 3 hydroxyl groups on the sugar ring. Chain terminator a term for a dideoxynucleotide, which if incorporated into a growing daughter strand stops strand growth because there is no hydroxyl group to attack the incoming nucleotide. Mixture of small amount of chain terminators and larger amounts of the 4 nucleotides, dna primer, dna template, dna polymerase can produce series of interrupted daughter strands gives us dna sequence. Possible because free 3" hydroxyl group is essential for each step in elongation site of nucleotide attack. Each of the 4 dideoxynucleotides is chemially labelled w/ different fluorescent dye, so they can be distinguished ina single reaction.
