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Lecture 8: DNA Sequencing - Coggle Diagram
Lecture 8: DNA Sequencing
Requirements
Denatured to serve as template (ie. ssDNA)
in vitro system for DNA replication / polymerisation
DNA fragments - from PCR or cloning
high through-put and accurate method
Dideoxy Chain Termination Method (Sanger Method)
N3 replaces OH at C3 à can not join to C5
Modified nucleotides used as drugs
Steps
DNA is synthesized in vitro in a mixture that contains ssDNA, DNA polymerase and the 4 dNTPs
If a ddNTP analog of one dNTP is present, it can become incorporated into growing chain
Because it lacks the 3’OH, the addition of the next dNTP is impossible
DNA chain terminates
Repeat for all dNTPs
Sequencing I
dsDNA -> ssDNA
one ssDNA used for sequencing
4 different ddNTPs are used in 4 separate DNA synthesis reactions
Each reaction produces a set of DNA copies that terminate at different points in the sequence
Sequencing II
Products separated by Gel electrophoresis in 4 lanes
Newly synthesized strands are detected by a label- usually isotope P32 or such
In each lane, the bands represent fragments that have terminated at a given nucleotide but at different positions in the DNA
Sequencing
Start from bottom, read across 4 lanes
REMEMBER: sequence is complementary to template strand
Modification of Sanger Method
Primer no longer radioactively labelled
4 ddNTPs each carry a different coloured fluorescent tag
reaction performed in single tube
fragments separated in single lane
automatic laser scanning and recording
Sequencing
Applications
Verify what you made is what you thought it was
primer bound correctly
NGS
no base insertions, deletions, substitutions
Primer Walking
sequence as if shorter fragment
Design new primer complementary to the final 20 bases of the known sequence
Use universal primers to identify the first 1000 (approx.) bases
– Keep "walking" along the sequence
Shotgun Sequencing
works on shorter strands
Longer sequence divided into smaller ones
chain termination method
multiple overlapping reads
Use computer program
Enable full genome
Sanger VS NGS
Sanger
sequence single gene
1-100 amplicon targets
sequence 96 samples at a time
Microbial identification
Fragment analysis
Microsatellite or STR analysis
NGS confirmation
NGS
more than100 genes at a time
finding novel variants by expanding the no. of target sequenced in a single run
low input amounts of starting material
sequencing microbial genomes for pathogen subtyping to enable research of critical outbreak situations
The Human Genome
Genesweep
betting how many genes humans have
Where are genes?
one strand serves as template, but gene may be diff depending on which strand
The human chromosome
Large region of non-coding DNA
RNA processing (intron/exon)
Related genes on different chromosomes
How to determine function of gene?
Comparative genomics
Relationships within a family of genes: sequence homology
genetics/biochemistry
First member of a gene family: mutation analysis
Conservation of genes
critical genes highly conserved
Model organisms
Applications of HGP
Genome organisation
what % is proteins/repeats/etc?
Identify Gene Functions
study "knockout" mice
Population genetics
compare w diff diff and those in history
Product development
pharmacy
cost of DNA sequencing going down, personalised medicine?
Evolutionary Studies
compare genomes of diff species
Diagnostic
identify gene causing gene
GENOMICS
Last decade, massively parallel sequencing technologies
Ability to estimate relatedness among individuals using dense genetic markers
High throughput, relatively cheap, can
genotype many individuals simultaneously
Non-model organisms